Binding proteins enhance specific uptake rate by increasing the substrate-transporter encounter rate.

PubMed

Bosdriesz, Evert; Magnúsdóttir, Stefanía; Bruggeman, Frank J; Teusink, Bas; Molenaar, Douwe

2015-06-01

Microorganisms rely on binding-protein assisted, active transport systems to scavenge for scarce nutrients. Several advantages of using binding proteins in such uptake systems have been proposed. However, a systematic, rigorous and quantitative analysis of the function of binding proteins is lacking. By combining knowledge of selection pressure and physiochemical constraints, we derive kinetic, thermodynamic, and stoichiometric properties of binding-protein dependent transport systems that enable a maximal import activity per amount of transporter. Under the hypothesis that this maximal specific activity of the transport complex is the selection objective, binding protein concentrations should exceed the concentration of both the scarce nutrient and the transporter. This increases the encounter rate of transporter with loaded binding protein at low substrate concentrations, thereby enhancing the affinity and specific uptake rate. These predictions are experimentally testable, and a number of observations confirm them. © 2015 FEBS.

A universal small molecule, inorganic phosphate, restricts the substrate specificity of Dicer-2 in small RNA biogenesis

PubMed Central

Fukunaga, Ryuya; Zamore, Phillip D

2014-01-01

The enzyme Dicer is central to the production of small silencing RNAs such as microRNAs (miRNAs) and small interfering RNAs (siRNAs). Like other insects, Drosophila melanogaster uses different Dicers to make siRNAs and miRNAs: Dicer-1 produces miRNAs from pre-miRNAs, whereas Dicer-2 generates siRNAs from long double-stranded RNA (dsRNA). How do the 2 Dicers achieve their substrate specificity? Here, we review recent findings that inorganic phosphate restricts the substrate specificity of Dicer-2 to long dsRNA. Inorganic phosphate inhibits Dicer-2 from binding and cleaving pre-miRNAs, without affecting the processing of long dsRNA. Crystal structures of a fragment of human Dicer in complex with an RNA duplex identify a phosphate-binding pocket that recognizes both the 5′-monophosphate of a substrate RNA and inorganic phosphate. We propose that inorganic phosphate occupies the phosphate-binding pocket in the fly Dicer-2, blocking binding of pre-miRNA and restricting pre-miRNA processing to Dicer-1. Thus, a small molecule can alter the substrate specificity of a nucleic acid-processing enzyme. PMID:24787225

GSK3 controls axon growth via CLASP-mediated regulation of growth cone microtubules

PubMed Central

Hur, Eun-Mi; Saijilafu; Lee, Byoung Dae; Kim, Seong-Jin; Xu, Wen-Lin; Zhou, Feng-Quan

2011-01-01

Suppression of glycogen synthase kinase 3 (GSK3) activity in neurons yields pleiotropic outcomes, causing both axon growth promotion and inhibition. Previous studies have suggested that specific GSK3 substrates, such as adenomatous polyposis coli (APC) and collapsin response mediator protein 2 (CRMP2), support axon growth by regulating the stability of axonal microtubules (MTs), but the substrate(s) and mechanisms conveying axon growth inhibition remain elusive. Here we show that CLIP (cytoplasmic linker protein)-associated protein (CLASP), originally identified as a MT plus end-binding protein, displays both plus end-binding and lattice-binding activities in nerve growth cones, and reveal that the two MT-binding activities regulate axon growth in an opposing manner: The lattice-binding activity mediates axon growth inhibition induced by suppression of GSK3 activity via preventing MT protrusion into the growth cone periphery, whereas the plus end-binding property supports axon extension via stabilizing the growing ends of axonal MTs. We propose a model in which CLASP transduces GSK3 activity levels to differentially control axon growth by coordinating the stability and configuration of growth cone MTs. PMID:21937714

Mevalonate 5-diphosphate mediates ATP binding to the mevalonate diphosphate decarboxylase from the bacterial pathogen Enterococcus faecalis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Chun-Liang; Mermoud, James C.; Paul, Lake N.

The mevalonate pathway produces isopentenyl diphosphate (IPP), a building block for polyisoprenoid synthesis, and is a crucial pathway for growth of the human bacterial pathogen Enterococcus faecalis. The final enzyme in this pathway, mevalonate diphosphate decarboxylase (MDD), acts on mevalonate diphosphate (MVAPP) to produce IPP while consuming ATP. This essential enzyme has been suggested as a therapeutic target for the treatment of drug-resistant bacterial infections. Here, we report functional and structural studies on the mevalonate diphosphate decarboxylase from E. faecalis (MDDEF). The MDDEF crystal structure in complex with ATP (MDDEF–ATP) revealed that the phosphate-binding loop (amino acids 97–105) is notmore » involved in ATP binding and that the phosphate tail of ATP in this structure is in an outward-facing position pointing away from the active site. This suggested that binding of MDDEF to MVAPP is necessary to guide ATP into a catalytically favorable position. Enzymology experiments show that the MDDEF performs a sequential ordered bi-substrate reaction with MVAPP as the first substrate, consistent with the isothermal titration calorimetry (ITC) experiments. On the basis of ITC results, we propose that this initial prerequisite binding of MVAPP enhances ATP binding. In summary, our findings reveal a substrate-induced substrate-binding event that occurs during the MDDEF-catalyzed reaction. The disengagement of the phosphate-binding loop concomitant with the alternative ATP-binding configuration may provide the structural basis for antimicrobial design against these pathogenic enterococci.« less

UFD4 lacking the proteasome-binding region catalyses ubiquitination but is impaired in proteolysis.

PubMed

Xie, Youming; Varshavsky, Alexander

2002-12-01

The ubiquitin system recognizes degradation signals of protein substrates through E3-E2 ubiquitin ligases, which produce a substrate-linked multi-ubiquitin chain. Ubiquitinated substrates are degraded by the 26S proteasome, which consists of the 20S protease and two 19S particles. We previously showed that UBR1 and UFD4, two E3 ligases of the yeast Saccharomyces cerevisiae, interact with specific proteasomal subunits. Here we advance this analysis for UFD4 and show that it interacts with RPT4 and RPT6, two subunits of the 19S particle. The 201-residue amino-terminal region of UFD4 is essential for its binding to RPT4 and RPT6. UFD4(DeltaN), which lacks this N-terminal region, adds ubiquitin to test substrates with apparently wild-type activity, but is impaired in conferring short half-lives on these substrates. We propose that interaction of a targeted substrate with the 26S proteasome involves contacts of specific proteasomal subunits with the substrate-bound ubiquitin ligase, with the substrate-linked multi-ubiquitin chain and with the substrate itself. This multiple-site binding may function to slow down dissociation of the substrate from the proteasome and to facilitate the unfolding of substrate through ATP-dependent movements of the chaperone subunits of the 19S particle.

Prediction of the binding mode of N2-phenylguanine derivative inhibitors to herpes simplex virus type 1 thymidine kinase

NASA Astrophysics Data System (ADS)

Gaudio, Anderson Coser; Takahata, Yuji; Richards, William Graham

1998-01-01

The probable binding mode of the herpes simplex virus thymidine kinase (HSV1 TK) N2-[substituted]-phenylguanine inhibitors is proposed. A computational experiment was designed to check some qualitative binding parameters and to calculate the interaction binding energies of alternative binding modes of N2-phenylguanines. The known binding modes of the HSV1 TK natural substrate deoxythymidine and one of its competitive inhibitors ganciclovir were used as templates. Both the qualitative and quantitative parts of the computational experiment indicated that the N2-phenylguanine derivatives bind to the HSV1 TK active site in the deoxythymidine-like binding mode. An experimental observation that N2-phenylguanosine derivatives are not phosphorylated during the interaction with the HSV1 TK gives support to the proposed binding mode.

A kinetic and thermodynamic framework for the Azoarcus group I ribozyme reaction

PubMed Central

Gleitsman, Kristin R.

2014-01-01

Determination of quantitative thermodynamic and kinetic frameworks for ribozymes derived from the Azoarcus group I intron and comparisons to their well-studied analogs from the Tetrahymena group I intron reveal similarities and differences between these RNAs. The guanosine (G) substrate binds to the Azoarcus and Tetrahymena ribozymes with similar equilibrium binding constants and similar very slow association rate constants. These and additional literature observations support a model in which the free ribozyme is not conformationally competent to bind G and in which the probability of assuming the binding-competent state is determined by tertiary interactions of peripheral elements. As proposed previously, the slow binding of guanosine may play a role in the specificity of group I intron self-splicing, and slow binding may be used analogously in other biological processes. The internal equilibrium between ribozyme-bound substrates and products is similar for these ribozymes, but the Azoarcus ribozyme does not display the coupling in the binding of substrates that is observed with the Tetrahymena ribozyme, suggesting that local preorganization of the active site and rearrangements within the active site upon substrate binding are different for these ribozymes. Our results also confirm the much greater tertiary binding energy of the 5′-splice site analog with the Azoarcus ribozyme, binding energy that presumably compensates for the fewer base-pairing interactions to allow the 5′-exon intermediate in self splicing to remain bound subsequent to 5′-exon cleavage and prior to exon ligation. Most generally, these frameworks provide a foundation for design and interpretation of experiments investigating fundamental properties of these and other structured RNAs. PMID:25246656

Structure of an XPF endonuclease with and without DNA suggests a model for substrate recognition

PubMed Central

Newman, Matthew; Murray-Rust, Judith; Lally, John; Rudolf, Jana; Fadden, Andrew; Knowles, Philip P; White, Malcolm F; McDonald, Neil Q

2005-01-01

The XPF/Mus81 structure-specific endonucleases cleave double-stranded DNA (dsDNA) within asymmetric branched DNA substrates and play an essential role in nucleotide excision repair, recombination and genome integrity. We report the structure of an archaeal XPF homodimer alone and bound to dsDNA. Superposition of these structures reveals a large domain movement upon binding DNA, indicating how the (HhH)2 domain and the nuclease domain are coupled to allow the recognition of double-stranded/single-stranded DNA junctions. We identify two nonequivalent DNA-binding sites and propose a model in which XPF distorts the 3′ flap substrate in order to engage both binding sites and promote strand cleavage. The model rationalises published biochemical data and implies a novel role for the ERCC1 subunit of eukaryotic XPF complexes. PMID:15719018

Crystallography Coupled with Kinetic Analysis Provide Mechanistic Underpinnings of a Nicotine-Degrading Enzyme.

PubMed

Tararina, Margarita A; Xue, Song; Smith, Lauren C; Muellers, Samantha N; Miranda, Pedro O; Janda, Kim D; Allen, Karen N

2018-05-29

Nicotine oxidoreductase (NicA2) is a bacterial flavoenzyme, which catalyzes the first step of nicotine catabolism by oxidizing S-nicotine into N-methyl-myosmine. Its use has been proposed as a biotherapeutic for nicotine addiction due to its nanomolar substrate binding affinity. The first crystal structure of NicA2 has been reported, establishing NicA2 as a member of the monoamine oxidase (MAO) family. However, substrate specificity and structural determinants of substrate binding/catalysis have not been explored. Herein, analysis of pH-rate profile, single-turnover kinetics and binding data establish that pH does not significantly affect catalytic rate and product release is not rate limiting. The X-ray crystal structure of NicA2 with S-nicotine refined to 2.65 Å resolution reveals a hydrophobic binding site with a solvent exclusive cavity. Hydrophobic interactions predominantly orient the substrate, promoting the binding of a deprotonated species and supporting a hydride-transfer mechanism. Notably, NicA2 showed no activity against neurotransmitters oxidized by the two isoforms of human MAO. To further probe the substrate range of NicA2, enzyme activity was evaluated using a series of substrate analogs, indicating that S-nicotine is the optimal substrate and substitutions within the pyridyl ring abolish NicA2 activity. Moreover, mutagenesis and kinetic analysis of active-site residues reveal that removal of a hydrogen bond between the pyridyl ring of S-nicotine and the hydroxyl group of T381 has a 10-fold effect on KM, supporting the role of this bond in positioning the catalytically competent form of the substrate. Together, crystallography combined with kinetic analysis provide a deeper understanding of this enzyme's remarkable specificity.

Structures and Mechanism of the Monoamine Oxidase Family

PubMed Central

Gaweska, Helena; Fitzpatrick, Paul F.

2011-01-01

Members of the monoamine oxidase family of flavoproteins catalyze the oxidation of primary and secondary amines, polyamines, amino acids, and methylated lysine side chains in proteins. The enzymes have similar overall structures, with conserved FAD-binding domains and varied substrate-binding sites. Multiple mechanisms have been proposed for the catalytic reactions of these enzymes. The present review compares the structures of different members of the family and the various mechanistic proposals. PMID:22022344

Lipid Microarray Biosensor for Biotoxin Detection.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Singh, Anup K.; Throckmorton, Daniel J.; Moran-Mirabal, Jose C.

2006-05-01

We present the use of micron-sized lipid domains, patterned onto planar substrates and within microfluidic channels, to assay the binding of bacterial toxins via total internal reflection fluorescence microscopy (TIRFM). The lipid domains were patterned using a polymer lift-off technique and consisted of ganglioside-populated DSPC:cholesterol supported lipid bilayers (SLBs). Lipid patterns were formed on the substrates by vesicle fusion followed by polymer lift-off, which revealed micron-sized SLBs containing either ganglioside GT1b or GM1. The ganglioside-populated SLB arrays were then exposed to either Cholera toxin subunit B (CTB) or Tetanus toxin fragment C (TTC). Binding was assayed on planar substrates bymore » TIRFM down to 1 nM concentration for CTB and 100 nM for TTC. Apparent binding constants extracted from three different models applied to the binding curves suggest that binding of a protein to a lipid-based receptor is strongly affected by the lipid composition of the SLB and by the substrate on which the bilayer is formed. Patterning of SLBs inside microfluidic channels also allowed the preparation of lipid domains with different compositions on a single device. Arrays within microfluidic channels were used to achieve segregation and selective binding from a binary mixture of the toxin fragments in one device. The binding and segregation within the microfluidic channels was assayed with epifluorescence as proof of concept. We propose that the method used for patterning the lipid microarrays on planar substrates and within microfluidic channels can be easily adapted to proteins or nucleic acids and can be used for biosensor applications and cell stimulation assays under different flow conditions. KEYWORDS. Microarray, ganglioside, polymer lift-off, cholera toxin, tetanus toxin, TIRFM, binding constant.4« less

Self-energy effect and Coulomb potential modulation of the exciton in monolayer MoS2 on polar substrate

NASA Astrophysics Data System (ADS)

Wang, Zi-Wu; Xiao, Yao; Li, Run-Ze; Li, Wei-Ping; Li, Zhi-Qing

2017-11-01

We theoretically investigate the correction of exciton binding energy in monolayer MoS2 resulting from the exciton couples with surface optical (SO) phonons induced by polar substrate. The total correction of binding energy can be divided into the self-energy effect and modification of Coulomb potential using the unitary transformation method. We find that both the self-energy and Coulomb potential vary from tens of meV to several hundreds of meV depending on the cut-off wave vector of SO phonon modes, polarizability of substrate materials and internal distance between the monolayer MoS2 and polar substrate. An effective Coulomb potential is obtained by combining the modified term into the Coulomb potential. This potentially could be widely used in various two-dimensional materials. Our theoretical results not only propose the ways to externally control the exciton binding energy in experiment, but also enrich the understanding of the exciton properties in the dielectric environment.

STN1 OB Fold Mutation Alters DNA Binding and Affects Selective Aspects of CST Function

PubMed Central

Bhattacharjee, Anukana; Stewart, Jason; Chaiken, Mary; Price, Carolyn M.

2016-01-01

Mammalian CST (CTC1-STN1-TEN1) participates in multiple aspects of telomere replication and genome-wide recovery from replication stress. CST resembles Replication Protein A (RPA) in that it binds ssDNA and STN1 and TEN1 are structurally similar to RPA2 and RPA3. Conservation between CTC1 and RPA1 is less apparent. Currently the mechanism underlying CST action is largely unknown. Here we address CST mechanism by using a DNA-binding mutant, (STN1 OB-fold mutant, STN1-OBM) to examine the relationship between DNA binding and CST function. In vivo, STN1-OBM affects resolution of endogenous replication stress and telomere duplex replication but telomeric C-strand fill-in and new origin firing after exogenous replication stress are unaffected. These selective effects indicate mechanistic differences in CST action during resolution of different replication problems. In vitro binding studies show that STN1 directly engages both short and long ssDNA oligonucleotides, however STN1-OBM preferentially destabilizes binding to short substrates. The finding that STN1-OBM affects binding to only certain substrates starts to explain the in vivo separation of function observed in STN1-OBM expressing cells. CST is expected to engage DNA substrates of varied length and structure as it acts to resolve different replication problems. Since STN1-OBM will alter CST binding to only some of these substrates, the mutant should affect resolution of only a subset of replication problems, as was observed in the STN1-OBM cells. The in vitro studies also provide insight into CST binding mechanism. Like RPA, CST likely contacts DNA via multiple OB folds. However, the importance of STN1 for binding short substrates indicates differences in the architecture of CST and RPA DNA-protein complexes. Based on our results, we propose a dynamic DNA binding model that provides a general mechanism for CST action at diverse forms of replication stress. PMID:27690379

Synthesis, crystal structure and investigation of mononuclear copper(II) and zinc(II) complexes of a new carboxylate rich tripodal ligand and their interaction with carbohydrates in alkaline aqueous solution

PubMed Central

Stewart, Christopher D.; Pedraza, Mayra; Arman, Hadi; Fan, Hua-Jun; Schilling, Eduardo Luiz; Szpoganicz, Bruno; Musie, Ghezai T.

2016-01-01

A new carboxylate rich asymmetric tripodal ligand, N-[2-carboxybenzomethyl]-N-[carboxymethyl]-β-alanine (H3camb), and its di-copper(II), (NH4)2[1]2, and di-zinc(II), ((CH3)4 N)2[2]2, complexes have been synthesized as carbohydrate binding models in aqueous solutions. The ligand and complexes have been fully characterized using several techniques, including single crystal X-ray diffraction. The interactions of (NH4)2[1]2 and ((CH3)4 N)2[2]2 with D-glucose, D-mannose, D-xylose and xylitol in aqueous alkaline media were investigated using UV–Vis and 13C-NMR spectroscopic techniques, respectively. The molar conductance, NMR and ESI–MS studies indicate that the complexes dissociate in solution to produce the respective complex anions, 1− and 2−. Complexes 1− and 2− showed chelating ability towards the naturally abundant and biologically relevant sugars, D-glucose, D-mannose, D-xylose, and xylitol. The complex ions bind to one molar equivalent of the sugars, even in the presence of stoichiometric excess of the substrates, in solution. Experimentally obtained spectroscopic data and computational results suggest that the substrates bind to the metal center in a bidentate fashion. Apparent binding constant values, pKapp, between the complexes and the substrates were determined and a specific mode of substrate binding is proposed. The pKapp and relativistic density functional theory (DFT) calculated Gibbs free energy values indicate that D-mannose displayed the strongest interaction with the complexes. Syntheses, characterizations, detailed substrate binding studies using spectroscopic techniques, single crystal X-ray diffraction and geometry optimizations of the complex-substrates with DFT calculations are also reported. PMID:25969174

ATP binding and hydrolysis by Saccharomyces cerevisiae Msh2-Msh3 are differentially modulated by mismatch and double-strand break repair DNA substrates.

PubMed

Kumar, Charanya; Eichmiller, Robin; Wang, Bangchen; Williams, Gregory M; Bianco, Piero R; Surtees, Jennifer A

2014-06-01

In Saccharomyces cerevisiae, Msh2-Msh3-mediated mismatch repair (MMR) recognizes and targets insertion/deletion loops for repair. Msh2-Msh3 is also required for 3' non-homologous tail removal (3'NHTR) in double-strand break repair. In both pathways, Msh2-Msh3 binds double-strand/single-strand junctions and initiates repair in an ATP-dependent manner. However, we recently demonstrated that the two pathways have distinct requirements with respect to Msh2-Msh3 activities. We identified a set of aromatic residues in the nucleotide binding pocket (FLY motif) of Msh3 that, when mutated, disrupted MMR, but left 3'NHTR largely intact. One of these mutations, msh3Y942A, was predicted to disrupt the nucleotide sandwich and allow altered positioning of ATP within the pocket. To develop a mechanistic understanding of the differential requirements for ATP binding and/or hydrolysis in the two pathways, we characterized Msh2-Msh3 and Msh2-msh3Y942A ATP binding and hydrolysis activities in the presence of MMR and 3'NHTR DNA substrates. We observed distinct, substrate-dependent ATP hydrolysis and nucleotide turnover by Msh2-Msh3, indicating that the MMR and 3'NHTR DNA substrates differentially modify the ATP binding/hydrolysis activities of Msh2-Msh3. Msh2-msh3Y942A retained the ability to bind DNA and ATP but exhibited altered ATP hydrolysis and nucleotide turnover. We propose that both ATP and structure-specific repair substrates cooperate to direct Msh2-Msh3-mediated repair and suggest an explanation for the msh3Y942A separation-of-function phenotype. Copyright © 2014 Elsevier B.V. All rights reserved.

ATP binding and hydrolysis by Saccharomyces cerevisiae Msh2-Msh3 are differentially modulated by Mismatch and Double-strand Break Repair DNA substrates

PubMed Central

Kumar, Charanya; Eichmiller, Robin; Wang, Bangchen; Williams, Gregory M.; Bianco, Piero R.; Surtees, Jennifer A.

2014-01-01

In Saccharomyces cerevisiae, Msh2-Msh3-mediated mismatch repair (MMR) recognizes and targets insertion/deletion loops for repair. Msh2-Msh3 is also required for 3′ non-homologous tail removal (3′NHTR) in double-strand break repair. In both pathways, Msh2-Msh3 binds double-strand/single-strand junctions and initiates repair in an ATP-dependent manner. However, we recently demonstrated that the two pathways have distinct requirements with respect to Msh2-Msh3 activities. We identified a set of aromatic residues in the nucleotide binding pocket (FLY motif) of Msh3 that, when mutated, disrupted MMR, but left 3′ NHTR largely intact. One of these mutations, msh3Y942A, was predicted to disrupt the nucleotide sandwich and allow altered positioning of ATP within the pocket. To develop a mechanistic understanding of the differential requirements for ATP binding and/or hydrolysis in the two pathways, we characterized Msh2-Msh3 and Msh2-msh3Y942A ATP binding and hydrolysis activities in the presence of MMR and 3′ NHTR DNA substrates. We observed distinct, substrate-dependent ATP hydrolysis and nucleotide turnover by Msh2-Msh3, indicating that the MMR and 3′ NHTR DNA substrates differentially modify the ATP binding/hydrolysis activities of Msh2-Msh3. Msh2-msh3Y942A retained the ability to bind DNA and ATP but exhibited altered ATP hydrolysis and nucleotide turnover. We propose that both ATP and structure-specific repair substrates cooperate to direct Msh2-Msh3-mediated repair and suggest an explanation for the msh3Y942A separation-of-function phenotype. PMID:24746922

Tyrosinase autoactivation and the problem of the lag period.

PubMed

Naish-Byfield, S; Riley, P A

1998-06-01

Evidence is presented for the binding of the quinone oxidation product of the monohydric phenol substrate, 4-hydroxyanisole, to mushroom tyrosinase. Column chromatography and SDS-PAGE separation showed labelling of the enzyme when incubated with 14C ring-labelled 4-hydroxyanisole. It is proposed that covalent binding to the enzyme and other proteins is through reaction of accessible nucleophilic groups, including thiols and amino groups, with the anisylquinone. This reductive addition enables the indirect generation of the catecholic substrate, which acts as an electron donor for the bicupric active site of met-tyrosinase and explains the lag kinetics of tyrosinase oxidation of non-cyclizing substrates. The effects of diluting the enzyme or the addition of amino acids on the lag period was consistent with a mechanism involving indirect generation of the dihydric phenol, which acts as the met-enzyme-recruiting substrate.

The UL5 and UL52 subunits of the herpes simplex virus type 1 helicase-primase subcomplex exhibit a complex interdependence for DNA binding.

PubMed

Biswas, N; Weller, S K

2001-05-18

Herpes simplex virus type 1 encodes a heterotrimeric helicase-primase complex composed of the products of the UL5, UL52, and UL8 genes. The UL5 protein contains seven motifs found in all members of helicase Superfamily 1 (SF1), and the UL52 protein contains several conserved motifs found in primases; however, the contributions of each subunit to the biochemical activities of the subcomplex are not clear. In this work, the DNA binding properties of wild type and mutant subcomplexes were examined using single-stranded, duplex, and forked substrates. A gel mobility shift assay indicated that the UL5-UL52 subcomplex binds more efficiently to the forked substrate than to either single strand or duplex DNA. Although nucleotides are not absolutely required for DNA binding, ADP stimulated the binding of UL5-UL52 to single strand DNA whereas ATP, ADP, and adenosine 5'-O-(thiotriphosphate) stimulated the binding to a forked substrate. We have previously shown that both subunits contact single-stranded DNA in a photocross-linking assay (Biswas, N., and Weller, S. K. (1999) J. Biol. Chem. 274, 8068-8076). In this study, photocross-linking assays with forked substrates indicate that the UL5 and UL52 subunits contact the forked substrates at different positions, UL52 at the single-stranded DNA tail and UL5 near the junction between single-stranded and double-stranded DNA. Neither subunit was able to cross-link a forked substrate when 5-iododeoxyuridine was located within the duplex portion. Photocross-linking experiments with subcomplexes containing mutant versions of UL5 and wild type UL52 indicated that the integrity of the ATP binding region is important for DNA binding of both subunits. These results support our previous proposal that UL5 and UL52 exhibit a complex interdependence for DNA binding (Biswas, N., and Weller, S. K. (1999) J. Biol. Chem. 274, 8068-8076) and indicate that the UL52 subunit may play a more active role in helicase activity than had previously been thought.

Substrate inhibition kinetic model for West Nile virus NS2B-NS3 protease.

PubMed

Tomlinson, Suzanne M; Watowich, Stanley J

2008-11-11

West Nile virus (WNV) has recently emerged in North America as a significant disease threat to humans and animals. Unfortunately, no approved antiviral drugs exist to combat WNV or other members of the genus Flavivirus in humans. The WNV NS2B-NS3 protease has been one of the primary targets for anti-WNV drug discovery and design since it is required for virus replication. As part of our efforts to develop effective WNV inhibitors, we reexamined the reaction kinetics of the NS2B-NS3 protease and the inhibition mechanisms of newly discovered inhibitors. The WNV protease showed substrate inhibition in assays utilizing fluorophore-linked peptide substrates GRR, GKR, and DFASGKR. Moreover, a substrate inhibition reaction step was required to accurately model kinetic data generated from protease assays with a peptide inhibitor. The substrate inhibition model suggested that peptide substrates could bind to two binding sites on the protease. Reaction product analogues also showed inhibition of the protease, demonstrating product inhibition in addition to and distinct from substrate inhibition. We propose that small peptide substrates and inhibitors may interact with protease residues that form either the P3-P1 binding surface (i.e., the S3-S1 sites) or the P1'-P3' interaction surface (i.e., the S1'-S3' sites). Optimization of substrate analogue inhibitors that target these two independent sites may lead to novel anti-WNV drugs.

Multiple ligand simultaneous docking: orchestrated dancing of ligands in binding sites of protein.

PubMed

Li, Huameng; Li, Chenglong

2010-07-30

Present docking methodologies simulate only one single ligand at a time during docking process. In reality, the molecular recognition process always involves multiple molecular species. Typical protein-ligand interactions are, for example, substrate and cofactor in catalytic cycle; metal ion coordination together with ligand(s); and ligand binding with water molecules. To simulate the real molecular binding processes, we propose a novel multiple ligand simultaneous docking (MLSD) strategy, which can deal with all the above processes, vastly improving docking sampling and binding free energy scoring. The work also compares two search strategies: Lamarckian genetic algorithm and particle swarm optimization, which have respective advantages depending on the specific systems. The methodology proves robust through systematic testing against several diverse model systems: E. coli purine nucleoside phosphorylase (PNP) complex with two substrates, SHP2NSH2 complex with two peptides and Bcl-xL complex with ABT-737 fragments. In all cases, the final correct docking poses and relative binding free energies were obtained. In PNP case, the simulations also capture the binding intermediates and reveal the binding dynamics during the recognition processes, which are consistent with the proposed enzymatic mechanism. In the other two cases, conventional single-ligand docking fails due to energetic and dynamic coupling among ligands, whereas MLSD results in the correct binding modes. These three cases also represent potential applications in the areas of exploring enzymatic mechanism, interpreting noisy X-ray crystallographic maps, and aiding fragment-based drug design, respectively. 2010 Wiley Periodicals, Inc.

Insights into the glycyl radical enzyme active site of benzylsuccinate synthase: a computational study.

PubMed

Bharadwaj, Vivek S; Dean, Anthony M; Maupin, C Mark

2013-08-21

The fumarate addition reaction, catalyzed by the enzyme benzylsuccinate synthase (BSS), is considered to be one of the most intriguing and energetically challenging reactions in biology. BSS belongs to the glycyl radical enzyme family and catalyzes the fumarate addition reaction, which enables microorganisms to utilize hydrocarbons as an energy source under anaerobic conditions. Unfortunately, the extreme sensitivity of the glycyl radical to oxygen has hampered the structural and kinetic characterization of BSS, thereby limiting our knowledge on this enzyme. To enhance our molecular-level understanding of BSS, a computational approach involving homology modeling, docking studies, and molecular dynamics (MD) simulations has been used to deduce the structure of BSS's catalytic subunit (BSSα) and illuminate the molecular basis for the fumarate addition reaction. We have identified two conserved and distinct binding pockets at the BSSα active site: a hydrophobic pocket for toluene binding and a polar pocket for fumaric acid binding. Subsequent dynamical and energetic evaluations have identified Glu509, Ser827, Leu390, and Phe384 as active site residues critical for substrate binding. The orientation of substrates at the active site observed in MD simulations is consistent with experimental observations of the syn addition of toluene to fumaric acid. It is also found that substrate binding tightens the active site and restricts the conformational flexibility of the thiyl radical, leading to hydrogen transfer distances conducive to the proposed reaction mechanism. The stability of substrates at the active site and the occurrence of feasible radical transfer distances between the thiyl radical, substrates, and the active site glycine indicate a substrate-assisted radical transfer pathway governing fumarate addition.

Frataxin Directly Stimulates Mitochondrial Cysteine Desulfurase by Exposing Substrate-binding Sites, and a Mutant Fe-S Cluster Scaffold Protein with Frataxin-bypassing Ability Acts Similarly*♦

PubMed Central

Pandey, Alok; Gordon, Donna M.; Pain, Jayashree; Stemmler, Timothy L.; Dancis, Andrew; Pain, Debkumar

2013-01-01

For iron-sulfur (Fe-S) cluster synthesis in mitochondria, the sulfur is derived from the amino acid cysteine by the cysteine desulfurase activity of Nfs1. The enzyme binds the substrate cysteine in the pyridoxal phosphate-containing site, and a persulfide is formed on the active site cysteine in a manner depending on the accessory protein Isd11. The persulfide is then transferred to the scaffold Isu, where it combines with iron to form the Fe-S cluster intermediate. Frataxin is implicated in the process, although it is unclear where and how, and deficiency causes Friedreich ataxia. Using purified proteins and isolated mitochondria, we show here that the yeast frataxin homolog (Yfh1) directly and specifically stimulates cysteine binding to Nfs1 by exposing substrate-binding sites. This novel function of frataxin does not require iron, Isu1, or Isd11. Once bound to Nfs1, the substrate cysteine is the source of the Nfs1 persulfide, but this step is independent of frataxin and strictly dependent on Isd11. Recently, a point mutation in Isu1 was found to bypass many frataxin functions. The data presented here show that the Isu1 suppressor mimics the frataxin effects on Nfs1, explaining the bypassing activity. We propose a regulatory mechanism for the Nfs1 persulfide-forming activity. Specifically, at least two separate conformational changes must occur in the enzyme for optimum activity as follows: one is mediated by frataxin interaction that exposes the “buried” substrate-binding sites, and the other is mediated by Isd11 interaction that brings the bound substrate cysteine and the active site cysteine in proximity for persulfide formation. PMID:24217246

Structural characterization of tartrate dehydrogenase: a versatile enzyme catalyzing multiple reactions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Malik, Radhika; Viola, Ronald E.

2010-10-28

The first structure of an NAD-dependent tartrate dehydrogenase (TDH) has been solved to 2 {angstrom} resolution by single anomalous diffraction (SAD) phasing as a complex with the intermediate analog oxalate, Mg{sup 2+} and NADH. This TDH structure from Pseudomonas putida has a similar overall fold and domain organization to other structurally characterized members of the hydroxy-acid dehydrogenase family. However, there are considerable differences between TDH and these functionally related enzymes in the regions connecting the core secondary structure and in the relative positioning of important loops and helices. The active site in these complexes is highly ordered, allowing the identificationmore » of the substrate-binding and cofactor-binding groups and the ligands to the metal ions. Residues from the adjacent subunit are involved in both the substrate and divalent metal ion binding sites, establishing a dimer as the functional unit and providing structural support for an alternating-site reaction mechanism. The divalent metal ion plays a prominent role in substrate binding and orientation, together with several active-site arginines. Functional groups from both subunits form the cofactor-binding site and the ammonium ion aids in the orientation of the nicotinamide ring of the cofactor. A lysyl amino group (Lys192) is the base responsible for the water-mediated proton abstraction from the C2 hydroxyl group of the substrate that begins the catalytic reaction, followed by hydride transfer to NAD. A tyrosyl hydroxyl group (Tyr141) functions as a general acid to protonate the enolate intermediate. Each substrate undergoes the initial hydride transfer, but differences in substrate orientation are proposed to account for the different reactions catalyzed by TDH.« less

Frataxin directly stimulates mitochondrial cysteine desulfurase by exposing substrate-binding sites, and a mutant Fe-S cluster scaffold protein with frataxin-bypassing ability acts similarly.

PubMed

Pandey, Alok; Gordon, Donna M; Pain, Jayashree; Stemmler, Timothy L; Dancis, Andrew; Pain, Debkumar

2013-12-27

For iron-sulfur (Fe-S) cluster synthesis in mitochondria, the sulfur is derived from the amino acid cysteine by the cysteine desulfurase activity of Nfs1. The enzyme binds the substrate cysteine in the pyridoxal phosphate-containing site, and a persulfide is formed on the active site cysteine in a manner depending on the accessory protein Isd11. The persulfide is then transferred to the scaffold Isu, where it combines with iron to form the Fe-S cluster intermediate. Frataxin is implicated in the process, although it is unclear where and how, and deficiency causes Friedreich ataxia. Using purified proteins and isolated mitochondria, we show here that the yeast frataxin homolog (Yfh1) directly and specifically stimulates cysteine binding to Nfs1 by exposing substrate-binding sites. This novel function of frataxin does not require iron, Isu1, or Isd11. Once bound to Nfs1, the substrate cysteine is the source of the Nfs1 persulfide, but this step is independent of frataxin and strictly dependent on Isd11. Recently, a point mutation in Isu1 was found to bypass many frataxin functions. The data presented here show that the Isu1 suppressor mimics the frataxin effects on Nfs1, explaining the bypassing activity. We propose a regulatory mechanism for the Nfs1 persulfide-forming activity. Specifically, at least two separate conformational changes must occur in the enzyme for optimum activity as follows: one is mediated by frataxin interaction that exposes the "buried" substrate-binding sites, and the other is mediated by Isd11 interaction that brings the bound substrate cysteine and the active site cysteine in proximity for persulfide formation.

Label-free and substrate-free potentiometric aptasensing using polycation-sensitive membrane electrodes.

PubMed

Ding, Jiawang; Chen, Yan; Wang, Xuewei; Qin, Wei

2012-02-21

A potentiometric label-free and substrate-free (LFSF) aptasensing strategy which eliminates the labeling, separation, and immobilization steps is described in this paper. An aptamer binds specifically to a target molecule via reaction incubation, which could induce a change in the aptamer conformation from a random coil-like configuration to a rigid folded structure. Such a target binding-induced aptamer conformational change effectively prevents the aptamer from electrostatically interacting with the protamine binding domain. This could either shift the response curve for the potentiometric titration of the aptamer with protamine as monitored by a conventional polycation-sensitive membrane electrode or change the current-dependent potential detected by a protamine-conditioned polycation-sensitive electrode with the pulsed current-driven ion fluxes of protamine across the polymeric membrane. Using adenosine triphosphate (ATP) as a model analyte, the proposed concept offers potentiometric detection of ATP down to the submicromolar concentration range and has been applied to the determination of ATP in HeLa cells. In contrast to the current LFSF aptasensors based on optical detection, the proposed strategy allows the LFSF biosensing of aptamer/target binding events in a homogeneous solution via electrochemical transduction. It is anticipated that the proposed strategy will lay a foundation for development of potentiometric sensors for LFSF aptasensing of a variety of analytes where target binding-induced conformational changes such as the formation of folded structures and the opening of DNA hairpin loops are involved.

A new structural framework for integrating replication protein A into DNA processing machinery

PubMed Central

Brosey, Chris A.; Yan, Chunli; Tsutakawa, Susan E.; Heller, William T.; Rambo, Robert P.; Tainer, John A.; Ivanov, Ivaylo; Chazin, Walter J.

2013-01-01

By coupling the protection and organization of single-stranded DNA (ssDNA) with recruitment and alignment of DNA processing factors, replication protein A (RPA) lies at the heart of dynamic multi-protein DNA processing machinery. Nevertheless, how RPA coordinates biochemical functions of its eight domains remains unknown. We examined the structural biochemistry of RPA’s DNA-binding activity, combining small-angle X-ray and neutron scattering with all-atom molecular dynamics simulations to investigate the architecture of RPA’s DNA-binding core. The scattering data reveal compaction promoted by DNA binding; DNA-free RPA exists in an ensemble of states with inter-domain mobility and becomes progressively more condensed and less dynamic on binding ssDNA. Our results contrast with previous models proposing RPA initially binds ssDNA in a condensed state and becomes more extended as it fully engages the substrate. Moreover, the consensus view that RPA engages ssDNA in initial, intermediate and final stages conflicts with our data revealing that RPA undergoes two (not three) transitions as it binds ssDNA with no evidence for a discrete intermediate state. These results form a framework for understanding how RPA integrates the ssDNA substrate into DNA processing machinery, provides substrate access to its binding partners and promotes the progression and selection of DNA processing pathways. PMID:23303776

Quantitative Comparison of Human Parainfluenza Virus Hemagglutinin-Neuraminidase Receptor Binding and Receptor Cleavage

PubMed Central

Tappert, Mary M.; Porterfield, J. Zachary; Mehta-D'Souza, Padmaja; Gulati, Shelly

2013-01-01

The human parainfluenza virus (hPIV) hemagglutinin-neuraminidase (HN) protein binds (H) oligosaccharide receptors that contain N-acetylneuraminic acid (Neu5Ac) and cleaves (N) Neu5Ac from these oligosaccharides. In order to determine if one of HN′s two functions is predominant, we measured the affinity of H for its ligands by a solid-phase binding assay with two glycoprotein substrates and by surface plasmon resonance with three monovalent glycans. We compared the dissociation constant (Kd) values from these experiments with previously determined Michaelis-Menten constants (Kms) for the enzyme activity. We found that glycoprotein substrates and monovalent glycans containing Neu5Acα2-3Galβ1-4GlcNAc bind HN with Kd values in the 10 to 100 μM range. Km values for HN were previously determined to be on the order of 1 mM (M. M. Tappert, D. F. Smith, and G. M. Air, J. Virol. 85:12146–12159, 2011). A Km value greater than the Kd value indicates that cleavage occurs faster than the dissociation of binding and will dominate under N-permissive conditions. We propose, therefore, that HN is a neuraminidase that can hold its substrate long enough to act as a binding protein. The N activity can therefore regulate binding by reducing virus-receptor interactions when the concentration of receptor is high. PMID:23740997

A new structural framework for integrating replication protein A into DNA processing machinery

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brosey, Chris; Yan, Chunli; Tsutakawa, Susan

2013-01-17

By coupling the protection and organization of single-stranded DNA (ssDNA) with recruitment and alignment of DNA processing factors, replication protein A (RPA) lies at the heart of dynamic multi-protein DNA processing machinery. Nevertheless, how RPA coordinates biochemical functions of its eight domains remains unknown. We examined the structural biochemistry of RPA's DNA-binding activity, combining small-angle X-ray and neutron scattering with all-atom molecular dynamics simulations to investigate the architecture of RPA's DNA-binding core. The scattering data reveal compaction promoted by DNA binding; DNA-free RPA exists in an ensemble of states with inter-domain mobility and becomes progressively more condensed and less dynamicmore » on binding ssDNA. Our results contrast with previous models proposing RPA initially binds ssDNA in a condensed state and becomes more extended as it fully engages the substrate. Moreover, the consensus view that RPA engages ssDNA in initial, intermediate and final stages conflicts with our data revealing that RPA undergoes two (not three) transitions as it binds ssDNA with no evidence for a discrete intermediate state. These results form a framework for understanding how RPA integrates the ssDNA substrate into DNA processing machinery, provides substrate access to its binding partners and promotes the progression and selection of DNA processing pathways.« less

Adenosine Monophosphate Binding Stabilizes the KTN Domain of the Shewanella denitrificans Kef Potassium Efflux System.

PubMed

Pliotas, Christos; Grayer, Samuel C; Ekkerman, Silvia; Chan, Anthony K N; Healy, Jess; Marius, Phedra; Bartlett, Wendy; Khan, Amjad; Cortopassi, Wilian A; Chandler, Shane A; Rasmussen, Tim; Benesch, Justin L P; Paton, Robert S; Claridge, Timothy D W; Miller, Samantha; Booth, Ian R; Naismith, James H; Conway, Stuart J

2017-08-15

Ligand binding is one of the most fundamental properties of proteins. Ligand functions fall into three basic types: substrates, regulatory molecules, and cofactors essential to protein stability, reactivity, or enzyme-substrate complex formation. The regulation of potassium ion movement in bacteria is predominantly under the control of regulatory ligands that gate the relevant channels and transporters, which possess subunits or domains that contain Rossmann folds (RFs). Here we demonstrate that adenosine monophosphate (AMP) is bound to both RFs of the dimeric bacterial Kef potassium efflux system (Kef), where it plays a structural role. We conclude that AMP binds with high affinity, ensuring that the site is fully occupied at all times in the cell. Loss of the ability to bind AMP, we demonstrate, causes protein, and likely dimer, instability and consequent loss of function. Kef system function is regulated via the reversible binding of comparatively low-affinity glutathione-based ligands at the interface between the dimer subunits. We propose this interfacial binding site is itself stabilized, at least in part, by AMP binding.

Adenosine Monophosphate Binding Stabilizes the KTN Domain of the Shewanella denitrificans Kef Potassium Efflux System

PubMed Central

2017-01-01

Ligand binding is one of the most fundamental properties of proteins. Ligand functions fall into three basic types: substrates, regulatory molecules, and cofactors essential to protein stability, reactivity, or enzyme–substrate complex formation. The regulation of potassium ion movement in bacteria is predominantly under the control of regulatory ligands that gate the relevant channels and transporters, which possess subunits or domains that contain Rossmann folds (RFs). Here we demonstrate that adenosine monophosphate (AMP) is bound to both RFs of the dimeric bacterial Kef potassium efflux system (Kef), where it plays a structural role. We conclude that AMP binds with high affinity, ensuring that the site is fully occupied at all times in the cell. Loss of the ability to bind AMP, we demonstrate, causes protein, and likely dimer, instability and consequent loss of function. Kef system function is regulated via the reversible binding of comparatively low-affinity glutathione-based ligands at the interface between the dimer subunits. We propose this interfacial binding site is itself stabilized, at least in part, by AMP binding. PMID:28656748

Structural insight into catalytic mechanism of PET hydrolase.

PubMed

Han, Xu; Liu, Weidong; Huang, Jian-Wen; Ma, Jiantao; Zheng, Yingying; Ko, Tzu-Ping; Xu, Limin; Cheng, Ya-Shan; Chen, Chun-Chi; Guo, Rey-Ting

2017-12-13

PET hydrolase (PETase), which hydrolyzes polyethylene terephthalate (PET) into soluble building blocks, provides an attractive avenue for the bioconversion of plastics. Here we present the structures of a novel PETase from the PET-consuming microbe Ideonella sakaiensis in complex with substrate and product analogs. Through structural analyses, mutagenesis, and activity measurements, a substrate-binding mode is proposed, and several features critical for catalysis are elucidated.

Slow Off-rates and Strong Product Binding Are Required for Processivity and Efficient Degradation of Recalcitrant Chitin by Family 18 Chitinases*

PubMed Central

Kurašin, Mihhail; Kuusk, Silja; Kuusk, Piret; Sørlie, Morten; Väljamäe, Priit

2015-01-01

Processive glycoside hydrolases are the key components of enzymatic machineries that decompose recalcitrant polysaccharides, such as chitin and cellulose. The intrinsic processivity (PIntr) of cellulases has been shown to be governed by the rate constant of dissociation from polymer chain (koff). However, the reported koff values of cellulases are strongly dependent on the method used for their measurement. Here, we developed a new method for determining koff, based on measuring the exchange rate of the enzyme between a non-labeled and a 14C-labeled polymeric substrate. The method was applied to the study of the processive chitinase ChiA from Serratia marcescens. In parallel, ChiA variants with weaker binding of the N-acetylglucosamine unit either in substrate-binding site −3 (ChiA-W167A) or the product-binding site +1 (ChiA-W275A) were studied. Both ChiA variants showed increased off-rates and lower apparent processivity on α-chitin. The rate of the production of insoluble reducing groups on the reduced α-chitin was an order of magnitude higher than koff, suggesting that the enzyme can initiate several processive runs without leaving the substrate. On crystalline chitin, the general activity of the wild type enzyme was higher, and the difference was magnifying with hydrolysis time. On amorphous chitin, the variants clearly outperformed the wild type. A model is proposed whereby strong interactions with polymer in the substrate-binding sites (low off-rates) and strong binding of the product in the product-binding sites (high pushing potential) are required for the removal of obstacles, like disintegration of chitin microfibrils. PMID:26468285

Slow Off-rates and Strong Product Binding Are Required for Processivity and Efficient Degradation of Recalcitrant Chitin by Family 18 Chitinases.

PubMed

Kurašin, Mihhail; Kuusk, Silja; Kuusk, Piret; Sørlie, Morten; Väljamäe, Priit

2015-11-27

Processive glycoside hydrolases are the key components of enzymatic machineries that decompose recalcitrant polysaccharides, such as chitin and cellulose. The intrinsic processivity (P(Intr)) of cellulases has been shown to be governed by the rate constant of dissociation from polymer chain (koff). However, the reported koff values of cellulases are strongly dependent on the method used for their measurement. Here, we developed a new method for determining koff, based on measuring the exchange rate of the enzyme between a non-labeled and a (14)C-labeled polymeric substrate. The method was applied to the study of the processive chitinase ChiA from Serratia marcescens. In parallel, ChiA variants with weaker binding of the N-acetylglucosamine unit either in substrate-binding site -3 (ChiA-W167A) or the product-binding site +1 (ChiA-W275A) were studied. Both ChiA variants showed increased off-rates and lower apparent processivity on α-chitin. The rate of the production of insoluble reducing groups on the reduced α-chitin was an order of magnitude higher than koff, suggesting that the enzyme can initiate several processive runs without leaving the substrate. On crystalline chitin, the general activity of the wild type enzyme was higher, and the difference was magnifying with hydrolysis time. On amorphous chitin, the variants clearly outperformed the wild type. A model is proposed whereby strong interactions with polymer in the substrate-binding sites (low off-rates) and strong binding of the product in the product-binding sites (high pushing potential) are required for the removal of obstacles, like disintegration of chitin microfibrils. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Ammonia binding to the oxygen-evolving complex of photosystem II identifies the solvent-exchangeable oxygen bridge (μ-oxo) of the manganese tetramer

PubMed Central

Pérez Navarro, Montserrat; Ames, William M.; Nilsson, Håkan; Lohmiller, Thomas; Pantazis, Dimitrios A.; Rapatskiy, Leonid; Nowaczyk, Marc M.; Neese, Frank; Boussac, Alain; Messinger, Johannes; Lubitz, Wolfgang; Cox, Nicholas

2013-01-01

The assignment of the two substrate water sites of the tetra-manganese penta-oxygen calcium (Mn4O5Ca) cluster of photosystem II is essential for the elucidation of the mechanism of biological O-O bond formation and the subsequent design of bio-inspired water-splitting catalysts. We recently demonstrated using pulsed EPR spectroscopy that one of the five oxygen bridges (μ-oxo) exchanges unusually rapidly with bulk water and is thus a likely candidate for one of the substrates. Ammonia, a water analog, was previously shown to bind to the Mn4O5Ca cluster, potentially displacing a water/substrate ligand [Britt RD, et al. (1989) J Am Chem Soc 111(10):3522–3532]. Here we show by a combination of EPR and time-resolved membrane inlet mass spectrometry that the binding of ammonia perturbs the exchangeable μ-oxo bridge without drastically altering the binding/exchange kinetics of the two substrates. In combination with broken-symmetry density functional theory, our results show that (i) the exchangable μ-oxo bridge is O5 {using the labeling of the current crystal structure [Umena Y, et al. (2011) Nature 473(7345):55–60]}; (ii) ammonia displaces a water ligand to the outer manganese (MnA4-W1); and (iii) as W1 is trans to O5, ammonia binding elongates the MnA4-O5 bond, leading to the perturbation of the μ-oxo bridge resonance and to a small change in the water exchange rates. These experimental results support O-O bond formation between O5 and possibly an oxyl radical as proposed by Siegbahn and exclude W1 as the second substrate water. PMID:24023065

Structural studies of human histone deacetylase 8 and its site-specific variants complexed with substrate and inhibitors.

PubMed

Dowling, Daniel P; Gantt, Stephanie L; Gattis, Samuel G; Fierke, Carol A; Christianson, David W

2008-12-23

Metal-dependent histone deacetylases (HDACs) require Zn(2+) or Fe(2+) to regulate the acetylation of lysine residues in histones and other proteins in eukaryotic cells. Isozyme HDAC8 is perhaps the archetypical member of the class I HDAC family and serves as a paradigm for studying structure-function relationships. Here, we report the structures of HDAC8 complexes with trichostatin A and 3-(1-methyl-4-phenylacetyl-1H-2-pyrrolyl)-N-hydroxy-2-propenamide (APHA) in a new crystal form. The structure of the APHA complex reveals that the hydroxamate CO group accepts a hydrogen bond from Y306 but does not coordinate to Zn(2+) with favorable geometry, perhaps due to the constraints of its extended pi system. Additionally, since APHA binds to only two of the three protein molecules in the asymmetric unit of this complex, the structure of the third monomer represents the first structure of HDAC8 in the unliganded state. Comparison of unliganded and liganded structures illustrates ligand-induced conformational changes in the L2 loop that likely accompany substrate binding and catalysis. Furthermore, these structures, along with those of the D101N, D101E, D101A, and D101L variants, support the proposal that D101 is critical for the function of the L2 loop. However, amino acid substitutions for D101 can also trigger conformational changes of Y111 and W141 that perturb the substrate binding site. Finally, the structure of H143A HDAC8 complexed with an intact acetylated tetrapeptide substrate molecule confirms the importance of D101 for substrate binding and reveals how Y306 and the active site zinc ion together bind and activate the scissile amide linkage of acetyllysine.

Insight into the mechanism of action and selectivity of caspase-3 reversible inhibitors through in silico studies

NASA Astrophysics Data System (ADS)

Minini, Lucía; Ferraro, Florencia; Cancela, Saira; Merlino, Alicia

2017-11-01

Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder worldwide for which there is currently no cure. Recently, caspase-3 has been proposed as a potential therapeutic target for treating AD. Since this enzyme is overexpressed in brains from AD patients its selective modulation by non-covalent inhibitors becomes an interesting strategy in the search of potential drugs against this neuropathology. With this in mind, we have combined molecular docking, molecular dynamics simulations and QM calculations of unliganded caspase-3 and caspase-7 and in complex with a series of known inhibitors of caspase-3 described in the literature in order to assess the structural features responsible for good inhibitory activity and selectivity against this potential target. This work has allowed us to identify hotspots for drug binding as well as the importance of shape and charge distribution for interacting into the substrate binding cleft or into the dimer interface in each enzyme. Our results showed that most selective compounds against caspsase-3 bind into the substrate binding cleft acting as competitive inhibitors whereas in caspase-7 they bind close to an allosteric site at the dimer interface but since they are weakly bound their presence would not be affecting enzyme dynamics or function. In addition, for both enzymes we have found evidence indicating that differences in shape and accessibility exist between the substrate binding site of each monomer which could be modulating the binding affinity of non-covalent molecules.

Effects of Mutations and Ligands on the Thermostability of the l-Arginine/Agmatine Antiporter AdiC and Deduced Insights into Ligand-Binding of Human l-Type Amino Acid Transporters

PubMed Central

Ilgü, Hüseyin; Jeckelmann, Jean-Marc; Colas, Claire; Ucurum, Zöhre; Schlessinger, Avner; Fotiadis, Dimitrios

2018-01-01

The l-arginine/agmatine transporter AdiC is a prokaryotic member of the SLC7 family, which enables pathogenic enterobacteria to survive the extremely acidic gastric environment. Wild-type AdiC from Escherichia coli, as well as its previously reported point mutants N22A and S26A, were overexpressed homologously and purified to homogeneity. A size-exclusion chromatography-based thermostability assay was used to determine the melting temperatures (Tms) of the purified AdiC variants in the absence and presence of the selected ligands l-arginine (Arg), agmatine, l-arginine methyl ester, and l-arginine amide. The resulting Tms indicated stabilization of AdiC variants upon ligand binding, in which Tms and ligand binding affinities correlated positively. Considering results from this and previous studies, we revisited the role of AdiC residue S26 in Arg binding and proposed interactions of the α-carboxylate group of Arg exclusively with amide groups of the AdiC backbone. In the context of substrate binding in the human SLC7 family member l-type amino acid transporter-1 (LAT1; SLC7A5), an analogous role of S66 in LAT1 to S26 in AdiC is discussed based on homology modeling and amino acid sequence analysis. Finally, we propose a binding mechanism for l-amino acid substrates to LATs from the SLC7 family. PMID:29558430

Effects of Mutations and Ligands on the Thermostability of the l-Arginine/Agmatine Antiporter AdiC and Deduced Insights into Ligand-Binding of Human l-Type Amino Acid Transporters.

PubMed

Ilgü, Hüseyin; Jeckelmann, Jean-Marc; Colas, Claire; Ucurum, Zöhre; Schlessinger, Avner; Fotiadis, Dimitrios

2018-03-20

The l-arginine/agmatine transporter AdiC is a prokaryotic member of the SLC7 family, which enables pathogenic enterobacteria to survive the extremely acidic gastric environment. Wild-type AdiC from Escherichia coli, as well as its previously reported point mutants N22A and S26A, were overexpressed homologously and purified to homogeneity. A size-exclusion chromatography-based thermostability assay was used to determine the melting temperatures ( T m s) of the purified AdiC variants in the absence and presence of the selected ligands l-arginine (Arg), agmatine, l-arginine methyl ester, and l-arginine amide. The resulting T m s indicated stabilization of AdiC variants upon ligand binding, in which T m s and ligand binding affinities correlated positively. Considering results from this and previous studies, we revisited the role of AdiC residue S26 in Arg binding and proposed interactions of the α-carboxylate group of Arg exclusively with amide groups of the AdiC backbone. In the context of substrate binding in the human SLC7 family member l-type amino acid transporter-1 (LAT1; SLC7A5), an analogous role of S66 in LAT1 to S26 in AdiC is discussed based on homology modeling and amino acid sequence analysis. Finally, we propose a binding mechanism for l-amino acid substrates to LATs from the SLC7 family.

The early mature part of bacterial twin-arginine translocation (Tat) precursor proteins contributes to TatBC receptor binding.

PubMed

Ulfig, Agnes; Freudl, Roland

2018-05-11

The twin-arginine translocation (Tat) pathway transports folded proteins across bacterial membranes. Tat precursor proteins possess a conserved twin-arginine (RR) motif in their signal peptides that is involved in the binding of the proteins to the membrane-associated TatBC receptor complex. In addition, the hydrophobic region in the Tat signal peptides also contributes to TatBC binding, but whether regions beyond the signal-peptide cleavage site are involved in this process is unknown. Here, we analyzed the contribution of the early mature protein part of the Escherichia coli trimethylamine N -oxide reductase (TorA) to productive TatBC receptor binding. We identified substitutions in the 30 amino acids immediately following the TorA signal peptide (30aa-region) that restored export of a transport-defective TorA[KQ]-30aa-MalE precursor, in which the RR residues had been replaced by a lysine-glutamine pair. Some of these substitutions increased the hydrophobicity of the N-terminal part of the 30aa-region and thereby likely enhanced hydrophobic substrate-receptor interactions within the hydrophobic TatBC substrate-binding cavity. Another class of substitutions increased the positive net charge of the region's C-terminal part, presumably leading to strengthened electrostatic interactions between the mature substrate part and the cytoplasmic TatBC regions. Furthermore, we identified substitutions in the C-terminal domains of TatB following the transmembrane segment that restored transport of various transport-defective TorA-MalE derivatives. Some of these substitutions most likely affected the orientation or conformation of the flexible, carboxy-proximal helices of TatB. Therefore, we propose that a tight accommodation of the folded mature region by TatB contributes to productive binding of Tat substrates to TatBC. © 2018 Ulfig and Freudl.

Probing the substrate specificity of Golgi alpha-mannosidase II by use of synthetic oligosaccharides and a catalytic nucleophile mutant.

PubMed

Zhong, Wei; Kuntz, Douglas A; Ember, Brian; Singh, Harminder; Moremen, Kelley W; Rose, David R; Boons, Geert-Jan

2008-07-16

Inhibition of Golgi alpha-mannosidase II (GMII), which acts late in the N-glycan processing pathway, provides a route to blocking cancer-induced changes in cell surface oligosaccharide structures. To probe the substrate requirements of GMII, oligosaccharides were synthesized that contained an alpha(1,3)- or alpha(1,6)-linked 1-thiomannoside. Surprisingly, these oligosaccharides were not observed in X-ray crystal structures of native Drosophila GMII (dGMII). However, a mutant enzyme in which the catalytic nucleophilic aspartate was changed to alanine (D204A) allowed visualization of soaked oligosaccharides and led to the identification of the binding site for the alpha(1,3)-linked mannoside of the natural substrate. These studies also indicate that the conformational change of the bound mannoside to a high-energy B 2,5 conformation is facilitated by steric hindrance from, and the formation of strong hydrogen bonds to, Asp204. The observation that 1-thio-linked mannosides are not well tolerated by the catalytic site of dGMII led to the synthesis of a pentasaccharide containing the alpha(1,6)-linked Man of the natural substrate and the beta(1,2)-linked GlcNAc moiety proposed to be accommodated by the extended binding site of the enzyme. A cocrystal structure of this compound with the D204A enzyme revealed the molecular interactions with the beta(1,2)-linked GlcNAc. The structure is consistent with the approximately 80-fold preference of dGMII for the cleavage of substrates containing a nonreducing beta(1,2)-linked GlcNAc. By contrast, the lysosomal mannosidase lacks an equivalent GlcNAc binding site and kinetic analysis indicates oligomannoside substrates without non-reducing-terminal GlcNAc modifications are preferred, suggesting that selective inhibitors for GMII could exploit the additional binding specificity of the GlcNAc binding site.

Substrate sequence selectivity of APOBEC3A implicates intra-DNA interactions.

PubMed

Silvas, Tania V; Hou, Shurong; Myint, Wazo; Nalivaika, Ellen; Somasundaran, Mohan; Kelch, Brian A; Matsuo, Hiroshi; Kurt Yilmaz, Nese; Schiffer, Celia A

2018-05-14

The APOBEC3 (A3) family of human cytidine deaminases is renowned for providing a first line of defense against many exogenous and endogenous retroviruses. However, the ability of these proteins to deaminate deoxycytidines in ssDNA makes A3s a double-edged sword. When overexpressed, A3s can mutate endogenous genomic DNA resulting in a variety of cancers. Although the sequence context for mutating DNA varies among A3s, the mechanism for substrate sequence specificity is not well understood. To characterize substrate specificity of A3A, a systematic approach was used to quantify the affinity for substrate as a function of sequence context, length, secondary structure, and solution pH. We identified the A3A ssDNA binding motif as (T/C)TC(A/G), which correlated with enzymatic activity. We also validated that A3A binds RNA in a sequence specific manner. A3A bound tighter to substrate binding motif within a hairpin loop compared to linear oligonucleotide, suggesting A3A affinity is modulated by substrate structure. Based on these findings and previously published A3A-ssDNA co-crystal structures, we propose a new model with intra-DNA interactions for the molecular mechanism underlying A3A sequence preference. Overall, the sequence and structural preferences identified for A3A leads to a new paradigm for identifying A3A's involvement in mutation of endogenous or exogenous DNA.

Molecular clamp mechanism of substrate binding by hydrophobic coiled-coil residues of the archaeal chaperone prefoldin

PubMed Central

Lundin, Victor F.; Stirling, Peter C.; Gomez-Reino, Juan; Mwenifumbo, Jill C.; Obst, Jennifer M.; Valpuesta, José M.; Leroux, Michel R.

2004-01-01

Prefoldin (PFD) is a jellyfish-shaped molecular chaperone that has been proposed to play a general role in de novo protein folding in archaea and is known to assist the biogenesis of actins, tubulins, and potentially other proteins in eukaryotes. Using point mutants, chimeras, and intradomain swap variants, we show that the six coiledcoil tentacles of archaeal PFD act in concert to bind and stabilize nonnative proteins near the opening of the cavity they form. Importantly, the interaction between chaperone and substrate depends on the mostly buried interhelical hydrophobic residues of the coiled coils. We also show by electron microscopy that the tentacles can undergo an en bloc movement to accommodate an unfolded substrate. Our data reveal how archael PFD uses its unique architecture and intrinsic coiled-coil properties to interact with nonnative polypeptides. PMID:15070724

Mechanism and Substrate Recognition of Human Holo ACP Synthase

PubMed Central

Bunkoczi, Gabor; Pasta, Saloni; Joshi, Anil; Wu, Xiaoqiu; Kavanagh, Kathryn L.; Smith, Stuart; Oppermann, Udo

2007-01-01

Summary Mammals utilize a single phosphopantetheinyl transferase for the posttranslational modification of at least three different apoproteins: the carrier protein components of cytosolic and mitochondrial fatty acid synthases and the aminoadipate semialdehyde reductase involved in lysine degradation. We determined the crystal structure of the human phosphopantetheinyl transferase, a eukaryotic phosphopantetheinyl transferase characterized, complexed with CoA and Mg2+, and in ternary complex with CoA and ACP. The involvement of key residues in ligand binding and catalysis was confirmed by mutagenesis and kinetic analysis. Human phosphopantetheinyl transferase exhibits an α/β fold and 2-fold pseudosymmetry similar to the Sfp phosphopantetheinyl transferase from Bacillus subtilis. Although the bound ACP exhibits a typical four-helix structure, its binding is unusual in that it is facilitated predominantly by hydrophobic interactions. A detailed mechanism is proposed describing the substrate binding and catalytic process. PMID:18022563

The fast release of sticky protons: Kinetics of substrate binding and proton release in a multidrug transporter

PubMed Central

Adam, Yoav; Tayer, Naama; Rotem, Dvir; Schreiber, Gideon; Schuldiner, Shimon

2007-01-01

EmrE is an Escherichia coli H+-coupled multidrug transporter that provides a unique experimental paradigm because of its small size and stability, and because its activity can be studied in detergent solution. In this work, we report a study of the transient kinetics of substrate binding and substrate-induced proton release in EmrE. For this purpose, we measured transient changes in the tryptophan fluorescence upon substrate binding and the rates of substrate-induced proton release. The fluorescence of the essential and fully conserved Trp residue at position 63 is sensitive to the occupancy of the binding site with either protons or substrate. The maximal rate of binding to detergent-solubilized EmrE of TPP+, a high-affinity substrate, is 2 × 107 M−1·s−1, a rate typical of diffusion-limited reactions. Rate measurements with medium- and low-affinity substrates imply that the affinity is determined mainly by the koff of the substrate. The rates of substrate binding and substrate-induced release of protons are faster at basic pHs and slower at lower pHs. These findings imply that the substrate-binding rates are determined by the generation of the species capable of binding; this is controlled by the high affinity to protons of the glutamate at position 14, because an Asp replacement with a lower pK is faster at the same pHs. PMID:17984053

A structure-based catalytic mechanism for the xanthine oxidase family of molybdenum enzymes.

PubMed Central

Huber, R; Hof, P; Duarte, R O; Moura, J J; Moura, I; Liu, M Y; LeGall, J; Hille, R; Archer, M; Romão, M J

1996-01-01

The crystal structure of the xanthine oxidase-related molybdenum-iron protein aldehyde oxido-reductase from the sulfate reducing anaerobic Gram-negative bacterium Desulfovibrio gigas (Mop) was analyzed in its desulfo-, sulfo-, oxidized, reduced, and alcohol-bound forms at 1.8-A resolution. In the sulfo-form the molybdenum molybdopterin cytosine dinucleotide cofactor has a dithiolene-bound fac-[Mo, = O, = S, ---(OH2)] substructure. Bound inhibitory isopropanol in the inner compartment of the substrate binding tunnel is a model for the Michaelis complex of the reaction with aldehydes (H-C = O,-R). The reaction is proposed to proceed by transfer of the molybdenum-bound water molecule as OH- after proton transfer to Glu-869 to the carbonyl carbon of the substrate in concert with hydride transfer to the sulfido group to generate [MoIV, = O, -SH, ---(O-C = O, -R)). Dissociation of the carboxylic acid product may be facilitated by transient binding of Glu-869 to the molybdenum. The metal-bound water is replenished from a chain of internal water molecules. A second alcohol binding site in the spacious outer compartment may cause the strong substrate inhibition observed. This compartment is the putative binding site of large inhibitors of xanthine oxidase. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:8799115

A computational analysis of SARS cysteine proteinase-octapeptide substrate interaction: implication for structure and active site binding mechanism

PubMed Central

Phakthanakanok, Krongsakda; Ratanakhanokchai, Khanok; Kyu, Khin Lay; Sompornpisut, Pornthep; Watts, Aaron; Pinitglang, Surapong

2009-01-01

Background SARS coronavirus main proteinase (SARS CoVMpro) is an important enzyme for the replication of Severe Acute Respiratory Syndrome virus. The active site region of SARS CoVMpro is divided into 8 subsites. Understanding the binding mode of SARS CoVMpro with a specific substrate is useful and contributes to structural-based drug design. The purpose of this research is to investigate the binding mode between the SARS CoVMpro and two octapeptides, especially in the region of the S3 subsite, through a molecular docking and molecular dynamics (MD) simulation approach. Results The one turn α-helix chain (residues 47–54) of the SARS CoVMpro was directly involved in the induced-fit model of the enzyme-substrate complex. The S3 subsite of the enzyme had a negatively charged region due to the presence of Glu47. During MD simulations, Glu47 of the enzyme was shown to play a key role in electrostatic bonding with the P3Lys of the octapeptide. Conclusion MD simulations were carried out on the SARS CoVMpro-octapeptide complex. The hypothesis proposed that Glu47 of SARS CoVMpro is an important residue in the S3 subsite and is involved in binding with P3Lys of the octapeptide. PMID:19208150

Enzyme activation through the utilization of intrinsic dianion binding energy.

PubMed

Amyes, T L; Malabanan, M M; Zhai, X; Reyes, A C; Richard, J P

2017-03-01

We consider 'the proposition that the intrinsic binding energy that results from the noncovalent interaction of a specific substrate with the active site of the enzyme is considerably larger than is generally believed. An important part of this binding energy may be utilized to provide the driving force for catalysis, so that the observed binding energy represents only what is left over after this utilization' [Jencks,W.P. (1975) Adv. Enzymol. Relat. Areas. Mol. Biol. , , 219-410]. The large ~12 kcal/mol intrinsic substrate phosphodianion binding energy for reactions catalyzed by triosephosphate isomerase (TIM), orotidine 5'-monophosphate decarboxylase and glycerol-3-phosphate dehydrogenase is divided into 4-6 kcal/mol binding energy that is expressed on the formation of the Michaelis complex in anchoring substrates to the respective enzyme, and 6-8 kcal/mol binding energy that is specifically expressed at the transition state in activating the respective enzymes for catalysis. A structure-based mechanism is described where the dianion binding energy drives a conformational change that activates these enzymes for catalysis. Phosphite dianion plays the active role of holding TIM in a high-energy closed active form, but acts as passive spectator in showing no effect on transition-state structure. The result of studies on mutant enzymes is presented, which support the proposal that the dianion-driven enzyme conformational change plays a role in enhancing the basicity of side chain of E167, the catalytic base, by clamping the base between a pair of hydrophobic side chains. The insight these results provide into the architecture of enzyme active sites and the development of strategies for the de novo design of protein catalysts is discussed. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

Structural insights into the catalytic mechanism of a family 18 exo-chitinase

PubMed Central

van Aalten, D. M. F.; Komander, D.; Synstad, B.; Gåseidnes, S.; Peter, M. G.; Eijsink, V. G. H.

2001-01-01

Chitinase B (ChiB) from Serratia marcescens is a family 18 exo-chitinase whose catalytic domain has a TIM-barrel fold with a tunnel-shaped active site. We have solved structures of three ChiB complexes that reveal details of substrate binding, substrate-assisted catalysis, and product displacement. The structure of an inactive ChiB mutant (E144Q) complexed with a pentameric substrate (binding in subsites −2 to +3) shows closure of the “roof” of the active site tunnel. It also shows that the sugar in the −1 position is distorted to a boat conformation, thus providing structural evidence in support of a previously proposed catalytic mechanism. The structures of the active enzyme complexed to allosamidin (an analogue of a proposed reaction intermediate) and of the active enzyme soaked with pentameric substrate show events after cleavage of the glycosidic bond. The latter structure shows reopening of the roof of the active site tunnel and enzyme-assisted product displacement in the +1 and +2 sites, allowing a water molecule to approach the reaction center. Catalysis is accompanied by correlated structural changes in the core of the TIM barrel that involve conserved polar residues whose functions were hitherto unknown. These changes simultaneously contribute to stabilization of the reaction intermediate and alternation of the pKa of the catalytic acid during the catalytic cycle. PMID:11481469

Kinetics of bacterial phospholipase C activity at micellar interfaces: effect of substrate aggregate microstructure and a model for the kinetic parameters.

PubMed

Singh, Jasmeet; Ranganathan, Radha; Hajdu, Joseph

2008-12-25

Activity at micellar interfaces of bacterial phospholipase C from Bacillus cereus on phospholipids solubilized in micelles was investigated with the goal of elucidating the role of the interface microstructure and developing further an existing kinetic model. Enzyme kinetics and physicochemical characterization of model substrate aggregates were combined, thus enabling the interpretation of kinetics in the context of the interface. Substrates were diacylphosphatidylcholine of different acyl chain lengths in the form of mixed micelles with dodecyldimethylammoniopropanesulfonate. An early kinetic model, reformulated to reflect the interfacial nature of the kinetics, was applied to the kinetic data. A better method of data treatment is proposed, use of which makes the presence of microstructure effects quite transparent. Models for enzyme-micelle binding and enzyme-lipid binding are developed, and expressions incorporating the microstructural properties are derived for the enzyme-micelle dissociation constant K(s) and the interface Michaelis-Menten constant, K(M). Use of these expressions in the interface kinetic model brings excellent agreement between the kinetic data and the model. Numerical values for the thermodynamic and kinetic parameters are determined. Enzyme-lipid binding is found to be an activated process with an acyl chain length dependent free energy of activation that decreases with micelle lipid molar fraction with a coefficient of about -15RT and correlates with the tightness of molecular packing in the substrate aggregate. Thus, the physical insight obtained includes a model for the kinetic parameters that shows that these parameters depend on the substrate concentration and acyl chain length of the lipid. Enzyme-micelle binding is indicated to be hydrophobic and solvent mediated with a dissociation constant of 1.2 mM.

The mechanism of hydrolysis of beta-glycerophosphate by kidney alkaline phosphatase.

PubMed Central

Ahlers, J

1975-01-01

1. To identify the functional groups that are involved in the conversion of beta-glycerophosphate by alkaline phosphatase (EC 3.1.3.1) from pig kidney, the kinetics of alkaline phosphatase were investigated in the pH range 6.6-10.3 at substrate concentrations of 3 muM-30 mM. From the plots of log VH+ against pH and log VH+/KH+m against pH one functional group with pK = 7.0 and two functional groups with pK = 9.1 were identified. These groups are involved in substrate binding. Another group with pK = 8.8 was found, which in its unprotonated form catalyses substrate conversion. 2. GSH inhibits the alkaline phosphatase reversibly and non-competitively by attacking the bound Zn(II). 3. The influence of the H+ concentration on the activation by Mg2+ ions of alkaline pig kidney phosphate was investigated between pH 8.4 and 10.0. The binding of substrate and activating Mg2+ ions occurs independently at all pH values between 8.4 and 10.0. The activation mechanism is not affected by the H+ concentration. The Mg2+ ions are bound by a functional group with a pK of 10.15. 4. A scheme is proposed for the reaction between enzyme, substrate, Mg2+ and H+ and the overall rate equation is derived. 5. The mechanism of substrate binding and splitting by the functional groups of the active centre is discussed on the basis of a model. Mg2+ seems to play a role as an autosteric effector. PMID:995

Modified 5-fluorouracil: Uridine phosphorylase inhibitor

NASA Astrophysics Data System (ADS)

Lashkov, A. A.; Shchekotikhin, A. A.; Shtil, A. A.; Sotnichenko, S. E.; Mikhailov, A. M.

2016-09-01

5-Fluorouracil (5-FU) is a medication widely used in chemotherapy to treat various types of cancer. Being a substrate for the reverse reaction catalyzed by uridine phosphorylase (UPase), 5-FU serves as a promising prototype molecule (molecular scaffold) for the design of a selective UPase inhibitor that enhances the antitumor activity of 5-FU and exhibits intrinsic cytostatic effects on cancer cells. The chemical formula of the new compound, which binds to the uracil-binding site and, in the presence of a phosphate anion, to the phosphate-binding site of UPase, is proposed and investigated by molecular simulation methods.

Structural basis of Na(+)-independent and cooperative substrate/product antiport in CaiT.

PubMed

Schulze, Sabrina; Köster, Stefan; Geldmacher, Ulrike; Terwisscha van Scheltinga, Anke C; Kühlbrandt, Werner

2010-09-09

Transport of solutes across biological membranes is performed by specialized secondary transport proteins in the lipid bilayer, and is essential for life. Here we report the structures of the sodium-independent carnitine/butyrobetaine antiporter CaiT from Proteus mirabilis (PmCaiT) at 2.3-A and from Escherichia coli (EcCaiT) at 3.5-A resolution. CaiT belongs to the family of betaine/carnitine/choline transporters (BCCT), which are mostly Na(+) or H(+) dependent, whereas EcCaiT is Na(+) and H(+) independent. The three-dimensional architecture of CaiT resembles that of the Na(+)-dependent transporters LeuT and BetP, but in CaiT a methionine sulphur takes the place of the Na(+) ion to coordinate the substrate in the central transport site, accounting for Na(+)-independent transport. Both CaiT structures show the fully open, inward-facing conformation, and thus complete the set of functional states that describe the alternating access mechanism. EcCaiT contains two bound butyrobetaine substrate molecules, one in the central transport site, the other in an extracellular binding pocket. In the structure of PmCaiT, a tryptophan side chain occupies the transport site, and access to the extracellular site is blocked. Binding of both substrates to CaiT reconstituted into proteoliposomes is cooperative, with Hill coefficients up to 1.7, indicating that the extracellular site is regulatory. We propose a mechanism whereby the occupied regulatory site increases the binding affinity of the transport site and initiates substrate translocation.

An Aromatic Cap Seals the Substrate Binding Site in an ECF-Type S Subunit for Riboflavin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Karpowich, Nathan K.; Song, Jinmei; Wang, Da-Neng

2016-06-13

ECF transporters are a family of active membrane transporters for essential micronutrients, such as vitamins and trace metals. Found exclusively in archaea and bacteria, these transporters are composed of four subunits: an integral membrane substrate-binding subunit (EcfS), a transmembrane coupling subunit (EcfT), and two ATP-binding cassette ATPases (EcfA and EcfA'). We have characterized the structural basis of substrate binding by the EcfS subunit for riboflavin from Thermotoga maritima, TmRibU. TmRibU binds riboflavin with high affinity, and the protein–substrate complex is exceptionally stable in solution. The crystal structure of riboflavin-bound TmRibU reveals an electronegative binding pocket at the extracellular surface inmore » which the substrate is completely buried. Analysis of the intermolecular contacts indicates that nearly every available substrate hydrogen bond is satisfied. A conserved aromatic residue at the extracellular end of TM5, Tyr130, caps the binding site to generate a substrate-bound, occluded state, and non-conservative mutation of Tyr130 reduces the stability of this conformation. Using a novel fluorescence binding assay, we find that an aromatic residue at this position is essential for high-affinity substrate binding. Comparison with other S subunit structures suggests that TM5 and Loop5-6 contain a dynamic, conserved motif that plays a key role in gating substrate entry and release by S subunits of ECF transporters.« less

Inorganic phosphate blocks binding of pre-miRNA to Dicer-2 via its PAZ domain

PubMed Central

Fukunaga, Ryuya; Colpan, Cansu; Han, Bo W; Zamore, Phillip D

2014-01-01

In Drosophila, Dicer-1 produces microRNAs (miRNAs) from pre-miRNAs, whereas Dicer-2 generates small interfering RNAs from long double-stranded RNA (dsRNA), a process that requires ATP hydrolysis. We previously showed that inorganic phosphate inhibits Dicer-2 cleavage of pre-miRNAs, but not long dsRNAs. Here, we report that phosphate-dependent substrate discrimination by Dicer-2 reflects dsRNA substrate length. Efficient processing by Dicer-2 of short dsRNA requires a 5′ terminal phosphate and a two-nucleotide, 3′ overhang, but does not require ATP. Phosphate inhibits cleavage of such short substrates. In contrast, cleavage of longer dsRNA requires ATP but no specific end structure: phosphate does not inhibit cleavage of these substrates. Mutation of a pair of conserved arginine residues in the Dicer-2 PAZ domain blocked cleavage of short, but not long, dsRNA. We propose that inorganic phosphate occupies a PAZ domain pocket required to bind the 5′ terminal phosphate of short substrates, blocking their use and restricting pre-miRNA processing in flies to Dicer-1. Our study helps explain how a small molecule can alter the substrate specificity of a nucleic acid processing enzyme. PMID:24488111

Squeezing at Entrance of Proton Transport Pathway in Proton-translocating Pyrophosphatase upon Substrate Binding*

PubMed Central

Huang, Yun-Tzu; Liu, Tseng-Huang; Lin, Shih-Ming; Chen, Yen-Wei; Pan, Yih-Jiuan; Lee, Ching-Hung; Sun, Yuh-Ju; Tseng, Fan-Gang; Pan, Rong-Long

2013-01-01

Homodimeric proton-translocating pyrophosphatase (H+-PPase; EC 3.6.1.1) is indispensable for many organisms in maintaining organellar pH homeostasis. This unique proton pump couples the hydrolysis of PPi to proton translocation across the membrane. H+-PPase consists of 14–16 relatively hydrophobic transmembrane domains presumably for proton translocation and hydrophilic loops primarily embedding a catalytic site. Several highly conserved polar residues located at or near the entrance of the transport pathway in H+-PPase are essential for proton pumping activity. In this investigation single molecule FRET was employed to dissect the action at the pathway entrance in homodimeric Clostridium tetani H+-PPase upon ligand binding. The presence of the substrate analog, imidodiphosphate mediated two sites at the pathway entrance moving toward each other. Moreover, single molecule FRET analyses after the mutation at the first proton-carrying residue (Arg-169) demonstrated that conformational changes at the entrance are conceivably essential for the initial step of H+-PPase proton translocation. A working model is accordingly proposed to illustrate the squeeze at the entrance of the transport pathway in H+-PPase upon substrate binding. PMID:23720778

A molecular dynamics study of the complete binding process of meropenem to New Delhi metallo-β-lactamase 1.

PubMed

Duan, Juan; Hu, Chuncai; Guo, Jiafan; Guo, Lianxian; Sun, Jia; Zhao, Zuguo

2018-02-28

The mechanism of substrate hydrolysis of New Delhi metallo-β-lactamase 1 (NDM-1) has been reported, but the process in which NDM-1 captures and transports the substrate into its active center remains unknown. In this study, we investigated the process of the substrate entry into the NDM-1 activity center through long unguided molecular dynamics simulations using meropenem as the substrate. A total of 550 individual simulations were performed, each of which for 200 ns, and 110 of them showed enzyme-substrate binding events. The results reveal three categories of relatively persistent and noteworthy enzyme-substrate binding configurations, which we call configurations A, B, and C. We performed binding free energy calculations of the enzyme-substrate complexes of different configurations using the molecular mechanics Poisson-Boltzmann surface area method. The role of each residue of the active site in binding the substrate was investigated using energy decomposition analysis. The simulated trajectories provide a continuous atomic-level view of the entire binding process, revealing potentially valuable regions where the enzyme and the substrate interact persistently and five possible pathways of the substrate entering into the active center, which were validated using well-tempered metadynamics. These findings provide important insights into the binding mechanism of meropenem to NDM-1, which may provide new prospects for the design of novel metallo-β-lactamase inhibitors and enzyme-resistant antibiotics.

Fiber optic detector and method for using same for detecting chemical species

DOEpatents

Baylor, Lewis C.; Buchanan, Bruce R.

1995-01-01

An optical sensing device for uranyl and other substances, a method for making an optical sensing device and a method for chemically binding uranyl and other indicators to glass, quartz, cellulose and similar substrates. The indicator, such as arsenazo III, is immobilized on the substrate using a chemical binding process. The immobilized arsenazo III causes uranyl from a fluid sample to bind irreversibly to the substrate at its active sites, thus causing absorption of a portion of light transmitted through the substrate. Determination of the amount of light absorbed, using conventional means, yields the concentration of uranyl present in the sample fluid. The binding of uranyl on the substrate can be reversed by subsequent exposure of the substrate to a solution of 2,6-pyridinedicarboxylic acid. The chemical binding process is suitable for similarly binding other indicators, such as bromocresol green.

Structure-Function Relationship of a Plant NCS1 Member – Homology Modeling and Mutagenesis Identified Residues Critical for Substrate Specificity of PLUTO, a Nucleobase Transporter from Arabidopsis

PubMed Central

Witz, Sandra; Panwar, Pankaj; Schober, Markus; Deppe, Johannes; Pasha, Farhan Ahmad; Lemieux, M. Joanne; Möhlmann, Torsten

2014-01-01

Plastidic uracil salvage is essential for plant growth and development. So far, PLUTO, the plastidic nucleobase transporter from Arabidopsis thaliana is the only known uracil importer at the inner plastidic membrane which represents the permeability barrier of this organelle. We present the first homology model of PLUTO, the sole plant NCS1 member from Arabidopsis based on the crystal structure of the benzyl hydantoin transporter MHP1 from Microbacterium liquefaciens and validated by molecular dynamics simulations. Polar side chains of residues Glu-227 and backbones of Val-145, Gly-147 and Thr-425 are proposed to form the binding site for the three PLUTO substrates uracil, adenine and guanine. Mutational analysis and competition studies identified Glu-227 as an important residue for uracil and to a lesser extent for guanine transport. A differential response in substrate transport was apparent with PLUTO double mutants E227Q G147Q and E227Q T425A, both of which most strongly affected adenine transport, and in V145A G147Q, which markedly affected guanine transport. These differences could be explained by docking studies, showing that uracil and guanine exhibit a similar binding mode whereas adenine binds deep into the catalytic pocket of PLUTO. Furthermore, competition studies confirmed these results. The present study defines the molecular determinants for PLUTO substrate binding and demonstrates key differences in structure-function relations between PLUTO and other NCS1 family members. PMID:24621654

Structure-function relationship of a plant NCS1 member--homology modeling and mutagenesis identified residues critical for substrate specificity of PLUTO, a nucleobase transporter from Arabidopsis.

PubMed

Witz, Sandra; Panwar, Pankaj; Schober, Markus; Deppe, Johannes; Pasha, Farhan Ahmad; Lemieux, M Joanne; Möhlmann, Torsten

2014-01-01

Plastidic uracil salvage is essential for plant growth and development. So far, PLUTO, the plastidic nucleobase transporter from Arabidopsis thaliana is the only known uracil importer at the inner plastidic membrane which represents the permeability barrier of this organelle. We present the first homology model of PLUTO, the sole plant NCS1 member from Arabidopsis based on the crystal structure of the benzyl hydantoin transporter MHP1 from Microbacterium liquefaciens and validated by molecular dynamics simulations. Polar side chains of residues Glu-227 and backbones of Val-145, Gly-147 and Thr-425 are proposed to form the binding site for the three PLUTO substrates uracil, adenine and guanine. Mutational analysis and competition studies identified Glu-227 as an important residue for uracil and to a lesser extent for guanine transport. A differential response in substrate transport was apparent with PLUTO double mutants E227Q G147Q and E227Q T425A, both of which most strongly affected adenine transport, and in V145A G147Q, which markedly affected guanine transport. These differences could be explained by docking studies, showing that uracil and guanine exhibit a similar binding mode whereas adenine binds deep into the catalytic pocket of PLUTO. Furthermore, competition studies confirmed these results. The present study defines the molecular determinants for PLUTO substrate binding and demonstrates key differences in structure-function relations between PLUTO and other NCS1 family members.

Discovery of the target for immunomodulatory drugs (IMiDs).

PubMed

Ito, Takumi; Ando, Hideki; Handa, Hiroshi

2016-05-01

Half a century ago, the sedative thalidomide caused a serious drug disaster because of its teratogenicity and was withdrawn from the market. However, thalidomide, which has returned to the market, is now used for the treatment of leprosy and multiple myeloma (MM) under strict control. The mechanism of thalidomide action had been a long-standing question. We developed a new affinity bead technology and identified cereblon (CRBN) as a thalidomide-binding protein. We found that CRBN functions as a substrate receptor of an E3 cullin-Ring ligase complex 4 (CRL4) and is a primary target of thalidomide teratogenicity. Recently, new thalidomide derivatives, called immunomodulatory drugs (IMiDs), have been developed by Celgene. Among them, lenalidomide (Len) and pomalidomide (Pom) were shown to exert strong therapeutic effects against MM. It was found that Len and Pom both bind CRBN-CRL4 and recruit neomorphic substrates (Ikaros and Aiolos). More recently it was reported that casein kinase 1a (Ck1a) was identified as a substrate for CRBN-CRL4 in the presence of Len, but not Pom. Ck1a breakdown explains why Len is specifically effective for myelodysplastic syndrome with 5q deletion. It is now proposed that binding of IMiDs to CRBN appears to alter the substrate specificity of CRBN-CRL4. In this review, we introduce recent findings on IMiDs.

Structure of the Trehalose-6-phosphate Phosphatase from Brugia malayi Reveals Key Design Principles for Anthelmintic Drugs

PubMed Central

Farelli, Jeremiah D.; Galvin, Brendan D.; Li, Zhiru; Liu, Chunliang; Aono, Miyuki; Garland, Megan; Hallett, Olivia E.; Causey, Thomas B.; Ali-Reynolds, Alana; Saltzberg, Daniel J.; Carlow, Clotilde K. S.; Dunaway-Mariano, Debra; Allen, Karen N.

2014-01-01

Parasitic nematodes are responsible for devastating illnesses that plague many of the world's poorest populations indigenous to the tropical areas of developing nations. Among these diseases is lymphatic filariasis, a major cause of permanent and long-term disability. Proteins essential to nematodes that do not have mammalian counterparts represent targets for therapeutic inhibitor discovery. One promising target is trehalose-6-phosphate phosphatase (T6PP) from Brugia malayi. In the model nematode Caenorhabditis elegans, T6PP is essential for survival due to the toxic effect(s) of the accumulation of trehalose 6-phosphate. T6PP has also been shown to be essential in Mycobacterium tuberculosis. We determined the X-ray crystal structure of T6PP from B. malayi. The protein structure revealed a stabilizing N-terminal MIT-like domain and a catalytic C-terminal C2B-type HAD phosphatase fold. Structure-guided mutagenesis, combined with kinetic analyses using a designed competitive inhibitor, trehalose 6-sulfate, identified five residues important for binding and catalysis. This structure-function analysis along with computational mapping provided the basis for the proposed model of the T6PP-trehalose 6-phosphate complex. The model indicates a substrate-binding mode wherein shape complementarity and van der Waals interactions drive recognition. The mode of binding is in sharp contrast to the homolog sucrose-6-phosphate phosphatase where extensive hydrogen-bond interactions are made to the substrate. Together these results suggest that high-affinity inhibitors will be bi-dentate, taking advantage of substrate-like binding to the phosphoryl-binding pocket while simultaneously utilizing non-native binding to the trehalose pocket. The conservation of the key residues that enforce the shape of the substrate pocket in T6PP enzymes suggest that development of broad-range anthelmintic and antibacterial therapeutics employing this platform may be possible. PMID:24992307

Structure of the streptococcal endopeptidase IdeS, a cysteine proteinase with strict specificity for IgG.

PubMed

Wenig, Katja; Chatwell, Lorenz; von Pawel-Rammingen, Ulrich; Björck, Lars; Huber, Robert; Sondermann, Peter

2004-12-14

Pathogenic bacteria have developed complex and diverse virulence mechanisms that weaken or disable the host immune defense system. IdeS (IgG-degrading enzyme of Streptococcus pyogenes) is a secreted cysteine endopeptidase from the human pathogen S. pyogenes with an extraordinarily high degree of substrate specificity, catalyzing a single proteolytic cleavage at the lower hinge of human IgG. This proteolytic degradation promotes inhibition of opsonophagocytosis and interferes with the killing of group A Streptococcus. We have determined the crystal structure of the catalytically inactive mutant IdeS-C94S by x-ray crystallography at 1.9-A resolution. Despite negligible sequence homology to known proteinases, the core of the structure resembles the canonical papain fold although with major insertions and a distinct substrate-binding site. Therefore IdeS belongs to a unique family within the CA clan of cysteine proteinases. Based on analogy with inhibitor complexes of papain-like proteinases, we propose a model for substrate binding by IdeS.

Computational design and experimental study of tighter binding peptides to an inactivated mutant of HIV-1 protease

PubMed Central

Altman, Michael D.; Nalivaika, Ellen A.; Prabu-Jeyabalan, Moses; Schiffer, Celia A.; Tidor, Bruce

2009-01-01

Drug resistance in HIV-1 protease, a barrier to effective treatment, is generally caused by mutations in the enzyme that disrupt inhibitor binding but still allow for substrate processing. Structural studies with mutant, inactive enzyme, have provided detailed information regarding how the substrates bind to the protease yet avoid resistance mutations; insights obtained inform the development of next generation therapeutics. Although structures have been obtained of complexes between substrate peptide and inactivated (D25N) protease, thermodynamic studies of peptide binding have been challenging due to low affinity. Peptides that bind tighter to the inactivated protease than the natural substrates would be valuable for thermodynamic studies as well as to explore whether the structural envelope observed for substrate peptides is a function of weak binding. Here, two computational methods — namely, charge optimization and protein design — were applied to identify peptide sequences predicted to have higher binding affinity to the inactivated protease, starting from an RT–RH derived substrate peptide. Of the candidate designed peptides, three were tested for binding with isothermal titration calorimetry, with one, containing a single threonine to valine substitution, measured to have more than a ten-fold improvement over the tightest binding natural substrate. Crystal structures were also obtained for the same three designed peptide complexes; they show good agreement with computational prediction. Thermodynamic studies show that binding is entropically driven, more so for designed affinity enhanced variants than for the starting substrate. Structural studies show strong similarities between natural and tighter-binding designed peptide complexes, which may have implications in understanding the molecular mechanisms of drug resistance in HIV-1 protease. PMID:17729291

Mutational Insights into the Roles of Amino Acid Residues in Ligand Binding for Two Closely Related Family 16 Carbohydrate Binding Modules

DOE Office of Scientific and Technical Information (OSTI.GOV)

Su, Xiaoyun; Agarwal, Vinayak; Dodd, Dylan

2010-11-22

Carbohydrate binding modules (CBMs) are specialized proteins that bind to polysaccharides and oligosaccharides. Caldanaerobius polysaccharolyticus Man5ACBM16-1/CBM16-2 bind to glucose-, mannose-, and glucose/mannose-configured substrates. The crystal structures of the two proteins represent the only examples in CBM family 16, and studies that evaluate the roles of amino acid residues in ligand binding in this family are lacking. In this study, we probed the roles of amino acids (selected based on CBM16-1/ligand co-crystal structures) on substrate binding. Two tryptophan (Trp-20 and Trp-125) and two glutamine (Gln-81 and Gln-93) residues are shown to be critical in ligand binding. Additionally, several polar residues thatmore » flank the critical residues also contribute to ligand binding. The CBM16-1 Q121E mutation increased affinity for all substrates tested, whereas the Q21G and N97R mutants exhibited decreased substrate affinity. We solved CBM/substrate co-crystal structures to elucidate the molecular basis of the increased substrate binding by CBM16-1 Q121E. The Gln-121, Gln-21, and Asn-97 residues can be manipulated to fine-tune ligand binding by the Man5A CBMs. Surprisingly, none of the eight residues investigated was absolutely conserved in CBM family 16. Thus, the critical residues in the Man5A CBMs are either not essential for substrate binding in the other members of this family or the two CBMs are evolutionarily distinct from the members available in the current protein database. Man5A is dependent on its CBMs for robust activity, and insights from this study should serve to enhance our understanding of the interdependence of its catalytic and substrate binding modules.« less

Substrate binding stoichiometry and kinetics of the norepinephrine transporter.

PubMed

Schwartz, Joel W; Novarino, Gaia; Piston, David W; DeFelice, Louis J

2005-05-13

The human norepinephrine (NE) transporter (hNET) attenuates neuronal signaling by rapid NE clearance from the synaptic cleft, and NET is a target for cocaine and amphetamines as well as therapeutics for depression, obsessive-compulsive disorder, and post-traumatic stress disorder. In spite of its central importance in the nervous system, little is known about how NET substrates, such as NE, 1-methyl-4-tetrahydropyridinium (MPP+), or amphetamine, interact with NET at the molecular level. Nor do we understand the mechanisms behind the transport rate. Previously we introduced a fluorescent substrate similar to MPP+, which allowed separate and simultaneous binding and transport measurement (Schwartz, J. W., Blakely, R. D., and DeFelice, L. J. (2003) J. Biol. Chem. 278, 9768-9777). Here we use this substrate, 4-(4-(dimethylamino)styrl)-N-methyl-pyridinium (ASP+), in combination with green fluorescent protein-tagged hNETs to measure substrate-transporter stoichiometry and substrate binding kinetics. Calibrated confocal microscopy and fluorescence correlation spectroscopy reveal that hNETs, which are homomultimers, bind one substrate molecule per transporter subunit. Substrate residence at the transporter, obtained from rapid on-off kinetics revealed in fluorescence correlation spectroscopy, is 526 micros. Substrate residence obtained by infinite dilution is 1000 times slower. This novel examination of substrate-transporter kinetics indicates that a single ASP+ molecule binds and unbinds thousands of times before being transported or ultimately dissociated from hNET. Calibrated fluorescent images combined with mass spectroscopy give a transport rate of 0.06 ASP+/hNET-protein/s, thus 36,000 on-off binding events (and 36 actual departures) occur for one transport event. Therefore binding has a low probability of resulting in transport. We interpret these data to mean that inefficient binding could contribute to slow transport rates.

Structural insights into conserved L-arabinose metabolic enzymes reveal the substrate binding site of a thermophilic L-arabinose isomerase.

PubMed

Lee, Yong-Jik; Lee, Sang-Jae; Kim, Seong-Bo; Lee, Sang Jun; Lee, Sung Haeng; Lee, Dong-Woo

2014-03-18

Structural genomics demonstrates that despite low levels of structural similarity of proteins comprising a metabolic pathway, their substrate binding regions are likely to be conserved. Herein based on the 3D-structures of the α/β-fold proteins involved in the ara operon, we attempted to predict the substrate binding residues of thermophilic Geobacillus stearothermophilus L-arabinose isomerase (GSAI) with no 3D-structure available. Comparison of the structures of L-arabinose catabolic enzymes revealed a conserved feature to form the substrate-binding modules, which can be extended to predict the substrate binding site of GSAI (i.e., D195, E261 and E333). Moreover, these data implicated that proteins in the l-arabinose metabolic pathway might retain their substrate binding niches as the modular structure through conserved molecular evolution even with totally different structural scaffolds. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Specificity of Processing α-glucosidase I is guided by the substrate conformation: crystallographic and in silico studies.

PubMed

Barker, Megan K; Rose, David R

2013-05-10

The enzyme “GluI” is key to the synthesis of critical glycoproteins in the cell. We have determined the structure of GluI, and modeled binding with its unique sugar substrate. The specificity of this interaction derives from a unique conformation of the substrate. Understanding the mechanism of the enzyme is of basic importance and relevant to potential development of antiviral inhibitors. Processing α-glucosidase I (GluI) is a key member of the eukaryotic N-glycosylation processing pathway, selectively catalyzing the first glycoprotein trimming step in the endoplasmic reticulum. Inhibition of GluI activity impacts the infectivity of enveloped viruses; however, despite interest in this protein from a structural, enzymatic, and therapeutic standpoint, little is known about its structure and enzymatic mechanism in catalysis of the unique glycan substrate Glc3Man9GlcNAc2. The first structural model of eukaryotic GluI is here presented at 2-Å resolution. Two catalytic residues are proposed, mutations of which result in catalytically inactive, properly folded protein. Using Autodocking methods with the known substrate and inhibitors as ligands, including a novel inhibitor characterized in this work, the active site of GluI was mapped. From these results, a model of substrate binding has been formulated, which is most likely conserved in mammalian GluI.

Testing Geometrical Discrimination within an Enzyme Active Site: Constrained Hydrogen Bonding in the Ketosteroid Isomerase Oxyanion Hole

PubMed Central

Sigala, Paul A.; Kraut, Daniel A.; Caaveiro, Jose M. M.; Pybus, Brandon; Ruben, Eliza A.; Ringe, Dagmar; Petsko, Gregory A.; Herschlag, Daniel

2009-01-01

Enzymes are classically proposed to accelerate reactions by binding substrates within active site environments that are structurally preorganized to optimize binding interactions with reaction transition states rather than ground states. This is a remarkably formidable task considering the limited 0.1 – 1 Å scale of most substrate rearrangements. The flexibility of active site functional groups along the coordinate of substrate rearrangement, the distance scale on which enzymes can distinguish structural rearrangement, and the energetic significance of discrimination on that scale remain open questions that are fundamental to a basic physical understanding of enzyme active sites and catalysis. We bring together high resolution X-ray crystallography, 1H and 19F NMR spectroscopy, quantum mechanical calculations, and transition state analog binding measurements to test the distance scale on which non-covalent forces can constrain side chain and ligand relaxation or translation along a specific coordinate and the energetic consequences of such geometric constraints within the active site of bacterial ketosteroid isomerase (KSI). Our results strongly suggest that packing and binding interactions within the KSI active site can constrain local side chain reorientation and prevent hydrogen bond shortening by 0.1 Å or less. Further, this constraint has substantial energetic effects on ligand binding and stabilization of negative charge within the oxyanion hole. These results provide evidence that subtle geometric effects, indistinguishable in most X-ray crystallographic structures, can have significant energetic consequences and highlight the importance of using synergistic experimental approaches to dissect enzyme function. PMID:18808119

Substrate Binding Drives Active-Site Closing of Human Blood Group B Galactosyltransferase as Revealed by Hot-Spot Labeling and NMR Spectroscopy Experiments.

PubMed

Weissbach, Sophie; Flügge, Friedemann; Peters, Thomas

2018-05-04

Crystallography has shown that human blood group A (GTA) and B (GTB) glycosyltransferases undergo transitions between "open", "semiclosed", and "closed" conformations upon substrate binding. However, the timescales of the corresponding conformational reorientations are unknown. Crystal structures show that the Trp and Met residues are located at "conformational hot spots" of the enzymes. Therefore, we utilized 15 N side-chain labeling of Trp residues and 13 C-methyl labeling of Met residues to study substrate-induced conformational transitions of GTB. Chemical-shift perturbations (CSPs) of Met and Trp residues in direct contact with substrate ligands reflect binding kinetics, whereas the CSPs of Met and Trp residues at remote sites reflect conformational changes of the enzyme upon substrate binding. Acceptor binding is fast on the chemical-shift timescale with rather small CSPs in the range of less than approximately 20 Hz. Donor binding matches the intermediate exchange regime to yield an estimate for exchange rate constants of approximately 200-300 Hz. Donor or acceptor binding to GTB saturated with acceptor or donor substrate, respectively, is slow (<10 Hz), as are coupled protein motions, reflecting mutual allosteric control of donor and acceptor binding. Remote CSPs suggest that substrate binding drives the enzyme into the closed state required for catalysis. These findings should contribute to better understanding of the mechanism of glycosyl transfer of GTA and GTB. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Determination of the Structure and Catalytic Mechanism of Sorghum bicolor Caffeic Acid O-Methyltransferase and the Structural Impact of Three brown midrib12 Mutations1[W

PubMed Central

Green, Abigail R.; Lewis, Kevin M.; Barr, John T.; Jones, Jeffrey P.; Lu, Fachuang; Ralph, John; Vermerris, Wilfred; Sattler, Scott E.; Kang, ChulHee

2014-01-01

Using S-adenosyl-methionine as the methyl donor, caffeic acid O-methyltransferase from sorghum (Sorghum bicolor; SbCOMT) methylates the 5-hydroxyl group of its preferred substrate, 5-hydroxyconiferaldehyde. In order to determine the mechanism of SbCOMT and understand the observed reduction in the lignin syringyl-to-guaiacyl ratio of three brown midrib12 mutants that carry COMT gene missense mutations, we determined the apo-form and S-adenosyl-methionine binary complex SbCOMT crystal structures and established the ternary complex structure with 5-hydroxyconiferaldehyde by molecular modeling. These structures revealed many features shared with monocot ryegrass (Lolium perenne) and dicot alfalfa (Medicago sativa) COMTs. SbCOMT steady-state kinetic and calorimetric data suggest a random bi-bi mechanism. Based on our structural, kinetic, and thermodynamic results, we propose that the observed reactivity hierarchy among 4,5-dihydroxy-3-methoxycinnamyl (and 3,4-dihydroxycinnamyl) aldehyde, alcohol, and acid substrates arises from the ability of the aldehyde to stabilize the anionic intermediate that results from deprotonation of the 5-hydroxyl group by histidine-267. Additionally, despite the presence of other phenylpropanoid substrates in vivo, sinapaldehyde is the preferential product, as demonstrated by its low Km for 5-hydroxyconiferaldehyde. Unlike its acid and alcohol substrates, the aldehydes exhibit product inhibition, and we propose that this is due to nonproductive binding of the S-cis-form of the aldehydes inhibiting productive binding of the S-trans-form. The S-cis-aldehydes most likely act only as inhibitors, because the high rotational energy barrier around the 2-propenyl bond prevents S-trans-conversion, unlike alcohol substrates, whose low 2-propenyl bond rotational energy barrier enables rapid S-cis/S-trans-interconversion. PMID:24948836

Cooperative mechanism of RNA packaging motor.

PubMed

Lísal, Jirí; Tuma, Roman

2005-06-17

P4 is a hexameric ATPase that serves as the RNA packaging motor in double-stranded RNA bacteriophages from the Cystoviridae family. P4 shares sequence and structural similarities with hexameric helicases. A structure-based mechanism for mechano-chemical coupling has recently been proposed for P4 from bacteriophage phi12. However, coordination of ATP hydrolysis among the subunits and coupling with RNA translocation remains elusive. Here we present detailed kinetic study of nucleotide binding, hydrolysis, and product release by phi12 P4 in the presence of different RNA and DNA substrates. Whereas binding affinities for ATP and ADP are not affected by RNA binding, the hydrolysis step is accelerated and the apparent cooperativity is increased. No nucleotide binding cooperativity is observed. We propose a stochastic-sequential cooperativity model to describe the coordination of ATP hydrolysis within the hexamer. In this model the apparent cooperativity is a result of hydrolysis stimulation by ATP and RNA binding to neighboring subunits rather than cooperative nucleotide binding. The translocation step appears coupled to hydrolysis, which is coordinated among three neighboring subunits. Simultaneous interaction of neighboring subunits with RNA makes the otherwise random hydrolysis sequential and processive.

Sensitive and fast detection of fructose in complex media via symmetry breaking and signal amplification using surface-enhanced Raman spectroscopy.

PubMed

Sun, Fang; Bai, Tao; Zhang, Lei; Ella-Menye, Jean-Rene; Liu, Sijun; Nowinski, Ann K; Jiang, Shaoyi; Yu, Qiuming

2014-03-04

A new strategy is proposed to sensitively and rapidly detect analytes with weak Raman signals in complex media using surface-enhanced Raman spectroscopy (SERS) via detecting the SERS signal changes of the immobilized probe molecules on SERS-active substrates upon binding of the analytes. In this work, 4-mercaptophenylboronic acid (4-MPBA) was selected as the probe molecule which was immobilized on the gold surface of a quasi-three-dimensional plasmonic nanostructure array (Q3D-PNA) SERS substrate to detect fructose. The molecule of 4-MPBA possesses three key functions: molecule recognition and reversible binding of the analyte via the boronic acid group, amplification of SERS signals by the phenyl group and thus shielding of the background noise of complex media, and immobilization on the surface of SERS-active substrates via the thiol group. Most importantly, the symmetry breaking of the 4-MPBA molecule upon fructose binding leads to the change of area ratio between totally symmetric 8a ring mode and nontotally symmetric 8b ring mode, which enables the detection. The detection curves were obtained in phosphate-buffered saline (PBS) and in undiluted artificial urine at clinically relevant concentrations, and the limit of detection of 0.05 mM was achieved.

Non-competitive inhibition by active site binders.

PubMed

Blat, Yuval

2010-06-01

Classical enzymology has been used for generations to understand the interactions of inhibitors with their enzyme targets. Enzymology tools enabled prediction of the biological impact of inhibitors as well as the development of novel, more potent, ones. Experiments designed to examine the competition between the tested inhibitor and the enzyme substrate(s) are the tool of choice to identify inhibitors that bind in the active site. Competition between an inhibitor and a substrate is considered a strong evidence for binding of the inhibitor in the active site, while the lack of competition suggests binding to an alternative site. Nevertheless, exceptions to this notion do exist. Active site-binding inhibitors can display non-competitive inhibition patterns. This unusual behavior has been observed with enzymes utilizing an exosite for substrate binding, isomechanism enzymes, enzymes with multiple substrates and/or products and two-step binding inhibitors. In many of these cases, the mechanisms underlying the lack of competition between the substrate and the inhibitor are well understood. Tools like alternative substrates, testing the enzyme reaction in the reverse direction and monitoring inhibition time dependence can be applied to enable distinction between 'badly behaving' active site binders and true exosite inhibitors.

Na+ Interactions with the Neutral Amino Acid Transporter ASCT1*

PubMed Central

Scopelliti, Amanda J.; Heinzelmann, Germano; Kuyucak, Serdar; Ryan, Renae M.; Vandenberg, Robert J.

2014-01-01

The alanine, serine, cysteine transporters (ASCTs) belong to the solute carrier family 1A (SLC1A), which also includes the excitatory amino acid transporters (EAATs) and the prokaryotic aspartate transporter GltPh. Acidic amino acid transport by the EAATs is coupled to the co-transport of three Na+ ions and one proton, and the counter-transport of one K+ ion. In contrast, neutral amino acid exchange by the ASCTs does not require protons or the counter-transport of K+ ions and the number of Na+ ions required is not well established. One property common to SLC1A family members is a substrate-activated anion conductance. We have investigated the number and location of Na+ ions required by ASCT1 by mutating residues in ASCT1 that correspond to residues in the EAATs and GltPh that are involved in Na+ binding. Mutations to all three proposed Na+ sites influence the binding of substrate and/or Na+, or the rate of substrate exchange. A G422S mutation near the Na2 site reduced Na+ affinity, without affecting the rate of exchange. D467T and D467A mutations in the Na1 site reduce Na+ and substrate affinity and also the rate of substrate exchange. T124A and D380A mutations in the Na3 site selectively reduce the affinity for Na+ and the rate of substrate exchange without affecting substrate affinity. In many of the mutants that reduce the rate of substrate transport the amplitudes of the substrate-activated anion conductances are not substantially affected indicating altered ion dependence for channel activation compared with substrate exchange. PMID:24808181

Domain wise docking analyses of the modular chitin binding protein CBP50 from Bacillus thuringiensis serovar konkukian S4.

PubMed

Sehar, Ujala; Mehmood, Muhammad Aamer; Hussain, Khadim; Nawaz, Salman; Nadeem, Shahid; Siddique, Muhammad Hussnain; Nadeem, Habibullah; Gull, Munazza; Ahmad, Niaz; Sohail, Iqra; Gill, Saba Shahid; Majeed, Summera

2013-01-01

This paper presents an in silico characterization of the chitin binding protein CBP50 from B. thuringiensis serovar konkukian S4 through homology modeling and molecular docking. The CBP50 has shown a modular structure containing an N-terminal CBM33 domain, two consecutive fibronectin-III (Fn-III) like domains and a C-terminal CBM5 domain. The protein presented a unique modular structure which could not be modeled using ordinary procedures. So, domain wise modeling using MODELLER and docking analyses using Autodock Vina were performed. The best conformation for each domain was selected using standard procedure. It was revealed that four amino acid residues Glu-71, Ser-74, Glu-76 and Gln-90 from N-terminal domain are involved in protein-substrate interaction. Similarly, amino acid residues Trp-20, Asn-21, Ser-23 and Val-30 of Fn-III like domains and Glu-15, Ala-17, Ser-18 and Leu-35 of C-terminal domain were involved in substrate binding. Site-directed mutagenesis of these proposed amino acid residues in future will elucidate the key amino acids involved in chitin binding activity of CBP50 protein.

Inhibition of SCF ubiquitin ligases by engineered ubiquitin variants that target the Cul1 binding site on the Skp1–F-box interface

DOE PAGES

Gorelik, Maryna; Orlicky, Stephen; Sartori, Maria A.; ...

2016-03-14

Skp1–Cul1–F-box (SCF) E3 ligases play key roles in multiple cellular processes through ubiquitination and subsequent degradation of substrate proteins. Although Skp1 and Cul1 are invariant components of all SCF complexes, the 69 different human F-box proteins are variable substrate binding modules that determine specificity. SCF E3 ligases are activated in many cancers and inhibitors could have therapeutic potential. Here, we used phage display to develop specific ubiquitin-based inhibitors against two F-box proteins, Fbw7 and Fbw11. Unexpectedly, the ubiquitin variants bind at the interface of Skp1 and F-box proteins and inhibit ligase activity by preventing Cul1 binding to the same surface.more » Using structure-based design and phage display, we modified the initial inhibitors to generate broad-spectrum inhibitors that targeted many SCF ligases, or conversely, a highly specific inhibitor that discriminated between even the close homologs Fbw11 and Fbw1. We propose that most F-box proteins can be targeted by this approach for basic research and for potential cancer therapies.« less

Analysis of DNA binding by human factor xeroderma pigmentosum complementation group A (XPA) provides insight into its interactions with nucleotide excision repair substrates.

PubMed

Sugitani, Norie; Voehler, Markus W; Roh, Michelle S; Topolska-Woś, Agnieszka M; Chazin, Walter J

2017-10-13

Xeroderma pigmentosum (XP) complementation group A (XPA) is an essential scaffolding protein in the multiprotein nucleotide excision repair (NER) machinery. The interaction of XPA with DNA is a core function of this protein; a number of mutations in the DNA-binding domain (DBD) are associated with XP disease. Although structures of the central globular domain of human XPA and data on binding of DNA substrates have been reported, the structural basis for XPA's DNA-binding activity remains unknown. X-ray crystal structures of the central globular domain of yeast XPA (Rad14) with lesion-containing DNA duplexes have provided valuable insights, but the DNA substrates used for this study do not correspond to the substrates of XPA as it functions within the NER machinery. To better understand the DNA-binding activity of human XPA in NER, we used NMR to investigate the interaction of its DBD with a range of DNA substrates. We found that XPA binds different single-stranded/double-stranded junction DNA substrates with a common surface. Comparisons of our NMR-based mapping of binding residues with the previously reported Rad14-DNA crystal structures revealed similarities and differences in substrate binding between XPA and Rad14. This includes direct evidence for DNA contacts to the residues extending C-terminally from the globular core, which are lacking in the Rad14 construct. Moreover, mutation of the XPA residue corresponding to Phe-262 in Rad14, previously reported as being critical for DNA binding, had only a moderate effect on the DNA-binding activity of XPA. The DNA-binding properties of several disease-associated mutations in the DBD were investigated. These results suggest that for XPA mutants exhibiting altered DNA-binding properties, a correlation exists between the extent of reduction in DNA-binding affinity and the severity of symptoms in XP patients. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Unique structural modulation of a non-native substrate by cochaperone DnaJ.

PubMed

Tiwari, Satyam; Kumar, Vignesh; Jayaraj, Gopal Gunanathan; Maiti, Souvik; Mapa, Koyeli

2013-02-12

The role of bacterial DnaJ protein as a cochaperone of DnaK is strongly appreciated. Although DnaJ unaccompanied by DnaK can bind unfolded as well as native substrate proteins, its role as an individual chaperone remains elusive. In this study, we demonstrate that DnaJ binds a model non-native substrate with a low nanomolar dissociation constant and, more importantly, modulates the structure of its non-native state. The structural modulation achieved by DnaJ is different compared to that achieved by the DnaK-DnaJ complex. The nature of structural modulation exerted by DnaJ is suggestive of a unique unfolding activity on the non-native substrate by the chaperone. Furthermore, we demonstrate that the zinc binding motif along with the C-terminal substrate binding domain of DnaJ is necessary and sufficient for binding and the subsequent binding-induced structural alterations of the non-native substrate. We hypothesize that this hitherto unknown structural alteration of non-native states by DnaJ might be important for its chaperoning activity by removing kinetic traps of the folding intermediates.

pKa Modulation of the Acid/Base Catalyst within GH32 and GH68: A Role in Substrate/Inhibitor Specificity?

PubMed Central

Yuan, Shuguang; Le Roy, Katrien; Venken, Tom; Lammens, Willem; Van den Ende, Wim; De Maeyer, Marc

2012-01-01

Glycoside hydrolases of families 32 (GH32) and 68 (GH68) belong to clan GH-J, containing hydrolytic enzymes (sucrose/fructans as donor substrates) and fructosyltransferases (sucrose/fructans as donor and acceptor substrates). In GH32 members, some of the sugar substrates can also function as inhibitors, this regulatory aspect further adding to the complexity in enzyme functionalities within this family. Although 3D structural information becomes increasingly available within this clan and huge progress has been made on structure-function relationships, it is not clear why some sugars bind as inhibitors without being catalyzed. Conserved aspartate and glutamate residues are well known to act as nucleophile and acid/bases within this clan. Based on the available 3D structures of enzymes and enzyme-ligand complexes as well as docking simulations, we calculated the pKa of the acid-base before and after substrate binding. The obtained results strongly suggest that most GH-J members show an acid-base catalyst that is not sufficiently protonated before ligand entrance, while the acid-base can be fully protonated when a substrate, but not an inhibitor, enters the catalytic pocket. This provides a new mechanistic insight aiming at understanding the complex substrate and inhibitor specificities observed within the GH-J clan. Moreover, besides the effect of substrate entrance on its own, we strongly suggest that a highly conserved arginine residue (in the RDP motif) rather than the previously proposed Tyr motif (not conserved) provides the proton to increase the pKa of the acid-base catalyst. PMID:22662155

Molecular simulations bring new insights into flavonoid/quercetinase interaction modes.

PubMed

Fiorucci, Sébastien; Golebiowski, Jérôme; Cabrol-Bass, Daniel; Antonczak, Serge

2007-06-01

Molecular dynamics simulations, using the AMBER force field, were performed to study Quercetin 2,3-Dioxygenase enzyme (Quercetinase or 2,3QD). We have analyzed the structural modifications of the active site and of the linker region between the native enzyme and the enzyme-substrate complex. New structural informations, such as an allosteric effect in the presence of the substrate, as well as description of the enzyme-substrate interactions and values of binding free energies were brought out. All these results confirm the idea that the linker encloses the substrate in the active site and also enlighten the recognition role of the substrate B-ring by the enzyme. Moreover, a specific interaction scheme has been proposed to explain the relative degradation rate of various flavonoid compounds under the oxygenolysis reaction catalyzed by the Quercetin 2,3-Dioxygenase enzyme. 2007 Wiley-Liss, Inc.

A differential scanning calorimetric study of the effects of metal ions, substrate/product, substrate analogues and chaotropic anions on the thermal denaturation of yeast enolase 1.

PubMed

Brewer, J M; Wampler, J E

2001-03-14

The thermal denaturation of yeast enolase 1 was studied by differential scanning calorimetry (DSC) under conditions of subunit association/dissociation, enzymatic activity or substrate binding without turnover and substrate analogue binding. Subunit association stabilizes the enzyme, that is, the enzyme dissociates before denaturing. The conformational change produced by conformational metal ion binding increases thermal stability by reducing subunit dissociation. 'Substrate' or analogue binding additionally stabilizes the enzyme, irrespective of whether turnover is occurring, perhaps in part by the same mechanism. More strongly bound metal ions also stabilize the enzyme more, which we interpret as consistent with metal ion loss before denaturation, though possibly the denaturation pathway is different in the absence of metal ion. We suggest that some of the stabilization by 'substrate' and analogue binding is owing to the closure of moveable polypeptide loops about the active site, producing a more 'closed' and hence thermostable conformation.

Substrate binding interferes with active site conformational dynamics in endoglucanase Cel5A from Thermobifida fusca.

PubMed

Jiang, Xukai; Wang, Yuying; Xu, Limei; Chen, Guanjun; Wang, Lushan

2017-09-09

The role of protein dynamics in enzyme catalysis is one of the most active areas in current enzymological research. Here, using endoglucanase Cel5A from Thermobifida fusca (TfCel5A) as a model, we applied molecular dynamics simulations to explore the dynamic behavior of the enzyme upon substrate binding. The collective motions of the active site revealed that the mechanism of TfCel5A substrate binding can likely be described by the conformational-selection model; however, we observed that the conformations of active site residues changed differently along with substrate binding. Although most active site residues retained their native conformational ensemble, some (Tyr163 and Glu355) generated newly induced conformations, whereas others (Phe162 and Tyr189) exhibited shifts in the equilibration of their conformational distributions. These results showed that TfCel5A substrate binding relied on a hybrid mechanism involving induced fit and conformational selection. Interestingly, we found that TfCel5A active site could only partly rebalance its conformational dynamics upon substrate dissociation within the same simulation time, which implies that the conformational rebalance upon substrate dissociation is likely more difficult than the conformational selection upon substrate binding at least in the view of the time required. Our findings offer new insight into enzyme catalysis and potential applications for future protein engineering. Copyright © 2017 Elsevier Inc. All rights reserved.

Uncoupling metallonuclease metal ion binding sites via nudge mutagenesis.

PubMed

Papadakos, Grigorios A; Nastri, Horacio; Riggs, Paul; Dupureur, Cynthia M

2007-05-01

The hydrolysis of phosphodiester bonds by nucleases is critical to nucleic acid processing. Many nucleases utilize metal ion cofactors, and for a number of these enzymes two active-site metal ions have been detected. Testing proposed mechanistic roles for individual bound metal ions has been hampered by the similarity between the sites and cooperative behavior. In the homodimeric PvuII restriction endonuclease, the metal ion dependence of DNA binding is sigmoidal and consistent with two classes of coupled metal ion binding sites. We reasoned that a conservative active-site mutation would perturb the ligand field sufficiently to observe the titration of individual metal ion binding sites without significantly disturbing enzyme function. Indeed, mutation of a Tyr residue 5.5 A from both metal ions in the enzyme-substrate crystal structure (Y94F) renders the metal ion dependence of DNA binding biphasic: two classes of metal ion binding sites become distinct in the presence of DNA. The perturbation in metal ion coordination is supported by 1H-15N heteronuclear single quantum coherence spectra of enzyme-Ca(II) and enzyme-Ca(II)-DNA complexes. Metal ion binding by free Y94F is basically unperturbed: through multiple experiments with different metal ions, the data are consistent with two alkaline earth metal ion binding sites per subunit of low millimolar affinity, behavior which is very similar to that of the wild type. The results presented here indicate a role for the hydroxyl group of Tyr94 in the coupling of metal ion binding sites in the presence of DNA. Its removal causes the affinities for the two metal ion binding sites to be resolved in the presence of substrate. Such tuning of metal ion affinities will be invaluable to efforts to ascertain the contributions of individual bound metal ions to metallonuclease function.

Electrostatic steering and ionic tethering in enzyme-ligand binding: insights from simulations.

PubMed

Wade, R C; Gabdoulline, R R; Lüdemann, S K; Lounnas, V

1998-05-26

To bind at an enzyme's active site, a ligand must diffuse or be transported to the enzyme's surface, and, if the binding site is buried, the ligand must diffuse through the protein to reach it. Although the driving force for ligand binding is often ascribed to the hydrophobic effect, electrostatic interactions also influence the binding process of both charged and nonpolar ligands. First, electrostatic steering of charged substrates into enzyme active sites is discussed. This is of particular relevance for diffusion-influenced enzymes. By comparing the results of Brownian dynamics simulations and electrostatic potential similarity analysis for triose-phosphate isomerases, superoxide dismutases, and beta-lactamases from different species, we identify the conserved features responsible for the electrostatic substrate-steering fields. The conserved potentials are localized at the active sites and are the primary determinants of the bimolecular association rates. Then we focus on a more subtle effect, which we will refer to as "ionic tethering." We explore, by means of molecular and Brownian dynamics simulations and electrostatic continuum calculations, how salt links can act as tethers between structural elements of an enzyme that undergo conformational change upon substrate binding, and thereby regulate or modulate substrate binding. This is illustrated for the lipase and cytochrome P450 enzymes. Ionic tethering can provide a control mechanism for substrate binding that is sensitive to the electrostatic properties of the enzyme's surroundings even when the substrate is nonpolar.

Mismatch repair factor MSH2-MSH3 binds and alters the conformation of branched DNA structures predicted to form during genetic recombination.

PubMed

Surtees, Jennifer A; Alani, Eric

2006-07-14

Genetic studies in Saccharomyces cerevisiae predict that the mismatch repair (MMR) factor MSH2-MSH3 binds and stabilizes branched recombination intermediates that form during single strand annealing and gene conversion. To test this model, we constructed a series of DNA substrates that are predicted to form during these recombination events. We show in an electrophoretic mobility shift assay that S. cerevisiae MSH2-MSH3 specifically binds branched DNA substrates containing 3' single-stranded DNA and that ATP stimulates its release from these substrates. Chemical footprinting analyses indicate that MSH2-MSH3 specifically binds at the double-strand/single-strand junction of branched substrates, alters its conformation and opens up the junction. Therefore, MSH2-MSH3 binding to its substrates creates a unique nucleoprotein structure that may signal downstream steps in repair that include interactions with MMR and nucleotide excision repair factors.

Deconstructing the DGAT1 enzyme: membrane interactions at substrate binding sites.

PubMed

Lopes, Jose L S; Beltramini, Leila M; Wallace, Bonnie A; Araujo, Ana P U

2015-01-01

Diacylglycerol acyltransferase 1 (DGAT1) is a key enzyme in the triacylglyceride synthesis pathway. Bovine DGAT1 is an endoplasmic reticulum membrane-bound protein associated with the regulation of fat content in milk and meat. The aim of this study was to evaluate the interaction of DGAT1 peptides corresponding to putative substrate binding sites with different types of model membranes. Whilst these peptides are predicted to be located in an extramembranous loop of the membrane-bound protein, their hydrophobic substrates are membrane-bound molecules. In this study, peptides corresponding to the binding sites of the two substrates involved in the reaction were examined in the presence of model membranes in order to probe potential interactions between them that might influence the subsequent binding of the substrates. Whilst the conformation of one of the peptides changed upon binding several types of micelles regardless of their surface charge, suggesting binding to hydrophobic domains, the other peptide bound strongly to negatively-charged model membranes. This binding was accompanied by a change in conformation, and produced leakage of the liposome-entrapped dye calcein. The different hydrophobic and electrostatic interactions observed suggest the peptides may be involved in the interactions of the enzyme with membrane surfaces, facilitating access of the catalytic histidine to the triacylglycerol substrates.

Structural basis of protein translocation by the Vps4-Vta1 AAA ATPase

PubMed Central

Monroe, Nicole; Han, Han; Shen, Peter S; Sundquist, Wesley I; Hill, Christopher P

2017-01-01

Many important cellular membrane fission reactions are driven by ESCRT pathways, which culminate in disassembly of ESCRT-III polymers by the AAA ATPase Vps4. We report a 4.3 Å resolution cryo-EM structure of the active Vps4 hexamer with its cofactor Vta1, ADP·BeFx, and an ESCRT-III substrate peptide. Four Vps4 subunits form a helix whose interfaces are consistent with ATP binding, is stabilized by Vta1, and binds the substrate peptide. The fifth subunit approximately continues this helix but appears to be dissociating. The final Vps4 subunit completes a notched-washer configuration as if transitioning between the ends of the helix. We propose that ATP binding propagates growth at one end of the helix while hydrolysis promotes disassembly at the other end, so that Vps4 ‘walks’ along ESCRT-III until it encounters the ordered N-terminal domain to destabilize the ESCRT-III lattice. This model may be generally applicable to other protein-translocating AAA ATPases. DOI: http://dx.doi.org/10.7554/eLife.24487.001 PMID:28379137

Structural Elements Regulating AAA+ Protein Quality Control Machines.

PubMed

Chang, Chiung-Wen; Lee, Sukyeong; Tsai, Francis T F

2017-01-01

Members of the ATPases Associated with various cellular Activities (AAA+) superfamily participate in essential and diverse cellular pathways in all kingdoms of life by harnessing the energy of ATP binding and hydrolysis to drive their biological functions. Although most AAA+ proteins share a ring-shaped architecture, AAA+ proteins have evolved distinct structural elements that are fine-tuned to their specific functions. A central question in the field is how ATP binding and hydrolysis are coupled to substrate translocation through the central channel of ring-forming AAA+ proteins. In this mini-review, we will discuss structural elements present in AAA+ proteins involved in protein quality control, drawing similarities to their known role in substrate interaction by AAA+ proteins involved in DNA translocation. Elements to be discussed include the pore loop-1, the Inter-Subunit Signaling (ISS) motif, and the Pre-Sensor I insert (PS-I) motif. Lastly, we will summarize our current understanding on the inter-relationship of those structural elements and propose a model how ATP binding and hydrolysis might be coupled to polypeptide translocation in protein quality control machines.

Native mitochondrial RNA-binding complexes in kinetoplastid RNA editing differ in guide RNA composition

PubMed Central

Madina, Bhaskara R.; Kumar, Vikas; Metz, Richard; Mooers, Blaine H.M.; Bundschuh, Ralf; Cruz-Reyes, Jorge

2014-01-01

Mitochondrial mRNAs in kinetoplastids require extensive U-insertion/deletion editing that progresses 3′-to-5′ in small blocks, each directed by a guide RNA (gRNA), and exhibits substrate and developmental stage-specificity by unsolved mechanisms. Here, we address compositionally related factors, collectively known as the mitochondrial RNA-binding complex 1 (MRB1) or gRNA-binding complex (GRBC), that contain gRNA, have a dynamic protein composition, and transiently associate with several mitochondrial factors including RNA editing core complexes (RECC) and ribosomes. MRB1 controls editing by still unknown mechanisms. We performed the first next-generation sequencing study of native subcomplexes of MRB1, immunoselected via either RNA helicase 2 (REH2), that binds RNA and associates with unwinding activity, or MRB3010, that affects an early editing step. The particles contain either REH2 or MRB3010 but share the core GAP1 and other proteins detected by RNA photo-crosslinking. Analyses of the first editing blocks indicate an enrichment of several initiating gRNAs in the MRB3010-purified complex. Our data also indicate fast evolution of mRNA 3′ ends and strain-specific alternative 3′ editing within 3′ UTR or C-terminal protein-coding sequence that could impact mitochondrial physiology. Moreover, we found robust specific copurification of edited and pre-edited mRNAs, suggesting that these particles may bind both mRNA and gRNA editing substrates. We propose that multiple subcomplexes of MRB1 with different RNA/protein composition serve as a scaffold for specific assembly of editing substrates and RECC, thereby forming the editing holoenzyme. The MRB3010-subcomplex may promote early editing through its preferential recruitment of initiating gRNAs. PMID:24865612

Motor-substrate interactions in mycoplasma motility explains non-Arrhenius temperature dependence.

PubMed

Chen, Jing; Neu, John; Miyata, Makoto; Oster, George

2009-12-02

Mycoplasmas exhibit a novel, substrate-dependent gliding motility that is driven by approximately 400 "leg" proteins. The legs interact with the substrate and transmit the forces generated by an assembly of ATPase motors. The velocity of the cell increases linearly by nearly 10-fold over a narrow temperature range of 10-40 degrees C. This corresponds to an Arrhenius factor that decreases from approximately 45 k(B)T at 10 degrees C to approximately 10 k(B)T at 40 degrees C. On the other hand, load-velocity curves at different temperatures extrapolate to nearly the same stall force, suggesting a temperature-insensitive force-generation mechanism near stall. In this article, we propose a leg-substrate interaction mechanism that explains the intriguing temperature sensitivity of this motility. The large Arrhenius factor at low temperature comes about from the addition of many smaller energy barriers arising from many substrate-binding sites at the distal end of the leg protein. The Arrhenius dependence attenuates at high temperature due to two factors: 1), the reduced effective multiplicity of energy barriers intrinsic to the multiple-site binding mechanism; and 2), the temperature-sensitive weakly facilitated leg release that curtails the power stroke. The model suggests an explanation for the similar steep, sub-Arrhenius temperature-velocity curves observed in many molecular motors, such as kinesin and myosin, wherein the temperature behavior is dominated not by the catalytic biochemistry, but by the motor-substrate interaction.

Motor-Substrate Interactions in Mycoplasma Motility Explains Non-Arrhenius Temperature Dependence

PubMed Central

Chen, Jing; Neu, John; Miyata, Makoto; Oster, George

2009-01-01

Abstract Mycoplasmas exhibit a novel, substrate-dependent gliding motility that is driven by ∼400 “leg” proteins. The legs interact with the substrate and transmit the forces generated by an assembly of ATPase motors. The velocity of the cell increases linearly by nearly 10-fold over a narrow temperature range of 10–40°C. This corresponds to an Arrhenius factor that decreases from ∼45 kBT at 10°C to ∼10 kBT at 40°C. On the other hand, load-velocity curves at different temperatures extrapolate to nearly the same stall force, suggesting a temperature-insensitive force-generation mechanism near stall. In this article, we propose a leg-substrate interaction mechanism that explains the intriguing temperature sensitivity of this motility. The large Arrhenius factor at low temperature comes about from the addition of many smaller energy barriers arising from many substrate-binding sites at the distal end of the leg protein. The Arrhenius dependence attenuates at high temperature due to two factors: 1), the reduced effective multiplicity of energy barriers intrinsic to the multiple-site binding mechanism; and 2), the temperature-sensitive weakly facilitated leg release that curtails the power stroke. The model suggests an explanation for the similar steep, sub-Arrhenius temperature-velocity curves observed in many molecular motors, such as kinesin and myosin, wherein the temperature behavior is dominated not by the catalytic biochemistry, but by the motor-substrate interaction. PMID:19948122

Overlapping and Specific Functions of the Hsp104 N Domain Define Its Role in Protein Disaggregation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Jungsoon; Sung, Nuri; Mercado, Jonathan M.

Hsp104 is a ring-forming protein disaggregase that rescues stress-damaged proteins from an aggregated state. To facilitate protein disaggregation, Hsp104 cooperates with Hsp70 and Hsp40 chaperones (Hsp70/40) to form a bi-chaperone system. How Hsp104 recognizes its substrates, particularly the importance of the N domain, remains poorly understood and multiple, seemingly conficting mechanisms have been proposed. Although the N domain is dispensable for protein disaggregation, it is sensitive to point mutations that abolish the function of the bacterial Hsp104 homolog in vitro, and is essential for curing yeast prions by Hsp104 overexpression in vivo. Here, we present the crystal structure of anmore » N-terminal fragment of Saccharomyces cerevisiae Hsp104 with the N domain of one molecule bound to the C-terminal helix of the neighboring D1 domain. Consistent with mimicking substrate interaction, mutating the putative substrate-binding site in a constitutively active Hsp104 variant impairs the recovery of functional protein from aggregates. We fnd that the observed substrate-binding defect can be rescued by Hsp70/40 chaperones, providing a molecular explanation as to why the N domain is dispensable for protein disaggregation when Hsp70/40 is present, yet essential for the dissolution of Hsp104-specifc substrates, such as yeast prions, which likely depends on a direct N domain interaction.« less

Overlapping and Specific Functions of the Hsp104 N Domain Define Its Role in Protein Disaggregation

DOE PAGES

Lee, Jungsoon; Sung, Nuri; Mercado, Jonathan M.; ...

2017-09-11

Hsp104 is a ring-forming protein disaggregase that rescues stress-damaged proteins from an aggregated state. To facilitate protein disaggregation, Hsp104 cooperates with Hsp70 and Hsp40 chaperones (Hsp70/40) to form a bi-chaperone system. How Hsp104 recognizes its substrates, particularly the importance of the N domain, remains poorly understood and multiple, seemingly conficting mechanisms have been proposed. Although the N domain is dispensable for protein disaggregation, it is sensitive to point mutations that abolish the function of the bacterial Hsp104 homolog in vitro, and is essential for curing yeast prions by Hsp104 overexpression in vivo. Here, we present the crystal structure of anmore » N-terminal fragment of Saccharomyces cerevisiae Hsp104 with the N domain of one molecule bound to the C-terminal helix of the neighboring D1 domain. Consistent with mimicking substrate interaction, mutating the putative substrate-binding site in a constitutively active Hsp104 variant impairs the recovery of functional protein from aggregates. We fnd that the observed substrate-binding defect can be rescued by Hsp70/40 chaperones, providing a molecular explanation as to why the N domain is dispensable for protein disaggregation when Hsp70/40 is present, yet essential for the dissolution of Hsp104-specifc substrates, such as yeast prions, which likely depends on a direct N domain interaction.« less

Substrate-bound structure of the E. coli multidrug resistance transporter MdfA

PubMed Central

Heng, Jie; Zhao, Yan; Liu, Ming; Liu, Yue; Fan, Junping; Wang, Xianping; Zhao, Yongfang; Zhang, Xuejun C

2015-01-01

Multidrug resistance is a serious threat to public health. Proton motive force-driven antiporters from the major facilitator superfamily (MFS) constitute a major group of multidrug-resistance transporters. Currently, no reports on crystal structures of MFS antiporters in complex with their substrates exist. The E. coli MdfA transporter is a well-studied model system for biochemical analyses of multidrug-resistance MFS antiporters. Here, we report three crystal structures of MdfA-ligand complexes at resolutions up to 2.0 Å, all in the inward-facing conformation. The substrate-binding site sits proximal to the conserved acidic residue, D34. Our mutagenesis studies support the structural observations of the substrate-binding mode and the notion that D34 responds to substrate binding by adjusting its protonation status. Taken together, our data unveil the substrate-binding mode of MFS antiporters and suggest a mechanism of transport via this group of transporters. PMID:26238402

The oxidative half-reaction of Old Yellow Enzyme. The role of tyrosine 196.

PubMed

Kohli, R M; Massey, V

1998-12-04

Tyrosine 196 in Old Yellow Enzyme (OYE) was mutated to phenylalanine, and the resulting mutant enzyme was characterized to evaluate the mechanistic role of the residue. The residue demonstrates little effect on ligand binding and the reductive half-reaction, but a dramatic slowing by nearly 6 orders of magnitude of its oxidative half-reaction with 2-cyclohexenone. Observation of the oxidative half-reaction with a series of substrates allows us to propose a model describing the mechanism of the oxidative half-reaction. In addition, the curtailed reactivity with enones allows for characterization of the manner in which reduced enzyme primes the substrate for the redox reaction by observation of the Michaelis complex with reduced enzyme bound to substrate.

A Brownian motor mechanism of translocation and strand separation by hepatitis C virus helicase.

PubMed

Levin, Mikhail K; Gurjar, Madhura; Patel, Smita S

2005-05-01

Helicases translocate along their nucleic acid substrates using the energy of ATP hydrolysis and by changing conformations of their nucleic acid-binding sites. Our goal is to characterize the conformational changes of hepatitis C virus (HCV) helicase at different stages of ATPase cycle and to determine how they lead to translocation. We have reported that ATP binding reduces HCV helicase affinity for nucleic acid. Now we identify the stage of the ATPase cycle responsible for translocation and unwinding. We show that a rapid directional movement occurs upon helicase binding to DNA in the absence of ATP, resulting in opening of several base pairs. We propose that HCV helicase translocates as a Brownian motor with a simple two-stroke cycle. The directional movement step is fueled by single-stranded DNA binding energy while ATP binding allows for a brief period of random movement that prepares the helicase for the next cycle.

Specificity of a protein-protein interface: local dynamics direct substrate recognition of effector caspases.

PubMed

Fuchs, Julian E; von Grafenstein, Susanne; Huber, Roland G; Wallnoefer, Hannes G; Liedl, Klaus R

2014-04-01

Proteases are prototypes of multispecific protein-protein interfaces. Proteases recognize and cleave protein and peptide substrates at a well-defined position in a substrate binding groove and a plethora of experimental techniques provide insights into their substrate recognition. We investigate the caspase family of cysteine proteases playing a key role in programmed cell death and inflammation, turning caspases into interesting drug targets. Specific ligand binding to one particular caspase is difficult to achieve, as substrate specificities of caspase isoforms are highly similar. In an effort to rationalize substrate specificity of two closely related caspases, we investigate the substrate promiscuity of the effector Caspases 3 and 7 by data mining (cleavage entropy) and by molecular dynamics simulations. We find a strong correlation between binding site rigidity and substrate readout for individual caspase subpockets explaining more stringent substrate readout of Caspase 7 via its narrower conformational space. Caspase 3 subpockets S3 and S4 show elevated local flexibility explaining the more unspecific substrate readout of that isoform in comparison to Caspase 7. We show by in silico exchange mutations in the S3 pocket of the proteases that a proline residue in Caspase 7 contributes to the narrowed conformational space of the binding site. These findings explain the substrate specificities of caspases via a mechanism of conformational selection and highlight the crucial importance of binding site local dynamics in substrate recognition of proteases. Proteins 2014; 82:546-555. © 2013 Wiley Periodicals, Inc. Copyright © 2013 The Authors Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.

Structure of tropinone reductase-II complexed with NADP+ and pseudotropine at 1.9 A resolution: implication for stereospecific substrate binding and catalysis.

PubMed

Yamashita, A; Kato, H; Wakatsuki, S; Tomizaki, T; Nakatsu, T; Nakajima, K; Hashimoto, T; Yamada, Y; Oda, J

1999-06-15

Tropinone reductase-II (TR-II) catalyzes the NADPH-dependent reduction of the carbonyl group of tropinone to a beta-hydroxyl group. The crystal structure of TR-II complexed with NADP+ and pseudotropine (psi-tropine) has been determined at 1.9 A resolution. A seven-residue peptide near the active site, disordered in the unliganded structure, is fixed in the ternary complex by participation of the cofactor and substrate binding. The psi-tropine molecule is bound in an orientation which satisfies the product configuration and the stereochemical arrangement toward the cofactor. The substrate binding site displays a complementarity to the bound substrate (psi-tropine) in its correct orientation. In addition, electrostatic interactions between the substrate and Glu156 seem to specify the binding position and orientation of the substrate. A comparison between the active sites in TR-II and TR-I shows that they provide different van der Waals surfaces and electrostatic features. These differences likely contribute to the correct binding mode of the substrates, which are in opposite orientations in TR-II and TR-I, and to different reaction stereospecificities. The active site structure in the TR-II ternary complex also suggests that the arrangement of the substrate, cofactor, and catalytic residues is stereoelectronically favorable for the reaction.

Trimethylation of histone H3 lysine 4 impairs methylation of histone H3 lysine 9

PubMed Central

LeRoy, Gary; Bua, Dennis J; Garcia, Benjamin A; Gozani, Or; Richard, Stéphane

2010-01-01

Chromatin is broadly compartmentalized in two defined states: euchromatin and heterochromatin. Generally, euchromatin is trimethylated on histone H3 lysine 4 (H3K4me3) while heterochromatin contains the H3K9me3 mark. The H3K9me3 modification is added by lysine methyltransferases (KMTs) such as SETDB1. Herein, we show that SETDB1 interacts with its substrate H3, but only in the absence of the euchromatic mark H3K4me3. In addition, we show that SETDB1 fails to methylate substrates containing the H3K4me3 mark. Likewise, the functionally related H3K9 KMTs G9A, GLP and SUV39H1 also fail to bind and to methylate H3K4me3 substrates. Accordingly, we provide in vivo evidence that H3K9me2-enriched histones are devoid of H3K4me2/3 and that histones depleted of H3K4me2/3 have elevated H3K9me2/3. The correlation between the loss of interaction of these KMTs with H3K4me3 and concomitant methylation impairment leads to the postulate that at least these four KMTs require stable interaction with their respective substrates for optimal activity. Thus, novel substrates could be discovered via the identification of KMT interacting proteins. Indeed, we find that SETDB1 binds to and methylates a novel substrate, the inhibitor of growth protein ING2, while SUV39H1 binds to and methylates the heterochromatin protein HP1α. Thus, our observations suggest a mechanism of post-translational regulation of lysine methylation and propose a potential mechanism for the segregation of the biologically opposing marks, H3K4me3 and H3K9me3. Furthermore, the correlation between H3-KMTs interaction and substrate methylation highlights that the identification of novel KMT substrates may be facilitated by the identification of interaction partners. PMID:21124070

GUN4-Porphyrin Complexes Bind the ChlH/GUN5 Subunit of Mg-Chelatase and Promote Chlorophyll Biosynthesis in Arabidopsis[W

PubMed Central

Adhikari, Neil D.; Froehlich, John E.; Strand, Deserah D.; Buck, Stephanie M.; Kramer, David M.; Larkin, Robert M.

2011-01-01

The GENOMES UNCOUPLED4 (GUN4) protein stimulates chlorophyll biosynthesis by activating Mg-chelatase, the enzyme that commits protoporphyrin IX to chlorophyll biosynthesis. This stimulation depends on GUN4 binding the ChlH subunit of Mg-chelatase and the porphyrin substrate and product of Mg-chelatase. After binding porphyrins, GUN4 associates more stably with chloroplast membranes and was proposed to promote interactions between ChlH and chloroplast membranes—the site of Mg-chelatase activity. GUN4 was also proposed to attenuate the production of reactive oxygen species (ROS) by binding and shielding light-exposed porphyrins from collisions with O2. To test these proposals, we first engineered Arabidopsis thaliana plants that express only porphyrin binding–deficient forms of GUN4. Using these transgenic plants and particular mutants, we found that the porphyrin binding activity of GUN4 and Mg-chelatase contribute to the accumulation of chlorophyll, GUN4, and Mg-chelatase subunits. Also, we found that the porphyrin binding activity of GUN4 and Mg-chelatase affect the associations of GUN4 and ChlH with chloroplast membranes and have various effects on the expression of ROS-inducible genes. Based on our findings, we conclude that ChlH and GUN4 use distinct mechanisms to associate with chloroplast membranes and that mutant alleles of GUN4 and Mg-chelatase genes cause sensitivity to intense light by a mechanism that is potentially complex. PMID:21467578

Examination of the Mechanism of Human Brain Aspartoacylase through the Binding of an Intermediate Analogue†‡

PubMed Central

Le Coq, Johanne; Pavlovsky, Alexander; Malik, Radhika; Sanishvili, Ruslan; Xu, Chengfu; Viola, Ronald E.

2009-01-01

Canavan disease is a fatal neurological disorder caused by the malfunctioning of a single metabolic enzyme, aspartoacylase, that catalyzes the deacetylation of N-acetyl-l-aspartate to produce l-aspartate and acetate. The structure of human brain aspartoacylase has been determined in complex with a stable tetrahedral intermediate analogue, N-phosphonomethyl-l-aspartate. This potent inhibitor forms multiple interactions between each of its heteroatoms and the substrate binding groups arrayed within the active site. The binding of the catalytic intermediate analogue induces the conformational ordering of several substrate binding groups, thereby setting up the active site for catalysis. The highly ordered binding of this inhibitor has allowed assignments to be made for substrate binding groups and provides strong support for a carboxypeptidase-type mechanism for the hydrolysis of the amide bond of the substrate, N-acetyl-l-aspartate. PMID:18293939

Examination of the mechanism of human brain aspartoacylase through the binding of an intermediate analogue.

PubMed

Le Coq, Johanne; Pavlovsky, Alexander; Malik, Radhika; Sanishvili, Ruslan; Xu, Chengfu; Viola, Ronald E

2008-03-18

Canavan disease is a fatal neurological disorder caused by the malfunctioning of a single metabolic enzyme, aspartoacylase, that catalyzes the deacetylation of N-acetyl-L-aspartate to produce L-aspartate and acetate. The structure of human brain aspartoacylase has been determined in complex with a stable tetrahedral intermediate analogue, N-phosphonomethyl-L-aspartate. This potent inhibitor forms multiple interactions between each of its heteroatoms and the substrate binding groups arrayed within the active site. The binding of the catalytic intermediate analogue induces the conformational ordering of several substrate binding groups, thereby setting up the active site for catalysis. The highly ordered binding of this inhibitor has allowed assignments to be made for substrate binding groups and provides strong support for a carboxypeptidase-type mechanism for the hydrolysis of the amide bond of the substrate, N-acetyl- l-aspartate.

Mutations in the C-terminal fragment of DnaK affecting peptide binding.

PubMed Central

Burkholder, W F; Zhao, X; Zhu, X; Hendrickson, W A; Gragerov, A; Gottesman, M E

1996-01-01

Escherichia coli DnaK acts as a molecular chaperone through its ATP-regulated binding and release of polypeptide substrates. Overexpressing a C-terminal fragment (CTF) of DnaK (Gly-384 to Lys-638) containing the polypeptide substrate binding domain is lethal in wild-type E. coli. This dominant-negative phenotype may result from the nonproductive binding of CTF to cellular polypeptide targets of DnaK. Mutations affecting DnaK substrate binding were identified by selecting noncytotoxic CTF mutants followed by in vitro screening. The clustering of such mutations in the three-dimensional structure of CTF suggests the model that loops L1,2 and L4,5 form a rigid core structure critical for interactions with substrate. Images Fig. 1 Fig. 2 Fig. 3 PMID:8855230

Structural, kinetic, and thermodynamic studies of specificity designed HIV-1 protease.

PubMed

Alvizo, Oscar; Mittal, Seema; Mayo, Stephen L; Schiffer, Celia A

2012-07-01

HIV-1 protease recognizes and cleaves more than 12 different substrates leading to viral maturation. While these substrates share no conserved motif, they are specifically selected for and cleaved by protease during viral life cycle. Drug resistant mutations evolve within the protease that compromise inhibitor binding but allow the continued recognition of all these substrates. While the substrate envelope defines a general shape for substrate recognition, successfully predicting the determinants of substrate binding specificity would provide additional insights into the mechanism of altered molecular recognition in resistant proteases. We designed a variant of HIV protease with altered specificity using positive computational design methods and validated the design using X-ray crystallography and enzyme biochemistry. The engineered variant, Pr3 (A28S/D30F/G48R), was designed to preferentially bind to one out of three of HIV protease's natural substrates; RT-RH over p2-NC and CA-p2. In kinetic assays, RT-RH binding specificity for Pr3 increased threefold compared to the wild-type (WT), which was further confirmed by isothermal titration calorimetry. Crystal structures of WT protease and the designed variant in complex with RT-RH, CA-p2, and p2-NC were determined. Structural analysis of the designed complexes revealed that one of the engineered substitutions (G48R) potentially stabilized heterogeneous flap conformations, thereby facilitating alternate modes of substrate binding. Our results demonstrate that while substrate specificity could be engineered in HIV protease, the structural pliability of protease restricted the propagation of interactions as predicted. These results offer new insights into the plasticity and structural determinants of substrate binding specificity of the HIV-1 protease. Copyright © 2012 The Protein Society.

Characterization of Carboxylic Acid Reductases as Enzymes in the Toolbox for Synthetic Chemistry.

PubMed

Finnigan, William; Thomas, Adam; Cromar, Holly; Gough, Ben; Snajdrova, Radka; Adams, Joseph P; Littlechild, Jennifer A; Harmer, Nicholas J

2017-03-20

Carboxylic acid reductase enzymes (CARs) meet the demand in synthetic chemistry for a green and regiospecific route to aldehydes from their respective carboxylic acids. However, relatively few of these enzymes have been characterized. A sequence alignment with members of the ANL (Acyl-CoA synthetase/ NRPS adenylation domain/Luciferase) superfamily of enzymes shed light on CAR functional dynamics. Four unstudied enzymes were selected by using a phylogenetic analysis of known and hypothetical CARs, and for the first time, a thorough biochemical characterization was performed. Kinetic analysis of these enzymes with various substrates shows that they have a broad but similar substrate specificity. Electron-rich acids are favored, which suggests that the first step in the proposed reaction mechanism, attack by the carboxylate on the α-phosphate of adenosine triphosphate (ATP), is the step that determines the substrate specificity and reaction kinetics. The effects of pH and temperature provide a clear operational window for the use of these CARs, whereas an investigation of product inhibition by NADP + , adenosine monophosphate, and pyrophosphate indicates that the binding of substrates at the adenylation domain is ordered with ATP binding first. This study consolidates CARs as important and exciting enzymes in the toolbox for sustainable chemistry and provides specifications for their use as a biocatalyst.

The structures of the horseradish peroxidase C-ferulic acid complex and the ternary complex with cyanide suggest how peroxidases oxidize small phenolic substrates.

PubMed

Henriksen, A; Smith, A T; Gajhede, M

1999-12-03

We have solved the x-ray structures of the binary horseradish peroxidase C-ferulic acid complex and the ternary horseradish peroxidase C-cyanide-ferulic acid complex to 2.0 and 1.45 A, respectively. Ferulic acid is a naturally occurring phenolic compound found in the plant cell wall and is an in vivo substrate for plant peroxidases. The x-ray structures demonstrate the flexibility and dynamic character of the aromatic donor binding site in horseradish peroxidase and emphasize the role of the distal arginine (Arg(38)) in both substrate oxidation and ligand binding. Arg(38) hydrogen bonds to bound cyanide, thereby contributing to the stabilization of the horseradish peroxidase-cyanide complex and suggesting that the distal arginine will be able to contribute with a similar interaction during stabilization of a bound peroxy transition state and subsequent O-O bond cleavage. The catalytic arginine is additionally engaged in an extensive hydrogen bonding network, which also includes the catalytic distal histidine, a water molecule and Pro(139), a proline residue conserved within the plant peroxidase superfamily. Based on the observed hydrogen bonding network and previous spectroscopic and kinetic work, a general mechanism of peroxidase substrate oxidation is proposed.

Proteomics Analysis of Nucleolar SUMO-1 Target Proteins upon Proteasome Inhibition*

PubMed Central

Matafora, Vittoria; D'Amato, Alfonsina; Mori, Silvia; Blasi, Francesco; Bachi, Angela

2009-01-01

Many cellular processes are regulated by the coordination of several post-translational modifications that allow a very fine modulation of substrates. Recently it has been reported that there is a relationship between sumoylation and ubiquitination. Here we propose that the nucleolus is the key organelle in which SUMO-1 conjugates accumulate in response to proteasome inhibition. We demonstrated that, upon proteasome inhibition, the SUMO-1 nuclear dot localization is redirected to nucleolar structures. To better understand this process we investigated, by quantitative proteomics, the effect of proteasome activity on endogenous nucleolar SUMO-1 targets. 193 potential SUMO-1 substrates were identified, and interestingly in several purified SUMO-1 conjugates ubiquitin chains were found to be present, confirming the coordination of these two modifications. 23 SUMO-1 targets were confirmed by an in vitro sumoylation reaction performed on nuclear substrates. They belong to protein families such as small nuclear ribonucleoproteins, heterogeneous nuclear ribonucleoproteins, ribosomal proteins, histones, RNA-binding proteins, and transcription factor regulators. Among these, histone H1, histone H3, and p160 Myb-binding protein 1A were further characterized as novel SUMO-1 substrates. The analysis of the nature of the SUMO-1 targets identified in this study strongly indicates that sumoylation, acting in coordination with the ubiquitin-proteasome system, regulates the maintenance of nucleolar integrity. PMID:19596686

Unbiased Simulations Reveal the Inward-Facing Conformation of the Human Serotonin Transporter and Na+ Ion Release

PubMed Central

Koldsø, Heidi; Noer, Pernille; Grouleff, Julie; Autzen, Henriette Elisabeth; Sinning, Steffen; Schiøtt, Birgit

2011-01-01

Monoamine transporters are responsible for termination of synaptic signaling and are involved in depression, control of appetite, and anxiety amongst other neurological processes. Despite extensive efforts, the structures of the monoamine transporters and the transport mechanism of ions and substrates are still largely unknown. Structural knowledge of the human serotonin transporter (hSERT) is much awaited for understanding the mechanistic details of substrate translocation and binding of antidepressants and drugs of abuse. The publication of the crystal structure of the homologous leucine transporter has resulted in homology models of the monoamine transporters. Here we present extended molecular dynamics simulations of an experimentally supported homology model of hSERT with and without the natural substrate yielding a total of more than 1.5 µs of simulation of the protein dimer. The simulations reveal a transition of hSERT from an outward-facing occluded conformation to an inward-facing conformation in a one-substrate-bound state. Simulations with a second substrate in the proposed symport effector site did not lead to conformational changes associated with translocation. The central substrate binding site becomes fully exposed to the cytoplasm leaving both the Na+-ion in the Na2-site and the substrate in direct contact with the cytoplasm through water interactions. The simulations reveal how sodium is released and show indications of early events of substrate transport. The notion that ion dissociation from the Na2-site drives translocation is supported by experimental studies of a Na2-site mutant. Transmembrane helices (TMs) 1 and 6 are identified as the helices involved in the largest movements during transport. PMID:22046120

Structural basis for the substrate selectivity of a HAD phosphatase from Thermococcus onnurineus NA1.

PubMed

Ngo, Tri Duc; Van Le, Binh; Subramani, Vinod Kumar; Thi Nguyen, Chi My; Lee, Hyun Sook; Cho, Yona; Kim, Kyeong Kyu; Hwang, Hye-Yeon

2015-05-22

Proteins in the haloalkaloic acid dehalogenase (HAD) superfamily, which is one of the largest enzyme families, is generally composed of a catalytic core domain and a cap domain. Although proteins in this family show broad substrate specificities, the mechanisms of their substrate recognition are not well understood. In this study, we identified a new substrate binding motif of HAD proteins from structural and functional analyses, and propose that this motif might be crucial for interacting with hydrophobic rings of substrates. The crystal structure of TON_0338, one of the 17 putative HAD proteins identified in a hyperthermophilic archaeon, Thermococcus onnurineus NA1, was determined as an apo-form at 2.0 Å resolution. In addition, we determined the crystal structure TON_0338 in complex with Mg(2+) or N-cyclohexyl-2-aminoethanesulfonic acid (CHES) at 1.7 Å resolution. Examination of the apo-form and CHES-bound structures revealed that CHES is sandwiched between Trp58 and Trp61, suggesting that this Trp sandwich might function as a substrate recognition motif. In the phosphatase assay, TON_0338 was shown to have high activity for flavin mononucleotide (FMN), and the docking analysis suggested that the flavin of FMN may interact with Trp58 and Trp61 in a way similar to that observed in the crystal structure. Moreover, the replacement of these tryptophan residues significantly reduced the phosphatase activity for FMN. Our results suggest that WxxW may function as a substrate binding motif in HAD proteins, and expand the diversity of their substrate recognition mode. Copyright © 2015 Elsevier Inc. All rights reserved.

Terminally Truncated Isopenicillin N Synthase Generates a Dithioester Product: Evidence for a Thioaldehyde Intermediate during Catalysis and a New Mode of Reaction for Non‐Heme Iron Oxidases

PubMed Central

McNeill, Luke A.; Brown, Toby J. N.; Sami, Malkit; Clifton, Ian J.; Burzlaff, Nicolai I.; Claridge, Timothy D. W.; Adlington, Robert M.; Baldwin, Jack E.

2017-01-01

Abstract Isopenicillin N synthase (IPNS) catalyses the four‐electron oxidation of a tripeptide, l‐δ‐(α‐aminoadipoyl)‐l‐cysteinyl‐d‐valine (ACV), to give isopenicillin N (IPN), the first‐formed β‐lactam in penicillin and cephalosporin biosynthesis. IPNS catalysis is dependent upon an iron(II) cofactor and oxygen as a co‐substrate. In the absence of substrate, the carbonyl oxygen of the side‐chain amide of the penultimate residue, Gln330, co‐ordinates to the active‐site metal iron. Substrate binding ablates the interaction between Gln330 and the metal, triggering rearrangement of seven C‐terminal residues, which move to take up a conformation that extends the final α‐helix and encloses ACV in the active site. Mutagenesis studies are reported, which probe the role of the C‐terminal and other aspects of the substrate binding pocket in IPNS. The hydrophobic nature of amino acid side‐chains around the ACV binding pocket is important in catalysis. Deletion of seven C‐terminal residues exposes the active site and leads to formation of a new type of thiol oxidation product. The isolated product is shown by LC‐MS and NMR analyses to be the ene‐thiol tautomer of a dithioester, made up from two molecules of ACV linked between the thiol sulfur of one tripeptide and the oxidised cysteinyl β‐carbon of the other. A mechanism for its formation is proposed, supported by an X‐ray crystal structure, which shows the substrate ACV bound at the active site, its cysteinyl β‐carbon exposed to attack by a second molecule of substrate, adjacent. Formation of this product constitutes a new mode of reaction for IPNS and non‐heme iron oxidases in general. PMID:28703303

Terminally Truncated Isopenicillin N Synthase Generates a Dithioester Product: Evidence for a Thioaldehyde Intermediate during Catalysis and a New Mode of Reaction for Non-Heme Iron Oxidases.

PubMed

McNeill, Luke A; Brown, Toby J N; Sami, Malkit; Clifton, Ian J; Burzlaff, Nicolai I; Claridge, Timothy D W; Adlington, Robert M; Baldwin, Jack E; Rutledge, Peter J; Schofield, Christopher J

2017-09-18

Isopenicillin N synthase (IPNS) catalyses the four-electron oxidation of a tripeptide, l-δ-(α-aminoadipoyl)-l-cysteinyl-d-valine (ACV), to give isopenicillin N (IPN), the first-formed β-lactam in penicillin and cephalosporin biosynthesis. IPNS catalysis is dependent upon an iron(II) cofactor and oxygen as a co-substrate. In the absence of substrate, the carbonyl oxygen of the side-chain amide of the penultimate residue, Gln330, co-ordinates to the active-site metal iron. Substrate binding ablates the interaction between Gln330 and the metal, triggering rearrangement of seven C-terminal residues, which move to take up a conformation that extends the final α-helix and encloses ACV in the active site. Mutagenesis studies are reported, which probe the role of the C-terminal and other aspects of the substrate binding pocket in IPNS. The hydrophobic nature of amino acid side-chains around the ACV binding pocket is important in catalysis. Deletion of seven C-terminal residues exposes the active site and leads to formation of a new type of thiol oxidation product. The isolated product is shown by LC-MS and NMR analyses to be the ene-thiol tautomer of a dithioester, made up from two molecules of ACV linked between the thiol sulfur of one tripeptide and the oxidised cysteinyl β-carbon of the other. A mechanism for its formation is proposed, supported by an X-ray crystal structure, which shows the substrate ACV bound at the active site, its cysteinyl β-carbon exposed to attack by a second molecule of substrate, adjacent. Formation of this product constitutes a new mode of reaction for IPNS and non-heme iron oxidases in general. © 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

Electrostatic steering and ionic tethering in enzyme–ligand binding: Insights from simulations

PubMed Central

Wade, Rebecca C.; Gabdoulline, Razif R.; Lüdemann, Susanna K.; Lounnas, Valère

1998-01-01

To bind at an enzyme’s active site, a ligand must diffuse or be transported to the enzyme’s surface, and, if the binding site is buried, the ligand must diffuse through the protein to reach it. Although the driving force for ligand binding is often ascribed to the hydrophobic effect, electrostatic interactions also influence the binding process of both charged and nonpolar ligands. First, electrostatic steering of charged substrates into enzyme active sites is discussed. This is of particular relevance for diffusion-influenced enzymes. By comparing the results of Brownian dynamics simulations and electrostatic potential similarity analysis for triose-phosphate isomerases, superoxide dismutases, and β-lactamases from different species, we identify the conserved features responsible for the electrostatic substrate-steering fields. The conserved potentials are localized at the active sites and are the primary determinants of the bimolecular association rates. Then we focus on a more subtle effect, which we will refer to as “ionic tethering.” We explore, by means of molecular and Brownian dynamics simulations and electrostatic continuum calculations, how salt links can act as tethers between structural elements of an enzyme that undergo conformational change upon substrate binding, and thereby regulate or modulate substrate binding. This is illustrated for the lipase and cytochrome P450 enzymes. Ionic tethering can provide a control mechanism for substrate binding that is sensitive to the electrostatic properties of the enzyme’s surroundings even when the substrate is nonpolar. PMID:9600896

Molecular dynamics and binding selectivity of nucleotides and polynucleotide substrates with EIF2C2/Ago2 PAZ domain.

PubMed

Kandeel, Mahmoud; Kitade, Yukio

2018-02-01

RNA interference (RNAi) constitutes a major target in drug discovery. Recently, we reported that the Argonaute protein 2 (Ago2) PAZ domain selectively binds with all ribonucleotides except adenine and poorly recognizes deoxyribonucleotides. The binding properties of the PAZ domain with polynucleotides and the molecular mechanisms of substrates' selectivity remains unclear. In this study, the binding potencies of polynucleotides and the associated conformational and dynamic changes in PAZ domain are investigated. Coinciding with nucleotides' binding profile with the PAZ domain, polyuridylate (PolyU) and polycytidylate (PolyC) were potent binders. However, K dPolyU and K dPolyC were 15.8 and 9.3μM, respectively. In contrast, polyadenylate (PolyA) binding was not detectable. Molecular dynamics (MD) simulation revealed the highest change in root mean square deviation (RMSD) with ApoPAZ or PAZ domain bound with experimentally approved, low affinity substrates, whereas stronger binding substrates such as UMP or PolyU showed minimal RMSD changes. The loop between α3 and β5 in the β-hairpin subdomain showed the most responsive change in RMSD, being highly movable in the ApoPAZ and PAZ-AMP complex. Favorable substrate recognition was associate with moderate change in secondary structure content. In conclusion, the PAZ domain retains differential substrate selectivity associated with corresponding dynamic and structural changes upon binding. Copyright © 2017 Elsevier B.V. All rights reserved.

Identification of the High-affinity Substrate-binding Site of the Multidrug and Toxic Compound Extrusion (MATE) Family Transporter from Pseudomonas stutzeri*

PubMed Central

Nie, Laiyin; Grell, Ernst; Malviya, Viveka Nand; Xie, Hao; Wang, Jingkang; Michel, Hartmut

2016-01-01

Multidrug and toxic compound extrusion (MATE) transporters exist in all three domains of life. They confer multidrug resistance by utilizing H+ or Na+ electrochemical gradients to extrude various drugs across the cell membranes. The substrate binding and the transport mechanism of MATE transporters is a fundamental process but so far not fully understood. Here we report a detailed substrate binding study of NorM_PS, a representative MATE transporter from Pseudomonas stutzeri. Our results indicate that NorM_PS is a proton-dependent multidrug efflux transporter. Detailed binding studies between NorM_PS and 4′,6-diamidino-2-phenylindole (DAPI) were performed by isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), and spectrofluorometry. Two exothermic binding events were observed from ITC data, and the high-affinity event was directly correlated with the extrusion of DAPI. The affinities are about 1 μm and 0.1 mm for the high and low affinity binding, respectively. Based on our homology model of NorM_PS, variants with mutations of amino acids that are potentially involved in substrate binding, were constructed. By carrying out the functional characterization of these variants, the critical amino acid residues (Glu-257 and Asp-373) for high-affinity DAPI binding were determined. Taken together, our results suggest a new substrate-binding site for MATE transporters. PMID:27235402

TERRA mimicking ssRNAs prevail over the DNA substrate for telomerase in vitro due to interactions with the alternative binding site.

PubMed

Azhibek, Dulat; Skvortsov, Dmitry; Andreeva, Anna; Zatsepin, Timofei; Arutyunyan, Alexandr; Zvereva, Maria; Dontsova, Olga

2016-06-01

Telomerase is a key component of the telomere length maintenance system in the majority of eukaryotes. Telomerase displays maximal activity in stem and cancer cells with high proliferative potential. In humans, telomerase activity is regulated by various mechanisms, including the interaction with telomere ssDNA overhangs that contain a repetitive G-rich sequence, and with noncoding RNA, Telomeric repeat-containing RNA (TERRA), that contains the same sequence. So these nucleic acids can compete for telomerase RNA templates in the cell. In this study, we have investigated the ability of different model substrates mimicking telomere DNA overhangs and TERRA RNA to compete for telomerase in vitro through a previously developed telomerase inhibitor assay. We have shown in this study that RNA oligonucleotides are better competitors for telomerase that DNA ones as RNA also use an alternative binding site on telomerase, and the presence of 2'-OH groups is significant in these interactions. In contrast to DNA, the possibility of forming intramolecular G-quadruplex structures has a minor effect for RNA binding to telomerase. Taking together our data, we propose that TERRA RNA binds better to telomerase compared with its native substrate - the 3'-end of telomere DNA overhang. As a result, some specific factor may exist that participates in switching telomerase from TERRA to the 3'-end of DNA for telomere elongation at the distinct period of a cell cycle in vivo. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Phosphate-binding pocket in Dicer-2 PAZ domain for high-fidelity siRNA production

PubMed Central

Kandasamy, Suresh K.

2016-01-01

The enzyme Dicer produces small silencing RNAs such as micro-RNAs (miRNAs) and small interfering RNAs (siRNAs). In Drosophila, Dicer-1 produces ∼22–24-nt miRNAs from pre-miRNAs, whereas Dicer-2 makes 21-nt siRNAs from long double-stranded RNAs (dsRNAs). How Dicer-2 precisely makes 21-nt siRNAs with a remarkably high fidelity is unknown. Here we report that recognition of the 5′-monophosphate of a long dsRNA substrate by a phosphate-binding pocket in the Dicer-2 PAZ (Piwi, Argonaute, and Zwille/Pinhead) domain is crucial for the length fidelity, but not the efficiency, in 21-nt siRNA production. Loss of the length fidelity, meaning increased length heterogeneity of siRNAs, caused by point mutations in the phosphate-binding pocket of the Dicer-2 PAZ domain decreased RNA silencing activity in vivo, showing the importance of the high fidelity to make 21-nt siRNAs. We propose that the 5′-monophosphate of a long dsRNA substrate is anchored by the phosphate-binding pocket in the Dicer-2 PAZ domain and the distance between the pocket and the RNA cleavage active site in the RNaseIII domain corresponds to the 21-nt pitch in the A-form duplex of a long dsRNA substrate, resulting in high-fidelity 21-nt siRNA production. This study sheds light on the molecular mechanism by which Dicer-2 produces 21-nt siRNAs with a remarkably high fidelity for efficient RNA silencing. PMID:27872309

Metabolic activation and nucleic acid binding of acetaminophen and related arylamine substrates by the respiratory burst of human granulocytes.

PubMed

Corbett, M D; Corbett, B R; Hannothiaux, M H; Quintana, S J

1989-01-01

Following stimulation with phorbol myristate acetate, human granulocytes were found to incorporate acetaminophen, p-phenetidine, p-aminophenol, and p-chloroaniline into cellular DNA and RNA. Phenacetin was not incorporated into nucleic acid or metabolized by such activated granulocytes. None of the substrates gave nucleic acid binding if the granulocyte cultures were not induced to undergo the respiratory burst. Additional studies on the binding of acetaminophen to DNA and RNA were made by use of both ring-14C-labeled and carbonyl-14C-labeled forms of this substrate. The finding that equivalent amounts of these two labeled acetaminophen substrates were bound to cellular DNA demonstrated that the intact acetaminophen molecule was incorporated into DNA. On the other hand, the finding that excess ring-14C-labeled acetaminophen was incorporated into cellular RNA implies partial hydrolysis of the acetaminophen substrate prior to RNA binding. Evidence was presented which strongly indicates that the nucleic acid binding of the substrates was covalent in nature. The inability of the respiratory burst to result in the binding of phenacetin to nucleic acid suggests that arylamides are not normally activated or metabolized by activated granulocytes. Acetaminophen is an exception to the recalcitrance of arylamides to such bioactivation processes because it also possesses the phenolic functional group, which, like the arylamine group, is oxidized by certain reactive oxygen species. Myeloperoxidase appears to be much more important in the binding of acetaminophen to DNA than it is in the DNA binding of arylamines in general. The role of the respiratory burst in causing the bioactivation of certain arylamines, which are not normally genotoxic via the more usual microsomal activation pathways, was extended to include certain amide substrates such as acetaminophen.

X-ray structure of the ternary MTX·NADPH complex of the anthrax dihydrofolate reductase: A pharmacophore for dual-site inhibitor design

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bennett, Brad C.; Wan, Qun; Ahmad, Md Faiz

2009-11-18

For reasons of bioterrorism and drug resistance, it is imperative to identify and develop new molecular points of intervention against anthrax. Dihydrofolate reductase (DHFR) is a highly conserved enzyme and an established target in a number of species for a variety of chemotherapeutic programs. Recently, the crystal structure of B. anthracis DHFR (baDHFR) in complex with methotrexate (MTX) was determined and, based on the structure, proposals were made for drug design strategies directed against the substrate binding site. However, little is gleaned about the binding site for NADPH, the cofactor responsible for hydride transfer in the catalytic mechanism. In themore » present study, X-ray crystallography at 100 K was used to determine the structure of baDHFR in complex with MTX and NADPH. Although the NADPH binding mode is nearly identical to that seen in other DHFR ternary complex structures, the adenine moiety adopts an off-plane tilt of nearly 90 deg. and this orientation is stabilized by hydrogen bonds to functionally conserved Arg residues. A comparison of the binding site, focusing on this region, between baDHFR and the human enzyme is discussed, with an aim at designing species-selective therapeutics. Indeed, the ternary model, refined to 2.3{angstrom} resolution, provides an accurate template for testing the feasibility of identifying dual-site inhibitors, compounds that target both the substrate and cofactor binding site. With the ternary model in hand, using in silico methods, several compounds were identified which could potentially form key bonding contacts in the substrate and cofactor binding sites. Ultimately, two structurally distinct compounds were verified that inhibit baDHFR at low {mu}M concentrations. The apparent K{sub d} for one of these, (2-(3-(2-(hydroxyimino)-2-(pyridine-4-yl)-6,7-dimethylquinoxalin-2-yl)-1-(pyridine-4-yl)ethanone oxime), was measured by fluorescence spectroscopy to be 5.3 {mu}M.« less

Evolution of the herpes thymidine kinase: identification and comparison of the equine herpesvirus 1 thymidine kinase gene reveals similarity to a cell-encoded thymidylate kinase.

PubMed Central

Robertson, G R; Whalley, J M

1988-01-01

We have identified the equine herpesvirus 1 (EHV-1) thymidine kinase gene (TK) by DNA-mediated transformation and by DNA sequencing. Alignment of the amino acid sequence of the EHV-1 TK with the TKs from 3 other herpesviruses revealed regions of homology, some of which correspond to the previously identified substrate binding sites, while others have as yet, no assigned function. In particular, the strict conservation of an aspartate within the proposed nucleoside binding site suggests a role in ATP binding for this residue. Comparison of 5 herpes TKs with the thymidylate kinase of yeast revealed significant similarity which was strongest in those regions important to catalytic activity of the herpes TKs, and, therefore we propose that the herpes TK may be derived from a cellular thymidylate kinase. The implications for the evolution of enzyme activities within a pathway of nucleotide metabolism are discussed. PMID:2849761

Structural insight into the mechanism of substrate specificity and catalytic activity of an HD domain phosphohydrolase: the 5′-deoxyribonucleotidase YfbR from Escherichia coli

PubMed Central

Zimmerman, Matthew D.; Proudfoot, Michael; Yakunin, Alexander; Minor, Wladek

2008-01-01

Summary HD-domain phosphohydrolases have nucleotidase and phosphodiesterase activities and play important roles in the metabolism of nucleotides and in signaling. We present three 2.1 Å resolution crystal structures (one in the free state and two complexed with natural substrates) of a HD-domain phosphohydrolase, the E. coli 5′-nucleotidase YfbR. The free-state structure of YfbR contains a large cavity accommodating the metal-coordinating HD motif (H33, H68, D69, and D137) and other conserved residues (R18, E72, and D77). Alanine scanning mutagenesis confirms that these residues are important for activity. Two structures of the catalytically inactive mutant E72A complexed with Co2+ and either TMP or dAMP disclose the novel binding mode of deoxyribonucleotides in the active site. Residue R18 stabilizes the phosphate on the Co2+, and residue D77 forms a strong hydrogen bond critical for binding the ribose. The indole side chain of W19 is located close to the 2′-carbon atom of the deoxyribose moiety and is proposed to act as the selectivity switch for deoxyribonucleotide, which is supported by comparison to YfdR, another 5′-nucleotidase in E. coli. The nucleotide bases of both dAMP and TMP make no specific hydrogen bonds with the protein, explaining the lack of nucleotide base selectivity. The YfbR E72A substrate complex structures also suggest a plausible single-step nucleophilic substitution mechanism. This is the first proposed molecular mechanism for a HD-domain phosphohydrolase based directly on substrate-bound crystal structures. PMID:18353368

Uncoupling binding of substrate CO from turnover by vanadium nitrogenase.

PubMed

Lee, Chi Chung; Fay, Aaron W; Weng, Tsu-Chien; Krest, Courtney M; Hedman, Britt; Hodgson, Keith O; Hu, Yilin; Ribbe, Markus W

2015-11-10

Biocatalysis by nitrogenase, particularly the reduction of N2 and CO by this enzyme, has tremendous significance in environment- and energy-related areas. Elucidation of the detailed mechanism of nitrogenase has been hampered by the inability to trap substrates or intermediates in a well-defined state. Here, we report the capture of substrate CO on the resting-state vanadium-nitrogenase in a catalytically competent conformation. The close resemblance of this active CO-bound conformation to the recently described structure of CO-inhibited molybdenum-nitrogenase points to the mechanistic relevance of sulfur displacement to the activation of iron sites in the cofactor for CO binding. Moreover, the ability of vanadium-nitrogenase to bind substrate in the resting-state uncouples substrate binding from subsequent turnover, providing a platform for generation of defined intermediate(s) of both CO and N2 reduction.

Microfabricated, flowthrough porous apparatus for discrete detection of binding reactions

DOEpatents

Beattie, Kenneth L.

1998-01-01

An improved microfabricated apparatus for conducting a multiplicity of individual and simultaneous binding reactions is described. The apparatus comprises a substrate on which are located discrete and isolated sites for binding reactions. The apparatus is characterized by discrete and isolated regions that extend through said substrate and terminate on a second surface thereof such that when a test sample is allowed to the substrate, it is capable of penetrating through each such region during the course of said binding reaction. The apparatus is especially useful for sequencing by hybridization of DNA molecules.

Common folds and transport mechanisms of secondary active transporters.

PubMed

Shi, Yigong

2013-01-01

Secondary active transporters exploit the electrochemical potential of solutes to shuttle specific substrate molecules across biological membranes, usually against their concentration gradient. Transporters of different functional families with little sequence similarity have repeatedly been found to exhibit similar folds, exemplified by the MFS, LeuT, and NhaA folds. Observations of multiple conformational states of the same transporter, represented by the LeuT superfamily members Mhp1, AdiC, vSGLT, and LeuT, led to proposals that structural changes are associated with substrate binding and transport. Despite recent biochemical and structural advances, our understanding of substrate recognition and energy coupling is rather preliminary. This review focuses on the common folds and shared transport mechanisms of secondary active transporters. Available structural information generally supports the alternating access model for substrate transport, with variations and extensions made by emerging structural, biochemical, and computational evidence.

Long-range electrostatic complementarity governs substrate recognition by human chymotrypsin C, a key regulator of digestive enzyme activation.

PubMed

Batra, Jyotica; Szabó, András; Caulfield, Thomas R; Soares, Alexei S; Sahin-Tóth, Miklós; Radisky, Evette S

2013-04-05

Human chymotrypsin C (CTRC) is a pancreatic serine protease that regulates activation and degradation of trypsinogens and procarboxypeptidases by targeting specific cleavage sites within their zymogen precursors. In cleaving these regulatory sites, which are characterized by multiple flanking acidic residues, CTRC shows substrate specificity that is distinct from that of other isoforms of chymotrypsin and elastase. Here, we report the first crystal structure of active CTRC, determined at 1.9-Å resolution, revealing the structural basis for binding specificity. The structure shows human CTRC bound to the small protein protease inhibitor eglin c, which binds in a substrate-like manner filling the S6-S5' subsites of the substrate binding cleft. Significant binding affinity derives from burial of preferred hydrophobic residues at the P1, P4, and P2' positions of CTRC, although acidic P2' residues can also be accommodated by formation of an interfacial salt bridge. Acidic residues may also be specifically accommodated in the P6 position. The most unique structural feature of CTRC is a ring of intense positive electrostatic surface potential surrounding the primarily hydrophobic substrate binding site. Our results indicate that long-range electrostatic attraction toward substrates of concentrated negative charge governs substrate discrimination, which explains CTRC selectivity in regulating active digestive enzyme levels.

Molecular modeling studies of substrate binding by penicillin acylase.

PubMed

Chilov, G G; Stroganov, O V; Svedas, V K

2008-01-01

Molecular modeling has revealed intimate details of the mechanism of binding of natural substrate, penicillin G (PG), in the penicillin acylase active center and solved questions raised by analysis of available X-ray structures, mimicking Michaelis complex, which substantially differ in the binding pattern of the PG leaving group. Three MD trajectories were launched, starting from PDB complexes of the inactive mutant enzyme with PG (1FXV) and native penicillin acylase with sluggishly hydrolyzed substrate analog penicillin G sulfoxide (1GM9), or from the complex obtained by PG docking. All trajectories converged to a similar PG binding mode, which represented the near-to-attack conformation, consistent with chemical criteria of how reactive Michaelis complex should look. Simulated dynamic structure of the enzyme-substrate complex differed significantly from 1FXV, resembling rather 1GM9; however, additional contacts with residues bG385, bS386, and bN388 have been found, which were missing in X-ray structures. Combination of molecular docking and molecular dynamics also clarified the nature of extremely effective phenol binding in the hydrophobic pocket of penicillin acylase, which lacked proper explanation from crystallographic experiments. Alternative binding modes of phenol were probed, and corresponding trajectories converged to a single binding pattern characterized by a hydrogen bond between the phenol hydroxyl and the main chain oxygen of bS67, which was not evident from the crystal structure. Observation of the trajectory, in which phenol moved from its steady bound to pre-dissociation state, mapped the consequence of molecular events governing the conformational transitions in a coil region a143-a146 coupled to substrate binding and release of the reaction products. The current investigation provided information on dynamics of the conformational transitions accompanying substrate binding and significance of poorly structured and flexible regions in maintaining catalytic framework.

The disorderly conduct of Hsc70 and its interaction with the Alzheimer's related Tau protein.

PubMed

Taylor, Isabelle R; Ahmad, Atta; Wu, Taia; Nordhues, Bryce A; Bhullar, Anup; Gestwicki, Jason E; Zuiderweg, Erik R P

2018-05-15

Hsp70 chaperones bind to various protein substrates for folding, trafficking, and degradation. Considerable structural information is available about how prokaryotic Hsp70 (DnaK) binds substrates, but less is known about mammalian Hsp70s, of which there are 13 isoforms encoded in the human genome. Here, we report the interaction between the human Hsp70 isoform heat shock cognate 71 KDa protein (Hsc70 or HSPA8) and peptides derived from the microtubule-associated protein tau, which is linked to Alzheimer's disease. For structural studies, we used an Hsc70 construct (called BETA) comprising the substrate-binding domain, but lacking the lid. Importantly, we found that truncating the lid does not significantly impair Hsc70's chaperone activity or allostery in vitro. Using NMR, we show that BETA is partially dynamically disordered in the absence of substrate and that binding of the tau sequence GKVQIINKKG (with a KD = 500 nM) causes dramatic rigidification of BETA. Nuclear Overhauser effect distance measurements revealed that tau binds to the canonical substrate-binding cleft, similar to the binding observed with DnaK. To further develop BETA as a tool for studying Hsc70 interactions, we also measured BETA binding in NMR and fluorescent competition assays to peptides derived from huntingtin, insulin, a second tau-recognition sequence, and a KFERQ-like sequence linked to chaperone-mediated autophagy. We found that the insulin C-peptide binds BETA with high affinity (KD < 100 nM), whereas the others do not (KD > 100 μM). Together, our findings reveal several similarities and differences in how prokaryotic and mammalian Hsp70 isoforms interact with different substrate peptides. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Substrate binding ability of chemically inactivated pectinase for the substrate pectic acid.

PubMed

Chiba, Y; Kobayashi, M

1995-07-01

Pectinase (polygalacturonase) was purified from a commercial pectinase preparation from a mold. Substrate binding of pectinase was measured by centrifugal affinity chromatography using an immobilized substrate, pectic acid. Desorption of pectinase from the affinity matrix with the substrate pectin and pectic acid gave Kd values of 5.3 and 8.5 mg/ml, respectively. Chemical modification of pectinase by 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide (EDC) and diethyl pyrocarbonate (DEP) caused a loss of most of the enzyme activity, but the substrate binding ability was not impaired. Thus, the pectinase preparation was digested with lysyl endopeptidase and the resulting peptides were treated with pectic acid-affinity gel. Three peptide fragments, which were recovered from the affinity column and sequenced, were identical to sequences in the second pectinase gene from Aspergillus niger. The first peptide contained 17 amino acids, Asp101-Ser117, and the second and third peptides corresponded to 18 amino acids of Asn152-Asp169. These results indicate that the inactivated pectinase retained substrate binding ability and would function as an acidic polysaccharide recognizing protein.

Binding constant of cell adhesion receptors and substrate-immobilized ligands depends on the distribution of ligands

NASA Astrophysics Data System (ADS)

Li, Long; Hu, Jinglei; Xu, Guangkui; Song, Fan

2018-01-01

Cell-cell adhesion and the adhesion of cells to tissues and extracellular matrix, which are pivotal for immune response, tissue development, and cell locomotion, depend sensitively on the binding constant of receptor and ligand molecules anchored on the apposing surfaces. An important question remains of whether the immobilization of ligands affects the affinity of binding with cell adhesion receptors. We have investigated the adhesion of multicomponent membranes to a flat substrate coated with immobile ligands using Monte Carlo simulations of a statistical mesoscopic model with biologically relevant parameters. We find that the binding of the adhesion receptors to ligands immobilized on the substrate is strongly affected by the ligand distribution. In the case of ligand clusters, the receptor-ligand binding constant can be significantly enhanced due to the less translational entropy loss of lipid-raft domains in the model cell membranes upon the formation of additional complexes. For ligands randomly or uniformly immobilized on the substrate, the binding constant is rather decreased since the receptors localized in lipid-raft domains have to pay an energetic penalty in order to bind ligands. Our findings help to understand why cell-substrate adhesion experiments for measuring the impact of lipid rafts on the receptor-ligand interactions led to contradictory results.

Structural basis of redox-dependent substrate binding of protein disulfide isomerase

PubMed Central

Yagi-Utsumi, Maho; Satoh, Tadashi; Kato, Koichi

2015-01-01

Protein disulfide isomerase (PDI) is a multidomain enzyme, operating as an essential folding catalyst, in which the b′ and a′ domains provide substrate binding sites and undergo an open–closed domain rearrangement depending on the redox states of the a′ domain. Despite the long research history of this enzyme, three-dimensional structural data remain unavailable for its ligand-binding mode. Here we characterize PDI substrate recognition using α-synuclein (αSN) as the model ligand. Our nuclear magnetic resonance (NMR) data revealed that the substrate-binding domains of PDI captured the αSN segment Val37–Val40 only in the oxidized form. Furthermore, we determined the crystal structure of an oxidized form of the b′–a′ domains in complex with an undecapeptide corresponding to this segment. The peptide-binding mode observed in the crystal structure with NMR validation, was characterized by hydrophobic interactions on the b′ domain in an open conformation. Comparison with the previously reported crystal structure indicates that the a′ domain partially masks the binding surface of the b′ domain, causing steric hindrance against the peptide in the reduced form of the b′–a′ domains that exhibits a closed conformation. These findings provide a structural basis for the mechanism underlying the redox-dependent substrate binding of PDI. PMID:26350503

Determination of the Structure and Catalytic Mechanism of Sorghum bicolor Caffeic Acid O-Methyltransferase and the Structural Impact of Three brown midrib12 Mutations.

PubMed

Green, Abigail R; Lewis, Kevin M; Barr, John T; Jones, Jeffrey P; Lu, Fachuang; Ralph, John; Vermerris, Wilfred; Sattler, Scott E; Kang, ChulHee

2014-08-01

Using S-adenosyl-methionine as the methyl donor, caffeic acid O-methyltransferase from sorghum (Sorghum bicolor; SbCOMT) methylates the 5-hydroxyl group of its preferred substrate, 5-hydroxyconiferaldehyde. In order to determine the mechanism of SbCOMT and understand the observed reduction in the lignin syringyl-to-guaiacyl ratio of three brown midrib12 mutants that carry COMT gene missense mutations, we determined the apo-form and S-adenosyl-methionine binary complex SbCOMT crystal structures and established the ternary complex structure with 5-hydroxyconiferaldehyde by molecular modeling. These structures revealed many features shared with monocot ryegrass (Lolium perenne) and dicot alfalfa (Medicago sativa) COMTs. SbCOMT steady-state kinetic and calorimetric data suggest a random bi-bi mechanism. Based on our structural, kinetic, and thermodynamic results, we propose that the observed reactivity hierarchy among 4,5-dihydroxy-3-methoxycinnamyl (and 3,4-dihydroxycinnamyl) aldehyde, alcohol, and acid substrates arises from the ability of the aldehyde to stabilize the anionic intermediate that results from deprotonation of the 5-hydroxyl group by histidine-267. Additionally, despite the presence of other phenylpropanoid substrates in vivo, sinapaldehyde is the preferential product, as demonstrated by its low K m for 5-hydroxyconiferaldehyde. Unlike its acid and alcohol substrates, the aldehydes exhibit product inhibition, and we propose that this is due to nonproductive binding of the S-cis-form of the aldehydes inhibiting productive binding of the S-trans-form. The S-cis-aldehydes most likely act only as inhibitors, because the high rotational energy barrier around the 2-propenyl bond prevents S-trans-conversion, unlike alcohol substrates, whose low 2-propenyl bond rotational energy barrier enables rapid S-cis/S-trans-interconversion. © 2014 American Society of Plant Biologists. All Rights Reserved.

Articles including thin film monolayers and multilayers

DOEpatents

Li, DeQuan; Swanson, Basil I.

1995-01-01

Articles of manufacture including: (a) a base substrate having an oxide surface layer, and a multidentate ligand, capable of binding a metal ion, attached to the oxide surface layer of the base substrate, (b) a base substrate having an oxide surface layer, a multidentate ligand, capable of binding a metal ion, attached to the oxide surface layer of the base substrate, and a metal species attached to the multidentate ligand, (c) a base substrate having an oxide surface layer, a multidentate ligand, capable of binding a metal ion, attached to the oxide surface layer of the base substrate, a metal species attached to the multidentate ligand, and a multifunctional organic ligand attached to the metal species, and (d) a base substrate having an oxide surface layer, a multidentate ligand, capable of binding a metal ion, attached to the oxide surface layer of the base substrate, a metal species attached to the multidentate ligand, a multifunctional organic ligand attached to the metal species, and a second metal species attached to the multifunctional organic ligand, are provided, such articles useful in detecting the presence of a selected target species, as nonliear optical materials, or as scavengers for selected target species.

Dissection of structural and functional requirements that underlie the interaction of ERdj3 protein with substrates in the endoplasmic reticulum.

PubMed

Otero, Joel H; Lizák, Beata; Feige, Matthias J; Hendershot, Linda M

2014-10-03

ERdj3, a mammalian endoplasmic reticulum (ER) Hsp40/DnaJ family member, binds unfolded proteins, transfers them to BiP, and concomitantly stimulates BiP ATPase activity. However, the requirements for ERdj3 binding to and release from substrates in cells are not well understood. We found that ERdj3 homodimers that cannot stimulate the ATPase activity of BiP (QPD mutants) bound to unfolded ER proteins under steady state conditions in much greater amounts than wild-type ERdj3. This was due to reduced release from these substrates as opposed to enhanced binding, although in both cases dimerization was strictly required for substrate binding. Conversely, heterodimers consisting of one wild-type and one mutant ERdj3 subunit bound substrates at levels comparable with wild-type ERdj3 homodimers, demonstrating that release requires only one protomer to be functional in stimulating BiP ATPase activity. Co-expressing wild-type ERdj3 and a QPD mutant, which each exclusively formed homodimers, revealed that the release rate of wild-type ERdj3 varied according to the relative half-lives of substrates, suggesting that ERdj3 release is an important step in degradation of unfolded client proteins in the ER. Furthermore, pulse-chase experiments revealed that the binding of QPD mutant homodimers remained constant as opposed to increasing, suggesting that ERdj3 does not normally undergo reiterative binding cycles with substrates. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Dissection of Structural and Functional Requirements That Underlie the Interaction of ERdj3 Protein with Substrates in the Endoplasmic Reticulum*

PubMed Central

Otero, Joel H.; Lizák, Beata; Feige, Matthias J.; Hendershot, Linda M.

2014-01-01

ERdj3, a mammalian endoplasmic reticulum (ER) Hsp40/DnaJ family member, binds unfolded proteins, transfers them to BiP, and concomitantly stimulates BiP ATPase activity. However, the requirements for ERdj3 binding to and release from substrates in cells are not well understood. We found that ERdj3 homodimers that cannot stimulate the ATPase activity of BiP (QPD mutants) bound to unfolded ER proteins under steady state conditions in much greater amounts than wild-type ERdj3. This was due to reduced release from these substrates as opposed to enhanced binding, although in both cases dimerization was strictly required for substrate binding. Conversely, heterodimers consisting of one wild-type and one mutant ERdj3 subunit bound substrates at levels comparable with wild-type ERdj3 homodimers, demonstrating that release requires only one protomer to be functional in stimulating BiP ATPase activity. Co-expressing wild-type ERdj3 and a QPD mutant, which each exclusively formed homodimers, revealed that the release rate of wild-type ERdj3 varied according to the relative half-lives of substrates, suggesting that ERdj3 release is an important step in degradation of unfolded client proteins in the ER. Furthermore, pulse-chase experiments revealed that the binding of QPD mutant homodimers remained constant as opposed to increasing, suggesting that ERdj3 does not normally undergo reiterative binding cycles with substrates. PMID:25143379

Insights into the Interactions of Fasciola hepatica Cathepsin L3 with a Substrate and Potential Novel Inhibitors through In Silico Approaches

PubMed Central

Hernández Alvarez, Lilian; Naranjo Feliciano, Dany; Hernández González, Jorge Enrique; de Oliveira Soares, Rosemberg; Barreto Gomes, Diego Enry; Pascutti, Pedro Geraldo

2015-01-01

Background Fasciola hepatica is the causative agent of fascioliasis, a disease affecting grazing animals, causing economic losses in global agriculture and currently being an important human zoonosis. Overuse of chemotherapeutics against fascioliasis has increased the populations of drug resistant parasites. F. hepatica cathepsin L3 is a protease that plays important roles during the life cycle of fluke. Due to its particular collagenolytic activity it is considered an attractive target against the infective phase of F. hepatica. Methodology/Principal Findings Starting with a three dimensional model of FhCL3 we performed a structure-based design of novel inhibitors through a computational study that combined virtual screening, molecular dynamics simulations, and binding free energy (ΔGbind) calculations. Virtual screening was carried out by docking inhibitors obtained from the MYBRIDGE-HitFinder database inside FhCL3 and human cathepsin L substrate-binding sites. On the basis of dock-scores, five compounds were predicted as selective inhibitors of FhCL3. Molecular dynamic simulations were performed and, subsequently, an end-point method was employed to predict ΔGbind values. Two compounds with the best ΔGbind values (-10.68 kcal/mol and -7.16 kcal/mol), comparable to that of the positive control (-10.55 kcal/mol), were identified. A similar approach was followed to structurally and energetically characterize the interface of FhCL3 in complex with a peptidic substrate. Finally, through pair-wise and per-residue free energy decomposition we identified residues that are critical for the substrate/ligand binding and for the enzyme specificity. Conclusions/Significance The present study is the first computer-aided drug design approach against F. hepatica cathepsins. Here we predict the principal determinants of binding of FhCL3 in complex with a natural substrate by detailed energetic characterization of protease interaction surface. We also propose novel compounds as FhCL3 inhibitors. Overall, these results will foster the future rational design of new inhibitors against FhCL3, as well as other F. hepatica cathepsins. PMID:25978322

Insights into the Interactions of Fasciola hepatica Cathepsin L3 with a Substrate and Potential Novel Inhibitors through In Silico Approaches.

PubMed

Hernández Alvarez, Lilian; Naranjo Feliciano, Dany; Hernández González, Jorge Enrique; Soares, R O; Soares, Rosemberg de Oliveira; Barreto Gomes, Diego Enry; Pascutti, Pedro Geraldo

2015-05-01

Fasciola hepatica is the causative agent of fascioliasis, a disease affecting grazing animals, causing economic losses in global agriculture and currently being an important human zoonosis. Overuse of chemotherapeutics against fascioliasis has increased the populations of drug resistant parasites. F. hepatica cathepsin L3 is a protease that plays important roles during the life cycle of fluke. Due to its particular collagenolytic activity it is considered an attractive target against the infective phase of F. hepatica. Starting with a three dimensional model of FhCL3 we performed a structure-based design of novel inhibitors through a computational study that combined virtual screening, molecular dynamics simulations, and binding free energy (ΔGbind) calculations. Virtual screening was carried out by docking inhibitors obtained from the MYBRIDGE-HitFinder database inside FhCL3 and human cathepsin L substrate-binding sites. On the basis of dock-scores, five compounds were predicted as selective inhibitors of FhCL3. Molecular dynamic simulations were performed and, subsequently, an end-point method was employed to predict ΔGbind values. Two compounds with the best ΔGbind values (-10.68 kcal/mol and -7.16 kcal/mol), comparable to that of the positive control (-10.55 kcal/mol), were identified. A similar approach was followed to structurally and energetically characterize the interface of FhCL3 in complex with a peptidic substrate. Finally, through pair-wise and per-residue free energy decomposition we identified residues that are critical for the substrate/ligand binding and for the enzyme specificity. The present study is the first computer-aided drug design approach against F. hepatica cathepsins. Here we predict the principal determinants of binding of FhCL3 in complex with a natural substrate by detailed energetic characterization of protease interaction surface. We also propose novel compounds as FhCL3 inhibitors. Overall, these results will foster the future rational design of new inhibitors against FhCL3, as well as other F. hepatica cathepsins.

Structural basis for substrate recognition by the human N-terminal methyltransferase 1

DOE PAGES

Dong, Cheng; Mao, Yunfei; Tempel, Wolfram; ...

2015-11-05

α-N-terminal methylation represents a highly conserved and prevalent post-translational modification, yet its biological function has remained largely speculative. The recent discovery of α-N-terminal methyltransferase 1 (NTMT1) and its physiological substrates propels the elucidation of a general role of α-N-terminal methylation in mediating DNA-binding ability of the modified proteins. The phenotypes, observed from both NTMT1 knockdown in breast cancer cell lines and knockout mouse models, suggest the potential involvement of α-N-terminal methylation in DNA damage response and cancer development. In this study, we report the first crystal structures of human NTMT1 in complex with cofactor S-adenosyl-L-homocysteine (SAH) and six substrate peptides,more » respectively, and reveal that NTMT1 contains two characteristic structural elements (a β hairpin and an N-terminal extension) that contribute to its substrate specificity. Our complex structures, coupled with mutagenesis, binding, and enzymatic studies, also present the key elements involved in locking the consensus substrate motif XPK (X indicates any residue type other than D/E) into the catalytic pocket for α-N-terminal methylation and explain why NTMT1 prefers an XPK sequence motif. We propose a catalytic mechanism for α-N-terminal methylation. Overall, this study gives us the first glimpse of the molecular mechanism of α-N-terminal methylation and potentially contributes to the advent of therapeutic agents for human diseases associated with deregulated α-N-terminal methylation.« less

Key feature of the catalytic cycle of TNF-α converting enzyme involves communication between distal protein sites and the enzyme catalytic core

PubMed Central

Solomon, Ariel; Akabayov, Barak; Frenkel, Anatoly; Milla, Marcos E.; Sagi, Irit

2007-01-01

Despite their key roles in many normal and pathological processes, the molecular details by which zinc-dependent proteases hydrolyze their physiological substrates remain elusive. Advanced theoretical analyses have suggested reaction models for which there is limited and controversial experimental evidence. Here we report the structure, chemistry and lifetime of transient metal–protein reaction intermediates evolving during the substrate turnover reaction of a metalloproteinase, the tumor necrosis factor-α converting enzyme (TACE). TACE controls multiple signal transduction pathways through the proteolytic release of the extracellular domain of a host of membrane-bound factors and receptors. Using stopped-flow x-ray spectroscopy methods together with transient kinetic analyses, we demonstrate that TACE's catalytic zinc ion undergoes dynamic charge transitions before substrate binding to the metal ion. This indicates previously undescribed communication pathways taking place between distal protein sites and the enzyme catalytic core. The observed charge transitions are synchronized with distinct phases in the reaction kinetics and changes in metal coordination chemistry mediated by the binding of the peptide substrate to the catalytic metal ion and product release. Here we report key local charge transitions critical for proteolysis as well as long sought evidence for the proposed reaction model of peptide hydrolysis. This study provides a general approach for gaining critical insights into the molecular basis of substrate recognition and turnover by zinc metalloproteinases that may be used for drug design. PMID:17360351

The carbanion of nitroethane is an inhibitor of, and not a substrate for, flavocytochrome b2 [L-(+)-lactate dehydrogenase

PubMed Central

Genet, R; Lederer, F

1990-01-01

Although nitroethane does not bind to the active site of flavocytochrome b2, its anion, ethane nitronate, behaves as a competitive inhibitor, with a Ki of 2.2 mM. No electron transfer can be detected between the nitronate and the enzyme, in contrast with the observations of other workers on D-amino acid oxidase. Propionate is a competitive inhibitor, with a Ki of 28 mM. The significance of these results with respect to the proposed carbanion mechanism and the putative existence of a covalent enzyme-substrate intermediate is discussed. PMID:2178603

The initial step in the archaeal aspartate biosynthetic pathway catalyzed by a monofunctional aspartokinase

PubMed Central

Faehnle, Christopher R.; Liu, Xuying; Pavlovsky, Alexander; Viola, Ronald E.

2006-01-01

The activation of the β-carboxyl group of aspartate catalyzed by aspartokinase is the commitment step to amino-acid biosynthesis in the aspartate pathway. The first structure of a microbial aspartokinase, that from Methanococcus jannaschii, has been determined in the presence of the amino-acid substrate l-­aspartic acid and the nucleotide product MgADP. The enzyme assembles into a dimer of dimers, with the interfaces mediated by both the N- and C-terminal domains. The active-site functional groups responsible for substrate binding and specificity have been identified and roles have been proposed for putative catalytic functional groups. PMID:17012784

Probing Allosteric Inhibition Mechanisms of the Hsp70 Chaperone Proteins Using Molecular Dynamics Simulations and Analysis of the Residue Interaction Networks.

PubMed

Stetz, Gabrielle; Verkhivker, Gennady M

2016-08-22

Although molecular mechanisms of allosteric regulation in the Hsp70 chaperones have been extensively studied at both structural and functional levels, the current understanding of allosteric inhibition of chaperone activities by small molecules is still lacking. In the current study, using a battery of computational approaches, we probed allosteric inhibition mechanisms of E. coli Hsp70 (DnaK) and human Hsp70 proteins by small molecule inhibitors PET-16 and novolactone. Molecular dynamics simulations and binding free energy analysis were combined with network-based modeling of residue interactions and allosteric communications to systematically characterize and compare molecular signatures of the apo form, substrate-bound, and inhibitor-bound chaperone complexes. The results suggested a mechanism by which the allosteric inhibitors may leverage binding energy hotspots in the interaction networks to stabilize a specific conformational state and impair the interdomain allosteric control. Using the network-based centrality analysis and community detection, we demonstrated that substrate binding may strengthen the connectivity of local interaction communities, leading to a dense interaction network that can promote an efficient allosteric communication. In contrast, binding of PET-16 to DnaK may induce significant dynamic changes and lead to a fractured interaction network and impaired allosteric communications in the DnaK complex. By using a mechanistic-based analysis of distance fluctuation maps and allosteric propensities of protein residues, we determined that the allosteric network in the PET-16 complex may be small and localized due to the reduced communication and low cooperativity of the substrate binding loops, which may promote the higher rates of substrate dissociation and the decreased substrate affinity. In comparison with the significant effect of PET-16, binding of novolactone to HSPA1A may cause only moderate network changes and preserve allosteric coupling between the allosteric pocket and the substrate binding region. The impact of novolactone on the conformational dynamics and allosteric communications in the HSPA1A complex was comparable to the substrate effect, which is consistent with the experimental evidence that PET-16, but not novolactone binding, can significantly decrease substrate affinity. We argue that the unique dynamic and network signatures of PET-16 and novolactone may be linked with the experimentally observed functional effects of these inhibitors on allosteric regulation and substrate binding.

Selectivity of substrate binding and ionization of 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase.

PubMed

Luanloet, Thikumporn; Sucharitakul, Jeerus; Chaiyen, Pimchai

2015-08-01

2-Methyl-3-hydroxypyridine-5-carboxylic acid (MHPC) oxygenase (EC 1.14.12.4) from Pseudomonas sp. MA-1 is a flavin-dependent monooxygenase that catalyzes a hydroxylation and aromatic ring cleavage reaction. The functional roles of two residues, Tyr223 and Tyr82, located ~ 5 Å away from MHPC, were characterized using site-directed mutagenesis, along with ligand binding, product analysis and transient kinetic experiments. Mutation of Tyr223 resulted in enzyme variants that were impaired in their hydroxylation activity and had Kd values for substrate binding 5-10-fold greater than the wild-type enzyme. Because this residue is adjacent to the water molecule that is located next to the 3-hydroxy group of MHPC, the results indicate that the interaction between Tyr223, H2 O and the 3-hydroxyl group of MHPC are important for substrate binding and hydroxylation. By contrast, the Kd for substrate binding of Tyr82His and Tyr82Phe variants were similar to that of the wild-type enzyme. However, only ~ 40-50% of the substrate was hydroxylated in the reactions of both variants, whereas most of the substrate was hydroxylated in the wild-type enzyme reaction. In free solution, MHPC or 5-hydroxynicotinic acid exists in a mixture of monoanionic and tripolar ionic forms, whereas only the tripolar ionic form binds to the wild-type enzyme. The binding of tripolar ionic MHPC would allow efficient hydroxylation through an electrophilic aromatic substitution mechanism. For the Tyr82His and Tyr82Phe variants, both forms of substrates can bind to the enzymes, indicating that the mutation at Tyr82 abolished the selectivity of the enzyme towards the tripolar ionic form. Transient kinetic studies indicated that the hydroxylation rate constants of both Tyr82 variants are approximately two- to 2.5-fold higher than that of the wild-type enzyme. Altogether, our findings suggest that Tyr82 is important for the binding selectivity of MHPC oxygenase towards the tripolar ionic species, whereas the interaction between Tyr223 and the substrate is important for ensuring hydroxylation. These results highlight how the active site of a flavoenzyme is able to deal with the presence of multiple forms of a substrate in solution and ensure efficient hydroxylation. © 2015 FEBS.

Molecular Dynamics Investigation of the Substrate Binding Mechanism in Carboxylesterase

DOE PAGES

Chen, Qi; Luan, Zheng-Jiao; Cheng, Xiaolin; ...

2015-02-25

A recombinant carboxylesterase, cloned from Pseudomonas putida and designated as rPPE, is capable of catalyzing the bioresolution of racemic 2-acetoxy-2-(2 -chlorophenyl)acetate (rac-AcO-CPA) with excellent (S)-enantioselectivity. Semi-rational design of the enzyme showed that the W187H variant could increase the activity by ~100-fold compared to the wild type (WT) enzyme. In this study, we performed all-atom molecular dynamics (MD) simulations of both apo-rPPE and rPPE in complex with (S)-AcO-CPA to gain insights into the origin of the increased catalysis in the W187H mutant. Moreover, our results show differential binding of (S)-AcO-CPA in the WT and W187H enzymes, especially the interactions of themore » substrate with the two active site residues Ser159 and His286. The replacement of Trp187 by His leads to considerable structural rearrangement in the active site of W187H. Unlike in the WT rPPE, the cap domain in the W187 mutant shows an open conformation in the simulations of both apo and substrate-bound enzymes. This open conformation exposes the catalytic triad to the solvent through a water accessible channel, which may facilitate the entry of the substrate and/or the exit of the product. Binding free energy calculations confirmed that the substrate binds more strongly in W187H than in WT. Based on these computational results, furthermore, we predicted that the mutations W187Y and D287G might also be able to increase the substrate binding, thus improve the enzyme s catalytic efficiency. Experimental binding and kinetic assays on W187Y and D287G show improved catalytic efficiency over WT, but not W187H. Contrary to our prediction, W187Y shows slightly decreased substrate binding coupled with a 100 fold increase in turn-over rate, while in D287G the substrate binding is 8 times stronger but with a slightly reduced turn-over rate. Finally, our work provides important molecular-level insights into the binding of the (S)-AcO-CPA substrate to carboxylesterase rPPEs, which will help guide future development of more efficient rPPE variants.« less

Molecular Dynamics Investigation of the Substrate Binding Mechanism in Carboxylesterase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Qi; Luan, Zheng-Jiao; Cheng, Xiaolin

A recombinant carboxylesterase, cloned from Pseudomonas putida and designated as rPPE, is capable of catalyzing the bioresolution of racemic 2-acetoxy-2-(2 -chlorophenyl)acetate (rac-AcO-CPA) with excellent (S)-enantioselectivity. Semi-rational design of the enzyme showed that the W187H variant could increase the activity by ~100-fold compared to the wild type (WT) enzyme. In this study, we performed all-atom molecular dynamics (MD) simulations of both apo-rPPE and rPPE in complex with (S)-AcO-CPA to gain insights into the origin of the increased catalysis in the W187H mutant. Moreover, our results show differential binding of (S)-AcO-CPA in the WT and W187H enzymes, especially the interactions of themore » substrate with the two active site residues Ser159 and His286. The replacement of Trp187 by His leads to considerable structural rearrangement in the active site of W187H. Unlike in the WT rPPE, the cap domain in the W187 mutant shows an open conformation in the simulations of both apo and substrate-bound enzymes. This open conformation exposes the catalytic triad to the solvent through a water accessible channel, which may facilitate the entry of the substrate and/or the exit of the product. Binding free energy calculations confirmed that the substrate binds more strongly in W187H than in WT. Based on these computational results, furthermore, we predicted that the mutations W187Y and D287G might also be able to increase the substrate binding, thus improve the enzyme s catalytic efficiency. Experimental binding and kinetic assays on W187Y and D287G show improved catalytic efficiency over WT, but not W187H. Contrary to our prediction, W187Y shows slightly decreased substrate binding coupled with a 100 fold increase in turn-over rate, while in D287G the substrate binding is 8 times stronger but with a slightly reduced turn-over rate. Finally, our work provides important molecular-level insights into the binding of the (S)-AcO-CPA substrate to carboxylesterase rPPEs, which will help guide future development of more efficient rPPE variants.« less

Binding Isotope Effects for para-Aminobenzoic Acid with Dihydropteroate Synthase from Staphylococcus aureus and Plasmodium falciparum.

PubMed

Stratton, Christopher F; Namanja-Magliano, Hilda A; Cameron, Scott A; Schramm, Vern L

2015-10-16

Dihydropteroate synthase is a key enzyme in folate biosynthesis and is the target of the sulfonamide class of antimicrobials. Equilibrium binding isotope effects and density functional theory calculations indicate that the substrate binding sites for para-aminobenzoic acid on the dihydropteroate synthase enzymes from Staphylococcus aureus and Plasmodium falciparum present distinct chemical environments. Specifically, we show that para-aminobenzoic acid occupies a more sterically constrained vibrational environment when bound to dihydropteroate synthase from P. falciparum relative to that of S. aureus. Deletion of a nonhomologous, parasite-specific insert from the plasmodial dihydropteroate synthase abrogated the binding of para-aminobenzoic acid. The loop specific to P. falciparum is important for effective substrate binding and therefore plays a role in modulating the chemical environment at the substrate binding site.

Structure of L-Xylulose-5-Phosphate 3-Epimerase (UlaE) from the Anaerobic L-Ascorbate Utilization Pathway of Escherichia coli: Identification of a Novel Phosphate Binding Motif within a TIM Barrel Fold

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, Rong; Pineda, Marco; Ajamian, Eunice

2009-01-15

Three catabolic enzymes, UlaD, UlaE, and UlaF, are involved in a pathway leading to fermentation of L-ascorbate under anaerobic conditions. UlaD catalyzes a {beta}-keto acid decarboxylation reaction to produce L-xylulose-5-phosphate, which undergoes successive epimerization reactions with UlaE (L-xylulose-5-phosphate 3-epimerase) and UlaF (L-ribulose-5-phosphate 4-epimerase), yielding D-xylulose-5-phosphate, an intermediate in the pentose phosphate pathway. We describe here crystallographic studies of UlaE from Escherichia coli O157:H7 that complete the structural characterization of this pathway. UlaE has a triosephosphate isomerase (TIM) barrel fold and forms dimers. The active site is located at the C-terminal ends of the parallel {beta}-strands. The enzyme binds Zn{sup 2+},more » which is coordinated by Glu155, Asp185, His211, and Glu251. We identified a phosphate-binding site formed by residues from the {beta}1/{alpha}1 loop and {alpha}3' helix in the N-terminal region. This site differs from the well-characterized phosphate-binding motif found in several TIM barrel superfamilies that is located at strands {beta}7 and {beta}8. The intrinsic flexibility of the active site region is reflected by two different conformations of loops forming part of the substrate-binding site. Based on computational docking of the L-xylulose 5-phosphate substrate to UlaE and structural similarities of the active site of this enzyme to the active sites of other epimerases, a metal-dependent epimerization mechanism for UlaE is proposed, and Glu155 and Glu251 are implicated as catalytic residues. Mutation and activity measurements for structurally equivalent residues in related epimerases supported this mechanistic proposal.« less

Gibberellin Receptor GID1: Gibberellin Recognition and Molecular Evolution

NASA Astrophysics Data System (ADS)

Kato, Hiroaki; Sato, Tomomi; Ueguchi-Tanaka, Miyako

Gibberellins (GAs) are phytohormones essential for many developmental processes in plants. We analyzed the crystal structure of a nuclear GA receptor, GIBBERELLIN INSENSITIVE DWARF 1 (GID1) from Oryza sativa. As it was proposed from the sequence similarity, the overall structure of GID1 shows an α/β-hydrolase fold similar to that of the hormone-sensitive lipases (HSLs) except for an amino-terminal lid. The GA-binding site corresponds to the substrate-binding site of HSLs. Almost residues assigned for GA binding showed very little or no activity when they were replaced with Ala. The substitution of the residues corresponding to those of the lycophyte GID1s caused an increase in the binding affinity for GA34, a 2β-hydroxylated GA4. These findings indicate that GID1 originated from HSL and was tinkered to have the specificity for bioactive GAs in the course of plant evolution.

The Inhibitory Effect of Non-Substrate and Substrate DNA on the Ligation and Self-Adenylylation Reactions Catalyzed by T4 DNA Ligase

PubMed Central

Bauer, Robert J.; Evans, Thomas C.; Lohman, Gregory J. S.

2016-01-01

DNA ligases are essential both to in vivo replication, repair and recombination processes, and in vitro molecular biology protocols. Prior characterization of DNA ligases through gel shift assays has shown the presence of a nick site to be essential for tight binding between the enzyme and its dsDNA substrate, with no interaction evident on dsDNA lacking a nick. In the current study, we observed a significant substrate inhibition effect, as well as the inhibition of both the self-adenylylation and nick-sealing steps of T4 DNA ligase by non-nicked, non-substrate dsDNA. Inhibition by non-substrate DNA was dependent only on the total DNA concentration rather than the structure; with 1 μg/mL of 40-mers, 75-mers, or circular plasmid DNA all inhibiting ligation equally. A >15-fold reduction in T4 DNA ligase self-adenylylation rate when in the presence of high non-nicked dsDNA concentrations was observed. Finally, EMSAs were utilized to demonstrate that non-substrate dsDNA can compete with nicked dsDNA substrates for enzyme binding. Based upon these data, we hypothesize the inhibition of T4 DNA ligase by non-nicked dsDNA is direct evidence for a two-step nick-binding mechanism, with an initial, nick-independent, transient dsDNA-binding event preceding a transition to a stable binding complex in the presence of a nick site. PMID:26954034

The Inhibitory Effect of Non-Substrate and Substrate DNA on the Ligation and Self-Adenylylation Reactions Catalyzed by T4 DNA Ligase.

PubMed

Bauer, Robert J; Evans, Thomas C; Lohman, Gregory J S

2016-01-01

DNA ligases are essential both to in vivo replication, repair and recombination processes, and in vitro molecular biology protocols. Prior characterization of DNA ligases through gel shift assays has shown the presence of a nick site to be essential for tight binding between the enzyme and its dsDNA substrate, with no interaction evident on dsDNA lacking a nick. In the current study, we observed a significant substrate inhibition effect, as well as the inhibition of both the self-adenylylation and nick-sealing steps of T4 DNA ligase by non-nicked, non-substrate dsDNA. Inhibition by non-substrate DNA was dependent only on the total DNA concentration rather than the structure; with 1 μg/mL of 40-mers, 75-mers, or circular plasmid DNA all inhibiting ligation equally. A >15-fold reduction in T4 DNA ligase self-adenylylation rate when in the presence of high non-nicked dsDNA concentrations was observed. Finally, EMSAs were utilized to demonstrate that non-substrate dsDNA can compete with nicked dsDNA substrates for enzyme binding. Based upon these data, we hypothesize the inhibition of T4 DNA ligase by non-nicked dsDNA is direct evidence for a two-step nick-binding mechanism, with an initial, nick-independent, transient dsDNA-binding event preceding a transition to a stable binding complex in the presence of a nick site.

Interactions between Casein Kinase Iε (CKIε) and Two Substrates from Disparate Signaling Pathways Reveal Mechanisms for Substrate-Kinase Specificity

PubMed Central

Dahlberg, Caroline Lund; Nguyen, Elizabeth Z.; Goodlett, David; Kimelman, David

2009-01-01

Background Members of the Casein Kinase I (CKI) family of serine/threonine kinases regulate diverse biological pathways. The seven mammalian CKI isoforms contain a highly conserved kinase domain and divergent amino- and carboxy-termini. Although they share a preferred target recognition sequence and have overlapping expression patterns, individual isoforms often have specific substrates. In an effort to determine how substrates recognize differences between CKI isoforms, we have examined the interaction between CKIε and two substrates from different signaling pathways. Methodology/Principal Findings CKIε, but not CKIα, binds to and phosphorylates two proteins: Period, a transcriptional regulator of the circadian rhythms pathway, and Disheveled, an activator of the planar cell polarity pathway. We use GST-pull-down assays data to show that two key residues in CKIα's kinase domain prevent Disheveled and Period from binding. We also show that the unique C-terminus of CKIε does not determine Dishevelled's and Period's preference for CKIε nor is it essential for binding, but instead plays an auxillary role in stabilizing the interactions of CKIε with its substrates. We demonstrate that autophosphorylation of CKIε's C-terminal tail prevents substrate binding, and use mass spectrometry and chemical crosslinking to reveal how a phosphorylation-dependent interaction between the C-terminal tail and the kinase domain prevents substrate phosphorylation and binding. Conclusions/Significance The biochemical interactions between CKIε and Disheveled, Period, and its own C-terminus lead to models that explain CKIε's specificity and regulation. PMID:19274088

Investigation of the intermolecular recognition mechanism between the E3 ubiquitin ligase Keap1 and substrate based on multiple substrates analysis.

PubMed

Jiang, Zheng-Yu; Xu, Li-Li; Lu, Meng-Chen; Pan, Yang; Huang, Hao-Ze; Zhang, Xiao-Jin; Sun, Hao-Peng; You, Qi-Dong

2014-12-01

E3 ubiquitin ligases are attractive drug targets due to their specificity to the ubiquitin machinery. However, the development of E3 ligase inhibitors has proven challenging for the fact that they must disrupt protein-protein interactions (PPIs). The E3 ligase involved in interactome provide new hope for the discovery of the E3 ligase inhibitors. These currently known natural binding partners of the E3 ligase can benefit the discovery of other unknown substrates and also the E3 ligase inhibitors. Herein, we present a novel strategy that using multiple substrates to elucidate the molecular recognition mechanism of E3 ubiquitin ligase. Molecular dynamics simulation, molecular mechanics-generalized born surface area (MM-GBSA) binding energy calculation and energy decomposition scheme were incorporated to evaluate the quantitative contributions of sub-pocket and per-residue to binding. In this case, Kelch-like ECH-associated protein-1 (Keap1), a substrate adaptor component of the Cullin-RING ubiquitin ligases complex, is applied for the investigation of how it recognize its substrates, especially Nrf2, a master regulator of the antioxidant response. By analyzing multiple substrates binding determinants, we found that both the polar sub-pockets (P1 and P2) and the nonpolar sub-pockets (P4 and P5) of Keap1 can make remarkable contributions to intermolecular interactions. This finding stresses the requirement for substrates to interact with the polar and nonpolar sub-pockets simultaneously. The results discussed in this paper not only show the binding determinants of the Keap1 substrates but also provide valuable implications for both Keap1 substrate discovery and PPI inhibitor design.

Investigation of the intermolecular recognition mechanism between the E3 ubiquitin ligase Keap1 and substrate based on multiple substrates analysis

NASA Astrophysics Data System (ADS)

Jiang, Zheng-Yu; Xu, Li-Li; Lu, Meng-Chen; Pan, Yang; Huang, Hao-Ze; Zhang, Xiao-Jin; Sun, Hao-Peng; You, Qi-Dong

2014-12-01

E3 ubiquitin ligases are attractive drug targets due to their specificity to the ubiquitin machinery. However, the development of E3 ligase inhibitors has proven challenging for the fact that they must disrupt protein-protein interactions (PPIs). The E3 ligase involved in interactome provide new hope for the discovery of the E3 ligase inhibitors. These currently known natural binding partners of the E3 ligase can benefit the discovery of other unknown substrates and also the E3 ligase inhibitors. Herein, we present a novel strategy that using multiple substrates to elucidate the molecular recognition mechanism of E3 ubiquitin ligase. Molecular dynamics simulation, molecular mechanics-generalized born surface area (MM-GBSA) binding energy calculation and energy decomposition scheme were incorporated to evaluate the quantitative contributions of sub-pocket and per-residue to binding. In this case, Kelch-like ECH-associated protein-1 (Keap1), a substrate adaptor component of the Cullin-RING ubiquitin ligases complex, is applied for the investigation of how it recognize its substrates, especially Nrf2, a master regulator of the antioxidant response. By analyzing multiple substrates binding determinants, we found that both the polar sub-pockets (P1 and P2) and the nonpolar sub-pockets (P4 and P5) of Keap1 can make remarkable contributions to intermolecular interactions. This finding stresses the requirement for substrates to interact with the polar and nonpolar sub-pockets simultaneously. The results discussed in this paper not only show the binding determinants of the Keap1 substrates but also provide valuable implications for both Keap1 substrate discovery and PPI inhibitor design.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smith, Dayle; Danyal, Karamatullah; Raugei, Simone

Mo-dependent nitrogenase catalyzes the biological reduction of N 2 to 2NH 3 at the FeMo-cofactor buried deep inside the MoFe protein. Access of substrates, such as N 2, to the active site is likely restricted by the surrounding protein, requiring substrate channels that lead from the surface to the active site. Earlier studies on crystallographic structures of the MoFe protein have suggested three putative substrate channels. Here, we have utilized sub-microsecond atomistic molecular dynamics simulations to allow the nitrogenase MoFe protein to explore its conformational space in an aqueous solution at physiological ionic strength, revealing a putative substrate channel notmore » previously reported. The viability of the proposed channel was tested by examining the free energy of passage of N 2 from the surface through the channel to FeMo-cofactor, with discovery of a very low energy barrier. These studies point to a viable substrate channel in nitrogenase that appears during thermal motions of the protein in an aqueous environment that approaches a face of FeMo-cofactor earlier implicated in substrate binding.« less

Enzyme Architecture: Erection of Active Orotidine 5'-Monophosphate Decarboxylase by Substrate-Induced Conformational Changes.

PubMed

Reyes, Archie C; Amyes, Tina L; Richard, John P

2017-11-15

Orotidine 5'-monophosphate decarboxylase (OMPDC) catalyzes the decarboxylation of 5-fluoroorotate (FO) with k cat /K m = 1.4 × 10 -7 M -1 s -1 . Combining this and related kinetic parameters shows that the 31 kcal/mol stabilization of the transition state for decarboxylation of OMP provided by OMPDC represents the sum of 11.8 and 10.6 kcal/mol stabilization by the substrate phosphodianion and the ribosyl ring, respectively, and an 8.6 kcal/mol stabilization from the orotate ring. The transition state for OMPDC-catalyzed decarboxylation of FO is stabilized by 5.2, 7.2, and 9.0 kcal/mol, respectively, by 1.0 M phosphite dianion, d-glycerol 3-phosphate and d-erythritol 4-phosphate. The stabilization is due to the utilization of binding interactions of the substrate fragments to drive an enzyme conformational change, which locks the orotate ring of the whole substrate, or the substrate pieces in a caged complex. We propose that enzyme-activation is a possible, and perhaps probable, consequence of any substrate-induced enzyme conformational change.

Glutamate 338 is an electrostatic facilitator of C–Co bond breakage in a dynamic/electrostatic model of catalysis by ornithine aminomutase

PubMed Central

Menon, Binuraj R K; Menon, Navya; Fisher, Karl; Rigby, Stephen E J; Leys, David; Scrutton, Nigel S

2015-01-01

How cobalamin-dependent enzymes promote C–Co homolysis to initiate radical catalysis has been debated extensively. For the pyridoxal 5′-phosphate and cobalamin-dependent enzymes lysine 5,6-aminomutase and ornithine 4,5-aminomutase (OAM), large-scale re-orientation of the cobalamin-binding domain linked to C–Co bond breakage has been proposed. In these models, substrate binding triggers dynamic sampling of the B12-binding Rossmann domain to achieve a catalytically competent ‘closed’ conformational state. In ‘closed’ conformations of OAM, Glu338 is thought to facilitate C–Co bond breakage by close association with the cobalamin adenosyl group. We investigated this using stopped-flow continuous-wave photolysis, viscosity dependence kinetic measurements, and electron paramagnetic resonance spectroscopy of a series of Glu338 variants. We found that substrate-induced C–Co bond homolysis is compromised in Glu388 variant forms of OAM, although photolysis of the C–Co bond is not affected by the identity of residue 338. Electrostatic interactions of Glu338 with the 5′-deoxyadenosyl group of B12 potentiate C–Co bond homolysis in ‘closed’ conformations only; these conformations are unlocked by substrate binding. Our studies extend earlier models that identified a requirement for large-scale motion of the cobalamin domain. Our findings indicate that large-scale motion is required to pre-organize the active site by enabling transient formation of ‘closed’ conformations of OAM. In ‘closed’ conformations, Glu338 interacts with the 5′-deoxyadenosyl group of cobalamin. This interaction is required to potentiate C–Co homolysis, and is a crucial component of the approximately 1012 rate enhancement achieved by cobalamin-dependent enzymes for C–Co bond homolysis. PMID:25627283

Structural and biochemical studies of a fluoroacetyl-CoA-specific thioesterase reveal a molecular basis for fluorine selectivity.

PubMed

Weeks, Amy M; Coyle, Scott M; Jinek, Martin; Doudna, Jennifer A; Chang, Michelle C Y

2010-11-02

We have initiated a broad-based program aimed at understanding the molecular basis of fluorine specificity in enzymatic systems, and in this context, we report crystallographic and biochemical studies on a fluoroacetyl-coenzyme A (CoA) specific thioesterase (FlK) from Streptomyces cattleya. Our data establish that FlK is competent to protect its host from fluoroacetate toxicity in vivo and demonstrate a 10(6)-fold discrimination between fluoroacetyl-CoA (k(cat)/K(M) = 5 × 10⁷ M⁻¹ s⁻¹) and acetyl-CoA (k(cat)/K(M) = 30 M⁻¹ s⁻¹) based on a single fluorine substitution that originates from differences in both substrate reactivity and binding. We show that Thr 42, Glu 50, and His 76 are key catalytic residues and identify several factors that influence substrate selectivity. We propose that FlK minimizes interaction with the thioester carbonyl, leading to selection against acetyl-CoA binding that can be recovered in part by new C═O interactions in the T42S and T42C mutants. We hypothesize that the loss of these interactions is compensated by the entropic driving force for fluorinated substrate binding in a hydrophobic binding pocket created by a lid structure, containing Val 23, Leu 26, Phe 33, and Phe 36, that is not found in other structurally characterized members of this superfamily. We further suggest that water plays a critical role in fluorine specificity based on biochemical and structural studies focused on the unique Phe 36 "gate" residue, which functions to exclude water from the active site. Taken together, the findings from these studies offer molecular insights into organofluorine recognition and design of fluorine-specific enzymes.

Long-range Electrostatic Complementarity Governs Substrate Recognition by Human Chymotrypsin C, a Key Regulator of Digestive Enzyme Activation*

PubMed Central

Batra, Jyotica; Szabó, András; Caulfield, Thomas R.; Soares, Alexei S.; Sahin-Tóth, Miklós; Radisky, Evette S.

2013-01-01

Human chymotrypsin C (CTRC) is a pancreatic serine protease that regulates activation and degradation of trypsinogens and procarboxypeptidases by targeting specific cleavage sites within their zymogen precursors. In cleaving these regulatory sites, which are characterized by multiple flanking acidic residues, CTRC shows substrate specificity that is distinct from that of other isoforms of chymotrypsin and elastase. Here, we report the first crystal structure of active CTRC, determined at 1.9-Å resolution, revealing the structural basis for binding specificity. The structure shows human CTRC bound to the small protein protease inhibitor eglin c, which binds in a substrate-like manner filling the S6-S5′ subsites of the substrate binding cleft. Significant binding affinity derives from burial of preferred hydrophobic residues at the P1, P4, and P2′ positions of CTRC, although acidic P2′ residues can also be accommodated by formation of an interfacial salt bridge. Acidic residues may also be specifically accommodated in the P6 position. The most unique structural feature of CTRC is a ring of intense positive electrostatic surface potential surrounding the primarily hydrophobic substrate binding site. Our results indicate that long-range electrostatic attraction toward substrates of concentrated negative charge governs substrate discrimination, which explains CTRC selectivity in regulating active digestive enzyme levels. PMID:23430245

Structural basis of RND-type multidrug exporters

PubMed Central

Yamaguchi, Akihito; Nakashima, Ryosuke; Sakurai, Keisuke

2015-01-01

Bacterial multidrug exporters are intrinsic membrane transporters that act as cellular self-defense mechanism. The most notable characteristics of multidrug exporters is that they export a wide range of drugs and toxic compounds. The overexpression of these exporters causes multidrug resistance. Multidrug-resistant pathogens have become a serious problem in modern chemotherapy. Over the past decade, investigations into the structure of bacterial multidrug exporters have revealed the multidrug recognition and export mechanisms. In this review, we primarily discuss RND-type multidrug exporters particularly AcrAB-TolC, major drug exporter in Gram-negative bacteria. RND-type drug exporters are tripartite complexes comprising a cell membrane transporter, an outer membrane channel and an adaptor protein. Cell membrane transporters and outer membrane channels are homo-trimers; however, there is no consensus on the number of adaptor proteins in these tripartite complexes. The three monomers of a cell membrane transporter have varying conformations (access, binding, and extrusion) during transport. Drugs are exported following an ordered conformational change in these three monomers, through a functional rotation mechanism coupled with the proton relay cycle in ion pairs, which is driven by proton translocation. Multidrug recognition is based on a multisite drug-binding mechanism, in which two voluminous multidrug-binding pockets in cell membrane exporters recognize a wide range of substrates as a result of permutations at numerous binding sites that are specific for the partial structures of substrate molecules. The voluminous multidrug-binding pocket may have numerous binding sites even for a single substrate, suggesting that substrates may move between binding sites during transport, an idea named as multisite-drug-oscillation hypothesis. This hypothesis is consistent with the apparently broad substrate specificity of cell membrane exporters and their highly efficient ejection of drugs from the cell. Substrates are transported through dual multidrug-binding pockets via the peristaltic motion of the substrate translocation channel. Although there are no clinically available inhibitors of bacterial multidrug exporters, efforts to develop inhibitors based on structural information are underway. PMID:25941524

Structural basis of RND-type multidrug exporters.

PubMed

Yamaguchi, Akihito; Nakashima, Ryosuke; Sakurai, Keisuke

2015-01-01

Bacterial multidrug exporters are intrinsic membrane transporters that act as cellular self-defense mechanism. The most notable characteristics of multidrug exporters is that they export a wide range of drugs and toxic compounds. The overexpression of these exporters causes multidrug resistance. Multidrug-resistant pathogens have become a serious problem in modern chemotherapy. Over the past decade, investigations into the structure of bacterial multidrug exporters have revealed the multidrug recognition and export mechanisms. In this review, we primarily discuss RND-type multidrug exporters particularly AcrAB-TolC, major drug exporter in Gram-negative bacteria. RND-type drug exporters are tripartite complexes comprising a cell membrane transporter, an outer membrane channel and an adaptor protein. Cell membrane transporters and outer membrane channels are homo-trimers; however, there is no consensus on the number of adaptor proteins in these tripartite complexes. The three monomers of a cell membrane transporter have varying conformations (access, binding, and extrusion) during transport. Drugs are exported following an ordered conformational change in these three monomers, through a functional rotation mechanism coupled with the proton relay cycle in ion pairs, which is driven by proton translocation. Multidrug recognition is based on a multisite drug-binding mechanism, in which two voluminous multidrug-binding pockets in cell membrane exporters recognize a wide range of substrates as a result of permutations at numerous binding sites that are specific for the partial structures of substrate molecules. The voluminous multidrug-binding pocket may have numerous binding sites even for a single substrate, suggesting that substrates may move between binding sites during transport, an idea named as multisite-drug-oscillation hypothesis. This hypothesis is consistent with the apparently broad substrate specificity of cell membrane exporters and their highly efficient ejection of drugs from the cell. Substrates are transported through dual multidrug-binding pockets via the peristaltic motion of the substrate translocation channel. Although there are no clinically available inhibitors of bacterial multidrug exporters, efforts to develop inhibitors based on structural information are underway.

RING E3 mechanism for ubiquitin ligation to a disordered substrate visualized for human anaphase-promoting complex

DOE PAGES

Brown, Nicholas G.; VanderLinden, Ryan; Watson, Edmond R.; ...

2015-03-30

For many E3 ligases, a mobile RING (Really Interesting New Gene) domain stimulates ubiquitin (Ub) transfer from a thioester-linked E2~Ub intermediate to a lysine on a remotely bound disordered substrate. One such E3 is the gigantic, multisubunit 1.2-MDa anaphase-promoting complex/cyclosome (APC), which controls cell division by ubiquitinating cell cycle regulators to drive their timely degradation. Intrinsically disordered substrates are typically recruited via their KEN-box, D-box, and/or other motifs binding to APC and a coactivator such as CDH1. On the opposite side of the APC, the dynamic catalytic core contains the cullin-like subunit APC2 and its RING partner APC11, which collaboratesmore » with the E2 UBCH10 (UBE2C) to ubiquitinate substrates. However, how dynamic RING–E2~Ub catalytic modules such as APC11–UBCH10~Ub collide with distally tethered disordered substrates remains poorly understood. In this paper, we report structural mechanisms of UBCH10 recruitment to APC CDH1 and substrate ubiquitination. Unexpectedly, in addition to binding APC11’s RING, UBCH10 is corecruited via interactions with APC2, which we visualized in a trapped complex representing an APC CDH1–UBCH10~Ub–substrate intermediate by cryo-electron microscopy, and in isolation by X-ray crystallography. To our knowledge, this is the first structural view of APC, or any cullin–RING E3, with E2 and substrate juxtaposed, and it reveals how tripartite cullin–RING–E2 interactions establish APC’s specificity for UBCH10 and harness a flexible catalytic module to drive ubiquitination of lysines within an accessible zone. Finally, we propose that multisite interactions reduce the degrees of freedom available to dynamic RING E3–E2~Ub catalytic modules, condense the search radius for target lysines, increase the chance of active-site collision with conformationally fluctuating substrates, and enable regulation.« less

Structural Analysis of the Catalytic Mechanism and Substrate Specificity of Anabaena Alkaline Invertase InvA Reveals a Novel Glucosidase*

PubMed Central

Xie, Jin; Cai, Kun; Hu, Hai-Xi; Jiang, Yong-Liang; Yang, Feng; Hu, Peng-Fei; Cao, Dong-Dong; Li, Wei-Fang; Chen, Yuxing; Zhou, Cong-Zhao

2016-01-01

Invertases catalyze the hydrolysis of sucrose to glucose and fructose, thereby playing a key role in primary metabolism and plant development. According to the optimum pH, invertases are classified into acid invertases (Ac-Invs) and alkaline/neutral invertases (A/N-Invs), which share no sequence homology. Compared with Ac-Invs that have been extensively studied, the structure and catalytic mechanism of A/N-Invs remain unknown. Here we report the crystal structures of Anabaena alkaline invertase InvA, which was proposed to be the ancestor of modern plant A/N-Invs. These structures are the first in the GH100 family. InvA exists as a hexamer in both crystal and solution. Each subunit consists of an (α/α)6 barrel core structure in addition to an insertion of three helices. A couple of structures in complex with the substrate or products enabled us to assign the subsites −1 and +1 specifically binding glucose and fructose, respectively. Structural comparison combined with enzymatic assays indicated that Asp-188 and Glu-414 are putative catalytic residues. Further analysis of the substrate binding pocket demonstrated that InvA possesses a stringent substrate specificity toward the α1,2-glycosidic bond of sucrose. Together, we suggest that InvA and homologs represent a novel family of glucosidases. PMID:27777307

Structural Analysis of the Catalytic Mechanism and Substrate Specificity of Anabaena Alkaline Invertase InvA Reveals a Novel Glucosidase.

PubMed

Xie, Jin; Cai, Kun; Hu, Hai-Xi; Jiang, Yong-Liang; Yang, Feng; Hu, Peng-Fei; Cao, Dong-Dong; Li, Wei-Fang; Chen, Yuxing; Zhou, Cong-Zhao

2016-12-02

Invertases catalyze the hydrolysis of sucrose to glucose and fructose, thereby playing a key role in primary metabolism and plant development. According to the optimum pH, invertases are classified into acid invertases (Ac-Invs) and alkaline/neutral invertases (A/N-Invs), which share no sequence homology. Compared with Ac-Invs that have been extensively studied, the structure and catalytic mechanism of A/N-Invs remain unknown. Here we report the crystal structures of Anabaena alkaline invertase InvA, which was proposed to be the ancestor of modern plant A/N-Invs. These structures are the first in the GH100 family. InvA exists as a hexamer in both crystal and solution. Each subunit consists of an (α/α) 6 barrel core structure in addition to an insertion of three helices. A couple of structures in complex with the substrate or products enabled us to assign the subsites -1 and +1 specifically binding glucose and fructose, respectively. Structural comparison combined with enzymatic assays indicated that Asp-188 and Glu-414 are putative catalytic residues. Further analysis of the substrate binding pocket demonstrated that InvA possesses a stringent substrate specificity toward the α1,2-glycosidic bond of sucrose. Together, we suggest that InvA and homologs represent a novel family of glucosidases. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

A Novel Class of Modular Transporters for Vitamins in Prokaryotes ▿ †

PubMed Central

Rodionov, Dmitry A.; Hebbeln, Peter; Eudes, Aymerick; ter Beek, Josy; Rodionova, Irina A.; Erkens, Guus B.; Slotboom, Dirk J.; Gelfand, Mikhail S.; Osterman, Andrei L.; Hanson, Andrew D.; Eitinger, Thomas

2009-01-01

The specific and tightly controlled transport of numerous nutrients and metabolites across cellular membranes is crucial to all forms of life. However, many of the transporter proteins involved have yet to be identified, including the vitamin transporters in various human pathogens, whose growth depends strictly on vitamin uptake. Comparative analysis of the ever-growing collection of microbial genomes coupled with experimental validation enables the discovery of such transporters. Here, we used this approach to discover an abundant class of vitamin transporters in prokaryotes with an unprecedented architecture. These transporters have energy-coupling modules comprised of a conserved transmembrane protein and two nucleotide binding proteins similar to those of ATP binding cassette (ABC) transporters, but unlike ABC transporters, they use small integral membrane proteins to capture specific substrates. We identified 21 families of these substrate capture proteins, each with a different specificity predicted by genome context analyses. Roughly half of the substrate capture proteins (335 cases) have a dedicated energizing module, but in 459 cases distributed among almost 100 gram-positive bacteria, including numerous human pathogens, different and unrelated substrate capture proteins share the same energy-coupling module. The shared use of energy-coupling modules was experimentally confirmed for folate, thiamine, and riboflavin transporters. We propose the name energy-coupling factor transporters for the new class of membrane transporters. PMID:18931129

Systematic characterization of the specificity of the SH2 domains of cytoplasmic tyrosine kinases.

PubMed

Zhao, Bing; Tan, Pauline H; Li, Shawn S C; Pei, Dehua

2013-04-09

Cytoplasmic tyrosine kinases (CTK) generally contain a Src-homology 2 (SH2) domain, whose role in the CTK family is not fully understood. Here we report the determination of the specificity of 25 CTK SH2 domains by screening one-bead-one-compound (OBOC) peptide libraries. Based on the peptide sequences selected by the SH2 domains, we built Support Vector Machine (SVM) models for the prediction of binding ligands for the SH2 domains. These models yielded support for the progressive phosphorylation model for CTKs in which the overlapping specificity of the CTK SH2 and kinase domains has been proposed to facilitate targeting of the CTK substrates with at least two potential phosphotyrosine (pTyr) sites. We curated 93 CTK substrates with at least two pTyr sites catalyzed by the same CTK, and showed that 71% of these substrates had at least two pTyr sites predicted to bind a common CTK SH2 domain. More importantly, we found 34 instances where there was at least one pTyr site predicted to be recognized by the SH2 domain of the same CTK, suggesting that the SH2 and kinase domains of the CTKs may cooperate to achieve progressive phosphorylation of a protein substrate. This article is part of a Special Issue entitled: From protein structures to clinical applications. Copyright © 2012 Elsevier B.V. All rights reserved.

LeuT conformational sampling utilizing accelerated molecular dynamics and principal component analysis.

PubMed

Thomas, James R; Gedeon, Patrick C; Grant, Barry J; Madura, Jeffry D

2012-07-03

Monoamine transporters (MATs) function by coupling ion gradients to the transport of dopamine, norepinephrine, or serotonin. Despite their importance in regulating neurotransmission, the exact conformational mechanism by which MATs function remains elusive. To this end, we have performed seven 250 ns accelerated molecular dynamics simulations of the leucine transporter, a model for neurotransmitter MATs. By varying the presence of binding-pocket leucine substrate and sodium ions, we have sampled plausible conformational states representative of the substrate transport cycle. The resulting trajectories were analyzed using principal component analysis of transmembrane helices 1b and 6a. This analysis revealed seven unique structures: two of the obtained conformations are similar to the currently published crystallographic structures, one conformation is similar to a proposed open inward structure, and four conformations represent novel structures of potential importance to the transport cycle. Further analysis reveals that the presence of binding-pocket sodium ions is necessary to stabilize the locked-occluded and open-inward conformations. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Structure of the Deactive State of Mammalian Respiratory Complex I.

PubMed

Blaza, James N; Vinothkumar, Kutti R; Hirst, Judy

2018-02-06

Complex I (NADH:ubiquinone oxidoreductase) is central to energy metabolism in mammalian mitochondria. It couples NADH oxidation by ubiquinone to proton transport across the energy-conserving inner membrane, catalyzing respiration and driving ATP synthesis. In the absence of substrates, active complex I gradually enters a pronounced resting or deactive state. The active-deactive transition occurs during ischemia and is crucial for controlling how respiration recovers upon reperfusion. Here, we set a highly active preparation of Bos taurus complex I into the biochemically defined deactive state, and used single-particle electron cryomicroscopy to determine its structure to 4.1 Å resolution. We show that the deactive state arises when critical structural elements that form the ubiquinone-binding site become disordered, and we propose reactivation is induced when substrate binding to the NADH-reduced enzyme templates their reordering. Our structure both rationalizes biochemical data on the deactive state and offers new insights into its physiological and cellular roles. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Atomic and electronic structures of single-layer FeSe on SrTiO 3(001): The role of oxygen deficiency

DOE PAGES

Bang, Junhyeok; Li, Zhi; Sun, Y. Y.; ...

2013-06-06

Using first-principles calculation, we propose an interface structure for single triple-layer FeSe on the SrTiO 3(001) surface, a high-T c superconductor found recently. The key component of this structure is the oxygen deficiency on the top layer of the SrTiO 3 substrate, as a result of Se etching used in preparing the high-T c samples. The O vacancies strongly bind the FeSe triple layer to the substrate giving rise to a (2×1) reconstruction, as observed by scanning tunneling microscopy. The enhanced binding correlates to the significant increase of T c observed in experiment. The O vacancies also serve as themore » source of electron doping, which modifies the Fermi surface of the first FeSe layer by filling the hole pocket near the center of the surface Brillouin zone, as suggested from angle-resolved photoemission spectroscopy measurement.« less

Electrogenic Binding of Intracellular Cations Defines a Kinetic Decision Point in the Transport Cycle of the Human Serotonin Transporter.

PubMed

Hasenhuetl, Peter S; Freissmuth, Michael; Sandtner, Walter

2016-12-09

The plasmalemmal monoamine transporters clear the extracellular space from their cognate substrates and sustain cellular monoamine stores even during neuronal activity. In some instances, however, the transporters enter a substrate-exchange mode, which results in release of intracellular substrate. Understanding what determines the switch between these two transport modes demands time-resolved measurements of intracellular (co-)substrate binding and release. Here, we report an electrophysiological investigation of intracellular solute-binding to the human serotonin transporter (SERT) expressed in HEK-293 cells. We measured currents induced by rapid application of serotonin employing varying intracellular (co-)substrate concentrations and interpreted the data using kinetic modeling. Our measurements revealed that the induction of the substrate-exchange mode depends on both voltage and intracellular Na + concentrations because intracellular Na + release occurs before serotonin release and is highly electrogenic. This voltage dependence was blunted by electrogenic binding of intracellular K + and, notably, also H + In addition, our data suggest that Cl - is bound to SERT during the entire catalytic cycle. Our experiments, therefore, document an essential role of electrogenic binding of K + or of H + to the inward-facing conformation of SERT in (i) cancelling out the electrogenic nature of intracellular Na + release and (ii) in selecting the forward-transport over the substrate-exchange mode. Finally, the kinetics of intracellular Na + release and K + (or H + ) binding result in a voltage-independent rate-limiting step where SERT may return to the outward-facing state in a KCl- or HCl-bound form. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Allosteric response and substrate sensitivity in peptide binding of the signal recognition particle.

PubMed

Wang, Connie Y; Miller, Thomas F

2014-10-31

We characterize the conformational dynamics and substrate selectivity of the signal recognition particle (SRP) using a thermodynamic free energy cycle approach and microsecond timescale molecular dynamics simulations. The SRP is a central component of the co-translational protein targeting machinery that binds to the N-terminal signal peptide (SP) of nascent proteins. We determined the shift in relative conformational stability of the SRP upon substrate binding to quantify allosteric coupling between SRP domains. In particular, for dipeptidyl aminopeptidase, an SP that is recognized by the SRP for co-translational targeting, it is found that substrate binding induces substantial changes in the SRP toward configurations associated with targeting of the nascent protein, and it is found that the changes are modestly enhanced by a mutation that increases the hydrophobicity of the SP. However, for alkaline phosphatase, an SP that is recognized for post-translational targeting, substrate binding induces the reverse change in the SRP conformational distribution away from targeting configurations. Microsecond timescale trajectories reveal the intrinsic flexibility of the SRP conformational landscape and provide insight into recent single molecule studies by illustrating that 10-nm lengthscale changes between FRET pairs occur via the rigid-body movement of SRP domains connected by the flexible linker region. In combination, these results provide direct evidence for the hypothesis that substrate-controlled conformational switching in the SRP provides a mechanism for discriminating between different SPs and for connecting substrate binding to downstream steps in the protein targeting pathway. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Lactate Dehydrogenase Undergoes a Substantial Structural Change to Bind its Substrate

PubMed Central

Qiu, Linlin; Gulotta, Miriam; Callender, Robert

2007-01-01

Employing temperature-jump relaxation spectroscopy, we investigate the kinetics and thermodynamics of the formation of a very early ternary binding intermediate formed when lactate dehydrogenase (LDH) binds a substrate mimic on its way to forming the productive LDH/NADH·substrate Michaelis complex. Temperature-jump scans show two distinct submillisecond processes are involved in the formation of this ternary binding intermediate, called the encounter complex here. The on-rate of the formation of the encounter complex from LDH/NADH with oxamate (a substrate mimic) is determined as a function of temperature and in the presence of small concentrations of a protein destabilizer (urea) and protein stabilizer (TMAO). It shows a strong temperature dependence with inverse Arrhenius behavior and a temperature-dependent enthalpy (heat capacity of 610 ± 84 cal/Mol K), is slowed in the presence of TMAO and speeded up in the presence of urea. These results suggest that LDH/NADH occupies a range of conformations, some competent to bind substrate (open structure; a minority population) and others noncompetent (closed), in fast equilibrium with each other in accord with a select fit model of binding. From the thermodynamic results, the two species differ in the rearrangement of low energy hydrogen bonds as would arise from changes in internal hydrogen bonding and/or increases in the solvation of the protein structure. The binding-competent species can bind ligand at or very near diffusion-limited speeds, suggesting that the binding pocket is substantially exposed to solvent in these species. This would be in contrast to the putative closed structure where the binding pocket resides deep within the protein interior. PMID:17483169

Self-assembled nanospheres with multiple endohedral binding sites pre-organize catalysts and substrates for highly efficient reactions

NASA Astrophysics Data System (ADS)

Wang, Qi-Qiang; Gonell, Sergio; Leenders, Stefan H. A. M.; Dürr, Maximilian; Ivanović-Burmazović, Ivana; Reek, Joost N. H.

2016-03-01

Tuning reagent and catalyst concentrations is crucial in the development of efficient catalytic transformations. In enzyme-catalysed reactions the substrate is bound—often by multiple non-covalent interactions—in a well-defined pocket close to the active site of the enzyme; this pre-organization facilitates highly efficient transformations. Here we report an artificial system that co-encapsulates multiple catalysts and substrates within the confined space defined by an M12L24 nanosphere that contains 24 endohedral guanidinium-binding sites. Cooperative binding means that sulfonate guests are bound much more strongly than carboxylates. This difference has been used to fix gold-based catalysts firmly, with the remaining binding sites left to pre-organize substrates. This strategy was applied to a Au(I)-catalysed cyclization of acetylenic acid to enol lactone in which the pre-organization resulted in much higher reaction rates. We also found that the encapsulated sulfonate-containing Au(I) catalysts did not convert neutral (acid) substrates, and so could have potential in the development of substrate-selective catalysis and base-triggered on/off switching of catalysis.

The Structural Basis of ATP as an Allosteric Modulator

PubMed Central

Wang, Qi; Shen, Qiancheng; Li, Shuai; Nussinov, Ruth; Zhang, Jian

2014-01-01

Adenosine-5’-triphosphate (ATP) is generally regarded as a substrate for energy currency and protein modification. Recent findings uncovered the allosteric function of ATP in cellular signal transduction but little is understood about this critical behavior of ATP. Through extensive analysis of ATP in solution and proteins, we found that the free ATP can exist in the compact and extended conformations in solution, and the two different conformational characteristics may be responsible for ATP to exert distinct biological functions: ATP molecules adopt both compact and extended conformations in the allosteric binding sites but conserve extended conformations in the substrate binding sites. Nudged elastic band simulations unveiled the distinct dynamic processes of ATP binding to the corresponding allosteric and substrate binding sites of uridine monophosphate kinase, and suggested that in solution ATP preferentially binds to the substrate binding sites of proteins. When the ATP molecules occupy the allosteric binding sites, the allosteric trigger from ATP to fuel allosteric communication between allosteric and functional sites is stemmed mainly from the triphosphate part of ATP, with a small number from the adenine part of ATP. Taken together, our results provide overall understanding of ATP allosteric functions responsible for regulation in biological systems. PMID:25211773

Inhibition of ferric ion to oxalate oxidase shed light on the substrate binding site.

PubMed

Pang, Yu; Lan, Wanjun; Huang, Xuelei; Zuo, Guanke; Liu, Hui; Zhang, Jingyan

2015-10-01

Oxalate oxidase (OxOx), a well known enzyme catalyzes the cleavage of oxalate to carbon dioxide with reduction of dioxygen to hydrogen peroxide, however its catalytic process is not well understood. To define the substrate binding site, interaction of Fe(3+) ions with OxOx was systemically investigated using biochemical method, circular dichrosim spectroscopy, microscale thermophoresis, and computer modeling. We demonstrated that Fe(3+) is a non-competitive inhibitor with a milder binding affinity to OxOx, and the secondary structure of the OxOx was slightly altered upon its binding. On the basis of the structural properties of the OxOx and its interaction with Fe(3+) ions, two residue clusters of OxOx were assigned as potential Fe(3+) binding sites, the mechanism of the inhibition of Fe(3+) was delineated. Importantly, the residues that interact with Fe(3+) ions are involved in the substrate orienting based on computer docking. Consequently, the interaction of OxOx with Fe(3+) highlights insight into substrate binding site in OxOx.

Insight into the substrate specificity change caused by the Y227H mutation of α-glucosidase III from the European honeybee (Apis mellifera) through molecular dynamics simulations.

PubMed

Na Ayutthaya, Pratchaya Pramoj; Chanchao, Chanpen; Chunsrivirot, Surasak

2018-01-01

Honey from the European honeybee, Apis mellifera, is produced by α-glucosidases (HBGases) and is widely used in food, pharmaceutical, and cosmetic industries. Categorized by their substrate specificities, HBGases have three isoforms: HBGase I, II and III. Previous experimental investigations showed that wild-type HBGase III from Apis mellifera (WT) preferred sucrose to maltose as a substrate, while the Y227H mutant (MT) preferred maltose to sucrose. This mutant can potentially be used for malt hydrolysis because it can efficiently hydrolyze maltose. In this work, to elucidate important factors contributing to substrate specificity of this enzyme and gain insight into how the Y227H mutation causes substrate specificity change, WT and MT homology models were constructed, and sucrose/maltose was docked into active sites of the WT and MT. AMBER14 was employed to perform three independent molecular dynamics runs for these four complexes. Based on the relative binding free energies calculated by the MM-GBSA method, sucrose is better than maltose for WT binding, while maltose is better than sucrose for MT binding. These rankings support the experimentally observed substrate specificity that WT preferred sucrose to maltose as a substrate, while MT preferred maltose to sucrose, suggesting the importance of binding affinity for substrate specificity. We also found that the Y227H mutation caused changes in the proximities between the atoms necessary for sucrose/maltose hydrolysis that may affect enzyme efficiency in the hydrolysis of sucrose/maltose. Moreover, the per-residue binding free energy decomposition results show that Y227/H227 may be a key residue for preference binding of sucrose/maltose in the WT/MT active site. Our study provides important and novel insight into the binding of sucrose/maltose in the active site of Apis mellifera HBGase III and into how the Y227H mutation leads to the substrate specificity change at the molecular level. This knowledge could be beneficial in the design of this enzyme for increased production of desired products.

Effect of Binding on Enantioselectivity of Epoxide Hydrolase.

PubMed

Zaugg, Julian; Gumulya, Yosephine; Bodén, Mikael; Mark, Alan E; Malde, Alpeshkumar K

2018-03-26

Molecular dynamics simulations and free energy calculations have been used to investigate the effect of ligand binding on the enantioselectivity of an epoxide hydrolase (EH) from Aspergillus niger. Despite sharing a common mechanism, a wide range of alternative mechanisms have been proposed to explain the origin of enantiomeric selectivity in EHs. By comparing the interactions of ( R)- and ( S)-glycidyl phenyl ether (GPE) with both the wild type (WT, E = 3) and a mutant showing enhanced enantioselectivity to GPE (LW202, E = 193), we have examined whether enantioselectivity is due to differences in the binding pose, the affinity for the ( R)- or ( S)- enantiomers, or a kinetic effect. The two enantiomers were easily accommodated within the binding pockets of the WT enzyme and LW202. Free energy calculations suggested that neither enzyme had a preference for a given enantiomer. The two substrates sampled a wide variety of conformations in the simulations with the sterically hindered and unhindered carbon atoms of the GPE epoxide ring both coming in close proximity to the nucleophilic aspartic acid residue. This suggests that alternative pathways could lead to the formation of a ( S)- and ( R)-diol product. Together, the calculations suggest that the enantioselectivity is due to kinetic rather than thermodynamic effects and that the assumption that one substrate results in one product when interpreting the available experimental data and deriving E-values may be inappropriate in the case of EHs.

Structure of photosystem II and substrate binding at room temperature.

PubMed

Young, Iris D; Ibrahim, Mohamed; Chatterjee, Ruchira; Gul, Sheraz; Fuller, Franklin; Koroidov, Sergey; Brewster, Aaron S; Tran, Rosalie; Alonso-Mori, Roberto; Kroll, Thomas; Michels-Clark, Tara; Laksmono, Hartawan; Sierra, Raymond G; Stan, Claudiu A; Hussein, Rana; Zhang, Miao; Douthit, Lacey; Kubin, Markus; de Lichtenberg, Casper; Long Vo, Pham; Nilsson, Håkan; Cheah, Mun Hon; Shevela, Dmitriy; Saracini, Claudio; Bean, Mackenzie A; Seuffert, Ina; Sokaras, Dimosthenis; Weng, Tsu-Chien; Pastor, Ernest; Weninger, Clemens; Fransson, Thomas; Lassalle, Louise; Bräuer, Philipp; Aller, Pierre; Docker, Peter T; Andi, Babak; Orville, Allen M; Glownia, James M; Nelson, Silke; Sikorski, Marcin; Zhu, Diling; Hunter, Mark S; Lane, Thomas J; Aquila, Andy; Koglin, Jason E; Robinson, Joseph; Liang, Mengning; Boutet, Sébastien; Lyubimov, Artem Y; Uervirojnangkoorn, Monarin; Moriarty, Nigel W; Liebschner, Dorothee; Afonine, Pavel V; Waterman, David G; Evans, Gwyndaf; Wernet, Philippe; Dobbek, Holger; Weis, William I; Brunger, Axel T; Zwart, Petrus H; Adams, Paul D; Zouni, Athina; Messinger, Johannes; Bergmann, Uwe; Sauter, Nicholas K; Kern, Jan; Yachandra, Vittal K; Yano, Junko

2016-12-15

Light-induced oxidation of water by photosystem II (PS II) in plants, algae and cyanobacteria has generated most of the dioxygen in the atmosphere. PS II, a membrane-bound multi-subunit pigment protein complex, couples the one-electron photochemistry at the reaction centre with the four-electron redox chemistry of water oxidation at the Mn 4 CaO 5 cluster in the oxygen-evolving complex (OEC). Under illumination, the OEC cycles through five intermediate S-states (S 0 to S 4 ), in which S 1 is the dark-stable state and S 3 is the last semi-stable state before O-O bond formation and O 2 evolution. A detailed understanding of the O-O bond formation mechanism remains a challenge, and will require elucidation of both the structures of the OEC in the different S-states and the binding of the two substrate waters to the catalytic site. Here we report the use of femtosecond pulses from an X-ray free electron laser (XFEL) to obtain damage-free, room temperature structures of dark-adapted (S 1 ), two-flash illuminated (2F; S 3 -enriched), and ammonia-bound two-flash illuminated (2F-NH 3 ; S 3 -enriched) PS II. Although the recent 1.95 Å resolution structure of PS II at cryogenic temperature using an XFEL provided a damage-free view of the S 1 state, measurements at room temperature are required to study the structural landscape of proteins under functional conditions, and also for in situ advancement of the S-states. To investigate the water-binding site(s), ammonia, a water analogue, has been used as a marker, as it binds to the Mn 4 CaO 5 cluster in the S 2 and S 3 states. Since the ammonia-bound OEC is active, the ammonia-binding Mn site is not a substrate water site. This approach, together with a comparison of the native dark and 2F states, is used to discriminate between proposed O-O bond formation mechanisms.

A Conserved Surface Loop in Type I Dehydroquinate Dehydratases Positions an Active Site Arginine and Functions in Substrate Binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Light, Samuel H.; Minasov, George; Shuvalova, Ludmilla

2012-04-18

Dehydroquinate dehydratase (DHQD) catalyzes the third step in the biosynthetic shikimate pathway. We present three crystal structures of the Salmonella enterica type I DHQD that address the functionality of a surface loop that is observed to close over the active site following substrate binding. Two wild-type structures with differing loop conformations and kinetic and structural studies of a mutant provide evidence of both direct and indirect mechanisms of involvement of the loop in substrate binding. In addition to allowing amino acid side chains to establish a direct interaction with the substrate, closure of the loop necessitates a conformational change ofmore » a key active site arginine, which in turn positions the substrate productively. The absence of DHQD in humans and its essentiality in many pathogenic bacteria make the enzyme a target for the development of nontoxic antimicrobials. The structures and ligand binding insights presented here may inform the design of novel type I DHQD inhibiting molecules.« less

Structures of FolT in substrate-bound and substrate-released conformations reveal a gating mechanism for ECF transporters

NASA Astrophysics Data System (ADS)

Zhao, Qin; Wang, Chengcheng; Wang, Chengyuan; Guo, Hui; Bao, Zhihao; Zhang, Minhua; Zhang, Peng

2015-07-01

Energy-coupling factor (ECF) transporters are a new family of ABC transporters that consist of four subunits, two cytoplasmic ATPases EcfA and EcfA' and two transmembrane proteins namely EcfS for substrate-specific binding and EcfT for energy coupling. Here, we report the 3.2-Å resolution crystal structure of the EcfS protein of a folate ECF transporter from Enterococcus faecalis-EfFolT, a close homologue of FolT from Lactobacillus brevis-LbFolT. Structural and biochemical analyses reveal the residues constituting the folate-binding pocket and determining the substrate-binding specificity. Structural comparison of the folate-bound EfFolT with the folate-free LbFolT contained in the holotransporter complex discloses significant conformational change at the L1 loop, and reveals a gating mechanism of ECF transporters in which the L1 loop of EcfS acts as a gate in the substrate binding and release.

Monitoring conformational heterogeneity of the lid of DnaK substrate-binding domain during its chaperone cycle.

PubMed

Banerjee, Rupa; Jayaraj, Gopal Gunanathan; Peter, Joshua Jebakumar; Kumar, Vignesh; Mapa, Koyeli

2016-08-01

DnaK or Hsp70 of Escherichia coli is a master regulator of the bacterial proteostasis network. Allosteric communication between the two functional domains of DnaK, the N-terminal nucleotide-binding domain (NBD) and the C-terminal substrate- or peptide-binding domain (SBD) regulate its activity. X-ray crystallography and NMR studies have provided snapshots of distinct conformations of Hsp70 proteins in various physiological states; however, the conformational heterogeneity and dynamics of allostery-driven Hsp70 activity remains underexplored. In this work, we employed single-molecule Förster resonance energy transfer (sm-FRET) measurements to capture distinct intradomain conformational states of a region within the DnaK-SBD known as the lid. Our data conclusively demonstrate prominent conformational heterogeneity of the DnaK lid in ADP-bound states; in contrast, the ATP-bound open conformations are homogeneous. Interestingly, a nonhydrolysable ATP analogue, AMP-PNP, imparts heterogeneity to the lid conformations mimicking the ADP-bound state. The cochaperone DnaJ confers ADP-like heterogeneous lid conformations to DnaK, although the presence of the cochaperone accelerates the substrate-binding rate by a hitherto unknown mechanism. Irrespective of the presence of DnaJ, binding of a peptide substrate to the DnaK-SBD leads to prominent lid closure. Lid closure is only partial upon binding to molten globule-like authentic cellular substrates, probably to accommodate non-native substrate proteins of varied structures. © 2016 Federation of European Biochemical Societies.

Protein NMR Studies of Substrate Binding to Human Blood Group A and B Glycosyltransferases.

PubMed

Grimm, Lena Lisbeth; Weissbach, Sophie; Flügge, Friedemann; Begemann, Nora; Palcic, Monica M; Peters, Thomas

2017-07-04

Donor and acceptor substrate binding to human blood group A and B glycosyltransferases (GTA, GTB) has been studied by a variety of protein NMR experiments. Prior crystallographic studies had shown these enzymes to adopt an open conformation in the absence of substrates. Binding either of the donor substrate UDP-Gal or of UDP induces a semiclosed conformation. In the presence of both donor and acceptor substrates, the enzymes shift towards a closed conformation with ordering of an internal loop and the C-terminal residues, which then completely cover the donor-binding pocket. Chemical-shift titrations of uniformly 2 H, 15 N-labeled GTA or GTB with UDP affected about 20 % of all crosspeaks in 1 H, 15 N TROSY-HSQC spectra, reflecting substantial plasticity of the enzymes. On the other hand, it is this conformational flexibility that impedes NH backbone assignments. Chemical-shift-perturbation experiments with δ1-[ 13 C]methyl-Ile-labeled samples revealed two Ile residues-Ile123 at the bottom of the UDP binding pocket, and Ile192 as part of the internal loop-that were significantly disturbed upon stepwise addition of UDP and H-disaccharide, also revealing long-range perturbations. Finally, methyl TROSY-based relaxation dispersion experiments do not reveal micro- to millisecond timescale motions. Although this study reveals substantial conformational plasticity of GTA and GTB, the matter of how binding of substrates shifts the enzymes into catalytically competent states remains enigmatic. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Kinetic mechanism of Toxoplasma gondii adenosine kinase and the highly efficient utilization of adenosine

PubMed Central

Naguib, Fardos N. M.; Rais, Reem H.; Al Safarjalani, Omar N.; el Kouni, Mahmoud H.

2015-01-01

Toxoplasma gondii has an extraordinarily ability to utilize adenosine (Ado) as the primary source of all necessary purines in this parasite which lacks de novo purine biosynthesis. The activity of T. gondii adenosine kinase (TgAK, EC 2.7.1.20) is responsible for this efficient salvage of Ado in T. gondii. To fully understand this remarkable efficiency of TgAK in the utilization of Ado, complete kinetic parameters of this enzyme are necessary. Initial velocity and product inhibition studies of TgAK demonstrated that the basic mechanism of this enzyme is a hybrid random bi-uni ping-pong uni-bi. Initial velocity studies showed an intersecting pattern, consistent with substrate-enzyme-co-substrate complex formation and a binding pattern indicating that binding of the substrate interferes with the binding of the co-substrate and vice versa. Estimated kinetic parameters were KAdo = 0.002 ± 0.0002 mM, KATP = 0.05 ± 0.008 mM, and Vmax = 920 ± 35 μmol/min/mg protein. Ado exhibited substrate inhibition suggesting the presence of more than one binding site for Ado on the enzyme. ATP relieved substrate inhibition by Ado. Thus, Ado also binds to the ATP binding site. AMP was competitive with ATP, inferring that AMP binds to the same site as ATP. AMP, ADP and ATP were non-competitive with Ado, therefore, none of these nucleotides binds to the Ado binding site. Combining ATP with ADP was additive. Therefore, the binding of either ATP or ADP does not interfere with the binding of the other. It is concluded that for every ATP consumed, TgAK generates three new AMPs. These findings along with the fact that a wide range of nucleoside 5′-mono, di, and triphosphates could substitute for ATP as phosphate donors in this reaction may explain the efficient and central role played by TgAK in the utilization of Ado as the major source from which all other purines can be synthesized in T. gondii. PMID:26112826

Surface enhanced Raman spectroscopy detection of biomolecules using EBL fabricated nanostructured substrates.

PubMed

Peters, Robert F; Gutierrez-Rivera, Luis; Dew, Steven K; Stepanova, Maria

2015-03-20

Fabrication and characterization of conjugate nano-biological systems interfacing metallic nanostructures on solid supports with immobilized biomolecules is reported. The entire sequence of relevant experimental steps is described, involving the fabrication of nanostructured substrates using electron beam lithography, immobilization of biomolecules on the substrates, and their characterization utilizing surface-enhanced Raman spectroscopy (SERS). Three different designs of nano-biological systems are employed, including protein A, glucose binding protein, and a dopamine binding DNA aptamer. In the latter two cases, the binding of respective ligands, D-glucose and dopamine, is also included. The three kinds of biomolecules are immobilized on nanostructured substrates by different methods, and the results of SERS imaging are reported. The capabilities of SERS to detect vibrational modes from surface-immobilized proteins, as well as to capture the protein-ligand and aptamer-ligand binding are demonstrated. The results also illustrate the influence of the surface nanostructure geometry, biomolecules immobilization strategy, Raman activity of the molecules and presence or absence of the ligand binding on the SERS spectra acquired.

The conformational free-energy landscape of β-D-mannopyranose: evidence for a (1)S(5) → B(2,5) → (O)S(2) catalytic itinerary in β-mannosidases.

PubMed

Ardèvol, Albert; Biarnés, Xevi; Planas, Antoni; Rovira, Carme

2010-11-17

The mechanism of glycosidic bond cleavage by glycosidases involves substrate ring distortions in the Michaelis complex that favor catalysis. Retaining β-mannosidases bind the substrate in a (1)S(5) conformation, and recent experiments have proposed an unusual substrate conformational pathway ((1)S(5) → B(2,5) → (O)S(2)) for the hydrolysis reaction. By means of Car-Parrinello metadynamics simulations, we have obtained the conformational free-energy surface (FES) of a β-d-mannopyranose molecule associated with the ideal Stoddart conformational diagram. We have found that (1)S(5) is among the most stable conformers and simultaneously is the most preactivated conformation in terms of elongation/shortening of the C1-O1/C1-O5 bonds, C1-O1 orientation, and charge development at the anomeric carbon. Analysis of the computed FES gives support to the proposed (1)S(5) → B(2,5) → (O)S(2) catalytic itinerary, showing that the degree of preactivation of the substrate in glycoside hydrolases (GHs) is related to the properties of an isolated sugar ring. We introduce a simple preactivation index integrating several structural, electronic, and energetic properties that can be used to predict the conformation of the substrate in the Michaelis complex of any GH.

Mapping substrate interactions of the human membrane-associated neuraminidase, NEU3, using STD NMR.

PubMed

Albohy, Amgad; Richards, Michele R; Cairo, Christopher W

2015-03-01

Saturation transfer difference (STD) nuclear magnetic resonance (NMR) is a powerful technique which can be used to investigate interactions between proteins and their substrates. The method identifies specific sites of interaction found on a small molecule ligand when in complex with a protein. The ability of STD NMR to provide specific insight into binding interactions in the absence of other structural data is an attractive feature for its use with membrane proteins. We chose to employ STD NMR in our ongoing investigations of the human membrane-associated neuraminidase NEU3 and its interaction with glycolipid substrates (e.g., GM3). In order to identify critical substrate-enzyme interactions, we performed STD NMR with a catalytically inactive form of the enzyme, NEU3(Y370F), containing an N-terminal maltose-binding protein (MBP)-affinity tag. In the absence of crystallographic data on the enzyme, these data represent a critical experimental test of proposed homology models, as well as valuable new structural data. To aid interpretation of the STD NMR data, we compared the results with molecular dynamics (MD) simulations of the enzyme-substrate complexes. We find that the homology model is able to predict essential features of the experimental data, including close contact of the hydrophobic aglycone and the Neu5Ac residue with the enzyme. Additionally, the model and STD NMR data agree on the facial recognition of the galactose and glucose residues of the GM3-analog studied. We conclude that the homology model of NEU3 can be used to predict substrate recognition, but our data indicate that unstructured portions of the NEU3 model may require further refinement. © The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Comparison of crystal structures of human type 3 3α-hydroxysteroid dehydrogenase reveals an “induced-fit” mechanism and a conserved basic motif involved in the binding of androgen

PubMed Central

Couture, Jean-François; Pereira De Jésus-Tran, Karine; Roy, Anne-Marie; Cantin, Line; Côté, Pierre-Luc; Legrand, Pierre; Luu-The, Van; Labrie, Fernand; Breton, Rock

2005-01-01

The aldo-keto reductase (AKR) human type 3 3α-hydroxysteroid dehydrogenase (h3α–HSD3, AKR1C2) plays a crucial role in the regulation of the intracellular concentrations of testosterone and 5α-dihydrotestosterone (5α-DHT), two steroids directly linked to the etiology and the progression of many prostate diseases and cancer. This enzyme also binds many structurally different molecules such as 4-hydroxynonenal, polycyclic aromatic hydrocarbons, and indanone. To understand the mechanism underlying the plasticity of its substrate-binding site, we solved the binary complex structure of h3α–HSD3-NADP(H) at 1.9 Å resolution. During the refinement process, we found acetate and citrate molecules deeply engulfed in the steroid-binding cavity. Superimposition of this structure with the h3α–HSD3-NADP(H)-testosterone/acetate ternary complex structure reveals that one of themobile loops forming the binding cavity operates a slight contraction movement against the citrate molecule while the side chains of many residues undergo numerous conformational changes, probably to create an optimal binding site for the citrate. These structural changes, which altogether cause a reduction of the substrate-binding cavity volume (from 776 Å3 in the presence of testosterone/acetate to 704 Å3 in the acetate/citratecomplex), are reminiscent of the “induced-fit” mechanism previously proposed for the aldose reductase, another member of the AKR superfamily. We also found that the replacement of residues Arg301 and Arg304, localized near the steroid-binding cavity, significantly affects the 3α–HSD activity of this enzyme toward 5α-DHT and completely abolishes its 17β–HSD activity on 4-dione. All these results have thus been used to reevaluate the binding mode of this enzyme for androgens. PMID:15929998

The I domain of the AAA+ HslUV protease coordinates substrate binding, ATP hydrolysis, and protein degradation

PubMed Central

Sundar, Shankar; Baker, Tania A; Sauer, Robert T

2012-01-01

In the AAA+ HslUV protease, substrates are bound and unfolded by a ring hexamer of HslU, before translocation through an axial pore and into the HslV degradation chamber. Here, we show that the N-terminal residues of an Arc substrate initially bind in the HslU axial pore, with key contacts mediated by a pore loop that is highly conserved in all AAA+ unfoldases. Disordered loops from the six intermediate domains of the HslU hexamer project into a funnel-shaped cavity above the pore and are positioned to contact protein substrates. Mutations in these I-domain loops increase KM and decrease Vmax for degradation, increase the mobility of bound substrates, and prevent substrate stimulation of ATP hydrolysis. HslU-ΔI has negligible ATPase activity. Thus, the I domain plays an active role in coordinating substrate binding, ATP hydrolysis, and protein degradation by the HslUV proteolytic machine. PMID:22102327

Identification of an Electrostatic Ruler Motif for Sequence-Specific Binding of Collagenase to Collagen.

PubMed

Subramanian, Sundar Raman; Singam, Ettayapuram Ramaprasad Azhagiya; Berinski, Michael; Subramanian, Venkatesan; Wade, Rebecca C

2016-08-25

Sequence-specific cleavage of collagen by mammalian collagenase plays a pivotal role in cell function. Collagenases are matrix metalloproteinases that cleave the peptide bond at a specific position on fibrillar collagen. The collagenase Hemopexin-like (HPX) domain has been proposed to be responsible for substrate recognition, but the mechanism by which collagenases identify the cleavage site on fibrillar collagen is not clearly understood. In this study, Brownian dynamics simulations coupled with atomic-detail and coarse-grained molecular dynamics simulations were performed to dock matrix metalloproteinase-1 (MMP-1) on a collagen IIIα1 triple helical peptide. We find that the HPX domain recognizes the collagen triple helix at a conserved R-X11-R motif C-terminal to the cleavage site to which the HPX domain of collagen is guided electrostatically. The binding of the HPX domain between the two arginine residues is energetically stabilized by hydrophobic contacts with collagen. From the simulations and analysis of the sequences and structural flexibility of collagen and collagenase, a mechanistic scheme by which MMP-1 can recognize and bind collagen for proteolysis is proposed.

Mechanism of substrate specificity in 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidases

PubMed Central

Siu, Karen K.W.; Asmus, Kyle; Zhang, Allison N.; Horvatin, Cathy; Li, Sheng; Liu, Tong; Moffatt, Barbara; Woods, Virgil L.; Howell, P. Lynne

2010-01-01

5′-Methylthioadenosine/S-adenosylhomocysteine (MTA/SAH) nucleosidase (MTAN) plays a key role in the methionine-recycling pathway of bacteria and plants. Despite extensive structural and biochemical studies, the molecular mechanism of substrate specificity for MTAN remains an outstanding question. Bacterial MTANs show comparable efficiency in hydrolyzing MTA and SAH, while the plant enzymes select preferentially for MTA, with either no or significantly reduced activity towards SAH. Bacterial and plant MTANs show significant conservation in the overall structure, and the adenine- and ribose-binding sites. The observation of a more constricted 5′-alkylthio binding site in Arabidopsis thaliana AtM-TAN1 and AtMTAN2, two plant MTAN homologues, led to the hypothesis that steric hindrance may play a role in substrate selection in plant MTANs. We show using isothermal titration calorimetry that SAH binds to both Escherichia coli MTAN (EcMTAN) and AtMTAN1 with comparable micromolar affinity. To understand why AtMTAN1 can bind but not hydrolyze SAH, we determined the structure of the protein–SAH complex at 2.2 Å resolution. The lack of catalytic activity appears to be related to the enzyme’s inability to bind the substrate in a catalytically competent manner. The role of dynamics in substrate selection was also examined by probing the amide proton exchange rates of EcMTAN and AtMTAN1 via deuterium–hydrogen exchange coupled mass spectrometry. These results correlate with the B factors of available structures and the thermodynamic parameters associated with substrate binding, and suggest a higher level of conformational flexibility in the active site of EcMTAN. Our results implicate dynamics as an important factor in substrate selection in MTAN. PMID:20554051

Design of fluidic self-assembly bonds for precise component positioning

NASA Astrophysics Data System (ADS)

Ramadoss, Vivek; Crane, Nathan B.

2008-02-01

Self Assembly is a promising alternative to conventional pick and place robotic assembly of micro components. Its benefits include parallel integration of parts with low equipment costs. Various approaches to self assembly have been demonstrated, yet demanding applications like assembly of micro-optical devices require increased positioning accuracy. This paper proposes a new method for design of self assembly bonds that addresses this need. Current methods have zero force at the desired assembly position and low stiffness. This allows small disturbance forces to create significant positioning errors. The proposed method uses a substrate assembly feature to provide a high accuracy alignment guide to the part. The capillary bond region of the part and substrate are then modified to create a non-zero positioning force to maintain the part in the desired assembly position. Capillary force models show that this force aligns the part to the substrate assembly feature and reduces sensitivity of part position to process variation. Thus, the new configuration can substantially improve positioning accuracy of capillary self-assembly. This will result in a dramatic decrease in positioning errors in the micro parts. Various binding site designs are analyzed and guidelines are proposed for the design of an effective assembly bond using this new approach.

Bivalent phenethylamines as novel dopamine transporter inhibitors: evidence for multiple substrate-binding sites in a single transporter.

PubMed

Schmitt, Kyle C; Mamidyala, Sreeman; Biswas, Swati; Dutta, Aloke K; Reith, Maarten E A

2010-03-01

Bivalent ligands--compounds incorporating two receptor-interacting moieties linked by a flexible chain--often exhibit profoundly enhanced binding affinity compared with their monovalent components, implying concurrent binding to multiple sites on the target protein. It is generally assumed that neurotransmitter sodium symporter (NSS) proteins, such as the dopamine transporter (DAT), contain a single domain responsible for recognition of substrate molecules. In this report, we show that molecules possessing two substrate-like phenylalkylamine moieties linked by a progressively longer aliphatic spacer act as progressively more potent DAT inhibitors (rather than substrates). One compound bearing two dopamine (DA)-like pharmacophoric 'heads' separated by an 8-carbon linker achieved an 82-fold gain in inhibition of [(3)H] 2beta-carbomethoxy-3beta-(4-fluorophenyl)-tropane (CFT) binding compared with DA itself; bivalent compounds with a 6-carbon linker and heterologous combinations of DA-, amphetamine- and beta-phenethylamine-like heads all resulted in considerable and comparable gains in DAT affinity. A series of short-chain bivalent-like compounds with a single N-linkage was also identified, the most potent of which displayed a 74-fold gain in binding affinity. Computational modelling of the DAT protein and docking of the two most potent bivalent (-like) ligands suggested simultaneous occupancy of two discrete substrate-binding domains. Assays with the DAT mutants W84L and D313N--previously employed by our laboratory to probe conformation-specific binding of different structural classes of DAT inhibitors--indicated a bias of the bivalent ligands for inward-facing transporters. Our results strongly indicate the existence of multiple DAT substrate-interaction sites, implying that it is possible to design novel types of DAT inhibitors based upon the 'multivalent ligand' strategy.

Structural basis for high substrate-binding affinity and enantioselectivity of 3-quinuclidinone reductase AtQR

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hou, Feng; Miyakawa, Takuya; Kataoka, Michihiko

2014-04-18

Highlights: • Crystal structure of AtQR has been determined at 1.72 Å. • NADH binding induces the formation of substrate binding site. • AtQR possesses a conserved hydrophobic wall for stereospecific binding of substrate. • Additional Glu197 residue is critical to the high binding affinity. - Abstract: (R)-3-Quinuclidinol, a useful compound for the synthesis of various pharmaceuticals, can be enantioselectively produced from 3-quinuclidinone by 3-quinuclidinone reductase. Recently, a novel NADH-dependent 3-quinuclidionone reductase (AtQR) was isolated from Agrobacterium tumefaciens, and showed much higher substrate-binding affinity (>100 fold) than the reported 3-quinuclidionone reductase (RrQR) from Rhodotorula rubra. Here, we report the crystalmore » structure of AtQR at 1.72 Å. Three NADH-bound protomers and one NADH-free protomer form a tetrameric structure in an asymmetric unit of crystals. NADH not only acts as a proton donor, but also contributes to the stability of the α7 helix. This helix is a unique and functionally significant part of AtQR and is related to form a deep catalytic cavity. AtQR has all three catalytic residues of the short-chain dehydrogenases/reductases family and the hydrophobic wall for the enantioselective reduction of 3-quinuclidinone as well as RrQR. An additional residue on the α7 helix, Glu197, exists near the active site of AtQR. This acidic residue is considered to form a direct interaction with the amine part of 3-quinuclidinone, which contributes to substrate orientation and enhancement of substrate-binding affinity. Mutational analyses also support that Glu197 is an indispensable residue for the activity.« less

Protein dynamics and motions in relation to their functions: several case studies and the underlying mechanisms

PubMed Central

Yang, Li-Quan; Sang, Peng; Tao, Yan; Fu, Yun-Xin; Zhang, Ke-Qin; Xie, Yue-Hui; Liu, Shu-Qun

2013-01-01

Proteins are dynamic entities in cellular solution with functions governed essentially by their dynamic personalities. We review several dynamics studies on serine protease proteinase K and HIV-1 gp120 envelope glycoprotein to demonstrate the importance of investigating the dynamic behaviors and molecular motions for a complete understanding of their structure–function relationships. Using computer simulations and essential dynamic (ED) analysis approaches, the dynamics data obtained revealed that: (i) proteinase K has highly flexible substrate-binding site, thus supporting the induced-fit or conformational selection mechanism of substrate binding; (ii) Ca2+ removal from proteinase K increases the global conformational flexibility, decreases the local flexibility of substrate-binding region, and does not influence the thermal motion of catalytic triad, thus explaining the experimentally determined decreased thermal stability, reduced substrate affinity, and almost unchanged catalytic activity upon Ca2+ removal; (iii) substrate binding affects the large concerted motions of proteinase K, and the resulting dynamic pocket can be connected to substrate binding, orientation, and product release; (iv) amino acid mutations 375 S/W and 423 I/P of HIV-1 gp120 have distinct effects on molecular motions of gp120, facilitating 375 S/W mutant to assume the CD4-bound conformation, while 423 I/P mutant to prefer for CD4-unliganded state. The mechanisms underlying protein dynamics and protein–ligand binding, including the concept of the free energy landscape (FEL) of the protein–solvent system, how the ruggedness and variability of FEL determine protein's dynamics, and how the three ligand-binding models, the lock-and-key, induced-fit, and conformational selection are rationalized based on the FEL theory are discussed in depth. PMID:23527883

Protein dynamics and motions in relation to their functions: several case studies and the underlying mechanisms.

PubMed

Yang, Li-Quan; Sang, Peng; Tao, Yan; Fu, Yun-Xin; Zhang, Ke-Qin; Xie, Yue-Hui; Liu, Shu-Qun

2014-01-01

Proteins are dynamic entities in cellular solution with functions governed essentially by their dynamic personalities. We review several dynamics studies on serine protease proteinase K and HIV-1 gp120 envelope glycoprotein to demonstrate the importance of investigating the dynamic behaviors and molecular motions for a complete understanding of their structure-function relationships. Using computer simulations and essential dynamic (ED) analysis approaches, the dynamics data obtained revealed that: (i) proteinase K has highly flexible substrate-binding site, thus supporting the induced-fit or conformational selection mechanism of substrate binding; (ii) Ca(2+) removal from proteinase K increases the global conformational flexibility, decreases the local flexibility of substrate-binding region, and does not influence the thermal motion of catalytic triad, thus explaining the experimentally determined decreased thermal stability, reduced substrate affinity, and almost unchanged catalytic activity upon Ca(2+) removal; (iii) substrate binding affects the large concerted motions of proteinase K, and the resulting dynamic pocket can be connected to substrate binding, orientation, and product release; (iv) amino acid mutations 375 S/W and 423 I/P of HIV-1 gp120 have distinct effects on molecular motions of gp120, facilitating 375 S/W mutant to assume the CD4-bound conformation, while 423 I/P mutant to prefer for CD4-unliganded state. The mechanisms underlying protein dynamics and protein-ligand binding, including the concept of the free energy landscape (FEL) of the protein-solvent system, how the ruggedness and variability of FEL determine protein's dynamics, and how the three ligand-binding models, the lock-and-key, induced-fit, and conformational selection are rationalized based on the FEL theory are discussed in depth.

Rate constants for proteins binding to substrates with multiple binding sites using a generalized forward flux sampling expression

NASA Astrophysics Data System (ADS)

Vijaykumar, Adithya; ten Wolde, Pieter Rein; Bolhuis, Peter G.

2018-03-01

To predict the response of a biochemical system, knowledge of the intrinsic and effective rate constants of proteins is crucial. The experimentally accessible effective rate constant for association can be decomposed in a diffusion-limited rate at which proteins come into contact and an intrinsic association rate at which the proteins in contact truly bind. Reversely, when dissociating, bound proteins first separate into a contact pair with an intrinsic dissociation rate, before moving away by diffusion. While microscopic expressions exist that enable the calculation of the intrinsic and effective rate constants by conducting a single rare event simulation of the protein dissociation reaction, these expressions are only valid when the substrate has just one binding site. If the substrate has multiple binding sites, a bound enzyme can, besides dissociating into the bulk, also hop to another binding site. Calculating transition rate constants between multiple states with forward flux sampling requires a generalized rate expression. We present this expression here and use it to derive explicit expressions for all intrinsic and effective rate constants involving binding to multiple states, including rebinding. We illustrate our approach by computing the intrinsic and effective association, dissociation, and hopping rate constants for a system in which a patchy particle model enzyme binds to a substrate with two binding sites. We find that these rate constants increase as a function of the rotational diffusion constant of the particles. The hopping rate constant decreases as a function of the distance between the binding sites. Finally, we find that blocking one of the binding sites enhances both association and dissociation rate constants. Our approach and results are important for understanding and modeling association reactions in enzyme-substrate systems and other patchy particle systems and open the way for large multiscale simulations of such systems.

The High-Affinity Binding Site for Tricyclic Antidepressants Resides in the Outer Vestibule of the Serotonin TransporterⓈ

PubMed Central

Sarker, Subhodeep; Weissensteiner, René; Steiner, Ilka; Sitte, Harald H.; Ecker, Gerhard F.; Freissmuth, Michael; Sucic, Sonja

2015-01-01

The structure of the bacterial leucine transporter from Aquifex aeolicus (LeuTAa) has been used as a model for mammalian Na+/Cl−-dependent transporters, in particular the serotonin transporter (SERT). The crystal structure of LeuTAa liganded to tricyclic antidepressants predicts simultaneous binding of inhibitor and substrate. This is incompatible with the mutually competitive inhibition of substrates and inhibitors of SERT. We explored the binding modes of tricyclic antidepressants by homology modeling and docking studies. Two approaches were used subsequently to differentiate between three clusters of potential docking poses: 1) a diagnostic SERTY95F mutation, which greatly reduced the affinity for [3H]imipramine but did not affect substrate binding; 2) competition binding experiments in the presence and absence of carbamazepine (i.e., a tricyclic imipramine analog with a short side chain that competes with [3H]imipramine binding to SERT). Binding of releasers (para-chloroamphetamine, methylene-dioxy-methamphetamine/ecstasy) and of carbamazepine were mutually exclusive, but Dixon plots generated in the presence of carbamazepine yielded intersecting lines for serotonin, MPP+, paroxetine, and ibogaine. These observations are consistent with a model, in which 1) the tricyclic ring is docked into the outer vestibule and the dimethyl-aminopropyl side chain points to the substrate binding site; 2) binding of amphetamines creates a structural change in the inner and outer vestibule that precludes docking of the tricyclic ring; 3) simultaneous binding of ibogaine (which binds to the inward-facing conformation) and of carbamazepine is indicative of a second binding site in the inner vestibule, consistent with the pseudosymmetric fold of monoamine transporters. This may be the second low-affinity binding site for antidepressants. PMID:20829432

Aptamer Recognition of Multiplexed Small-Molecule-Functionalized Substrates.

PubMed

Nakatsuka, Nako; Cao, Huan H; Deshayes, Stephanie; Melkonian, Arin Lucy; Kasko, Andrea M; Weiss, Paul S; Andrews, Anne M

2018-05-31

Aptamers are chemically synthesized oligonucleotides or peptides with molecular recognition capabilities. We investigated recognition of substrate-tethered small-molecule targets, using neurotransmitters as examples, and fluorescently labeled DNA aptamers. Substrate regions patterned via microfluidic channels with dopamine or L-tryptophan were selectively recognized by previously identified dopamine or L-tryptophan aptamers, respectively. The on-substrate dissociation constant determined for the dopamine aptamer was comparable to, though slightly greater than the previously determined solution dissociation constant. Using pre-functionalized neurotransmitter-conjugated oligo(ethylene glycol) alkanethiols and microfluidics patterning, we produced multiplexed substrates to capture and to sort aptamers. Substrates patterned with L-DOPA, L-DOPS, and L-5-HTP enabled comparison of the selectivity of the dopamine aptamer for different targets via simultaneous determination of in situ binding constants. Thus, beyond our previous demonstrations of recognition by protein binding partners (i.e., antibodies and G-protein-coupled receptors), strategically optimized small-molecule-functionalized substrates show selective recognition of nucleic acid binding partners. These substrates are useful for side-by-side target comparisons, and future identification and characterization of novel aptamers targeting neurotransmitters or other important small-molecules.

Characterization of substrate binding of the WW domains in human WWP2 protein.

PubMed

Jiang, Jiahong; Wang, Nan; Jiang, Yafei; Tan, Hongwei; Zheng, Jimin; Chen, Guangju; Jia, Zongchao

2015-07-08

WW domains harbor substrates containing proline-rich motifs, but the substrate specificity and binding mechanism remain elusive for those WW domains less amenable for structural studies, such as human WWP2 (hWWP2). Herein we have employed multiple techniques to investigate the second WW domain (WW2) in hWWP2. Our results show that hWWP2 is a specialized E3 for PPxY motif-containing substrates only and does not recognize other amino acids and phospho-residues. The strongest binding affinity of WW2, and the incompatibility between each WW domain, imply a novel relationship, and our SPR experiment reveals a dynamic binding mode in Class-I WW domains for the first time. The results from alanine-scanning mutagenesis and modeling further point to functionally conserved residues in WW2. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Resonance assignments for the substrate binding domain of Hsp70 chaperone Ssa1 from Saccharomyces cerevisiae.

PubMed

Hu, Wanhui; Wu, Huiwen; Zhang, Hong; Gong, Weibin; Perrett, Sarah

2015-10-01

Hsp70 chaperone proteins play crucial roles in the cell. Extensive structural and functional studies have been performed for bacterial and mammalian Hsp70s. Ssa1 from Saccharomyces cerevisiae is a member of the Hsp70 family. In vivo and biochemical studies on Ssa1 have revealed that it regulates prion propagation and the cell cycle. However, no structural data has been obtained for Ssa1 up to now. Here we report the almost complete (96 %) (1)H, (13)C, (15)N backbone and side chain NMR assignment of the 18.8 kDa Ssa1 substrate binding domain. The construct includes residues 382-554, which corresponds to the entire substrate binding domain and two following α-helices in homologous structures. The secondary structure predicted from the assigned chemical shifts is consistent with that of homologous Hsp70 substrate binding domains.

Understanding the molecular mechanism of aryl acylamidase activity of acetylcholinesterase - An in silico study.

PubMed

Chinnadurai, Raj Kumar; Saravanaraman, Ponne; Boopathy, Rathanam

2015-08-15

Acetylcholinesterase (AChE) exhibits two different activities, namely esterase and aryl acylamidase (AAA). Unlike esterase, AAA activity of AChE is inhibited by the active site inhibitors while remaining unaffected by the peripheral anionic site inhibitors. This differential inhibitory pattern of active and peripheral anionic site inhibitors on the AAA activity remains unanswered. To answer this, we investigated the mechanism of binding and trafficking of AAA substrates using in silico tools. Molecular docking of serotonin and AAA substrates (o-nitroacetanilide, and o-nitrotrifluoroacetanilide,) onto AChE shows that these compounds bind at the side door of AChE. Thus, we conceived that the AAA substrates prefer the side door to reach the active site for their catalysis. Further, steered molecular dynamics simulations show that the force required for binding and trafficking of the AAA substrate through the side door is comparatively lesser than their dissociation (900kJ/mol/nm). Among the two substrates, o-nitrotrifluoroacetanilide required lesser force (380kJ/mol/nm) than o-nitroacetanilide the (550kJ/mol/nm) for its binding, thus validating o-nitrotrifluoroacetanilide as a better substrate. With these observations, we resolve that the AAA activity of AChE is mediated through its side door. Therefore, binding of PAS inhibitors at the main door of AChE remain ineffective against AAA activity. Copyright © 2015 Elsevier Inc. All rights reserved.

Steady-state kinetics of substrate binding and iron release in tomato ACC oxidase.

PubMed

Thrower, J S; Blalock, R; Klinman, J P

2001-08-14

1-Aminocyclopropane-1-carboxylate oxidase (ACC oxidase) catalyzes the last step in the biosynthetic pathway of the plant hormone, ethylene. This unusual reaction results in the oxidative ring cleavage of 1-aminocyclopropane carboxylate (ACC) into ethylene, cyanide, and CO2 and requires ferrous ion, ascorbate, and molecular oxygen for catalysis. A new purification procedure and assay method have been developed for tomato ACC oxidase that result in greatly increased enzymatic activity. This method allowed us to determine the rate of iron release from the enzyme and the effect of the activator, CO2, on this rate. Initial velocity studies support an ordered kinetic mechanism where ACC binds first followed by O2; ascorbate can bind after O2 or possibly before ACC. This kinetic mechanism differs from one recently proposed for the ACC oxidase from avocado.

Structure of the MacAB-TolC ABC-type tripartite multidrug efflux pump

PubMed Central

Llabrés, Salomé; Neuberger, Arthur; Blaza, James N.; Bai, Xiao-chen; Okada, Ui; Murakami, Satoshi; van Veen, Hendrik W.; Zachariae, Ulrich; Scheres, Sjors H.W.; Luisi, Ben F.

2017-01-01

The MacA-MacB-TolC assembly of Escherichia coli is a transmembrane machine that spans the cell envelope and actively extrudes substrates, including macrolide antibiotics and polypeptide virulence factors. These transport processes are energized by the ATPase MacB, a member of the ATP-binding cassette (ABC) superfamily. We present an electron cryo-microscopy structure of the ABC-type tripartite assembly at near-atomic resolution. A hexamer of the periplasmic protein MacA bridges between a TolC trimer in the outer membrane and a MacB dimer in the inner membrane, generating a quaternary structure with a central channel for substrate translocation. A gating ring found in MacA is proposed to act as a one-way valve in substrate transport. The MacB structure features an atypical transmembrane domain (TMD) with a closely packed dimer interface and a periplasmic opening that is the likely portal for substrate entry from the periplasm, with subsequent displacement through an allosteric transport mechanism. PMID:28504659

Nonlocality and Short-Range Wetting Phenomena

NASA Astrophysics Data System (ADS)

Parry, A. O.; Romero-Enrique, J. M.; Lazarides, A.

2004-08-01

We propose a nonlocal interfacial model for 3D short-range wetting at planar and nonplanar walls. The model is characterized by a binding-potential functional depending only on the bulk Ornstein-Zernike correlation function, which arises from different classes of tubelike fluctuations that connect the interface and the substrate. The theory provides a physical explanation for the origin of the effective position-dependent stiffness and binding potential in approximate local theories and also obeys the necessary classical wedge covariance relationship between wetting and wedge filling. Renormalization group and computer simulation studies reveal the strong nonperturbative influence of nonlocality at critical wetting, throwing light on long-standing theoretical problems regarding the order of the phase transition.

Nonlocality and short-range wetting phenomena.

PubMed

Parry, A O; Romero-Enrique, J M; Lazarides, A

2004-08-20

We propose a nonlocal interfacial model for 3D short-range wetting at planar and nonplanar walls. The model is characterized by a binding-potential functional depending only on the bulk Ornstein-Zernike correlation function, which arises from different classes of tubelike fluctuations that connect the interface and the substrate. The theory provides a physical explanation for the origin of the effective position-dependent stiffness and binding potential in approximate local theories and also obeys the necessary classical wedge covariance relationship between wetting and wedge filling. Renormalization group and computer simulation studies reveal the strong nonperturbative influence of nonlocality at critical wetting, throwing light on long-standing theoretical problems regarding the order of the phase transition.

Structures of a Na+-coupled, substrate-bound MATE multidrug transporter

PubMed Central

Lu, Min; Symersky, Jindrich; Radchenko, Martha; Koide, Akiko; Guo, Yi; Nie, Rongxin; Koide, Shohei

2013-01-01

Multidrug transporters belonging to the multidrug and toxic compound extrusion (MATE) family expel dissimilar lipophilic and cationic drugs across cell membranes by dissipating a preexisting Na+ or H+ gradient. Despite its clinical relevance, the transport mechanism of MATE proteins remains poorly understood, largely owing to a lack of structural information on the substrate-bound transporter. Here we report crystal structures of a Na+-coupled MATE transporter NorM from Neisseria gonorrheae in complexes with three distinct translocation substrates (ethidium, rhodamine 6G, and tetraphenylphosphonium), as well as Cs+ (a Na+ congener), all captured in extracellular-facing and drug-bound states. The structures revealed a multidrug-binding cavity festooned with four negatively charged amino acids and surprisingly limited hydrophobic moieties, in stark contrast to the general belief that aromatic amino acids play a prominent role in multidrug recognition. Furthermore, we discovered an uncommon cation–π interaction in the Na+-binding site located outside the drug-binding cavity and validated the biological relevance of both the substrate- and cation-binding sites by conducting drug resistance and transport assays. Additionally, we uncovered potential rearrangement of at least two transmembrane helices upon Na+-induced drug export. Based on our structural and functional analyses, we suggest that Na+ triggers multidrug extrusion by inducing protein conformational changes rather than by directly competing for the substrate-binding amino acids. This scenario is distinct from the canonical antiport mechanism, in which both substrate and counterion compete for a shared binding site in the transporter. Collectively, our findings provide an important step toward a detailed and mechanistic understanding of multidrug transport. PMID:23341609

Rpn1 provides adjacent receptor sites for substrate binding and deubiquitination by the proteasome

PubMed Central

Shi, Yuan; Chen, Xiang; Elsasser, Suzanne; Stocks, Bradley B.; Tian, Geng; Lee, Byung-Hoon; Shi, Yanhong; Zhang, Naixia; de Poot, Stefanie A. H.; Tuebing, Fabian; Sun, Shuangwu; Vannoy, Jacob; Tarasov, Sergey G.; Engen, John R.; Finley, Daniel; Walters, Kylie J.

2016-01-01

Structured Abstract INTRODUCTION The ubiquitin-proteasome system comprises hundreds of distinct pathways of degradation, which converge at the step of ubiquitin recognition by the proteasome. Five proteasomal ubiquitin receptors have been identified, two that are intrinsic to the proteasome (Rpn10 and Rpn13) and three reversibly associated proteasomal ubiquitin receptors (Rad23, Dsk2, and Ddi1). RATIONALE We found that the five known proteasomal ubiquitin receptors of yeast are collectively nonessential for ubiquitin recognition by the proteasome. We therefore screened for additional ubiquitin receptors in the proteasome and identified subunit Rpn1 as a candidate. We used nuclear magnetic resonance (NMR) spectroscopy to characterize the structure of the binding site within Rpn1, which we term the T1 site. Mutational analysis of this site showed its functional importance within the context of intact proteasomes. T1 binds both ubiquitin and ubiquitin-like (UBL) proteins, in particular the substrate-delivering shuttle factor Rad23. A second site within the Rpn1 toroid, T2, recognizes the UBL domain of deubiquitinating enzyme Ubp6, as determined by hydrogen-deuterium exchange mass spectrometry analysis and validated by amino acid substitution and functional assays. The Rpn1 toroid thus serves a critical scaffolding role within the proteasome, helping to assemble multiple proteasome cofactors as well as substrates. RESULTS Our results indicate that proteasome subunit Rpn1 can recognize both ubiquitin and UBL domains of substrate shuttling factors that themselves bind ubiquitin and function as reversibly-associated proteasomal ubiquitin receptors. Recognition is mediated by the T1 site within the Rpn1 toroid, which supports proteasome function in vivo. We found that the capacity of T1 to recognize both ubiquitin and UBL proteins was shared with Rpn10 and Rpn13. The surprising multiplicity of ubiquitin-recognition domains within the proteasome may promote enhanced, multipoint binding of ubiquitin chains. The structures of the T1 site in its free state and complexed with monoubiquitin or K48-linked diubiquitin were solved, revealing that three neighboring outer helices from the T1 toroid engage two ubiquitins. This binding mode leads to a preference for certain ubiquitin chain types, especially K6- and K48-linked chains, in a distinct configuration that can position substrates close to the entry port of the proteasome. The fate of proteasome-docked ubiquitin conjugates is determined by a competition between deubiquitination and substrate degradation. We find that proximal to the T1 site within the Rpn1 toroid is a second UBL-binding site, T2, that does not assist in ubiquitin chain recognition, but rather in chain disassembly, by binding to the UBL domain of deubiquitinating enzyme Ubp6. Importantly, the UBL interactors at T1 and T2 are distinct, assigning substrate localization to T1 and substrate deubiquitination to T2. CONCLUSION A ligand-binding hotspot was identified in the Rpn1 toroid, consisting of two adjacent receptor sites, T1 and T2. The Rpn1 toroid represents a novel class of binding domains for ubiquitin and UBL proteins. This study thus defines a novel two-site recognition domain intrinsic to the proteasome that uses homologous ubiquitin/UBL-class ligands to assemble substrates, substrate shuttling factors, and a deubiquitinating enzyme in close proximity. A ligand-binding hotspot in the proteasome for assembling substrates and cofactors Schematic (top) and model structure (bottom, left) mapping the UBL-binding Rpn1 T1 (indigo) and T2 (orange) sites. (Bottom, right) Enlarged region of the proteasome to illustrate the Rpn1 T1 and T2 sites bound to a ubiquitin chain (yellow) and deubiquitinating enzyme Ubp6 (green), respectively. PDB 4CR2 and 2B9R were used for this figure. Hundreds of pathways for degradation converge at ubiquitin recognition by proteasome. Here we found that the five known proteasomal ubiquitin receptors are collectively nonessential for ubiquitin recognition, and identified a sixth receptor, Rpn1. A site (T1) in the Rpn1 toroid recognized ubiquitin and ubiquitin-like (UBL) domains of substrate shuttling factors. T1 structures with monoubiquitin or K48 diubiquitin show three neighboring outer helices engaging two ubiquitins. T1 contributes a distinct substrate-binding pathway with preference for K48-linked chains. Proximal to T1 within the Rpn1 toroid is a second UBL-binding site (T2) that assists in ubiquitin chain disassembly, by binding the UBL of deubiquitinating enzyme Ubp6. Thus a two-site recognition domain intrinsic to the proteasome uses homologous ubiquitin/UBL-class ligands to assemble substrates, shuttling factors, and a deubiquitinating enzyme. PMID:26912900

Allosteric regulation of rhomboid intramembrane proteolysis.

PubMed

Arutyunova, Elena; Panwar, Pankaj; Skiba, Pauline M; Gale, Nicola; Mak, Michelle W; Lemieux, M Joanne

2014-09-01

Proteolysis within the lipid bilayer is poorly understood, in particular the regulation of substrate cleavage. Rhomboids are a family of ubiquitous intramembrane serine proteases that harbour a buried active site and are known to cleave transmembrane substrates with broad specificity. In vitro gel and Förster resonance energy transfer (FRET)-based kinetic assays were developed to analyse cleavage of the transmembrane substrate psTatA (TatA from Providencia stuartii). We demonstrate significant differences in catalytic efficiency (kcat/K0.5) values for transmembrane substrate psTatA (TatA from Providencia stuartii) cleavage for three rhomboids: AarA from P. stuartii, ecGlpG from Escherichia coli and hiGlpG from Haemophilus influenzae demonstrating that rhomboids specifically recognize this substrate. Furthermore, binding of psTatA occurs with positive cooperativity. Competitive binding studies reveal an exosite-mediated mode of substrate binding, indicating allostery plays a role in substrate catalysis. We reveal that exosite formation is dependent on the oligomeric state of rhomboids, and when dimers are dissociated, allosteric substrate activation is not observed. We present a novel mechanism for specific substrate cleavage involving several dynamic processes including positive cooperativity and homotropic allostery for this interesting class of intramembrane proteases. © 2014 The Authors.

Allosteric regulation of rhomboid intramembrane proteolysis

PubMed Central

Arutyunova, Elena; Panwar, Pankaj; Skiba, Pauline M; Gale, Nicola; Mak, Michelle W; Lemieux, M Joanne

2014-01-01

Proteolysis within the lipid bilayer is poorly understood, in particular the regulation of substrate cleavage. Rhomboids are a family of ubiquitous intramembrane serine proteases that harbour a buried active site and are known to cleave transmembrane substrates with broad specificity. In vitro gel and Förster resonance energy transfer (FRET)-based kinetic assays were developed to analyse cleavage of the transmembrane substrate psTatA (TatA from Providencia stuartii). We demonstrate significant differences in catalytic efficiency (kcat/K0.5) values for transmembrane substrate psTatA (TatA from Providencia stuartii) cleavage for three rhomboids: AarA from P. stuartii, ecGlpG from Escherichia coli and hiGlpG from Haemophilus influenzae demonstrating that rhomboids specifically recognize this substrate. Furthermore, binding of psTatA occurs with positive cooperativity. Competitive binding studies reveal an exosite-mediated mode of substrate binding, indicating allostery plays a role in substrate catalysis. We reveal that exosite formation is dependent on the oligomeric state of rhomboids, and when dimers are dissociated, allosteric substrate activation is not observed. We present a novel mechanism for specific substrate cleavage involving several dynamic processes including positive cooperativity and homotropic allostery for this interesting class of intramembrane proteases. PMID:25009246

Simultaneous binding to the tracking strand, displaced strand and the duplex of a DNA fork enhances unwinding by Dda helicase

PubMed Central

Aarattuthodiyil, Suja; Byrd, Alicia K.; Raney, Kevin D.

2014-01-01

Interactions between helicases and the tracking strand of a DNA substrate are well-characterized; however, the role of the displaced strand is a less understood characteristic of DNA unwinding. Dda helicase exhibited greater processivity when unwinding a DNA fork compared to a ss/ds DNA junction substrate. The lag phase in the unwinding progress curve was reduced for the forked DNA compared to the ss/ds junction. Fewer kinetic steps were required to unwind the fork compared to the ss/ds junction, suggesting that binding to the fork leads to disruption of the duplex. DNA footprinting confirmed that interaction of Dda with a fork leads to two base pairs being disrupted whereas no disruption of base pairing was observed with the ss/ds junction. Neutralization of the phosphodiester backbone resulted in a DNA-footprinting pattern similar to that observed with the ss/ds junction, consistent with disruption of the interaction between Dda and the displaced strand. Several basic residues in the 1A domain which were previously proposed to bind to the incoming duplex DNA were replaced with alanines, resulting in apparent loss of interaction with the duplex. Taken together, these results suggest that Dda interaction with the tracking strand, displaced strand and duplex coordinates DNA unwinding. PMID:25249618

An atomistic view of Hsp70 allosteric crosstalk: from the nucleotide to the substrate binding domain and back

PubMed Central

Chiappori, Federica; Merelli, Ivan; Milanesi, Luciano; Colombo, Giorgio; Morra, Giulia

2016-01-01

The Hsp70 is an allosterically regulated family of molecular chaperones. They consist of two structural domains, NBD and SBD, connected by a flexible linker. ATP hydrolysis at the NBD modulates substrate recognition at the SBD, while peptide binding at the SBD enhances ATP hydrolysis. In this study we apply Molecular Dynamics (MD) to elucidate the molecular determinants underlying the allosteric communication from the NBD to the SBD and back. We observe that local structural and dynamical modulation can be coupled to large-scale rearrangements, and that different combinations of ligands at NBD and SBD differently affect the SBD domain mobility. Substituting ADP with ATP in the NBD induces specific structural changes involving the linker and the two NBD lobes. Also, a SBD-bound peptide drives the linker docking by increasing the local dynamical coordination of its C-terminal end: a partially docked DnaK structure is achieved by combining ATP in the NBD and peptide in the SBD. We propose that the MD-based analysis of the inter domain dynamics and structure modulation could be used as a tool to computationally predict the allosteric behaviour and functional response of Hsp70 upon introducing mutations or binding small molecules, with potential applications for drug discovery. PMID:27025773

Bacterial protease uses distinct thermodynamic signatures for substrate recognition.

PubMed

Bezerra, Gustavo Arruda; Ohara-Nemoto, Yuko; Cornaciu, Irina; Fedosyuk, Sofiya; Hoffmann, Guillaume; Round, Adam; Márquez, José A; Nemoto, Takayuki K; Djinović-Carugo, Kristina

2017-06-06

Porphyromonas gingivalis and Porphyromonas endodontalis are important bacteria related to periodontitis, the most common chronic inflammatory disease in humans worldwide. Its comorbidity with systemic diseases, such as type 2 diabetes, oral cancers and cardiovascular diseases, continues to generate considerable interest. Surprisingly, these two microorganisms do not ferment carbohydrates; rather they use proteinaceous substrates as carbon and energy sources. However, the underlying biochemical mechanisms of their energy metabolism remain unknown. Here, we show that dipeptidyl peptidase 11 (DPP11), a central metabolic enzyme in these bacteria, undergoes a conformational change upon peptide binding to distinguish substrates from end products. It binds substrates through an entropy-driven process and end products in an enthalpy-driven fashion. We show that increase in protein conformational entropy is the main-driving force for substrate binding via the unfolding of specific regions of the enzyme ("entropy reservoirs"). The relationship between our structural and thermodynamics data yields a distinct model for protein-protein interactions where protein conformational entropy modulates the binding free-energy. Further, our findings provide a framework for the structure-based design of specific DPP11 inhibitors.

Temperature-dependent subunit exchange and chaperone-like activities of Hsp16.3, a small heat shock protein from Mycobacterium tuberculosis.

PubMed

Fu, Xinmiao; Chang, Zengyi

2004-04-02

Small heat shock proteins (sHsps) usually exist as oligomers that undergo dynamic oligomeric dissociation/re-association, with the dissociated oligomers as active forms to bind substrate proteins under heat shock conditions. In this study, however, we found that Hsp16.3, one sHsp from Mycobacterium tuberculosis, is able to sensitively modulate its chaperone-like activity in a range of physiological temperatures (from 25 to 37.5 degrees C) while its native oligomeric size is still maintained. Further analysis demonstrated that Hsp16.3 exposes higher hydrophobic surfaces upon temperatures increasing and that a large soluble complex between Hsp16.3 and substrate is formed only in the condition of heating temperature up to 35 and 37.5 degrees C. Structural analysis by fluorescence anisotropy showed that Hsp16.3 nonameric structure becomes more dynamic and variable at elevated temperatures. Moreover, subunit exchange between Hsp16.3 oligomers was found to occur faster upon temperatures increasing as revealed by fluorescence energy resonance transfer. These observations indicate that Hsp16.3 is able to modulate its chaperone activity by adjusting the dynamics of oligomeric dissociation/re-association process while maintaining its static oligomeric size unchangeable. A kinetic model is therefore proposed to explain the mechanism of sHsps-binding substrate proteins through oligomeric dissociation. The present study also implied that Hsp16.3 is at least capable of binding non-native proteins in vivo while expressing in the host organism that survives at 37 degrees C.

Neurotransmitter and psychostimulant recognition by the dopamine transporter

PubMed Central

Wang, Kevin H.; Penmatsa, Aravind; Gouaux, Eric

2015-01-01

Na+/Cl−-coupled biogenic amine transporters are the primary targets of therapeutic and abused drugs, ranging from antidepressants to the psychostimulants cocaine and amphetamines, and to their cognate substrates. Here we determine x-ray crystal structures of the Drosophila melanogaster dopamine transporter (dDAT) bound to its substrate dopamine (DA), a substrate analogue 3,4-dichlorophenethylamine, the psychostimulants D-amphetamine, methamphetamine, or to cocaine and cocaine analogues. All ligands bind to the central binding site, located approximately halfway across the membrane bilayer, in close proximity to bound sodium and chloride ions. The central binding site recognizes three chemically distinct classes of ligands via conformational changes that accommodate varying sizes and shapes, thus illustrating molecular principles that distinguish substrates from inhibitors in biogenic amine transporters. PMID:25970245

Structural determinants of species-selective substrate recognition in human and Drosophila serotonin transporters revealed through computational docking studies

PubMed Central

Kaufmann, Kristian W.; Dawson, Eric S.; Henry, L. Keith; Field, Julie R.; Blakely, Randy D.; Meiler, Jens

2009-01-01

To identify potential determinants of substrate selectivity in serotonin (5-HT) transporters (SERT), models of human and Drosophila serotonin transporters (hSERT, dSERT) were built based on the leucine transporter (LeuTAa) structure reported by Yamashita et al. (Nature 2005;437:215–223), PBDID 2A65. Although the overall amino acid identity between SERTs and the LeuTAa is only 17%, it increases to above 50% in the first shell of the putative 5-HT binding site, allowing de novo computational docking of tryptamine derivatives in atomic detail. Comparison of hSERT and dSERT complexed with substrates pinpoints likely structural determinants for substrate binding. Forgoing the use of experimental transport and binding data of tryptamine derivatives for construction of these models enables us to cHitically assess and validate their predictive power: A single 5-HT binding mode was identified that retains the amine placement observed in the LeuTAa structure, matches site-directed mutagenesis and substituted cysteine accessibility method (SCAM) data, complies with support vector machine derived relations activity relations, and predicts computational binding energies for 5-HT analogs with a significant correlation coefficient (R = 0.72). This binding mode places 5-HT deep in the binding pocket of the SERT with the 5-position near residue hSERT A169/dSERT D164 in transmembrane helix 3, the indole nitrogen next to residue Y176/Y171, and the ethylamine tail under residues F335/F327 and S336/S328 within 4 Å of residue D98. Our studies identify a number of potential contacts whose contribution to substrate binding and transport was previously unsuspected. PMID:18704946

Design and isolation of ribozyme-substrate pairs using RNase P-based ribozymes containing altered substrate binding sites.

PubMed Central

Mobley, E M; Pan, T

1999-01-01

Substrate recognition and cleavage by the bacterial RNase P RNA requires two domains, a specificity domain, or S-domain, and a catalytic domain, or C-domain. The S-domain binds the T stem-loop region in a pre-tRNA substrate to confer specificity for tRNA substrates. In this work, the entire S-domain of the Bacillus subtilis RNase P RNA is replaced with an artificial substrate binding module. New RNA substrates are isolated by in vitro selection using two libraries containing random regions of 60 nt. At the end of the selection, the cleavage rates of the substrate library are approximately 0.7 min(-1)in 10 mM MgCl(2)at 37 degrees C, approximately 4-fold better than the cleavage of a pre-tRNA substrate by the wild-type RNase P RNA under the same conditions. The contribution of the S-domain replacement to the catalytic efficiency is from 6- to 22 000-fold. Chemical and nuclease mapping of two ribozyme-product complexes shows that this contribution correlates with direct interactions between the S-domain replacement and the selected substrate. These results demonstrate the feasibility of design and isolation of RNase P-based, matching ribozyme-substrate pairs without prior knowledge of the sequence or structure of the interactive modules in the ribozyme or substrate. PMID:10518624

Microfluidic Channels on Nanopatterned Substrates: Monitoring Protein Binding to Lipid Bilayers with Surface-Enhanced Raman Spectroscopy

PubMed Central

Banerjee, Amrita; Perez-Castillejos, R.; Hahn, D.; Smirnov, Alex I.; Grebel, H.

2013-01-01

We used Surface Enhanced Raman Spectroscopy (SERS) to detect binding events between streptavidin and biotinylated lipid bilayers. The binding events took place at the surface between microfluidic channels and anodized aluminum oxide (AAO) with the latter serving as substrates. The bilayers were incorporated in the substrate pores. It was revealed that non-bound molecules were easily washed away and that large suspended cells (Salmonella enterica) are less likely to interfere with the monitoring process: when focusing to the lower surface of the channel, one may resolve mostly the bound molecules. PMID:24932024

Micro-fluidic channels on nanopatterned substrates: Monitoring protein binding to lipid bilayers with surface-enhanced Raman spectroscopy

NASA Astrophysics Data System (ADS)

Banerjee, Amrita; Perez-Castillejos, R.; Hahn, D.; Smirnov, Alex I.; Grebel, H.

2010-04-01

We used surface-enhanced Raman spectroscopy (SERS) to detect binding events between streptavidin and biotinylated lipid bilayers. The binding events took place at the surface between micro-fluidic channels and anodized aluminum oxide (AAO) with the latter serving as substrates. The bilayers were incorporated in the substrate pores. It was revealed that non-bound molecules were easily washed away and that large suspended cells ( Salmonella enterica) are less likely to interfere with the monitoring process: when focusing to the lower surface of the channel, one may resolve mostly the bound molecules.

A reentrant loop domain in the glutamate carrier EAAT1 participates in substrate binding and translocation.

PubMed

Seal, R P; Amara, S G

1998-12-01

To investigate the structural determinants underlying transport by the glutamate transporter EAAT1, we mutated each of 24 highly conserved residues (P392 to Q415) to cysteine. A majority of these substituted cysteines react with the sulfhydryl-modifying reagent MTSEA, suggesting that they reside in an aqueous environment. The impermeant reagents MTSES and MTSET react with residues at each end of the domain (A395C and A414C), supporting a model that places these residues near the extracellular surface. Substrates and inhibitors block the reaction between MTS derivatives and A395C, and the cosubstrate, sodium, slows reaction of MTSEA with Y405C and E406C. From these results, we propose that this domain forms a reentrant membrane loop at the cell surface and may comprise part of the translocation pore for substrates and cotransported ions.

Structural basis for phosphatidylinositol-phosphate biosynthesis

NASA Astrophysics Data System (ADS)

Clarke, Oliver B.; Tomasek, David; Jorge, Carla D.; Dufrisne, Meagan Belcher; Kim, Minah; Banerjee, Surajit; Rajashankar, Kanagalaghatta R.; Shapiro, Lawrence; Hendrickson, Wayne A.; Santos, Helena; Mancia, Filippo

2015-10-01

Phosphatidylinositol is critical for intracellular signalling and anchoring of carbohydrates and proteins to outer cellular membranes. The defining step in phosphatidylinositol biosynthesis is catalysed by CDP-alcohol phosphotransferases, transmembrane enzymes that use CDP-diacylglycerol as donor substrate for this reaction, and either inositol in eukaryotes or inositol phosphate in prokaryotes as the acceptor alcohol. Here we report the structures of a related enzyme, the phosphatidylinositol-phosphate synthase from Renibacterium salmoninarum, with and without bound CDP-diacylglycerol to 3.6 and 2.5 Å resolution, respectively. These structures reveal the location of the acceptor site, and the molecular determinants of substrate specificity and catalysis. Functional characterization of the 40%-identical ortholog from Mycobacterium tuberculosis, a potential target for the development of novel anti-tuberculosis drugs, supports the proposed mechanism of substrate binding and catalysis. This work therefore provides a structural and functional framework to understand the mechanism of phosphatidylinositol-phosphate biosynthesis.

Enzyme specificity under dynamic control

NASA Astrophysics Data System (ADS)

Ota, Nobuyuki; Agard, David A.

2002-03-01

The contributions of conformational dynamics to substrate specificity have been examined by the application of principal component analysis to molecular dynamics trajectories of alpha-lytic protease. The wild-type alpha-lytic protease is highly specific for substrates with small hydrophobic side chains at the specificity pocket, while the Met190Ala binding pocket mutant has a much broader specificity, actively hydrolyzing substrates ranging from Ala to Phe. We performed a principal component analysis using 1-nanosecond molecular dynamics simulations using solvent boundary condition. We found that the walls of the wild-type substrate binding pocket move in tandem with one another, causing the pocket size to remain fixed so that only small substrates are recognized. In contrast, the M190A mutant shows uncoupled movement of the binding pocket walls, allowing the pocket to sample both smaller and larger sizes, which appears to be the cause of the observed broad specificity. The results suggest that the protein dynamics of alpha-lytic protease may play a significant role in defining the patterns of substrate specificity.

Substrates Control Multimerization and Activation of the Multi-Domain ATPase Motor of Type VII Secretion

DOE PAGES

Rosenberg, Oren S.; Dovala, Dustin; Li, Xueming; ...

2015-04-09

We report that Mycobacterium tuberculosis and Staphylococcus aureus secrete virulence factors via type VII protein secretion (T7S), a system that intriguingly requires all of its secretion substrates for activity. To gain insights into T7S function, we used structural approaches to guide studies of the putative translocase EccC, a unique enzyme with three ATPase domains, and its secretion substrate EsxB. The crystal structure of EccC revealed that the ATPase domains are joined by linker/pocket interactions that modulate its enzymatic activity. EsxB binds via its signal sequence to an empty pocket on the C-terminal ATPase domain, which is accompanied by an increasemore » in ATPase activity. Surprisingly, substrate binding does not activate EccC allosterically but, rather, by stimulating its multimerization. Thus, the EsxB substrate is also an integral T7S component, illuminating a mechanism that helps to explain interdependence of substrates, and suggests a model in which binding of substrates modulates their coordinate release from the bacterium.« less

Methods of making functionalized nanorods

DOEpatents

Gur, Ilan [San Francisco, CA; Milliron, Delia [Berkeley, CA; Alivisatos, A Paul [Oakland, CA; Liu, Haitao [Berkeley, CA

2012-01-10

A process for forming functionalized nanorods. The process includes providing a substrate, modifying the substrate by depositing a self-assembled monolayer of a bi-functional molecule on the substrate, wherein the monolayer is chosen such that one side of the bi-functional molecule binds to the substrate surface and the other side shows an independent affinity for binding to a nanocrystal surface, so as to form a modified substrate. The process further includes contacting the modified substrate with a solution containing nanocrystal colloids, forming a bound monolayer of nanocrystals on the substrate surface, depositing a polymer layer over the monolayer of nanocrystals to partially cover the monolayer of nanocrystals, so as to leave a layer of exposed nanocrystals, functionalizing the exposed nanocrystals, to form functionalized nanocrystals, and then releasing the functionalized nanocrystals from the substrate.

Substrate-Assisted Catalysis in the Reaction Catalyzed by Salicylic Acid Binding Protein 2 (SABP2), a Potential Mechanism of Substrate Discrimination for Some Promiscuous Enzymes.

PubMed

Yao, Jianzhuang; Guo, Haobo; Chaiprasongsuk, Minta; Zhao, Nan; Chen, Feng; Yang, Xiaohan; Guo, Hong

2015-09-01

Although one of an enzyme's hallmarks is the high specificity for their natural substrates, substrate promiscuity has been reported more frequently. It is known that promiscuous enzymes generally show different catalytic efficiencies to different substrates, but our understanding of the origin of such differences is still lacking. Here we report the results of quantum mechanical/molecular mechanical simulations and an experimental study of salicylic acid binding protein 2 (SABP2). SABP2 has promiscuous esterase activity toward a series of substrates but shows a high activity toward its natural substrate, methyl salicylate (MeSA). Our results demonstrate that this enzyme may use substrate-assisted catalysis involving the hydroxyl group from MeSA to enhance the activity and achieve substrate discrimination.

Substrate-Assisted Catalysis in the Reaction Catalyzed by Salicylic Acid Binding Protein 2 (SABP2), a Potential Mechanism of Substrate Discrimination for Some Promiscuous Enzymes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yao, Jianzhuang; Guo, Haobo; Chaiprasongsuk, Minta

Although one of an enzyme’s hallmarks is the high specificity for their natural substrates, substrate promiscuity has been reported more frequently. We know that promiscuous enzymes generally show different catalytic efficiencies to different substrates, but our understanding of the origin of such differences is still lacking. We report the results of quantum mechanical/molecular mechanical simulations and an experimental study of salicylic acid binding protein 2 (SABP2). SABP2 has promiscuous esterase activity toward a series of substrates but shows a high activity toward its natural substrate, methyl salicylate (MeSA). Finally, our results demonstrate that this enzyme may use substrate-assisted catalysis involvingmore » the hydroxyl group from MeSA to enhance the activity and achieve substrate discrimination.« less

Substrate-Assisted Catalysis in the Reaction Catalyzed by Salicylic Acid Binding Protein 2 (SABP2), a Potential Mechanism of Substrate Discrimination for Some Promiscuous Enzymes

DOE PAGES

Yao, Jianzhuang; Guo, Haobo; Chaiprasongsuk, Minta; ...

2015-08-05

Although one of an enzyme’s hallmarks is the high specificity for their natural substrates, substrate promiscuity has been reported more frequently. We know that promiscuous enzymes generally show different catalytic efficiencies to different substrates, but our understanding of the origin of such differences is still lacking. We report the results of quantum mechanical/molecular mechanical simulations and an experimental study of salicylic acid binding protein 2 (SABP2). SABP2 has promiscuous esterase activity toward a series of substrates but shows a high activity toward its natural substrate, methyl salicylate (MeSA). Finally, our results demonstrate that this enzyme may use substrate-assisted catalysis involvingmore » the hydroxyl group from MeSA to enhance the activity and achieve substrate discrimination.« less

Active-site copper reduction promotes substrate binding of fungal lytic polysaccharide monooxygenase and reduces stability.

PubMed

Kracher, Daniel; Andlar, Martina; Furtmüller, Paul G; Ludwig, Roland

2018-02-02

Lytic polysaccharide monooxygenases (LPMOs) are a class of copper-containing enzymes that oxidatively degrade insoluble plant polysaccharides and soluble oligosaccharides. Upon reductive activation, they cleave the substrate and promote biomass degradation by hydrolytic enzymes. In this study, we employed LPMO9C from Neurospora crassa , which is active toward cellulose and soluble β-glucans, to study the enzyme-substrate interaction and thermal stability. Binding studies showed that the reduction of the mononuclear active-site copper by ascorbic acid increased the affinity and the maximum binding capacity of LPMO for cellulose. The reduced redox state of the active-site copper and not the subsequent formation of the activated oxygen species increased the affinity toward cellulose. The lower affinity of oxidized LPMO could support its desorption after catalysis and allow hydrolases to access the cleavage site. It also suggests that the copper reduction is not necessarily performed in the substrate-bound state of LPMO. Differential scanning fluorimetry showed a stabilizing effect of the substrates cellulose and xyloglucan on the apparent transition midpoint temperature of the reduced, catalytically active enzyme. Oxidative auto-inactivation and destabilization were observed in the absence of a suitable substrate. Our data reveal the determinants of LPMO stability under turnover and non-turnover conditions and indicate that the reduction of the active-site copper initiates substrate binding. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Predicting binding modes of reversible peptide-based inhibitors of falcipain-2 consistent with structure-activity relationships.

PubMed

Hernández González, Jorge Enrique; Hernández Alvarez, Lilian; Pascutti, Pedro Geraldo; Valiente, Pedro A

2017-09-01

Falcipain-2 (FP-2) is a major hemoglobinase of Plasmodium falciparum, considered an important drug target for the development of antimalarials. A previous study reported a novel series of 20 reversible peptide-based inhibitors of FP-2. However, the lack of tridimensional structures of the complexes hinders further optimization strategies to enhance the inhibitory activity of the compounds. Here we report the prediction of the binding modes of the aforementioned inhibitors to FP-2. A computational approach combining previous knowledge on the determinants of binding to the enzyme, docking, and postdocking refinement steps, is employed. The latter steps comprise molecular dynamics simulations and free energy calculations. Remarkably, this approach leads to the identification of near-native ligand conformations when applied to a validation set of protein-ligand structures. Overall, we proposed substrate-like binding modes of the studied compounds fulfilling the structural requirements for FP-2 binding and yielding free energy values that correlated well with the experimental data. Proteins 2017; 85:1666-1683. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Yeast enolase: mechanism of activation by metal ions.

PubMed

Brewer, J M

1981-01-01

Yeast enolase as prepared by current procedures is inherently chemically homogeneous, though deamidation and partial denaturation can produce electrophoretically distinct forms. A true isozyme of the enzyme exists but does not survive the purification procedure. The chemical sequence for both has been established. The enzyme behaves in solution like a compact, nearly spherical molecule of moderate hydration. Strong intramolecular forces maintain the structure of the individual subunits. The enzyme as isolated is dimeric. If dissociated in the presence of magnesium ions and substrate, then the subunits are active, but if the dissociation occurs in the absence of metal ions, they are inactive until they have reassociated and undergone a first order "annealing" process. Magnesium (II) enhances association. The interaction between the subunits is hydrophobic in character. The enzyme can bind up to 2 mol of most metal ions in "conformational" sites which then allows up to 2 mol of substrate or some substrate analogue to bind. This is not sufficient for catalysis, but conformational metal ions do more than just allow substrate binding. A change in the environment of the metal ions occurs on substrate or substrate analogue binding. There is an absolute correlation between the occurrence of a structural change undergone by the 3-amino analogue of phosphoenolpyruvate and whether the metal ions produce any level of enzymatic activity. For catalysis, two more moles of metal ions, called "catalytic", must bind. There is evidence that the enzymatic reaction involves a carbanion mechanism. It is likely that two more moles of metal ion can bind which inhibit the reaction. The requirement for 2 mol of metal ion per subunit which contribute in different ways to catalysis is exhibited by a number of other enzymes.

The Crystal Structure Analysis of Group B Streptococcus Sortase C1: A Model for the ;Lid; Movement upon Substrate Binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Khare, Baldeep; Fu, Zheng-Qing; Huang, I-Hsiu

2012-02-07

A unique feature of the class-C-type sortases, enzymes essential for Gram-positive pilus biogenesis, is the presence of a flexible 'lid' anchored in the active site. However, the mechanistic details of the 'lid' displacement, suggested to be a critical prelude for enzyme catalysis, are not yet known. This is partly due to the absence of enzyme-substrate and enzyme-inhibitor complex crystal structures. We have recently described the crystal structures of the Streptococcus agalactiae SAG2603 V/R sortase SrtC1 in two space groups (type II and type III) and that of its 'lid' mutant and proposed a role of the 'lid' as a protectormore » of the active-site hydrophobic environment. Here, we report the crystal structures of SAG2603 V/R sortase C1 in a different space group (type I) and that of its complex with a small-molecule cysteine protease inhibitor. We observe that the catalytic Cys residue is covalently linked to the small-molecule inhibitor without lid displacement. However, the type I structure provides a view of the sortase SrtC1 lid displacement while having structural elements similar to a substrate sorting motif suitably positioned in the active site. We propose that these major conformational changes seen in the presence of a substrate mimic in the active site may represent universal features of class C sortase substrate recognition and enzyme activation.« less

Clustering molecular dynamics trajectories for optimizing docking experiments.

PubMed

De Paris, Renata; Quevedo, Christian V; Ruiz, Duncan D; Norberto de Souza, Osmar; Barros, Rodrigo C

2015-01-01

Molecular dynamics simulations of protein receptors have become an attractive tool for rational drug discovery. However, the high computational cost of employing molecular dynamics trajectories in virtual screening of large repositories threats the feasibility of this task. Computational intelligence techniques have been applied in this context, with the ultimate goal of reducing the overall computational cost so the task can become feasible. Particularly, clustering algorithms have been widely used as a means to reduce the dimensionality of molecular dynamics trajectories. In this paper, we develop a novel methodology for clustering entire trajectories using structural features from the substrate-binding cavity of the receptor in order to optimize docking experiments on a cloud-based environment. The resulting partition was selected based on three clustering validity criteria, and it was further validated by analyzing the interactions between 20 ligands and a fully flexible receptor (FFR) model containing a 20 ns molecular dynamics simulation trajectory. Our proposed methodology shows that taking into account features of the substrate-binding cavity as input for the k-means algorithm is a promising technique for accurately selecting ensembles of representative structures tailored to a specific ligand.

Online assay of bone specific alkaline phosphatase with a flow injection-bead injection system.

PubMed

Hartwell, Supaporn Kradtap; Somprayoon, Duangporn; Kongtawelert, Prachya; Ongchai, Siriwan; Arppornchayanon, Olarn; Ganranoo, Lucksagoon; Lapanantnoppakhun, Somchai; Grudpan, Kate

2007-09-26

Alkaline phosphatase (ALP) has been used as one of the biomarkers for bone resorption and liver diseases. Normally, total alkaline phosphatase is quantified along with other symptoms to determine the releasing source of the alkaline phosphatase. A semi-automated flow injection-bead injection system was proposed to conveniently and selectively assay bone alkaline phosphatase (BALP) based on its specific binding to wheat germ coated beads. Amount of BALP in serum was determined from the intensity of the yellow product produced from bound BALP on the retained beads and its substrate pNPP. The used beads were discarded and the fresh ones were introduced for the next analysis. The reaction cell was designed to be opened and closed using a computer controlled solenoid valve for a precise incubation time. The performance of the proposed system was evaluated by using it to assay BALP in human serum. The results were compared to those obtained by using a commercial ELISA kit. The system is proposed to be an easy and cost effective system for quantification of BALP as an alternative to batch wise wheat germ specific binding technique.

In Silico Analyses of Substrate Interactions with Human Serum Paraoxonase 1

DTIC Science & Technology

2008-01-01

substrate interactions of HuPON1 remains elusive. In this study, we apply homology modeling, docking, and molecular dynamic (MD) simulations to probe the...mod- eling; docking; molecular dynamics simulations ; binding free energy decomposition. 486 PROTEINS Published 2008 WILEY-LISS, INC. yThis article is a...apply homology modeling, docking, and molecular dynamic (MD) simulations to probe the binding interactions of HuPON1 with representative substrates. The

Analysis of Substrate Access to Active Sites in Bacterial Multicomponent Monooxygenase Hydroxylases: X-ray Crystal Structure of Xenon-Pressurized Phenol Hydroxylase from Pseudomonas sp. OX1†,‡

PubMed Central

McCormick, Michael S.; Lippard, Stephen J.

2011-01-01

In all structurally characterized bacterial multicomponent monooxygenase (BMM) hydroxylase proteins, a series of hydrophobic cavities in the α-subunit trace a conserved path from the protein exterior to the carboxylate-bridged diiron active site. The present study examines these cavities as a potential route for dioxygen transport to the active site by crystallographic characterization of a xenon-pressurized sample of the hydroxylase component of phenol hydroxylase from Pseudomonas sp. OX1. Computational analyses of the hydrophobic cavities in the hydroxylase α-subunits of phenol hydroxylase (PHH), toluene/o-xylene monooxygenase (ToMOH), and soluble methane monooxygenase (sMMOH) are also presented. The results, together with previous findings from crystallographic studies of xenon-pressurized sMMO hydroxylase, clearly identify the propensity for these cavities to bind hydrophobic gas molecules in the protein interior. This proposed functional role is supported by recent stopped flow kinetic studies of ToMOH variants (Song, et al., 2011). In addition to information about the Xe sites, the structure determination revealed significantly reduced regulatory protein binding to the hydroxylase in comparison to the previously reported structure of PHH, as well as the presence of a newly identified metal binding site in the α-subunit that adopts a linear coordination environment consistent with Cu(I), and a glycerol molecule bound to Fe1 in a fashion that is unique among hydrocarbon-diiron site adducts reported to date in BMM hydroxylase structures. Finally, a comparative analysis of the α-subunit structures of MMOH, ToMOH, and PHH details proposed routes for the other three BMM substrates, the hydrocarbon, electrons, and protons, comprising cavities, channels, hydrogen-bonding networks, and pores in the structures of their α-subunits. PMID:22136180

Specificity and Versatility of Substrate Binding Sites in Four Catalytic Domains of Human N-Terminal Acetyltransferases

PubMed Central

Grauffel, Cédric; Abboud, Angèle; Liszczak, Glen; Marmorstein, Ronen; Arnesen, Thomas; Reuter, Nathalie

2012-01-01

Nt-acetylation is among the most common protein modifications in eukaryotes. Although thought for a long time to protect proteins from degradation, the role of Nt-acetylation is still debated. It is catalyzed by enzymes called N-terminal acetyltransferases (NATs). In eukaryotes, several NATs, composed of at least one catalytic domain, target different substrates based on their N-terminal sequences. In order to better understand the substrate specificity of human NATs, we investigated in silico the enzyme-substrate interactions in four catalytic subunits of human NATs (Naa10p, Naa20p, Naa30p and Naa50p). To date hNaa50p is the only human subunit for which X-ray structures are available. We used the structure of the ternary hNaa50p/AcCoA/MLG complex and a structural model of hNaa10p as a starting point for multiple molecular dynamics simulations of hNaa50p/AcCoA/substrate (substrate = MLG, EEE, MKG), hNaa10p/AcCoA/substrate (substrate = MLG, EEE). Nine alanine point-mutants of the hNaa50p/AcCoA/MLG complex were also simulated. Homology models of hNaa20p and hNaa30p were built and compared to hNaa50p and hNaa10p. The simulations of hNaa50p/AcCoA/MLG reproduce the interactions revealed by the X-ray data. We observed strong hydrogen bonds between MLG and tyrosines 31, 138 and 139. Yet the tyrosines interacting with the substrate’s backbone suggest that their role in specificity is limited. This is confirmed by the simulations of hNaa50p/AcCoA/EEE and hNaa10p/AcCoA/MLG, where these hydrogen bonds are still observed. Moreover these tyrosines are all conserved in hNaa20p and hNaa30p. Other amino acids tune the specificity of the S1’ sites that is different for hNaa10p (acidic), hNaa20p (hydrophobic/basic), hNaa30p (basic) and hNaa50p (hydrophobic). We also observe dynamic correlation between the ligand binding site and helix that tightens under substrate binding. Finally, by comparing the four structures we propose maps of the peptide-enzyme interactions that should help rationalizing substrate-specificity and lay the ground for inhibitor design. PMID:23285125

An allosteric conduit facilitates dynamic multisite substrate recognition by the SCFCdc4 ubiquitin ligase

NASA Astrophysics Data System (ADS)

Csizmok, Veronika; Orlicky, Stephen; Cheng, Jing; Song, Jianhui; Bah, Alaji; Delgoshaie, Neda; Lin, Hong; Mittag, Tanja; Sicheri, Frank; Chan, Hue Sun; Tyers, Mike; Forman-Kay, Julie D.

2017-01-01

The ubiquitin ligase SCFCdc4 mediates phosphorylation-dependent elimination of numerous substrates by binding one or more Cdc4 phosphodegrons (CPDs). Methyl-based NMR analysis of the Cdc4 WD40 domain demonstrates that Cyclin E, Sic1 and Ash1 degrons have variable effects on the primary Cdc4WD40 binding pocket. Unexpectedly, a Sic1-derived multi-CPD substrate (pSic1) perturbs methyls around a previously documented allosteric binding site for the chemical inhibitor SCF-I2. NMR cross-saturation experiments confirm direct contact between pSic1 and the allosteric pocket. Phosphopeptide affinity measurements reveal negative allosteric communication between the primary CPD and allosteric pockets. Mathematical modelling indicates that the allosteric pocket may enhance ultrasensitivity by tethering pSic1 to Cdc4. These results suggest negative allosteric interaction between two distinct binding pockets on the Cdc4WD40 domain may facilitate dynamic exchange of multiple CPD sites to confer ultrasensitive dependence on substrate phosphorylation.

Toward a mechanistic understanding of patterns in biomineralization and new insights for old dogmas in geological settings (Invited)

NASA Astrophysics Data System (ADS)

Dove, P. M.; Hamm, L.; Giuffre, A. J.; Han, N.; De Yoreo, J. J.

2013-12-01

The ability of organisms to mineralize tissues into skeletons and other functional structures is a remarkable achievement of biology. Yet, the physical basis for how macromolecules regulate the placement and onset of mineral formation is not well established. Efforts to understand nucleation onto organic substrates have produced two, seemingly contradictory, lines of thought: The biomineralization community widely assumes the organic matrix promotes nucleation through stereochemical matching to guide the organization of solute ions, while materials synthesis groups use simple binding assays to correlate high binding strength with good promoters of nucleation. This study reconciles the two views and provides a mechanistic explanation for template-directed nucleation by correlating heterogeneous nucleation barriers with crystal-substrate binding free energies. Using surface assembled monolayers (SAM) as simple model systems, we first measure the kinetics of calcite nucleation onto model substrates that present different functional group chemistries (carboxyl, thiol, phosphate, hydroxyl) and conformations (C11, C16 chain lengths). We find rates are substrate-specific and obey predictions of classical nucleation theory at supersaturations that extend above the solubility of amorphous calcium carbonate (ACC). Analysis of the kinetic data shows the thermodynamic barrier to nucleation is reduced by minimizing the interfacial free energy of the system, γ. We then use dynamic force spectroscopy to independently measure calcite-substrate binding free energies, ΔGb. Moreover, we show that within the classical theory of nucleation, γ and ΔGb should be linearly related. The results bear out this prediction and demonstrate that low energy barriers to nucleation correlate with strong crystal-substrate binding. This relationship is general to all functional group chemistries and conformations. These findings reconcile the long-standing concept of templated nucleation through stereochemical matching with the conventional wisdom that ';good binders are good nucleators'. Alternative perspectives become internally consistent when viewed through the lens of crystal-substrate binding and provide a physical basis for how organic chemistry can direct temporal and spatial patterns of carbonate nucleation.

Conformational Dynamics, Ligand Binding and Effects of Mutations in NirE an S-Adenosyl-L-Methionine Dependent Methyltransferase

NASA Astrophysics Data System (ADS)

Singh, Warispreet; Karabencheva-Christova, Tatyana G.; Black, Gary W.; Ainsley, Jon; Dover, Lynn; Christov, Christo Z.

2016-01-01

Heme d1, a vital tetrapyrrol involved in the denitrification processes is synthesized from its precursor molecule precorrin-2 in a chemical reaction catalysed by an S-adenosyl-L-methionine (SAM) dependent Methyltransferase (NirE). The NirE enzyme catalyses the transfer of a methyl group from the SAM to uroporphyrinogen III and serves as a novel potential drug target for the pharmaceutical industry. An important insight into the structure-activity relationships of NirE has been revealed by elucidating its crystal structure, but there is still no understanding about how conformational flexibility influences structure, cofactor and substrate binding by the enzyme as well as the structural effects of mutations of residues involved in binding and catalysis. In order to provide this missing but very important information we performed a comprehensive atomistic molecular dynamics study which revealed that i) the binding of the substrate contributes to the stabilization of the structure of the full complex; ii) conformational changes influence the orientation of the pyrrole rings in the substrate, iii) more open conformation of enzyme active site to accommodate the substrate as an outcome of conformational motions; and iv) the mutations of binding and active site residues lead to sensitive structural changes which influence binding and catalysis.

Substrate and pH-Dependent Kinetic Profile of 3-Mercaptopropionate Dioxygenase from Pseudomonas aeruginosa.

PubMed

Fellner, Matthias; Aloi, Sekotilani; Tchesnokov, Egor P; Wilbanks, Sigurd M; Jameson, Guy N L

2016-03-08

Thiol dioxygenases catalyze the synthesis of sulfinic acids in a range of organisms from bacteria to mammals. A thiol dioxygenase from the bacterium Pseudomonas aeruginosa oxidizes both 3-mercaptopropionic acid and cysteine, with a ∼70 fold preference for 3-mercaptopropionic acid over all pHs. This substrate reactivity is widened compared to other thiol dioxygenases and was exploited in this investigation of the residues important for activity. A simple model incorporating two protonation events was used to fit profiles of the Michaelis-Menten parameters determined at different pH values for both substrates. The pKs determined using plots of k(cat)/Km differ at low pH, but not in a way easily attributable to protonation of the substrate alone and share a common value at higher pH. Plots of k(cat) versus pH are also quite different at low pH showing the monoprotonated ES complexes with 3-mercaptopropionic acid and cysteine have different pKs. At higher pH, k(cat) decreases sigmoidally with a similar pK regardless of substrate. Loss of reactivity at high pH is attributed to deprotonation of tyrosine 159 and its influence on dioxygen binding. A mechanism is proposed by which deprotonation of tyrosine 159 both blocks oxygen binding and concomitantly promotes cystine formation. Finally, the role of tyrosine 159 was further probed by production of a G95C variant that is able to form a cysteine-tyrosine crosslink homologous to that found in mammalian cysteine dioxygenases. Activity of this variant is severely impaired. Crystallography shows that when un-crosslinked, the cysteine thiol excludes tyrosine 159 from its native position, while kinetic analysis shows that the thioether bond impairs reactivity of the crosslinked form.

Mechanistic and Structural Analysis of a Drosophila melanogaster Enzyme, Arylalkylamine N-Acetyltransferase Like 7, an Enzyme That Catalyzes the Formation of N-Acetylarylalkylamides and N-Acetylhistamine.

PubMed

Dempsey, Daniel R; Jeffries, Kristen A; Handa, Sumit; Carpenter, Anne-Marie; Rodriguez-Ospina, Santiago; Breydo, Leonid; Merkler, David J

2015-04-28

Arylalkylamine N-acetyltransferase like 7 (AANATL7) catalyzes the formation of N-acetylarylalkylamides and N-acetylhistamine from acetyl-CoA and the corresponding amine substrate. AANATL7 is a member of the GNAT superfamily of >10000 GCN5-related N-acetyltransferases, many members being linked to important roles in both human metabolism and disease. Drosophila melanogaster utilizes the N-acetylation of biogenic amines for the inactivation of neurotransmitters, the biosynthesis of melatonin, and the sclerotization of the cuticle. We have expressed and purified D. melanogaster AANATL7 in Escherichia coli and used the purified enzyme to define the substrate specificity for acyl-CoA and amine substrates. Information about the substrate specificity provides insight into the potential contribution made by AANATL7 to fatty acid amide biosynthesis because D. melanogaster has emerged as an important model system contributing to our understanding of fatty acid amide metabolism. Characterization of the kinetic mechanism of AANATL7 identified an ordered sequential mechanism, with acetyl-CoA binding first followed by histamine to generate an AANATL7·acetyl-CoA·histamine ternary complex prior to catalysis. Successive pH-activity profiling and site-directed mutagenesis experiments identified two ionizable groups: one with a pKa of 7.1 that is assigned to Glu-26 as a general base and a second pKa of 9.5 that is assigned to the protonation of the thiolate of the coenzyme A product. Using the data generated herein, we propose a chemical mechanism for AANATL7 and define functions for other important amino acid residues involved in substrate binding and regulation of catalysis.

Structural analysis of substrate recognition by glucose isomerase in Mn2+ binding mode at M2 site in S. rubiginosus.

PubMed

Bae, Ji-Eun; Hwang, Kwang Yeon; Nam, Ki Hyun

2018-06-16

Glucose isomerase (GI) catalyzes the reversible enzymatic isomerization of d-glucose and d-xylose to d-fructose and d-xylulose, respectively. This is one of the most important enzymes in the production of high-fructose corn syrup (HFCS) and biofuel. We recently determined the crystal structure of GI from S. rubiginosus (SruGI) complexed with a xylitol inhibitor in one metal binding mode. Although we assessed inhibitor binding at the M1 site, the metal binding at the M2 site and the substrate recognition mechanism for SruGI remains the unclear. Here, we report the crystal structure of the two metal binding modes of SruGI and its complex with glucose. This study provides a snapshot of metal binding at the SruGI M2 site in the presence of Mn 2+ , but not in the presence of Mg 2+ . Metal binding at the M2 site elicits a configuration change at the M1 site. Glucose molecule can only bind to the M1 site in presence of Mn 2+ at the M2 site. Glucose and Mn 2+ at the M2 site were bridged by water molecules using a hydrogen bonding network. The metal binding geometry of the M2 site indicates a distorted octahedral coordination with an angle of 55-110°, whereas the M1 site has a relatively stable octahedral coordination with an angle of 85-95°. We suggest a two-step sequential process for SruGI substrate recognition, in Mn 2+ binding mode, at the M2 site. Our results provide a better understanding of the molecular role of the M2 site in GI substrate recognition. Copyright © 2018. Published by Elsevier Inc.

Yeast ribonuclease III uses a network of multiple hydrogen bonds for RNA binding and cleavage.

PubMed

Lavoie, Mathieu; Abou Elela, Sherif

2008-08-19

Members of the bacterial RNase III family recognize a variety of short structured RNAs with few common features. It is not clear how this group of enzymes supports high cleavage fidelity while maintaining a broad base of substrates. Here we show that the yeast orthologue of RNase III (Rnt1p) uses a network of 2'-OH-dependent interactions to recognize substrates with different structures. We designed a series of bipartite substrates permitting the distinction between binding and cleavage defects. Each substrate was engineered to carry a single or multiple 2'- O-methyl or 2'-fluoro ribonucleotide substitutions to prevent the formation of hydrogen bonds with a specific nucleotide or group of nucleotides. Interestingly, introduction of 2'- O-methyl ribonucleotides near the cleavage site increased the rate of catalysis, indicating that 2'-OH are not required for cleavage. Substitution of nucleotides in known Rnt1p binding site with 2'- O-methyl ribonucleotides inhibited cleavage while single 2'-fluoro ribonucleotide substitutions did not. This indicates that while no single 2'-OH is essential for Rnt1p cleavage, small changes in the substrate structure are not tolerated. Strikingly, several nucleotide substitutions greatly increased the substrate dissociation constant with little or no effect on the Michaelis-Menten constant or rate of catalysis. Together, the results indicate that Rnt1p uses a network of nucleotide interactions to identify its substrate and support two distinct modes of binding. One mode is primarily mediated by the dsRNA binding domain and leads to the formation of stable RNA/protein complex, while the other requires the presence of the nuclease and N-terminal domains and leads to RNA cleavage.

Effect of substrate RNA sequence on the cleavage reaction by a short ribozyme.

PubMed Central

Ohmichi, T; Okumoto, Y; Sugimoto, N

1998-01-01

Leadzyme is a ribozyme that requires Pb2+. The catalytic sequence, CUGGGAGUCC, binds to an RNA substrate, GGACC downward arrowGAGCCAG, cleaving the RNA substrate at one site. We have investigated the effect of the substrate sequence on the cleavage activity of leadzyme using mutant substrates in order to structurally understand the RNA catalysis. The results showed that leadzyme acted as a catalyst for single site cleavage of a C5 deletion mutant substrate, GGAC downward arrowGAGCCAG, as well as the wild-type substrate. However, a mutant substrate GGACCGACCAG, which had G8 deleted from the wild-type substrate, was not cleaved. Kinetic studies by surface plasmon resonance indicated that the difference between active and inactive structures reflected the slow association and dissociation rate constants of complex formation induced by Pb2+rather than differences in complex stability. CD spectra showed that the active form of the substrate-leadzyme complex was rearranged by Pb2+binding. The G8 of the wild-type substrate, which was absent in the inactive complex, is not near the cleavage site. Thus, these results show that the active substrate-leadzyme complex has a Pb2+binding site at the junction between the unpaired region (asymmetric internal loop) and the stem region, which is distal to the cleavage site. Pb2+may play a role in rearranging the bases in the asymmetric internal loop to the correct position for catalysis. PMID:9837996

Inhibition of ATP Synthase by Chlorinated Adenosine Analogue

PubMed Central

Chen, Lisa S.; Nowak, Billie J.; Ayres, Mary L.; Krett, Nancy L.; Rosen, Steven T.; Zhang, Shuxing; Gandhi, Varsha

2009-01-01

8-Chloroadenosine (8-Cl-Ado) is a ribonucleoside analogue that is currently in clinical trial for chronic lymphocytic leukemia. Based on the decline in cellular ATP pool following 8-Cl-Ado treatment, we hypothesized that 8-Cl-ADP and 8-Cl-ATP may interfere with ATP synthase, a key enzyme in ATP production. Mitochondrial ATP synthase is composed of two major parts; FO intermembrane base and F1 domain, containing α and β subunits. Crystal structures of both α and β subunits that bind to the substrate, ADP, are known in tight binding (αdpβdp) and loose binding (αtpβtp) states. Molecular docking demonstrated that 8-Cl-ADP/8-Cl-ATP occupied similar binding modes as ADP/ATP in the tight and loose binding sites of ATP synthase, respectively, suggesting that the chlorinated nucleotide metabolites may be functional substrates and inhibitors of the enzyme. The computational predictions were consistent with our whole cell biochemical results. Oligomycin, an established pharmacological inhibitor of ATP synthase, decreased both ATP and 8-Cl-ATP formation from exogenous substrates, however, did not affect pyrimidine nucleoside analogue triphosphate accumulation. Synthesis of ATP from ADP was inhibited in cells loaded with 8-Cl-ATP. These biochemical studies are in consent with the computational modeling; in the αtpβtp state 8-Cl-ATP occupies similar binding as ANP, a non-hydrolyzable ATP mimic that is a known inhibitor. Similarly, in the substrate binding site (αdpβdp) 8-Cl-ATP occupies a similar position as ATP mimic ADP-BeF3 −. Collectively, our current work suggests that 8-Cl-ADP may serve as a substrate and the 8-Cl-ATP may be an inhibitor of ATP synthase. PMID:19477165

Enantioselective Hydroformylation of Aniline Derivatives

PubMed Central

Joe, Candice L.; Tan, Kian L.

2011-01-01

We have developed a ligand that reversibly binds to aniline substrates allowing for the control of regioselectivity and enantioselectivity in hydroformylation. In this paper we address how the electronics of the aniline ring affect both binding of the substrate to the ligand and the enantioselectivity in this reaction. PMID:21842847

In silico modeling of the cryptic E2∼ubiquitin-binding site of E6-associated protein (E6AP)/UBE3A reveals the mechanism of polyubiquitin chain assembly.

PubMed

Ronchi, Virginia P; Kim, Elizabeth D; Summa, Christopher M; Klein, Jennifer M; Haas, Arthur L

2017-11-03

To understand the mechanism for assembly of Lys 48 -linked polyubiquitin degradation signals, we previously demonstrated that the E6AP/UBE3A ligase harbors two functionally distinct E2∼ubiquitin-binding sites: a high-affinity Site 1 required for E6AP Cys 820 ∼ubiquitin thioester formation and a canonical Site 2 responsible for subsequent chain elongation. Ordered binding to Sites 1 and 2 is here revealed by observation of UbcH7∼ubiquitin-dependent substrate inhibition of chain formation at micromolar concentrations. To understand substrate inhibition, we exploited the PatchDock algorithm to model in silico UbcH7∼ubiquitin bound to Site 1, validated by chain assembly kinetics of selected point mutants. The predicted structure buries an extensive solvent-excluded surface bringing the UbcH7∼ubiquitin thioester bond within 6 Å of the Cys 820 nucleophile. Modeling onto the active E6AP trimer suggests that substrate inhibition arises from steric hindrance between Sites 1 and 2 of adjacent subunits. Confirmation that Sites 1 and 2 function in trans was demonstrated by examining the effect of E6APC820A on wild-type activity and single-turnover pulse-chase kinetics. A cyclic proximal indexation model proposes that Sites 1 and 2 function in tandem to assemble thioester-linked polyubiquitin chains from the proximal end attached to Cys 820 before stochastic en bloc transfer to the target protein. Non-reducing SDS-PAGE confirms assembly of the predicted Cys 820 -linked 125 I-polyubiquitin thioester intermediate. Other studies suggest that Glu 550 serves as a general base to generate the Cys 820 thiolate within the low dielectric binding interface and Arg 506 functions to orient Glu 550 and to stabilize the incipient anionic transition state during thioester exchange. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Structure and Mechanism of ArnA: Conformational Change Implies Ordered Dehydrogenase Mechanism in Key Enzyme for Polymyxin Resistance

PubMed Central

Gatzeva-Topalova, Petia Z.; May, Andrew P.; Sousa, Marcelo C.

2010-01-01

Summary The modification of lipid A with 4-amino-4-deoxy-L-arabinose (Ara4N) allows gram-negative bacteria to resist the antimicrobial activity of cationic antimicrobial peptides and antibiotics such as polymyxin. ArnA is the first enzyme specific to the lipid A-Ara4N pathway. It contains two functionally and physically separable domains: a dehydrogenase domain (ArnA_DH) catalyzing the NAD+-dependent oxidative decarboxylation of UDP-Glucuronic acid (UDP-GlcA), and a transformylase domain that formylates UDP-Ara4N. Here, we describe the crystal structure of the full-length bifunctional ArnA with UDP-GlcA and ATP bound to the dehydrogenase domain. Binding of UDP-GlcA triggers a 17 Å conformational change in ArnA_DH that opens the NAD+ binding site while trapping UDP-GlcA. We propose an ordered mechanism of substrate binding and product release. Mutation of residues R619 and S433 demonstrates their importance in catalysis and suggests that R619 functions as a general acid in catalysis. The proposed mechanism for ArnA_DH has important implications for the design of selective inhibitors. PMID:15939024

Substrate-Induced Facilitated Dissociation of the Competitive Inhibitor from the Active Site of O-Acetyl Serine Sulfhydrylase Reveals a Competitive-Allostery Mechanism.

PubMed

Singh, Appu Kumar; Ekka, Mary Krishna; Kaushik, Abhishek; Pandya, Vaibhav; Singh, Ravi P; Banerjee, Shrijita; Mittal, Monica; Singh, Vijay; Kumaran, S

2017-09-19

By classical competitive antagonism, a substrate and competitive inhibitor must bind mutually exclusively to the active site. The competitive inhibition of O-acetyl serine sulfhydrylase (OASS) by the C-terminus of serine acetyltransferase (SAT) presents a paradox, because the C-terminus of SAT binds to the active site of OASS with an affinity that is 4-6 log-fold (10 4 -10 6 ) greater than that of the substrate. Therefore, we employed multiple approaches to understand how the substrate gains access to the OASS active site under physiological conditions. Single-molecule and ensemble approaches showed that the active site-bound high-affinity competitive inhibitor is actively dissociated by the substrate, which is not consistent with classical views of competitive antagonism. We employed fast-flow kinetic approaches to demonstrate that substrate-mediated dissociation of full length SAT-OASS (cysteine regulatory complex) follows a noncanonical "facilitated dissociation" mechanism. To understand the mechanism by which the substrate induces inhibitor dissociation, we resolved the crystal structures of enzyme·inhibitor·substrate ternary complexes. Crystal structures reveal a competitive allosteric binding mechanism in which the substrate intrudes into the inhibitor-bound active site and disengages the inhibitor before occupying the site vacated by the inhibitor. In summary, here we reveal a new type of competitive allosteric binding mechanism by which one of the competitive antagonists facilitates the dissociation of the other. Together, our results indicate that "competitive allostery" is the general feature of noncanonical "facilitated/accelerated dissociation" mechanisms. Further understanding of the mechanistic framework of "competitive allosteric" mechanism may allow us to design a new family of "competitive allosteric drugs/small molecules" that will have improved selectivity and specificity as compared to their competitive and allosteric counterparts.

Real-time monitoring of hairpin ribozyme kinetics through base-specific quenching of fluorescein-labeled substrates.

PubMed Central

Walter, N G; Burke, J M

1997-01-01

Current methods for evaluating the kinetics of ribozyme-catalyzed reactions rely primarily on the use of radiolabeled RNA substrates, and so require tedious electrophoretic separation and quantitation of reaction products for each data point in any experiment. Here, we report the use of fluorescent substrates for real-time analysis of the time course of reactions of the hairpin ribozyme. Fluorescence of 3' fluorescein-labeled substrates was quenched upon binding to the hairpin ribozyme or its isolated substrate-binding strand (SBS), under conditions of ribozyme or SBS excess. This decrease was accompanied by an increase in anisotropy, and resulted from a base-specific quenching by a guanosine residue added to the 5' end of the SBS, close to fluorescein in the complex. Fluorescence quenching was used to determine rate constants for substrate binding (1.4 x 10(8) M(-1) min(-1)), cleavage (0.15 min(-1)), and substrate dissociation (0.010 min(-1)) by a structurally well-defined ribozyme at 25 degrees C in 50 mM Tris-HCI, pH 7.5, 12 mM MgCl2. These rates are in excellent agreement with those obtained using traditional radioisotopic methods. Measurements of dissociation rates provided physical support for interdomain interactions within the substrate-ribozyme complex. We estimate that 2.1 kcal/mol of additional substrate binding energy is provided by the B domain of the ribozyme. Part of this free energy apparently stems from coaxial stacking of helices in the hinge region between domains, and it is plausible that the remainder might be contributed by direct interactions with loop B. The fluorescence quenching and dequenching methods described here should be readily adaptable to studying a wide variety of RNA interactions and reactions using ribozymes and other model systems. PMID:9085846

Understanding transporter specificity and the discrete appearance of channel-like gating domains in transporters

PubMed Central

Diallinas, George

2014-01-01

Transporters are ubiquitous proteins mediating the translocation of solutes across cell membranes, a biological process involved in nutrition, signaling, neurotransmission, cell communication and drug uptake or efflux. Similarly to enzymes, most transporters have a single substrate binding-site and thus their activity follows Michaelis-Menten kinetics. Substrate binding elicits a series of structural changes, which produce a transporter conformer open toward the side opposite to the one from where the substrate was originally bound. This mechanism, involving alternate outward- and inward-facing transporter conformers, has gained significant support from structural, genetic, biochemical and biophysical approaches. Most transporters are specific for a given substrate or a group of substrates with similar chemical structure, but substrate specificity and/or affinity can vary dramatically, even among members of a transporter family that show high overall amino acid sequence and structural similarity. The current view is that transporter substrate affinity or specificity is determined by a small number of interactions a given solute can make within a specific binding site. However, genetic, biochemical and in silico modeling studies with the purine transporter UapA of the filamentous ascomycete Aspergillus nidulans have challenged this dogma. This review highlights results leading to a novel concept, stating that substrate specificity, but also transport kinetics and transporter turnover, are determined by subtle intramolecular interactions between a major substrate binding site and independent outward- or cytoplasmically-facing gating domains, analogous to those present in channels. This concept is supported by recent structural evidence from several, phylogenetically and functionally distinct transporter families. The significance of this concept is discussed in relationship to the role and potential exploitation of transporters in drug action. PMID:25309439

Role of Transmembrane Domain 8 in Substrate Selectivity and Translocation of SteT, a Member of the l-Amino Acid Transporter (LAT) Family*

PubMed Central

Bartoccioni, Paola; del Rio, César; Ratera, Merce; Kowalczyk, Lukasz; Baldwin, Jocelyn M.; Zorzano, Antonio; Quick, Matthias; Baldwin, Stephen A.; Vázquez-Ibar, José Luis; Palacín, Manuel

2010-01-01

System l-amino acid transporters (LAT) belong to the amino acid, polyamine, and organic cation superfamily of transporters and include the light subunits of heteromeric amino acid transporters and prokaryotic homologues. Cysteine reactivity of SteT (serine/threonine antiporter) has been used here to study the substrate-binding site of LAT transporters. Residue Cys-291, in transmembrane domain 8 (TM8), is inactivated by thiol reagents in a substrate protectable manner. Surprisingly, DTT activated the transporter by reducing residue Cys-291. Cysteine-scanning mutagenesis of TM8 showed DTT activation in the single-cysteine mutants S287C, G294C, and S298C, lining the same α-helical face. S-Thiolation in Escherichia coli cells resulted in complete inactivation of the single-cysteine mutant G294C. l-Serine blocked DTT activation with an EC50 similar to the apparent KM of this mutant. Thus, S-thiolation abolished substrate translocation but not substrate binding. Mutation of Lys-295, to Cys (K295C) broadened the profile of inhibitors and the spectrum of substrates with the exception of imino acids. A structural model of SteT based on the structural homologue AdiC (arginine/agmatine antiporter) positions residues Cys-291 and Lys-295 in the putative substrate binding pocket. All this suggests that Lys-295 is a main determinant in the recognition of the side chain of SteT substrates. In contrast, Gly-294 is not facing the surface, suggesting conformational changes involving TM8 during the transport cycle. Our results suggest that TM8 sculpts the substrate-binding site and undergoes conformational changes during the transport cycle of SteT. PMID:20610400

Cleavage-site specificity of prolyl endopeptidase FAP investigated with a full-length protein substrate.

PubMed

Huang, Chih-Hsiang; Suen, Ching-Shu; Lin, Ching-Ting; Chien, Chia-Hui; Lee, Hsin-Ying; Chung, Kuei-Min; Tsai, Ting-Yueh; Jiaang, Weir-Tong; Hwang, Ming-Jing; Chen, Xin

2011-06-01

Fibroblast activation protein (FAP) is a prolyl-cleaving endopeptidase proposed as an anti-cancer drug target. It is necessary to define its cleavage-site specificity to facilitate the identification of its in vivo substrates and to understand its biological functions. We found that the previously identified substrate of FAP, α(2)-anti-plasmin, is not a robust substrate in vitro. Instead, an intracellular protein, SPRY2, is cleavable by FAP and more suitable for investigation of its substrate specificity in the context of the full-length globular protein. FAP prefers uncharged residues, including small or bulky hydrophobic amino acids, but not charged amino acids, especially acidic residue at P1', P3 and P4 sites. Molecular modelling analysis shows that the substrate-binding site of FAP is surrounded by multiple tyrosine residues and some negatively charged residues, which may exert least preference for substrates with acidic residues. This provides an explanation why FAP cannot cleave interleukins, which have a glutamate at either P4 or P2', despite their P3-P2-P1 sites being identical to SPRY2 or α-AP. Our study provided new information on FAP cleavage-site specificity, which differs from the data obtained by profiling with a peptide library or with the denatured protein, gelatin, as the substrate. Furthermore, our study suggests that negatively charged residues should be avoided when designing FAP inhibitors.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aripirala, Srinivas; Gonzalez-Pacanowska, Dolores; Oldfield, Eric

Structural insights into L. major farnesyl diphosphate synthase, a key enzyme in the mevalonate pathway, are described. Farnesyl diphosphate synthase (FPPS) is an essential enzyme involved in the biosynthesis of sterols (cholesterol in humans and ergosterol in yeasts, fungi and trypanosomatid parasites) as well as in protein prenylation. It is inhibited by bisphosphonates, a class of drugs used in humans to treat diverse bone-related diseases. The development of bisphosphonates as antiparasitic compounds targeting ergosterol biosynthesis has become an important route for therapeutic intervention. Here, the X-ray crystallographic structures of complexes of FPPS from Leishmania major (the causative agent of cutaneousmore » leishmaniasis) with three bisphosphonates determined at resolutions of 1.8, 1.9 and 2.3 Å are reported. Two of the inhibitors, 1-(2-hydroxy-2,2-diphosphonoethyl)-3-phenylpyridinium (300B) and 3-butyl-1-(2,2-diphosphonoethyl)pyridinium (476A), co-crystallize with the homoallylic substrate isopentenyl diphosphate (IPP) and three Ca{sup 2+} ions. A third inhibitor, 3-fluoro-1-(2-hydroxy-2,2-diphosphonoethyl)pyridinium (46I), was found to bind two Mg{sup 2+} ions but not IPP. Calorimetric studies showed that binding of the inhibitors is entropically driven. Comparison of the structures of L. major FPPS (LmFPPS) and human FPPS provides new information for the design of bisphosphonates that will be more specific for inhibition of LmFPPS. The asymmetric structure of the LmFPPS–46I homodimer indicates that binding of the allylic substrate to both monomers of the dimer results in an asymmetric dimer with one open and one closed homoallylic site. It is proposed that IPP first binds to the open site, which then closes, opening the site on the other monomer, which closes after binding the second IPP, leading to the symmetric fully occupied FPPS dimer observed in other structures.« less

Asparagine 285 plays a key role in transition state stabilization in rabbit muscle creatine kinase

PubMed Central

Borders, Charles L.; MacGregor, Katherine M.; Edmiston, Paul L.; Gbeddy, Elikem R.K.; Thomenius, Michael J.; Mulligan, Guy B.; Snider, Mark J.

2003-01-01

To explore the possibility that asparagine 285 plays a key role in transition state stabilization in phosphagen kinase catalysis, the N285Q, N285D, and N285A site-directed mutants of recombinant rabbit muscle creatine kinase (rmCK) were prepared and characterized. Kinetic analysis of phosphocreatine formation showed that the catalytic efficiency of each N285 mutant was reduced by approximately four orders of magnitude, with the major cause of activity loss being a reduction in kcat in comparison to the recombinant native CK. The data for N285Q still fit a random-order, rapid-equilibrium mechanism, with either MgATP or creatine binding first with affinities very nearly equal to those for native CK. However, the affinity for the binding of the second substrate is reduced approximately 10-fold, suggesting that addition of a single methylene group at position 285 disrupts the symphony of substrate binding. The data for the N285A mutant only fit an ordered binding mechanism, with MgATP binding first. Isosteric replacement to form the N285D mutant has almost no effect on the KM values for either creatine or MgATP, thus the decrease in activity is due almost entirely to a 5000-fold reduction in kcat. Using the quenching of the intrinsic CK tryptophan fluorescence by added MgADP (Borders et al. 2002), it was found that, unlike native CK, none of the mutants have the ability to form a quaternary TSAC. We use these data to propose that asparagine 285 indeed plays a key role in transition state stabilization in the reaction catalyzed by creatine kinase and other phosphagen kinases. PMID:12592023

Asparagine 285 plays a key role in transition state stabilization in rabbit muscle creatine kinase.

PubMed

Borders, Charles L; MacGregor, Katherine M; Edmiston, Paul L; Gbeddy, Elikem R K; Thomenius, Michael J; Mulligan, Guy B; Snider, Mark J

2003-03-01

To explore the possibility that asparagine 285 plays a key role in transition state stabilization in phosphagen kinase catalysis, the N285Q, N285D, and N285A site-directed mutants of recombinant rabbit muscle creatine kinase (rmCK) were prepared and characterized. Kinetic analysis of phosphocreatine formation showed that the catalytic efficiency of each N285 mutant was reduced by approximately four orders of magnitude, with the major cause of activity loss being a reduction in k(cat) in comparison to the recombinant native CK. The data for N285Q still fit a random-order, rapid-equilibrium mechanism, with either MgATP or creatine binding first with affinities very nearly equal to those for native CK. However, the affinity for the binding of the second substrate is reduced approximately 10-fold, suggesting that addition of a single methylene group at position 285 disrupts the symphony of substrate binding. The data for the N285A mutant only fit an ordered binding mechanism, with MgATP binding first. Isosteric replacement to form the N285D mutant has almost no effect on the K(M) values for either creatine or MgATP, thus the decrease in activity is due almost entirely to a 5000-fold reduction in k(cat). Using the quenching of the intrinsic CK tryptophan fluorescence by added MgADP (Borders et al. 2002), it was found that, unlike native CK, none of the mutants have the ability to form a quaternary TSAC. We use these data to propose that asparagine 285 indeed plays a key role in transition state stabilization in the reaction catalyzed by creatine kinase and other phosphagen kinases.

Structure of photosystem II and substrate binding at room temperature

PubMed Central

Gul, Sheraz; Fuller, Franklin; Koroidov, Sergey; Brewster, Aaron S.; Tran, Rosalie; Alonso-Mori, Roberto; Kroll, Thomas; Michels-Clark, Tara; Laksmono, Hartawan; Sierra, Raymond G.; Stan, Claudiu A.; Hussein, Rana; Zhang, Miao; Douthit, Lacey; Kubin, Markus; de Lichtenberg, Casper; Long Vo, Pham; Nilsson, Håkan; Cheah, Mun Hon; Shevela, Dmitriy; Saracini, Claudio; Bean, Mackenzie A.; Seuffert, Ina; Sokaras, Dimosthenis; Weng, Tsu-Chien; Pastor, Ernest; Weninger, Clemens; Fransson, Thomas; Lassalle, Louise; Bräuer, Philipp; Aller, Pierre; Docker, Peter T.; Andi, Babak; Orville, Allen M.; Glownia, James M.; Nelson, Silke; Sikorski, Marcin; Zhu, Diling; Hunter, Mark S.; Lane, Thomas J.; Aquila, Andy; Koglin, Jason E.; Robinson, Joseph; Liang, Mengning; Boutet, Sébastien; Lyubimov, Artem Y.; Uervirojnangkoorn, Monarin; Moriarty, Nigel W.; Liebschner, Dorothee; Afonine, Pavel V.; Waterman, David G.; Evans, Gwyndaf; Wernet, Philippe; Dobbek, Holger; Weis, William I.; Brunger, Axel T.; Zwart, Petrus H.; Adams, Paul D.; Zouni, Athina; Messinger, Johannes; Bergmann, Uwe; Sauter, Nicholas K.; Kern, Jan; Yachandra, Vittal K.; Yano, Junko

2016-01-01

Light-induced oxidation of water by photosystem II (PS II) in plants, algae and cyanobacteria has generated most of the dioxygen in the atmosphere. PS II, a membrane-bound multi-subunit pigment-protein complex, couples the one-electron photochemistry at the reaction center with the four-electron redox chemistry of water oxidation at the Mn4CaO5 cluster in the oxygen-evolving complex (OEC) (Fig. 1a, Extended Data Fig. 1). Under illumination, the OEC cycles through five intermediate S-states (S0 to S4)1, where S1 is the dark stable state and S3 is the last semi-stable state before O-O bond formation and O2 evolution2,3. A detailed understanding of the O-O bond formation mechanism remains a challenge, and elucidating the structures of the OEC in the different S-states, as well as the binding of the two substrate waters to the catalytic site4-6, is a prerequisite for this purpose. Here we report the use of femtosecond pulses from an X-ray free electron laser (XFEL) to obtain damage free, room temperature (RT) structures of dark-adapted (S1), two-flash illuminated (2F; S3-enriched), and ammonia-bound two-flash illuminated (2F-NH3; S3-enriched) PS II. Although the recent 1.95 Å structure of PS II7 at cryogenic temperature using an XFEL provided a damage-free view of the S1 state, RT measurements are required to study the structural landscape of proteins under functional conditions8,9, and also for in situ advancement of the S-states. To investigate the water-binding site(s), ammonia, a water analog, has been used as a marker, as it binds to the Mn4CaO5 cluster in the S2 and S3 states10. Since the ammonia-bound OEC is active, the ammonia-binding Mn site is not a substrate water site10-13. Thus, this approach, together with a comparison of the native dark and 2F states, is used to discriminate between proposed O-O bond formation mechanisms. PMID:27871088

The Effects of Noncellulosic Compounds on the Nanoscale Interaction Forces Measured between Carbohydrate-Binding Module and Lignocellulosic Biomass.

PubMed

Arslan, Baran; Colpan, Mert; Ju, Xiaohui; Zhang, Xiao; Kostyukova, Alla; Abu-Lail, Nehal I

2016-05-09

The lack of fundamental understanding of the types of forces that govern how cellulose-degrading enzymes interact with cellulosic and noncellulosic components of lignocellulosic surfaces limits the design of new strategies for efficient conversion of biomass to bioethanol. In a step to improve our fundamental understanding of such interactions, nanoscale forces acting between a model cellulase-a carbohydrate-binding module (CBM) of cellobiohydrolase I (CBH I)-and a set of lignocellulosic substrates with controlled composition were measured using atomic force microscopy (AFM). The three model substrates investigated were kraft (KP), sulfite (SP), and organosolv (OPP) pulped substrates. These substrates varied in their surface lignin coverage, lignin type, and xylan and acetone extractives' content. Our results indicated that the overall adhesion forces of biomass to CBM increased linearly with surface lignin coverage with kraft lignin showing the highest forces among lignin types investigated. When the overall adhesion forces were decoupled into specific and nonspecific component forces via the Poisson statistical model, hydrophobic and Lifshitz-van der Waals (LW) forces dominated the binding forces of CBM to kraft lignin, whereas permanent dipole-dipole interactions and electrostatic forces facilitated the interactions of lignosulfonates to CBM. Xylan and acetone extractives' content increased the attractive forces between CBM and lignin-free substrates, most likely through hydrogen bonding forces. When the substrates treated differently were compared, it was found that both the differences in specific and nonspecific forces between lignin-containing and lignin-free substrates were the least for OPP. Therefore, cellulase enzymes represented by CBM would weakly bind to organosolv lignin. This will facilitate an easy enzyme recovery compared to other substrates treated with kraft or sulfite pulping. Our results also suggest that altering the surface hydrophobicity and the surface energy of lignin that facilitates the LW forces should be a priori to avoid nonproductive binding of cellulase to kraft lignin.

Binding of Substrates to the Central Pore of the Vps4 ATPase Is Autoinhibited by the Microtubule Interacting and Trafficking (MIT) Domain and Activated by MIT Interacting Motifs (MIMs).

PubMed

Han, Han; Monroe, Nicole; Votteler, Jörg; Shakya, Binita; Sundquist, Wesley I; Hill, Christopher P

2015-05-22

The endosomal sorting complexes required for transport (ESCRT) pathway drives reverse topology membrane fission events within multiple cellular pathways, including cytokinesis, multivesicular body biogenesis, repair of the plasma membrane, nuclear membrane vesicle formation, and HIV budding. The AAA ATPase Vps4 is recruited to membrane necks shortly before fission, where it catalyzes disassembly of the ESCRT-III lattice. The N-terminal Vps4 microtubule-interacting and trafficking (MIT) domains initially bind the C-terminal MIT-interacting motifs (MIMs) of ESCRT-III subunits, but it is unclear how the enzyme then remodels these substrates in response to ATP hydrolysis. Here, we report quantitative binding studies that demonstrate that residues from helix 5 of the Vps2p subunit of ESCRT-III bind to the central pore of an asymmetric Vps4p hexamer in a manner that is dependent upon the presence of flexible nucleotide analogs that can mimic multiple states in the ATP hydrolysis cycle. We also find that substrate engagement is autoinhibited by the Vps4p MIT domain and that this inhibition is relieved by binding of either Type 1 or Type 2 MIM elements, which bind the Vps4p MIT domain through different interfaces. These observations support the model that Vps4 substrates are initially recruited by an MIM-MIT interaction that activates the Vps4 central pore to engage substrates and generate force, thereby triggering ESCRT-III disassembly. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Binding of Substrates to the Central Pore of the Vps4 ATPase Is Autoinhibited by the Microtubule Interacting and Trafficking (MIT) Domain and Activated by MIT Interacting Motifs (MIMs)*

PubMed Central

Han, Han; Monroe, Nicole; Votteler, Jörg; Shakya, Binita; Sundquist, Wesley I.; Hill, Christopher P.

2015-01-01

The endosomal sorting complexes required for transport (ESCRT) pathway drives reverse topology membrane fission events within multiple cellular pathways, including cytokinesis, multivesicular body biogenesis, repair of the plasma membrane, nuclear membrane vesicle formation, and HIV budding. The AAA ATPase Vps4 is recruited to membrane necks shortly before fission, where it catalyzes disassembly of the ESCRT-III lattice. The N-terminal Vps4 microtubule-interacting and trafficking (MIT) domains initially bind the C-terminal MIT-interacting motifs (MIMs) of ESCRT-III subunits, but it is unclear how the enzyme then remodels these substrates in response to ATP hydrolysis. Here, we report quantitative binding studies that demonstrate that residues from helix 5 of the Vps2p subunit of ESCRT-III bind to the central pore of an asymmetric Vps4p hexamer in a manner that is dependent upon the presence of flexible nucleotide analogs that can mimic multiple states in the ATP hydrolysis cycle. We also find that substrate engagement is autoinhibited by the Vps4p MIT domain and that this inhibition is relieved by binding of either Type 1 or Type 2 MIM elements, which bind the Vps4p MIT domain through different interfaces. These observations support the model that Vps4 substrates are initially recruited by an MIM-MIT interaction that activates the Vps4 central pore to engage substrates and generate force, thereby triggering ESCRT-III disassembly. PMID:25833946

Structures of Saccharomyces cerevisiae D-arabinose dehydrogenase Ara1 and its complex with NADPH: implications for cofactor-assisted substrate recognition.

PubMed

Hu, Xiao-Qian; Guo, Peng-Chao; Ma, Jin-Di; Li, Wei-Fang

2013-11-01

The primary role of yeast Ara1, previously mis-annotated as a D-arabinose dehydrogenase, is to catalyze the reduction of a variety of toxic α,β-dicarbonyl compounds using NADPH as a cofactor at physiological pH levels. Here, crystal structures of Ara1 in apo and NADPH-complexed forms are presented at 2.10 and 2.00 Å resolution, respectively. Ara1 exists as a homodimer, each subunit of which adopts an (α/β)8-barrel structure and has a highly conserved cofactor-binding pocket. Structural comparison revealed that induced fit upon NADPH binding yielded an intact active-site pocket that recognizes the substrate. Moreover, the crystal structures combined with computational simulation defined an open substrate-binding site to accommodate various substrates that possess a dicarbonyl group.

The solution structure of the prototype foamy virus RNase H domain indicates an important role of the basic loop in substrate binding.

PubMed

Leo, Berit; Schweimer, Kristian; Rösch, Paul; Hartl, Maximilian J; Wöhrl, Birgitta M

2012-09-10

The ribonuclease H (RNase H) domains of retroviral reverse transcriptases play an essential role in the replication cycle of retroviruses. During reverse transcription of the viral genomic RNA, an RNA/DNA hybrid is created whose RNA strand needs to be hydrolyzed by the RNase H to enable synthesis of the second DNA strand by the DNA polymerase function of the reverse transcriptase. Here, we report the solution structure of the separately purified RNase H domain from prototype foamy virus (PFV) revealing the so-called C-helix and the adjacent basic loop, which both were suggested to be important in substrate binding and activity. The solution structure of PFV RNase H shows that it contains a mixed five-stranded β-sheet, which is sandwiched by four α-helices (A-D), including the C-helix, on one side and one α-helix (helix E) on the opposite side. NMR titration experiments demonstrate that upon substrate addition signal changes can be detected predominantly in the basic loop as well as in the C-helix. All these regions are oriented towards the bound substrate. In addition, signal intensities corresponding to residues in the B-helix and the active site decrease, while only minor or no changes of the overall structure of the RNase H are detectable upon substrate binding. Dynamic studies confirm the monomeric state of the RNase H domain. Structure comparisons with HIV-1 RNase H, which lacks the basic protrusion, indicate that the basic loop is relevant for substrate interaction, while the C-helix appears to fulfill mainly structural functions, i.e. positioning the basic loop in the correct orientation for substrate binding. The structural data of PFV RNase H demonstrate the importance of the basic loop, which contains four positively charged lysines, in substrate binding and the function of the C-helix in positioning of the loop. In the dimeric full length HIV-1 RT, the function of the basic loop is carried out by a different loop, which also harbors basic residues, derived from the connection domain of the p66 subunit. Our results suggest that RNases H which are also active as separate domains might need a functional basic loop for proper substrate binding.

p68 Sam is a substrate of the insulin receptor and associates with the SH2 domains of p85 PI3K.

PubMed

Sánchez-Margalet, V; Najib, S

1999-07-23

The 68 kDa Src substrate associated during mitosis is an RNA binding protein with Src homology 2 and 3 domain binding sites. A role for Src associated in mitosis 68 as an adaptor protein in signaling transduction has been proposed in different systems such as T-cell receptors. In the present work, we have sought to assess the possible role of Src associated in mitosis 68 in insulin receptor signaling. We performed in vivo studies in HTC-IR cells and in vitro studies using recombinant Src associated in mitosis 68, purified insulin receptor and fusion proteins containing either the N-terminal or the C-terminal Src homology 2 domain of p85 phosphatidylinositol-3-kinase. We have found that Src associated in mitosis 68 is a substrate of the insulin receptor both in vivo and in vitro. Moreover, tyrosine-phosphorylated Src associated in mitosis 68 was found to associate with p85 phosphatidylinositol-3-kinase in response to insulin, as assessed by co-immunoprecipitation studies. Therefore, Src associated in mitosis 68 may be part of the signaling complexes of insulin receptor along with p85. In vitro studies demonstrate that Src associated in mitosis 68 associates with the Src homology 2 domains of p85 after tyrosine phosphorylation by the activated insulin receptor. Moreover, tyr-phosphorylated Src associated in mitosis 68 binds with a higher affinity to the N-terminal Src homology 2 domain of p85 compared to the C-terminal Src homology 2 domain of p85, suggesting a preferential association of Src associated in mitosis 68 with the N-terminal Src homology 2 domain of p85. This association may be important for the link of the signaling with RNA metabolism.

Structural Basis for Recognition of L-lysine, L-ornithine, and L-2,4-diamino Butyric Acid by Lysine Cyclodeaminase.

PubMed

Min, Kyungjin; Yoon, Hye-Jin; Matsuura, Atsushi; Kim, Yong Hwan; Lee, Hyung Ho

2018-04-30

L-pipecolic acid is a non-protein amino acid commonly found in plants, animals, and microorganisms. It is a well-known precursor to numerous microbial secondary metabolites and pharmaceuticals, including anticancer agents, immunosuppressants, and several antibiotics. Lysine cyclodeaminase (LCD) catalyzes β-deamination of L-lysine into L-pipecolic acid using β-nicotinamide adenine dinucleotide as a cofactor. Expression of a human homolog of LCD, μ-crystallin, is elevated in prostate cancer patients. To understand the structural features and catalytic mechanisms of LCD, we determined the crystal structures of Streptomyces pristinaespiralis LCD (SpLCD) in (i) a binary complex with NAD + , (ii) a ternary complex with NAD + and L-pipecolic acid, (iii) a ternary complex with NAD + and L-proline, and (iv) a ternary complex with NAD + and L-2,4-diamino butyric acid. The overall structure of SpLCD was similar to that of ornithine cyclodeaminase from Pseudomonas putida . In addition, SpLCD recognized L-lysine, L-ornithine, and L-2,4-diamino butyric acid despite differences in the active site, including differences in hydrogen bonding by Asp236, which corresponds with Asp228 from Pseudomonas putida ornithine cyclodeaminase. The substrate binding pocket of SpLCD allowed substrates smaller than lysine to bind, thus enabling binding to ornithine and L-2,4-diamino butyric acid. Our structural and biochemical data facilitate a detailed understanding of substrate and product recognition, thus providing evidence for a reaction mechanism for SpLCD. The proposed mechanism is unusual in that NAD + is initially converted into NADH and then reverted back into NAD + at a late stage of the reaction.

Modeling of substrate and inhibitor binding to phospholipase A2.

PubMed

Sessions, R B; Dauber-Osguthorpe, P; Campbell, M M; Osguthorpe, D J

1992-09-01

Molecular graphics and molecular mechanics techniques have been used to study the mode of ligand binding and mechanism of action of the enzyme phospholipase A2. A substrate-enzyme complex was constructed based on the crystal structure of the apoenzyme. The complex was minimized to relieve initial strain, and the structural and energetic features of the resultant complex analyzed in detail, at the molecular and residue level. The minimized complex was then used as a basis for examining the action of the enzyme on modified substrates, binding of inhibitors to the enzyme, and possible reaction intermediate complexes. The model is compatible with the suggested mechanism of hydrolysis and with experimental data about stereoselectivity, efficiency of hydrolysis of modified substrates, and inhibitor potency. In conclusion, the model can be used as a tool in evaluating new ligands as possible substrates and in the rational design of inhibitors, for the therapeutic treatment of diseases such as rheumatoid arthritis, atherosclerosis, and asthma.

Mapping the signal peptide binding and oligomer contact sites of the core subunit of the pea twin arginine protein translocase.

PubMed

Ma, Xianyue; Cline, Kenneth

2013-03-01

Twin arginine translocation (Tat) systems of thylakoid and bacterial membranes transport folded proteins using the proton gradient as the sole energy source. Tat substrates have hydrophobic signal peptides with an essential twin arginine (RR) recognition motif. The multispanning cpTatC plays a central role in Tat operation: It binds the signal peptide, directs translocase assembly, and may facilitate translocation. An in vitro assay with pea (Pisum sativum) chloroplasts was developed to conduct mutagenesis and analysis of cpTatC functions. Ala scanning mutagenesis identified mutants defective in substrate binding and receptor complex assembly. Mutations in the N terminus (S1) and first stromal loop (S2) caused specific defects in signal peptide recognition. Cys matching between substrate and imported cpTatC confirmed that S1 and S2 directly and specifically bind the RR proximal region of the signal peptide. Mutations in four lumen-proximal regions of cpTatC were defective in receptor complex assembly. Copurification and Cys matching analyses suggest that several of the lumen proximal regions may be important for cpTatC-cpTatC interactions. Surprisingly, RR binding domains of adjacent cpTatCs directed strong cpTatC-cpTatC cross-linking. This suggests clustering of binding sites on the multivalent receptor complex and explains the ability of Tat to transport cross-linked multimers. Transport of substrate proteins cross-linked to the signal peptide binding site tentatively identified mutants impaired in the translocation step.

Analysis of the reaction of carbachol with acetylcholinesterase using thioflavin T as a coupled fluorescence reporter.

PubMed

Rosenberry, Terrone L; Sonoda, Leilani K; Dekat, Sarah E; Cusack, Bernadette; Johnson, Joseph L

2008-12-09

Acetylcholinesterase (AChE) contains a narrow and deep active site gorge with two sites of ligand binding, an acylation site (or A-site) at the base of the gorge and a peripheral site (or P-site) near the gorge entrance. The P-site contributes to catalytic efficiency by transiently binding substrates on their way to the acylation site, where a short-lived acylated enzyme intermediate is produced. Carbamates are very poor substrates that, like other AChE substrates, form an initial enzyme-substrate complex with free AChE (E) and proceed to an acylated enzyme intermediate (EC), which is then hydrolyzed. However, the hydrolysis of EC is slow enough to resolve the acylation and deacylation steps on the catalytic pathway. Here, we focus on the reaction of carbachol (carbamoylcholine) with AChE. The kinetics and thermodynamics of this reaction are of special interest because carbachol is an isosteric analogue of the physiological substrate acetylcholine. We show that the reaction can be monitored with thioflavin T as a fluorescent reporter group. The fluorescence of thioflavin T is strongly enhanced when it binds to the P-site of AChE, and this fluorescence is partially quenched when a second ligand binds to the A-site to form a ternary complex. Analysis of the fluorescence reaction profiles was challenging because four thermodynamic parameters and two fluorescence coefficients were fitted from the combined data both for E and for EC. Respective equilibrium dissociation constants of 6 and 26 mM were obtained for carbachol binding to the A- and P-sites in E and of 2 and 32 mM for carbachol binding to the A- and P-sites in EC. These constants for the binding of carbachol to the P-site are about an order of magnitude larger (i.e., indicating lower affinity) than previous estimates for the binding of acetylthiocholine to the P-site.

Analysis of the reaction of carbachol with acetylcholinesterase with thioflavin T as a coupled fluorescence reporter†

PubMed Central

Rosenberry, Terrone L.; Sonoda, Leilani K.; Dekat, Sarah E.; Cusack, Bernadette; Johnson, Joseph L.

2009-01-01

Acetylcholinesterase (AChE) contains a narrow and deep active site gorge with two sites of ligand binding, an acylation site (or A-site) at the base of the gorge and a peripheral site (or P-site) near the gorge entrance. The P-site contributes to catalytic efficiency by transiently binding substrates on their way to the acylation site, where a short-lived acylated enzyme intermediate is produced. Carbamates are very poor substrates that, like other AChE substrates, form an initial enzyme-substrate complex with free AChE (E) and proceed to an acylated enzyme intermediate (EC) which is then hydrolyzed. However, the hydrolysis of EC is slow enough to resolve the acylation and deacylation steps on the catalytic pathway. Here we focus on the reaction of carbachol (carbamoylcholine) with AChE. The kinetics and thermodynamics of this reaction are of special interest because carbachol is an isosteric analog of the physiological substrate acetylcholine. We show that the reaction can be monitored with thioflavin T as a fluorescent reporter group. The fluorescence of thioflavin T is strongly enhanced when it binds to the P-site of AChE, and this fluorescence is partially quenched when a second ligand binds to the A-site to form a ternary complex. Analysis of the fluorescence reaction profiles was challenging, because four thermodynamic parameters and two fluorescence coefficients were fitted from the combined data both for E and for EC. Respective equilibrium dissociation constants of 6 and 26 mM were obtained for carbachol binding to the A- and P-sites in E and of 2 and 32 mM for carbachol binding to the A- and P-sites in EC. These constants for the binding of carbachol to the P-site are about an order of magnitude larger (i.e., indicating lower affinity) than previous estimates for the binding of acetylthiocholine to the P-site. PMID:19006330

Method and apparatus for detection of fluorescently labeled materials

DOEpatents

Stern, David; Fiekowsky, Peter

2004-05-25

Fluorescently marked targets bind to a substrate 230 synthesized with polymer sequences at known locations. The targets are detected by exposing selected regions of the substrate 230 to light from a light source 100 and detecting the photons from the light fluoresced therefrom, and repeating the steps of exposure and detection until the substrate 230 is completely examined. The resulting data can be used to determine binding affinity of the targets to specific polymer sequences.

Cellular Uptake of Chloroquine Is Dependent on Binding to Ferriprotoporphyrin IX and Is Independent of NHE Activity in Plasmodium falciparum

PubMed Central

Bray, Patrick G.; Janneh, Omar; Raynes, Kaylene J.; Mungthin, Mathirut; Ginsburg, Hagai; Ward, Stephen A.

1999-01-01

Here we provide definitive evidence that chloroquine (CQ) uptake in Plasmodium falciparum is determined by binding to ferriprotoporphyrin IX (FPIX). Specific proteinase inhibitors that block the degradation of hemoglobin and stop the generation of FPIX also inhibit CQ uptake. Food vacuole enzymes can generate cell-free binding, using human hemoglobin as a substrate. This binding accounts for CQ uptake into intact cells and is subject to identical inhibitor specificity. Inhibition of CQ uptake by amiloride derivatives occurs because of inhibition of CQ–FPIX binding rather than inhibition of the Na+/H+ exchanger (NHE). Inhibition of parasite NHE using a sodium-free medium does not inhibit CQ uptake nor does it alter the ability of amilorides to inhibit uptake. CQ resistance is characterized by a reduced affinity of CQ–FPIX binding that is reversible by verapamil. Diverse compounds that are known to disrupt lysosomal pH can mimic the verapamil effect. These effects are seen in sodium-free medium and are not due to stimulation of the NHE. We propose that these compounds increase CQ accumulation and overcome CQ resistance by increasing the pH of lysosomes and endosomes, thereby causing an increased affinity of binding of CQ to FPIX. PMID:10209030

Quantum mechanical approaches to in silico enzyme characterization and drug design

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nilmeier, J P; Fattebert, J L; Jacobson, M P

2012-01-17

The astonishing, exponentially increasing rates of genome sequencing has led to one of the most significant challenges for the biological and computational sciences in the 21st century: assigning the likely functions of the encoded proteins. Enzymes represent a particular challenge, and a critical one, because the universe of enzymes is likely to contain many novel functions that may be useful for synthetic biology, or as drug targets. Current approaches to protein annotation are largely based on bioinformatics. At the simplest level, this annotation involves transferring the annotations of characterized enzymes to related sequences. In practice, however, there is no simple,more » sequence based criterion for transferring annotations, and bioinformatics alone cannot propose new enzymatic functions. Structure-based computational methods have the potential to address these limitations, by identifying potential substrates of enzymes, as we and others have shown. One successful approach has used in silico 'docking' methods, more commonly applied in structure-based drug design, to identify possible metabolite substrates. A major limitation of this approach is that it only considers substrate binding, and does not directly assess the potential of the enzyme to catalyze a particular reaction using a particular substrate. That is, substrate binding affinity is necessary but not sufficient to assign function. A reaction profile is ultimately what is needed for a more complete quantitative description of function. To address this rather fundamental limitation, they propose to use quantum mechanical methods to explicitly compute transition state barriers that govern the rates of catalysis. Although quantum mechanical, and mixed quantum/classical (QM/MM), methods have been used extensively to investigate enzymatic reactions, the focus has been primarily on elucidating complex reaction mechanisms. Here, the key catalytic steps are known, and they use these methods quantify substrate specificity. That is, we bring the power of quantum mechanics to bear on the problem of annotating enzyme function, which is a novel approach. Although it has been clear to us at the Jacobson group for some time that enzyme specificity may be encoded in transition states, rather than simply substrate recognition, the main limitation has always been computational expense. Using a hierarchy of different methods, they can reduce the list of plausible substrates of an enzyme to a small number in most cases, but even identifying the transition states for a dozen plausible substrates requires significant computational effort, beyond what is practical using standard QM/MM methods. For this project, they have chosen two enzyme superfamilies which they have used as 'model systems' for functional assignment. The enolase superfamily is a large group of {alpha}-{beta} barrel enzymes with highly diverse substrates and chemical transformations. Despite decades of work, over a third of the superfamily remains unassigned, which means that the remaining cases are by definition difficult to assign. They have focused on acid sugar dehydratases, and have considerable expertise on the matter. They are also interested in the isoprenoid synthase superfamily, which is of central interest to the synthetic biology community, because these enzymes are used by nature to create complex rare natural products of medicinal value. the most notable example of this is the artemisinin, an antimalarial compound that is found in trace amounts in the wormwod root. From the standpoint of enzyme function assignment, these enzymes are intriguing because they use a small number of chemically simple substrates to generate, potentially, tens of thousands of different products. Hence, substrate binding specificity is only a small part of the challenge; the key is determining how the enzyme directs the carbocation chemistry to specific products. These more complex modeling approaches clearly require quantum mechanical methods.« less

Allosteric Signaling Is Bidirectional in an Outer-Membrane Transport Protein.

PubMed

Sikora, Arthur; Joseph, Benesh; Matson, Morgan; Staley, Jacob R; Cafiso, David S

2016-11-01

In BtuB, the Escherichia coli TonB-dependent transporter for vitamin B 12 , substrate binding to the extracellular surface unfolds a conserved energy coupling motif termed the Ton box into the periplasm. This transmembrane signaling event facilitates an interaction between BtuB and the inner-membrane protein TonB. In this study, continuous-wave and pulse electron paramagnetic resonance in a native outer-membrane preparation demonstrate that signaling also occurs from the periplasmic to the extracellular surface in BtuB. The binding of a TonB fragment to the periplasmic interface alters the configuration of the second extracellular loop and partially dissociates a spin-labeled substrate analog. Moreover, mutants in the periplasmic Ton box that are transport-defective alter the binding site for vitamin B 12 in BtuB. This work demonstrates that the Ton box and the extracellular substrate binding site are allosterically coupled in BtuB, and that TonB binding may initiate a partial round of transport. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Methyl Transfer by Substrate Signaling from a Knotted Protein Fold

PubMed Central

Christian, Thomas; Sakaguchi, Reiko; Perlinska, Agata P.; Lahoud, Georges; Ito, Takuhiro; Taylor, Erika A.; Yokoyama, Shigeyuki; Sulkowska, Joanna I.; Hou, Ya-Ming

2017-01-01

Proteins with knotted configurations are restricted in conformational space relative to unknotted proteins. Little is known if knotted proteins have sufficient dynamics to communicate between spatially separated substrate-binding sites. In bacteria, TrmD is a methyl transferase that uses a knotted protein fold to catalyze methyl transfer from S-adenosyl methionine (AdoMet) to G37-tRNA. The product m1G37-tRNA is essential for life as a determinant to maintain protein synthesis reading-frame. Using an integrated approach of structure, kinetic, and computational analysis, we show here that the structurally constrained TrmD knot is required for its catalytic activity. Unexpectedly, the TrmD knot has complex internal movements that respond to AdoMet binding and signaling. Most of the signaling propagates the free energy of AdoMet binding to stabilize tRNA binding and to assemble the active site. This work demonstrates new principles of knots as an organized structure that captures the free energies of substrate binding to facilitate catalysis. PMID:27571175

Tyrosine phosphorylation switching of a G protein.

PubMed

Li, Bo; Tunc-Ozdemir, Meral; Urano, Daisuke; Jia, Haiyan; Werth, Emily G; Mowrey, David D; Hicks, Leslie M; Dokholyan, Nikolay V; Torres, Matthew P; Jones, Alan M

2018-03-30

Heterotrimeric G protein complexes are molecular switches relaying extracellular signals sensed by G protein-coupled receptors (GPCRs) to downstream targets in the cytoplasm, which effect cellular responses. In the plant heterotrimeric GTPase cycle, GTP hydrolysis, rather than nucleotide exchange, is the rate-limiting reaction and is accelerated by a receptor-like regulator of G signaling (RGS) protein. We hypothesized that posttranslational modification of the Gα subunit in the G protein complex regulates the RGS-dependent GTPase cycle. Our structural analyses identified an invariant phosphorylated tyrosine residue (Tyr 166 in the Arabidopsis Gα subunit AtGPA1) located in the intramolecular domain interface where nucleotide binding and hydrolysis occur. We also identified a receptor-like kinase that phosphorylates AtGPA1 in a Tyr 166 -dependent manner. Discrete molecular dynamics simulations predicted that phosphorylated Tyr 166 forms a salt bridge in this interface and potentially affects the RGS protein-accelerated GTPase cycle. Using a Tyr 166 phosphomimetic substitution, we found that the cognate RGS protein binds more tightly to the GDP-bound Gα substrate, consequently reducing its ability to accelerate GTPase activity. In conclusion, we propose that phosphorylation of Tyr 166 in AtGPA1 changes the binding pattern with AtRGS1 and thereby attenuates the steady-state rate of the GTPase cycle. We coin this newly identified mechanism "substrate phosphoswitching." © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Engineering Nucleotide Specificity of Succinyl-CoA Synthetase in Blastocystis: The Emerging Role of Gatekeeper Residues.

PubMed

Vashisht, Kapil; Verma, Sonia; Gupta, Sunita; Lynn, Andrew M; Dixit, Rajnikant; Mishra, Neelima; Valecha, Neena; Hamblin, Karleigh A; Maytum, Robin; Pandey, Kailash C; van der Giezen, Mark

2017-01-24

Charged, solvent-exposed residues at the entrance to the substrate binding site (gatekeeper residues) produce electrostatic dipole interactions with approaching substrates, and control their access by a novel mechanism called "electrostatic gatekeeper effect". This proof-of-concept study demonstrates that the nucleotide specificity can be engineered by altering the electrostatic properties of the gatekeeper residues outside the binding site. Using Blastocystis succinyl-CoA synthetase (SCS, EC 6.2.1.5), we demonstrated that the gatekeeper mutant (ED) resulted in ATP-specific SCS to show high GTP specificity. Moreover, nucleotide binding site mutant (LF) had no effect on GTP specificity and remained ATP-specific. However, via combination of the gatekeeper mutant with the nucleotide binding site mutant (ED+LF), a complete reversal of nucleotide specificity was obtained with GTP, but no detectable activity was obtained with ATP. This striking result of the combined mutant (ED+LF) was due to two changes; negatively charged gatekeeper residues (ED) favored GTP access, and nucleotide binding site residues (LF) altered ATP binding, which was consistent with the hypothesis of the "electrostatic gatekeeper effect". These results were further supported by molecular modeling and simulation studies. Hence, it is imperative to extend the strategy of the gatekeeper effect in a different range of crucial enzymes (synthetases, kinases, and transferases) to engineer substrate specificity for various industrial applications and substrate-based drug design.

Electrochemical biosensor based on enzyme substrate as a linker: Application for aldolase activity with pectin-thionine complex as recognization element and signal amplification probe.

PubMed

Wang, Xiaonan; Wang, Meiwen; Zhang, Yuanyuan; Miao, Xiaocao; Huang, Yuanyuan; Zhang, Juan; Sun, Lizhou

2016-09-15

A new strategy to fabricate electrochemical biosensor is reported based on the linkage of enzyme substrate, thereby an electrochemical method to detect aldolase activity is established using pectin-thionine complex (PTC) as recognization element and signal probe. The linkage effect of fructose-1,6-bisphosphate (FBP), the substrate of aldolase, can be achieved via its strong binding to magnetic nanoparticles (MNPs)/aminophenylboronic acid (APBA) and the formation of phosphoramidate bond derived from its reaction with p-phenylenediamine (PDA) on the surface of electrode. Aldolase can reversibly catalyze the substrates into the products which have no binding capacity with MNPs/APBA, resulting in the exposure of the corresponding binding sites and its subsequent recognization on signal probe. Meanwhile, signal amplification can be accomplished by using the firstly prepared PTC which can bind with MNPs/APBA, and accuracy can be strengthened through magnetic separation. With good precision and accuracy, the established sensor may be extended to other proteins with reversible catalyzed ability. Copyright © 2016 Elsevier B.V. All rights reserved.

The Structural Basis for Allosteric Inhibition of a Threonine-sensitive Aspartokinase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Xuying; Pavlovsky, Alexander G.; Viola, Ronald E.

2008-10-08

The commitment step to the aspartate pathway of amino acid biosynthesis is the phosphorylation of aspartic acid catalyzed by aspartokinase (AK). Most microorganisms and plants have multiple forms of this enzyme, and many of these isofunctional enzymes are subject to feedback regulation by the end products of the pathway. However, the archeal species Methanococcus jannaschii has only a single, monofunctional form of AK. The substrate l-aspartate binds to this recombinant enzyme in two different orientations, providing the first structural evidence supporting the relaxed regiospecificity previously observed with several alternative substrates of Escherichia coli AK. Binding of the nucleotide substrate triggersmore » significant domain movements that result in a more compact quaternary structure. In contrast, the highly cooperative binding of the allosteric regulator l-threonine to multiple sites on this dimer of dimers leads to an open enzyme structure. A comparison of these structures supports a mechanism for allosteric regulation in which the domain movements induced by threonine binding causes displacement of the substrates from the enzyme, resulting in a relaxed, inactive conformation.« less

Binding of mouse immunoglobulin G to polylysine-coated glass substrate for immunodiagnosis

NASA Astrophysics Data System (ADS)

Vashist, Sandeep Kumar; Tewari, Rupinder; Bajpai, Ram Prakash; Bharadwaj, Lalit Mohan; Raiteri, Roberto

2006-12-01

We report a method for immobilizing mouse immunoglobulin G (IgG) on polylysine-coated glass substrate for immunodiagnostic applications. Mouse IgG molecules were immobilized on polylysine-coated glass substrate employing 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and protein A. The amino groups of the polylysine-coated glass slide were cross linked to the carboxyl groups of protein A employing EDC crosslinker. Protein A was employed as it binds to the constant Fc region of antibodies keeping their antigen binding sites on the variable F ab region free to bind to antigens. The qualitative analysis of surface immobilized mouse IgG was done by fluorescent microscopy employing fluorescein isothiocyanate (FITC) labeled mouse IgG molecules. The immobilization densities of protein A and mouse IgG were determined by 3, 3', 4, 4'-tetramethyl benzidine (TMB) substrate assay employing horse radish peroxidise labelled molecules and were found to be 130 +/- 17 ng/cm2 and 596 +/- 31 ng/cm2 respectively. The biomolecular coatings analyzed by atomic force microscopy (AFM) were found to be uniform.

Structural Chemistry of Human RNA Methyltransferases.

PubMed

Schapira, Matthieu

2016-03-18

RNA methyltransferases (RNMTs) play important roles in RNA stability, splicing, and epigenetic mechanisms. They constitute a promising target class that is underexplored by the medicinal chemistry community. Information of relevance to drug design can be extracted from the rich structural coverage of human RNMTs. In this work, the structural chemistry of this protein family is analyzed in depth. Unlike most methyltransferases, RNMTs generally feature a substrate-binding site that is largely open on the cofactor-binding pocket, favoring the design of bisubstrate inhibitors. Substrate purine or pyrimidines are often sandwiched between hydrophobic walls that can accommodate planar ring systems. When the substrate base is laying on a shallow surface, a 5' flanking base is sometimes anchored in a druggable cavity. The cofactor-binding site is structurally more diverse than in protein methyltransferases and more druggable in SPOUT than in Rossman-fold enzymes. Finally, conformational plasticity observed both at the substrate and cofactor binding sites may be a challenge for structure-based drug design. The landscape drawn here may inform ongoing efforts toward the discovery of the first human RNMT inhibitors.

Maltase protein of Ogataea (Hansenula) polymorpha is a counterpart to the resurrected ancestor protein ancMALS of yeast maltases and isomaltases.

PubMed

Viigand, Katrin; Visnapuu, Triinu; Mardo, Karin; Aasamets, Anneli; Alamäe, Tiina

2016-08-01

Saccharomyces cerevisiae maltases use maltose, maltulose, turanose and maltotriose as substrates, isomaltases use isomaltose, α-methylglucoside and palatinose and both use sucrose. These enzymes are hypothesized to have evolved from a promiscuous α-glucosidase ancMALS through duplication and mutation of the genes. We studied substrate specificity of the maltase protein MAL1 from an earlier diverged yeast, Ogataea polymorpha (Op), in the light of this hypothesis. MAL1 has extended substrate specificity and its properties are strikingly similar to those of resurrected ancMALS. Moreover, amino acids considered to determine selective substrate binding are highly conserved between Op MAL1 and ancMALS. Op MAL1 represents an α-glucosidase in which both maltase and isomaltase activities are well optimized in a single enzyme. Substitution of Thr200 (corresponds to Val216 in S. cerevisiae isomaltase IMA1) with Val in MAL1 drastically reduced the hydrolysis of maltose-like substrates (α-1,4-glucosides), confirming the requirement of Thr at the respective position for this function. Differential scanning fluorimetry (DSF) of the catalytically inactive mutant Asp199Ala of MAL1 in the presence of its substrates and selected monosaccharides suggested that the substrate-binding pocket of MAL1 has three subsites (-1, +1 and +2) and that binding is strongest at the -1 subsite. The DSF assay results were in good accordance with affinity (Km ) and inhibition (Ki ) data of the enzyme for tested substrates, indicating the power of the method to predict substrate binding. Deletion of either the maltase (MAL1) or α-glucoside permease (MAL2) gene in Op abolished the growth of yeast on MAL1 substrates, confirming the requirement of both proteins for usage of these sugars. © 2016 The Authors. Yeast published by John Wiley & Sons, Ltd. © 2016 The Authors. Yeast published by John Wiley & Sons, Ltd.

insilico Characterization and Homology Modeling of Arabitol Dehydrogenase (ArDH) from Candida albican.

PubMed

Sarwar, Muhammad Waseem; Saleem, Irum Baddisha; Ali, Asif; Abbas, Farhat

2013-01-01

Arabitol dehydrogenase (ArDH) is involved in the production of different sugar alcohols like arabitol, sorbitol, mannitol, erythritol and xylitol by using five carbon sugars as substrate. Arabinose, d-ribose, d-ribulose, xylose and d-xylulose are known substrate of this enzyme. ArDH is mainly produced by osmophilic fungi for the conversion of ribulose to arabitol under stress conditions. Recently this enzyme has been used by various industries for the production of pharmaceutically important sugar alcohols form cheap source than glucose. But the information at structure level as well as its binding energy analysis with different substrates was missing. The present study was focused on sequence analysis, insilico characterization and substrate binding analysis of ArDH from a fungus specie candida albican. Sequence analysis and physicochemical properties showed that this protein is highly stable, negatively charged and having more hydrophilic regions, these properties made this enzyme to bind with number of five carbon sugars as substrate. The predicted 3D model will helpful for further structure based studies. Docking analysis provided free energies of binding of each substrate from a best pose as arabinose -9.8224calK/mol, dribose -11.3701Kcal/mol, d-ribulose -8.9230Kcal/mol, xylose -9.7007Kcal/mol and d-xylulose 9.7802Kcal/mol. Our study provided insight information of structure and interactions of ArDH with its substrate. These results obtained from this study clearly indicate that d-ribose is best substrate for ArDH for the production of sugar alcohols. This information will be helpful for better usage of this enzyme for hyper-production of sugar alcohols by different industries.

insilico Characterization and Homology Modeling of Arabitol Dehydrogenase (ArDH) from Candida albican

PubMed Central

Sarwar, Muhammad Waseem; Saleem, Irum Baddisha; Ali, Asif; Abbas, Farhat

2013-01-01

Background: Arabitol dehydrogenase (ArDH) is involved in the production of different sugar alcohols like arabitol, sorbitol, mannitol, erythritol and xylitol by using five carbon sugars as substrate. Arabinose, d-ribose, d-ribulose, xylose and d-xylulose are known substrate of this enzyme. ArDH is mainly produced by osmophilic fungi for the conversion of ribulose to arabitol under stress conditions. Recently this enzyme has been used by various industries for the production of pharmaceutically important sugar alcohols form cheap source than glucose. But the information at structure level as well as its binding energy analysis with different substrates was missing. Results: The present study was focused on sequence analysis, insilico characterization and substrate binding analysis of ArDH from a fungus specie candida albican. Sequence analysis and physicochemical properties showed that this protein is highly stable, negatively charged and having more hydrophilic regions, these properties made this enzyme to bind with number of five carbon sugars as substrate. The predicted 3D model will helpful for further structure based studies. Docking analysis provided free energies of binding of each substrate from a best pose as arabinose -9.8224calK/mol, dribose -11.3701Kcal/mol, d-ribulose -8.9230Kcal/mol, xylose -9.7007Kcal/mol and d-xylulose 9.7802Kcal/mol. Conclusion: Our study provided insight information of structure and interactions of ArDH with its substrate. These results obtained from this study clearly indicate that d-ribose is best substrate for ArDH for the production of sugar alcohols. This information will be helpful for better usage of this enzyme for hyper-production of sugar alcohols by different industries. PMID:24391356

Identification of a botulinum C3-like enzyme in bovine brain that catalyzes ADP-ribosylation of GTP-binding proteins.

PubMed

Maehama, T; Takahashi, K; Ohoka, Y; Ohtsuka, T; Ui, M; Katada, T

1991-06-05

A novel enzyme activity was found in bovine brain cytosol that transfers the ADP-ribosyl moiety of NAD to proteins with Mr values of 22,000 and 25,000. The substrates were the same GTP-binding proteins serving as the substrate of an ADP-ribosyltransferase C3 which was produced by a type C strain of Clostridium botulinum. The brain enzyme was partially purified from the cytosol and had a molecular mass of approximately 20,000 on a gel filtration column. The brain endogenous enzyme displayed unique properties similar to those observed with botulinum C3 enzyme. The enzyme activity was markedly stimulated by a protein factor that had been initially found in the cytosol as an activator for botulinum C3-catalyzed ADP-ribosylation (Ohtsuka, T., Nagata, K., Iiri, T., Nozawa, Y., Ueno, K., Ui, M., and Katada, T. (1989) J. Biol. Chem. 264, 15000-15005). The activity of the brain enzyme was also affected by certain types of detergents or phospholipids. The substrate of the brain enzyme was specific for GTP-binding proteins serving as the substrate of botulinum C3 enzyme; the alpha-subunits of trimeric GTP-binding proteins which served as the substrate of cholera or pertussis toxin were not ADP-ribosylated by the endogenous enzyme. Thus, this is the first report showing an endogenous enzyme in mammalian cells that catalyzes ADP-ribosylation of small molecular weight GTP-binding proteins.

The effect of the physical properties of the substrate on the kinetics of cell adhesion and crawling studied by an axisymmetric diffusion-energy balance coupled model.

PubMed

Samadi-Dooki, Aref; Shodja, Hossein M; Malekmotiei, Leila

2015-05-14

In this paper an analytical approach to study the effect of the substrate physical properties on the kinetics of adhesion and motility behavior of cells is presented. Cell adhesion is mediated by the binding of cell wall receptors and substrate's complementary ligands, and tight adhesion is accomplished by the recruitment of the cell wall binders to the adhesion zone. The binders' movement is modeled as their axisymmetric diffusion in the fluid-like cell membrane. In order to preserve the thermodynamic consistency, the energy balance for the cell-substrate interaction is imposed on the diffusion equation. Solving the axisymmetric diffusion-energy balance coupled equations, it turns out that the physical properties of the substrate (substrate's ligand spacing and stiffness) have considerable effects on the cell adhesion and motility kinetics. For a rigid substrate with uniform distribution of immobile ligands, the maximum ligand spacing which does not interrupt adhesion growth is found to be about 57 nm. It is also found that as a consequence of the reduction in the energy dissipation in the isolated adhesion system, cell adhesion is facilitated by increasing substrate's stiffness. Moreover, the directional movement of cells on a substrate with gradients in mechanical compliance is explored with an extension of the adhesion formulation. It is shown that cells tend to move from soft to stiff regions of the substrate, but their movement is decelerated as the stiffness of the substrate increases. These findings based on the proposed theoretical model are in excellent agreement with the previous experimental observations.

Aurone synthase is a catechol oxidase with hydroxylase activity and provides insights into the mechanism of plant polyphenol oxidases

PubMed Central

Molitor, Christian; Mauracher, Stephan Gerhard

2016-01-01

Tyrosinases and catechol oxidases belong to the family of polyphenol oxidases (PPOs). Tyrosinases catalyze the o-hydroxylation and oxidation of phenolic compounds, whereas catechol oxidases were so far defined to lack the hydroxylation activity and catalyze solely the oxidation of o-diphenolic compounds. Aurone synthase from Coreopsis grandiflora (AUS1) is a specialized plant PPO involved in the anabolic pathway of aurones. We present, to our knowledge, the first crystal structures of a latent plant PPO, its mature active and inactive form, caused by a sulfation of a copper binding histidine. Analysis of the latent proenzyme’s interface between the shielding C-terminal domain and the main core provides insights into its activation mechanisms. As AUS1 did not accept common tyrosinase substrates (tyrosine and tyramine), the enzyme is classified as a catechol oxidase. However, AUS1 showed hydroxylase activity toward its natural substrate (isoliquiritigenin), revealing that the hydroxylase activity is not correlated with the acceptance of common tyrosinase substrates. Therefore, we propose that the hydroxylase reaction is a general functionality of PPOs. Molecular dynamics simulations of docked substrate–enzyme complexes were performed, and a key residue was identified that influences the plant PPO’s acceptance or rejection of tyramine. Based on the evidenced hydroxylase activity and the interactions of specific residues with the substrates during the molecular dynamics simulations, a novel catalytic reaction mechanism for plant PPOs is proposed. The presented results strongly suggest that the physiological role of plant catechol oxidases were previously underestimated, as they might hydroxylate their—so far unknown—natural substrates in vivo. PMID:26976571

Binding of polychlorinated biphenyls to aquatic humic substances: The role of substrate and sorbate properties on partitioning

USGS Publications Warehouse

Uhle, M.E.; Chin, Y.-P.; Aiken, G.R.; McKnight, Diane M.

1999-01-01

Two ortho- (2,2',5 and 2,2',5,6') and a non-ortho- (3,3',4,4') substituted polychlorinated biphenyl (PCB) congeners were used to study the effects of sorbate structure in binding processes to two lacustrine fulvic acids. Binding constants were determined by solubility enhancement of the solutes by the fulvic acids. The binding of the ortho-trichlorobiphenyl was significantly less than the non-ortho-substituted tetrachlorobiphenyl to both fulvic acids. Surprisingly, the measured ortho-trichlorobiphenyl binding constant to both fulvic acids was approximately the same as the ortho- substituted tetrachlorobiphenyl. The effect of the chlorines in the ortho position inhibits free rotation around the 1,1' carbon bond, thereby making the molecule less able to interact effectively with the fulvic acid substrate relative to its non-ortho-substituted congeners. Finally, binding of all three PCBs to the Great Dismal Swamp fulvic acid was significantly higher than for the Pony Lake sample. This observation is attributable to the former substrate's higher degree of aromaticity and polarizability, which can potentially interact more favorably with the PCBs through an increase in van der Waals type interactions.Two ortho- (2,2???,5 and 2,2???,5,6???) and a non-ortho- (3,3???,4,4???) substituted polychlorinated biphenyl (PCB) congeners were used to study the effects of sorbate structure in binding processes to two lacustrine fulvic acids. Binding constants were determined by solubility enhancement of the solutes by the fulvic acids. The binding of the ortho-trichlorobiphenyl was significantly less than the non-ortho-substituted tetrachlorobiphenyl to both fulvic acids. Surprisingly, the measured ortho-trichlorobiphenyl binding constant to both fulvic acids was approximately the same as the ortho-substituted tetrachlorobiphenyl. The effect of the chlorines in the ortho position inhibits free rotation around the 1,1??? carbon bond, thereby making the molecule less able to interact effectively with the fulvic acid substrate relative to its non-ortho-substituted congeners. Finally, binding of all three PCBs to the Great Dismal Swamp fulvic acid was significantly higher than for the Pony Lake sample. This observation is attributable to the former substrate's higher degree of aromaticity and polarizability, which can potentially interact more favorably with the PCBs through an increase in van der Waals type interactions.

The bacterial dicarboxylate transporter, VcINDY, uses a two-domain elevator-type mechanism

PubMed Central

Mulligan, Christopher; Fenollar-Ferrer, Cristina; Fitzgerald, Gabriel A.; Vergara-Jaque, Ariela; Kaufmann, Desirée; Li, Yan; Forrest, Lucy R.; Mindell, Joseph A.

2016-01-01

Secondary transporters use alternating access mechanisms to couple uphill substrate movement to downhill ion flux. Most known transporters utilize a “rocking bundle” motion, where the protein moves around an immobile substrate binding site. However, the glutamate transporter homolog, GltPh, translocates its substrate binding site vertically across the membrane, an “elevator” mechanism. Here, we used the “repeat swap” approach to computationally predict the outward-facing state of the Na+/succinate transporter VcINDY, from Vibrio cholerae. Our model predicts a substantial “elevator”-like movement of vcINDY’s substrate binding site, with a vertical translation of ~15 Å and a rotation of ~43°; multiple disulfide crosslinks which completely inhibit transport provide experimental confirmation and demonstrate that such movement is essential. In contrast, crosslinks across the VcINDY dimer interface preserve transport, revealing an absence of large scale coupling between protomers. PMID:26828963

Mechanisms of pseudosubstrate inhibition of the anaphase promoting complex by Acm1

PubMed Central

Burton, Janet L; Xiong, Yong; Solomon, Mark J

2011-01-01

The anaphase promoting complex (APC) is a ubiquitin ligase that promotes the degradation of cell-cycle regulators by the 26S proteasome. Cdc20 and Cdh1 are WD40-containing APC co-activators that bind destruction boxes (DB) and KEN boxes within substrates to recruit them to the APC for ubiquitination. Acm1 is an APCCdh1 inhibitor that utilizes a DB and a KEN box to bind Cdh1 and prevent substrate binding, although Acm1 itself is not a substrate. We investigated what differentiates an APC substrate from an inhibitor. We identified the Acm1 A-motif that interacts with Cdh1 and together with the DB and KEN box is required for APCCdh1 inhibition. A genetic screen identified Cdh1 WD40 domain residues important for Acm1 A-motif interaction and inhibition that appears to reside near Cdh1 residues important for DB recognition. Specific lysine insertion mutations within Acm1 promoted its ubiquitination by APCCdh1 whereas lysine removal from the APC substrate Hsl1 converted it into a potent APCCdh1 inhibitor. These findings suggest that tight Cdh1 binding combined with the inaccessibility of ubiquitinatable lysines contributes to pseudosubstrate inhibition of APCCdh1. PMID:21460798

Assessing Carbon-Based Anodes for Lithium-Ion Batteries: A Universal Description of Charge-Transfer Binding

DOE PAGES

Liu, Yuanyue; Wang, Y. Morris; Yakobson, Boris I.; ...

2014-07-11

Many key performance characteristics of carbon-based lithium-ion battery anodes are largely determined by the strength of binding between lithium (Li) and sp 2 carbon (C), which can vary significantly with subtle changes in substrate structure, chemistry, and morphology. We use density functional theory calculations to investigate the interactions of Li with a wide variety of sp 2 C substrates, including pristine, defective, and strained graphene, planar C clusters, nanotubes, C edges, and multilayer stacks. In almost all cases, we find a universal linear relation between the Li-C binding energy and the work required to fill previously unoccupied electronic states withinmore » the substrate. This suggests that Li capacity is predominantly determined by two key factors—namely, intrinsic quantum capacitance limitations and the absolute placement of the Fermi level. This simple descriptor allows for straightforward prediction of the Li-C binding energy and related battery characteristics in candidate C materials based solely on the substrate electronic structure. It further suggests specific guidelines for designing more effective C-based anodes. Furthermore, this method should be broadly applicable to charge-transfer adsorption on planar substrates, and provides a phenomenological connection to established principles in supercapacitor and catalyst design.« less

Ligand Binding Phenomena that Pertain to the Metabolic Function of Renalase

PubMed Central

Beaupre, Brett A.; Roman, Joseph V.; Hoag, Matthew R.; Meneely, Kathleen M.; Silvaggi, Nicholas R.; Lamb, Audrey L.; Moran, Graham R.

2017-01-01

Renalase catalyzes the oxidation of isomers of β-NAD(P)H that carry the hydride in the 2 or 6 positions of the nicotinamide base to form β-NAD(P)+. This activity is thought to alleviate inhibition of multiple β-NAD(P)-dependent enzymes of primary and secondary metabolism by these isomers. Here we present evidence for a variety of ligand binding phenomena relevant to the function of renalase. We offer evidence of the potential for primary metabolism inhibition with structures of malate dehydrogenase and lactate dehydrogenase bound to the 6-dihydroNAD isomer. The previously observed preference of renalase from Pseudomonas for NAD-derived substrates over those derived from NADP is accounted for by the structure of the enzyme in complex with NADPH. We also show that nicotinamide nucleosides and mononucloetides reduced in the 2- and 6-positions are renalase substrates, but bind weakly. A seven-fold enhancement of acquisition (kred/Kd) for 6-dihydronicotinamide riboside was observed for human renalase in the presence of ADP. However, generally the addition of complement ligands, ADP for mononucloetide or AMP for nucleoside substrates, did not enhance the reductive half-reaction. Non-substrate nicotinamide nucleosides or nucleotides bind weakly suggesting that only β-NADH and β-NADPH compete with dinucleotide substrates for access to the active site. PMID:27769837

Effect of the F610A mutation on substrate extrusion in the AcrB transporter: explanation and rationale by molecular dynamics simulations.

PubMed

Vargiu, Attilio V; Collu, Francesca; Schulz, Robert; Pos, Klaas M; Zacharias, Martin; Kleinekathöfer, Ulrich; Ruggerone, Paolo

2011-07-20

The tripartite efflux pump AcrAB-TolC is responsible for the intrinsic and acquired multidrug resistance in Escherichia coli. Its active part, the homotrimeric transporter AcrB, is in charge of the selective binding of substrates and energy transduction. The mutation F610A has been shown to significantly reduce the minimum inhibitory concentration of doxorubicin and many other substrates, although F610 does not appear to interact strongly with them. Biochemical study of transport kinetics in AcrB is not yet possible, except for some β-lactams, and other techniques should supply this important information. Therefore, in this work, we assess the impact of the F610A mutation on the functionality of AcrB by means of computational techniques, using doxorubicin as substrate. We found that the compound slides deeply inside the binding pocket after mutation, increasing the strength of the interaction. During subsequent conformational alterations of the transporter, doxorubicin was either not extruded from the binding site or displaced along a direction other than the one associated with extrusion. Our study indicates how subtle interactions determine the functionality of multidrug transporters, since decreased transport might not be simplistically correlated to decreased substrate binding affinity.

Evidence for an allosteric mechanism of substrate release from membrane-transporter accessory binding proteins.

PubMed

Marinelli, Fabrizio; Kuhlmann, Sonja I; Grell, Ernst; Kunte, Hans-Jörg; Ziegler, Christine; Faraldo-Gómez, José D

2011-12-06

Numerous membrane importers rely on accessory water-soluble proteins to capture their substrates. These substrate-binding proteins (SBP) have a strong affinity for their ligands; yet, substrate release onto the low-affinity membrane transporter must occur for uptake to proceed. It is generally accepted that release is facilitated by the association of SBP and transporter, upon which the SBP adopts a conformation similar to the unliganded state, whose affinity is sufficiently reduced. Despite the appeal of this mechanism, however, direct supporting evidence is lacking. Here, we use experimental and theoretical methods to demonstrate that an allosteric mechanism of enhanced substrate release is indeed plausible. First, we report the atomic-resolution structure of apo TeaA, the SBP of the Na(+)-coupled ectoine TRAP transporter TeaBC from Halomonas elongata DSM2581(T), and compare it with the substrate-bound structure previously reported. Conformational free-energy landscape calculations based upon molecular dynamics simulations are then used to dissect the mechanism that couples ectoine binding to structural change in TeaA. These insights allow us to design a triple mutation that biases TeaA toward apo-like conformations without directly perturbing the binding cleft, thus mimicking the influence of the membrane transporter. Calorimetric measurements demonstrate that the ectoine affinity of the conformationally biased triple mutant is 100-fold weaker than that of the wild type. By contrast, a control mutant predicted to be conformationally unbiased displays wild-type affinity. This work thus demonstrates that substrate release from SBPs onto their membrane transporters can be facilitated by the latter through a mechanism of allosteric modulation of the former.

Trigger Factor and DnaK possess overlapping substrate pools and binding specificities.

PubMed

Deuerling, Elke; Patzelt, Holger; Vorderwülbecke, Sonja; Rauch, Thomas; Kramer, Günter; Schaffitzel, Elke; Mogk, Axel; Schulze-Specking, Agnes; Langen, Hanno; Bukau, Bernd

2003-03-01

Ribosome-associated Trigger Factor (TF) and the DnaK chaperone system assist the folding of newly synthesized proteins in Escherichia coli. Here, we show that DnaK and TF share a common substrate pool in vivo. In TF-deficient cells, deltatig, depleted for DnaK and DnaJ the amount of aggregated proteins increases with increasing temperature, amounting to 10% of total soluble protein (approximately 340 protein species) at 37 degrees C. A similar population of proteins aggregated in DnaK depleted tig+ cells, albeit to a much lower extent. Ninety-four aggregated proteins isolated from DnaK- and DnaJ-depleted deltatig cells were identified by mass spectrometry and found to include essential cytosolic proteins. Four potential in vivo substrates were screened for chaperone binding sites using peptide libraries. Although TF and DnaK recognize different binding motifs, 77% of TF binding peptides also associated with DnaK. In the case of the nascent polypeptides TF and DnaK competed for binding, however, with competitive advantage for TF. In vivo, the loss of TF is compensated by the induction of the heat shock response and thus enhanced levels of DnaK. In summary, our results demonstrate that the co-operation of the two mechanistically distinct chaperones in protein folding is based on their overlap in substrate specificities.

Structural insights into the neuroprotective-acting carbonyl reductase Sniffer of Drosophila melanogaster.

PubMed

Sgraja, Tanja; Ulschmid, Julia; Becker, Katja; Schneuwly, Stephan; Klebe, Gerhard; Reuter, Klaus; Heine, Andreas

2004-10-01

In vivo studies with the fruit-fly Drosophila melanogaster have shown that the Sniffer protein prevents age-dependent and oxidative stress-induced neurodegenerative processes. Sniffer is a NADPH-dependent carbonyl reductase belonging to the enzyme family of short-chain dehydrogenases/reductases (SDRs). The crystal structure of the homodimeric Sniffer protein from Drosophila melanogaster in complex with NADP+ has been determined by multiple-wavelength anomalous dispersion and refined to a resolution of 1.75 A. The observed fold represents a typical dinucleotide-binding domain as detected for other SDRs. With respect to the cofactor-binding site and the region referred to as substrate-binding loop, the Sniffer protein shows a striking similarity to the porcine carbonyl reductase (PTCR). This loop, in both Sniffer and PTCR, is substantially shortened compared to other SDRs. In most enzymes of the SDR family this loop adopts a well-defined conformation only after substrate binding and remains disordered in the absence of any bound ligands or even if only the dinucleotide cofactor is bound. In the structure of the Sniffer protein, however, the conformation of this loop is well defined, although no substrate is present. Molecular modeling studies provide an idea of how binding of substrate molecules to Sniffer could possibly occur.

Crystal Structures of Active Fully Assembled Substrate- and Product-Bound Complexes of UDP-N-Acetylmuramic Acid:l-Alanine Ligase (MurC) from Haemophilus influenzae

PubMed Central

Mol, Clifford D.; Brooun, Alexei; Dougan, Douglas R.; Hilgers, Mark T.; Tari, Leslie W.; Wijnands, Robert A.; Knuth, Mark W.; McRee, Duncan E.; Swanson, Ronald V.

2003-01-01

UDP-N-acetylmuramic acid:l-alanine ligase (MurC) catalyzes the addition of the first amino acid to the cytoplasmic precursor of the bacterial cell wall peptidoglycan. The crystal structures of Haemophilus influenzae MurC in complex with its substrate UDP-N-acetylmuramic acid (UNAM) and Mg2+ and of a fully assembled MurC complex with its product UDP-N-acetylmuramoyl-l-alanine (UMA), the nonhydrolyzable ATP analogue AMPPNP, and Mn2+ have been determined to 1.85- and 1.7-Å resolution, respectively. These structures reveal a conserved, three-domain architecture with the binding sites for UNAM and ATP formed at the domain interfaces: the N-terminal domain binds the UDP portion of UNAM, and the central and C-terminal domains form the ATP-binding site, while the C-terminal domain also positions the alanine. An active enzyme structure is thus assembled at the common domain interfaces when all three substrates are bound. The MurC active site clearly shows that the γ-phosphate of AMPPNP is positioned between two bound metal ions, one of which also binds the reactive UNAM carboxylate, and that the alanine is oriented by interactions with the positively charged side chains of two MurC arginine residues and the negatively charged alanine carboxyl group. These results indicate that significant diversity exists in binding of the UDP moiety of the substrate by MurC and the subsequent ligases in the bacterial cell wall biosynthesis pathway and that alterations in the domain packing and tertiary structure allow the Mur ligases to bind sequentially larger UNAM peptide substrates. PMID:12837790

Crystal structures of active fully assembled substrate- and product-bound complexes of UDP-N-acetylmuramic acid:L-alanine ligase (MurC) from Haemophilus influenzae.

PubMed

Mol, Clifford D; Brooun, Alexei; Dougan, Douglas R; Hilgers, Mark T; Tari, Leslie W; Wijnands, Robert A; Knuth, Mark W; McRee, Duncan E; Swanson, Ronald V

2003-07-01

UDP-N-acetylmuramic acid:L-alanine ligase (MurC) catalyzes the addition of the first amino acid to the cytoplasmic precursor of the bacterial cell wall peptidoglycan. The crystal structures of Haemophilus influenzae MurC in complex with its substrate UDP-N-acetylmuramic acid (UNAM) and Mg(2+) and of a fully assembled MurC complex with its product UDP-N-acetylmuramoyl-L-alanine (UMA), the nonhydrolyzable ATP analogue AMPPNP, and Mn(2+) have been determined to 1.85- and 1.7-A resolution, respectively. These structures reveal a conserved, three-domain architecture with the binding sites for UNAM and ATP formed at the domain interfaces: the N-terminal domain binds the UDP portion of UNAM, and the central and C-terminal domains form the ATP-binding site, while the C-terminal domain also positions the alanine. An active enzyme structure is thus assembled at the common domain interfaces when all three substrates are bound. The MurC active site clearly shows that the gamma-phosphate of AMPPNP is positioned between two bound metal ions, one of which also binds the reactive UNAM carboxylate, and that the alanine is oriented by interactions with the positively charged side chains of two MurC arginine residues and the negatively charged alanine carboxyl group. These results indicate that significant diversity exists in binding of the UDP moiety of the substrate by MurC and the subsequent ligases in the bacterial cell wall biosynthesis pathway and that alterations in the domain packing and tertiary structure allow the Mur ligases to bind sequentially larger UNAM peptide substrates.

Oxidations of N-(3-indoleethyl) cyclic aliphatic amines by horseradish peroxidase: the indole ring binds to the enzyme and mediates electron-transfer amine oxidation.

PubMed

Ling, Ke-Qing; Li, Wen-Shan; Sayre, Lawrence M

2008-01-23

Although oxidations of aromatic amines by horseradish peroxidase (HRP) are well-known, typical aliphatic amines are not substrates of HRP. In this study, the reactions of N-benzyl and N-methyl cyclic amines with HRP were found to be slow, but reactions of N-(3-indoleethyl) cyclic amines were 2-3 orders of magnitude faster. Analyses of pH-rate profiles revealed a dominant contribution to reaction by the amine-free base forms, the only species found to bind to the enzyme. A metabolic study on a family of congeneric N-(3-indoleethyl) cyclic amines indicated competition between amine and indole oxidation pathways. Amine oxidation dominated for the seven- and eight-membered azacycles, where ring size supports the change in hybridization from sp3 to sp2 that occurs upon one-electron amine nitrogen oxidation, whereas only indole oxidation was observed for the six-membered ring congener. Optical difference spectroscopic binding data and computational docking simulations suggest that all the arylalkylamine substrates bind to the enzyme through their aromatic termini with similar binding modes and binding affinities. Kinetic saturation was observed for a particularly soluble substrate, consistent with an obligatory role of an enzyme-substrate complexation preceding electron transfer. The significant rate enhancements seen for the indoleethylamine substrates suggest the ability of the bound indole ring to mediate what amounts to medium long-range electron-transfer oxidation of the tertiary amine center by the HRP oxidants. This is the first systematic investigation to document aliphatic amine oxidation by HRP at rates consistent with normal metabolic turnover, and the demonstration that this is facilitated by an auxiliary electron-rich aromatic ring.

Structural basis for the substrate specificity of PepA from Streptococcus pneumoniae, a dodecameric tetrahedral protease.

PubMed

Kim, Doyoun; San, Boi Hoa; Moh, Sang Hyun; Park, Hyejin; Kim, Dong Young; Lee, Sangho; Kim, Kyeong Kyu

2010-01-01

Regulated cytosolic proteolysis is one of the key cellular processes ensuring proper functioning of a cell. M42 family proteases show a broad spectrum of substrate specificities, but the structural basis for such diversity of the substrate specificities is lagging behind biochemical data. Here we report the crystal structure of PepA from Streptococcus pneumoniae, a glutamyl aminopeptidase belonging to M42 family (SpPepA). We found that Arg-257 in the substrate binding pocket is strategically positioned so that Arg-257 can make electrostatic interactions with the acidic residue of a substrate at its N-terminus. Structural comparison of the substrate binding pocket of the M42 family proteases, along with the structure-based multiple sequence alignment, argues that the appropriate electrostatic interactions contribute to the selective substrate specificity of SpPepA. Copyright 2009 Elsevier Inc. All rights reserved.

The Hsp70 interdomain linker is a dynamic switch that enables allosteric communication between two structured domains.

PubMed

English, Charles A; Sherman, Woody; Meng, Wenli; Gierasch, Lila M

2017-09-08

Hsp70 molecular chaperones play key roles in cellular protein homeostasis by binding to exposed hydrophobic regions of incompletely folded or aggregated proteins. This crucial Hsp70 function relies on allosteric communication between two well-structured domains: an N-terminal nucleotide-binding domain (NBD) and a C-terminal substrate-binding domain (SBD), which are tethered by an interdomain linker. ATP or ADP binding to the NBD alters the substrate-binding affinity of the SBD, triggering functionally essential cycles of substrate binding and release. The interdomain linker is a well-structured participant in the interdomain interface in ATP-bound Hsp70s. By contrast, in the ADP-bound state, exemplified by the Escherichia coli Hsp70 DnaK, the interdomain linker is flexible. Hsp70 interdomain linker sequences are highly conserved; moreover, mutations in this region compromise interdomain allostery. To better understand the role of this region in Hsp70 allostery, we used molecular dynamics simulations to explore the conformational landscape of the interdomain linker in ADP-bound DnaK and supported our simulations by strategic experimental data. We found that while the interdomain linker samples many conformations, it behaves as three relatively ordered segments connected by hinges. As a consequence, the distances and orientations between the NBD and SBD are limited. Additionally, the C-terminal region of the linker forms previously unreported, transient interactions with the SBD, and the predominant linker-docking site is available in only one allosteric state, that with high affinity for substrate. This preferential binding implicates the interdomain linker as a dynamic allosteric switch. The linker-binding site on the SBD is a potential target for small molecule modulators of the Hsp70 allosteric cycle. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Kinetic, Thermodynamic, and Structural Insight into the Mechanism of Phosphopantetheine Adenylyltransferase from Mycobacterium tuberculosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wubben, Thomas J.; Mesecar, Andrew D.; UIC)

Phosphopantetheine adenylyltransferase (PPAT) catalyzes the penultimate step in the coenzyme A (CoA) biosynthetic pathway, reversibly transferring an adenylyl group from ATP to 4'-phosphopantetheine (PhP) to form dephosphocoenzyme A. This reaction sits at the branch point between the de novo pathway and the salvage pathway, and has been shown to be a rate-limiting step in the biosynthesis of CoA. Importantly, bacterial and mammalian PPATs share little sequence homology, making the enzyme a potential target for antibiotic development. A series of steady-state kinetic, product inhibition, and direct binding studies with Mycobacterium tuberculosis PPAT (MtPPAT) was conducted and suggests that the enzyme utilizesmore » a nonrapid-equilibrium random bi-bi mechanism. The kinetic response of MtPPAT to the binding of ATP was observed to be sigmoidal under fixed PhP concentrations, but substrate inhibition was observed at high PhP concentrations under subsaturating ATP concentrations, suggesting a preferred pathway to ternary complex formation. Negative cooperativity in the kinetic response of MtPPAT to PhP binding was observed under certain conditions and confirmed thermodynamically by isothermal titration calorimetry, suggesting the formation of an asymmetric quaternary structure during sequential ligation of substrates. Asymmetry in binding was also observed in isothermal titration calorimetry experiments with dephosphocoenzyme A and CoA. X-ray structures of MtPPAT in complex with PhP and the nonhydrolyzable ATP analogue adenosine-5'-[({alpha},{beta})-methyleno]triphosphate were solved to 1.57 {angstrom} and 2.68 {angstrom}, respectively. These crystal structures reveal small conformational changes in enzyme structure upon ligand binding, which may play a role in the nonrapid-equilibrium mechanism. We suggest that the proposed kinetic mechanism and asymmetric character in MtPPAT ligand binding may provide a means of reaction and pathway regulation in addition to that of the previously determined CoA feedback.« less

Nucleotide-induced conformational dynamics in ABC transporters from structure-based coarse grained modelling.

NASA Astrophysics Data System (ADS)

Flechsig, Holger

2016-02-01

ATP-binding cassette (ABC) transporters are integral membrane proteins which mediate the exchange of diverse substrates across membranes powered by ATP molecules. Our understanding of their activity is still hampered since the conformational dynamics underlying the operation of such proteins cannot yet be resolved in detailed molecular dynamics studies. Here a coarse grained model which allows to mimic binding of nucleotides and follow subsequent conformational motions of full-length transporter structures in computer simulations is proposed and implemented. To justify its explanatory quality, the model is first applied to the maltose transporter system for which multiple conformations are known and we find that the model predictions agree remarkably well with the experimental data. For the MalK subunit the switching from open to the closed dimer configuration upon ATP binding is reproduced and, moreover, for the full-length maltose transporter, progression from inward-facing to the outward-facing state is correctly obtained. For the heme transporter HmuUV, for which only the free structure could yet be determined, the model was then applied to predict nucleotide-induced conformational motions. Upon binding of ATP-mimicking ligands the structure changed from a conformation in which the nucleotide-binding domains formed an open shape, to a conformation in which they were found in tight contact, while, at the same time, a pronounced rotation of the transmembrane domains was observed. This finding is supported by normal mode analysis, and, comparison with structural data of the homologous vitamin B12 transporter BtuCD suggests that the observed rotation mechanism may contribute a common functional aspect for this class of ABC transporters. Although in HmuuV noticeable rearrangement of essential transmembrane helices was detected, there are no indications from our simulations that ATP binding alone may facilitate propagation of substrate molecules in this transporter. Possible explanations are discussed in the light of currently debated transport scenarios of ABC transporters.

Mutations in the substrate binding site of human heat-shock protein 70 indicate specific interaction with HLA-DR outside the peptide binding groove

PubMed Central

Rohrer, Karin M; Haug, Markus; Schwörer, Daniela; Kalbacher, Hubert; Holzer, Ursula

2014-01-01

Heat-shock protein 70 (Hsp70)–peptide complexes are involved in MHC class I-and II-restricted antigen presentation, enabling enhanced activation of T cells. As shown previously, mammalian cytosolic Hsp70 (Hsc70) molecules interact specifically with HLA-DR molecules. This interaction might be of significance as Hsp70 molecules could transfer bound antigenic peptides in a ternary complex into the binding groove of HLA-DR molecules. The present study provides new insights into the distinct interaction of Hsp70 with HLA-DR molecules. Using a quantitative binding assay, it could be demonstrated that a point mutation of amino acids alanine 406 and valine 438 in the substrate binding pocket led to reduced peptide binding compared with the wild-type Hsp70 whereas HLA-DR binding remains unaffected. The removal of the C-terminal lid neither altered the substrate binding capacity nor the Hsp70 binding characteristics to HLA-DR. A truncated variant lacking the nucleotide binding domain showed no binding interactions with HLA-DR. Furthermore, the truncated ATPase subunit of constitutively expressed Hsc70 revealed similar binding affinities to HLA-DR compared with the complete Hsc70. Hence, it can be assumed that the Hsp70–HLA-DR interaction takes place outside the peptide binding groove and is attributed to the ATPase domain of HSP70 molecules. The Hsp70-chaperoned peptides might thereby be directly transferred into the binding groove of HLA-DR, so enabling enhanced presentation of the peptide on antigen-presenting cells and leading to an improved proliferation of responding T cells as shown previously. PMID:24428437

Kinetic studies and molecular modelling attribute a crucial role in the specificity and stereoselectivity of penicillin acylase to the pair ArgA145-ArgB263.

PubMed

Guncheva, Maya; Ivanov, Ivaylo; Galunsky, Boris; Stambolieva, Nicolina; Kaneti, Jose

2004-06-01

Kinetic experiments with a substrate series of phenylacetyl-arylamides reveal that at least one polar group in the amine moiety is required for the proper orientation of the substrate in the large nucleophile-binding subsite of penicillin acylase of Escherichia coli. Quantum mechanical molecular modelling of enzyme-substrate interactions in the enzyme active site shows that in the case of substrates lacking local symmetry, the productive binding implies two nonsymmetrical arrangements with respect to the two positively charged guanidinium residues of ArgA145 and ArgB263. This indicates a crucial role of the specified arginine pair in the substrate- and stereoselectivity of penicillin acylase.

Calmodulin fishing with a structurally disordered bait triggers CyaA catalysis

PubMed Central

O’Brien, Darragh P.; Durand, Dominique; Voegele, Alexis; Hourdel, Véronique; Davi, Marilyne; Chamot-Rooke, Julia; Vachette, Patrice; Brier, Sébastien; Ladant, Daniel

2017-01-01

Once translocated into the cytosol of target cells, the catalytic domain (AC) of the adenylate cyclase toxin (CyaA), a major virulence factor of Bordetella pertussis, is potently activated by binding calmodulin (CaM) to produce supraphysiological levels of cAMP, inducing cell death. Using a combination of small-angle X-ray scattering (SAXS), hydrogen/deuterium exchange mass spectrometry (HDX-MS), and synchrotron radiation circular dichroism (SR-CD), we show that, in the absence of CaM, AC exhibits significant structural disorder, and a 75-residue-long stretch within AC undergoes a disorder-to-order transition upon CaM binding. Beyond this local folding, CaM binding induces long-range allosteric effects that stabilize the distant catalytic site, whilst preserving catalytic loop flexibility. We propose that the high enzymatic activity of AC is due to a tight balance between the CaM-induced decrease of structural flexibility around the catalytic site and the preservation of catalytic loop flexibility, allowing for fast substrate binding and product release. The CaM-induced dampening of AC conformational disorder is likely relevant to other CaM-activated enzymes. PMID:29287065

L-tyrosine and L-DOPA as hormone-like regulators of melanocytes functions

PubMed Central

Slominski, Andrzej; Zmijewski, Michal; Pawelek, John

2011-01-01

Summary Evidence reveals that L-tyrosine and L-DOPA, besides serving as substrates and intermediates of melanogenesis, are also bioregulatory agents acting not only as inducers and positive regulators of melanogenesis but also as regulators of other cellular functions. These can be mediated through action on specific receptors or through non-receptor mediated mechanisms. The substrate induced (L-tyrosine and/or L-DOPA) melanogenic pathway would autoregulate itself as well as it would regulate the melanocyte functions through activity of its structural or regulatory proteins and through intermediates of melanogenesis and melanin itself. Dissection of regulatory and autoregulatory elements of this process may elucidate how substrate induced autoregulatory pathways have evolved from prokaryotic or simple eukaryotic organisms to complex systems in vertebrates. This could substantiate older theory proposing that receptors for amino-acid derived hormones arose from the receptors for those amino acids, and that nuclear receptors evolved from primitive intracellular receptors binding nutritional factors or metabolic intermediates. PMID:21834848

Structural Analysis of a Family 101 Glycoside Hydrolase in Complex with Carbohydrates Reveals Insights into Its Mechanism.

PubMed

Gregg, Katie J; Suits, Michael D L; Deng, Lehua; Vocadlo, David J; Boraston, Alisdair B

2015-10-16

O-Linked glycosylation is one of the most abundant post-translational modifications of proteins. Within the secretory pathway of higher eukaryotes, the core of these glycans is frequently an N-acetylgalactosamine residue that is α-linked to serine or threonine residues. Glycoside hydrolases in family 101 are presently the only known enzymes to be able to hydrolyze this glycosidic linkage. Here we determine the high-resolution structures of the catalytic domain comprising a fragment of GH101 from Streptococcus pneumoniae TIGR4, SpGH101, in the absence of carbohydrate, and in complex with reaction products, inhibitor, and substrate analogues. Upon substrate binding, a tryptophan lid (residues 724-WNW-726) closes on the substrate. The closing of this lid fully engages the substrate in the active site with Asp-764 positioned directly beneath C1 of the sugar residue bound within the -1 subsite, consistent with its proposed role as the catalytic nucleophile. In all of the bound forms of the enzyme, however, the proposed catalytic acid/base residue was found to be too distant from the glycosidic oxygen (>4.3 Å) to serve directly as a general catalytic acid/base residue and thereby facilitate cleavage of the glycosidic bond. These same complexes, however, revealed a structurally conserved water molecule positioned between the catalytic acid/base and the glycosidic oxygen. On the basis of these structural observations we propose a new variation of the retaining glycoside hydrolase mechanism wherein the intervening water molecule enables a Grotthuss proton shuttle between Glu-796 and the glycosidic oxygen, permitting this residue to serve as the general acid/base catalytic residue. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Pseudo-magnetic fields of strongly-curved graphene nanobubbles

NASA Astrophysics Data System (ADS)

Liu, Li-Chi

2018-04-01

We use the π-orbital axis vector (POAV) analysis to deal with large curvature effect of graphene in the tight-binding model. To test the validities of pseudo-magnetic fields (PMFs) derived from the tight-binding model and the model with Dirac equation coupled to a curved surface, we propose two types of spatially constant-field topographies for strongly-curved graphene nanobubbles, which correspond to these two models, respectively. It is shown from the latter model that the PMF induced by any spherical graphene nanobubble is always equivalent to the magnetic field caused by one magnetic monopole charge distributed on a complete spherical surface with the same radius. Such a PMF might be attributed to the isometry breaking of a graphene layer attached conformably to a spherical substrate with adhesion.

DNA/RNA hybrid substrates modulate the catalytic activity of purified AID.

PubMed

Abdouni, Hala S; King, Justin J; Ghorbani, Atefeh; Fifield, Heather; Berghuis, Lesley; Larijani, Mani

2018-01-01

Activation-induced cytidine deaminase (AID) converts cytidine to uridine at Immunoglobulin (Ig) loci, initiating somatic hypermutation and class switching of antibodies. In vitro, AID acts on single stranded DNA (ssDNA), but neither double-stranded DNA (dsDNA) oligonucleotides nor RNA, and it is believed that transcription is the in vivo generator of ssDNA targeted by AID. It is also known that the Ig loci, particularly the switch (S) regions targeted by AID are rich in transcription-generated DNA/RNA hybrids. Here, we examined the binding and catalytic behavior of purified AID on DNA/RNA hybrid substrates bearing either random sequences or GC-rich sequences simulating Ig S regions. If substrates were made up of a random sequence, AID preferred substrates composed entirely of DNA over DNA/RNA hybrids. In contrast, if substrates were composed of S region sequences, AID preferred to mutate DNA/RNA hybrids over substrates composed entirely of DNA. Accordingly, AID exhibited a significantly higher affinity for binding DNA/RNA hybrid substrates composed specifically of S region sequences, than any other substrates composed of DNA. Thus, in the absence of any other cellular processes or factors, AID itself favors binding and mutating DNA/RNA hybrids composed of S region sequences. AID:DNA/RNA complex formation and supporting mutational analyses suggest that recognition of DNA/RNA hybrids is an inherent structural property of AID. Copyright © 2017 Elsevier Ltd. All rights reserved.

The trimethylammonium headgroup of choline is a major determinant for substrate binding and specificity in choline oxidase.

PubMed

Gadda, Giovanni; Powell, Nichole L N; Menon, Prashanthi

2004-10-15

Choline oxidase catalyzes the oxidation of choline to glycine betaine via two sequential flavin-linked transfers of hydride equivalents to molecular oxygen and formation of a betaine aldehyde intermediate. In the present study, choline and glycine betaine analogs were used as substrates and inhibitors for the enzyme to investigate the structural determinants that are relevant for substrate recognition and specificity. Competitive inhibition patterns with respect to choline were determined for a number of substituted amines at pH 6.5 and 25 degrees C. The Kis values for the carboxylate-containing ligands glycine betaine, N,N-dimethylglycine, and N-methylglycine increased monotonically with decreasing number of methyl groups, consistent with the trimethylammonium portion of the ligand being important for binding. In contrast, the acetate portion of glycine betaine did not contribute to binding, as suggested by lack of changes in the Kis values upon substituting glycine betaine with inhibitors containing methyl, ethyl, allyl, and 2-amino-ethyl side chains. In agreement with the inhibition data, the specificity of the enzyme for the organic substrate (kcat/Km value) decreased when N,N-dimethylethanolamine, N-methylethanolamine, and the isosteric substrate 3,3-dimethyl-1-butanol were used as substrate instead of choline; a contribution of approximately 7 kcal mol(-1) toward substrate discrimination was estimated for the interaction of the trimethylammonium portion of the substrate with the active site of choline oxidase.

Structural Basis for Substrate Specificity in Phosphate Binding (beta/alpha)8-Barrels: D-Allulose 6-Phosphate 3-Epimerase from Escherichia coli K-12

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chan,K.; Fedorov, A.; Almo, S.

2008-01-01

Enzymes that share the ({beta}/{alpha})8-barrel fold catalyze a diverse range of reactions. Many utilize phosphorylated substrates and share a conserved C-terminal ({beta}/a)2-quarter barrel subdomain that provides a binding motif for the dianionic phosphate group. We recently reported functional and structural studies of d-ribulose 5-phosphate 3-epimerase (RPE) from Streptococcus pyogenes that catalyzes the equilibration of the pentulose 5-phosphates d-ribulose 5-phosphate and d-xylulose 5-phosphate in the pentose phosphate pathway [J. Akana, A. A. Fedorov, E. Fedorov, W. R. P. Novack, P. C. Babbitt, S. C. Almo, and J. A. Gerlt (2006) Biochemistry 45, 2493-2503]. We now report functional and structural studies ofmore » d-allulose 6-phosphate 3-epimerase (ALSE) from Escherichia coli K-12 that catalyzes the equilibration of the hexulose 6-phosphates d-allulose 6-phosphate and d-fructose 6-phosphate in a catabolic pathway for d-allose. ALSE and RPE prefer their physiological substrates but are promiscuous for each other's substrate. The active sites (RPE complexed with d-xylitol 5-phosphate and ALSE complexed with d-glucitol 6-phosphate) are superimposable (as expected from their 39% sequence identity), with the exception of the phosphate binding motif. The loop following the eighth {beta}-strand in ALSE is one residue longer than the homologous loop in RPE, so the binding site for the hexulose 6-phosphate substrate/product in ALSE is elongated relative to that for the pentulose 5-phosphate substrate/product in RPE. We constructed three single-residue deletion mutants of the loop in ALSE, ?T196, ?S197 and ?G198, to investigate the structural bases for the differing substrate specificities; for each, the promiscuity is altered so that d-ribulose 5-phosphate is the preferred substrate. The changes in kcat/Km are dominated by changes in kcat, suggesting that substrate discrimination results from differential transition state stabilization. In both ALSE and RPE, the phosphate group hydrogen bonds not only with the conserved motif but also with an active site loop following the sixth {beta}-strand, providing a potential structural mechanism for coupling substrate binding with catalysis.« less

Stochastic steps in secondary active sugar transport

PubMed Central

Adelman, Joshua L.; Ghezzi, Chiara; Bisignano, Paola; Loo, Donald D. F.; Choe, Seungho; Abramson, Jeff; Rosenberg, John M.; Wright, Ernest M.; Grabe, Michael

2016-01-01

Secondary active transporters, such as those that adopt the leucine-transporter fold, are found in all domains of life, and they have the unique capability of harnessing the energy stored in ion gradients to accumulate small molecules essential for life as well as expel toxic and harmful compounds. How these proteins couple ion binding and transport to the concomitant flow of substrates is a fundamental structural and biophysical question that is beginning to be answered at the atomistic level with the advent of high-resolution structures of transporters in different structural states. Nonetheless, the dynamic character of the transporters, such as ion/substrate binding order and how binding triggers conformational change, is not revealed from static structures, yet it is critical to understanding their function. Here, we report a series of molecular simulations carried out on the sugar transporter vSGLT that lend insight into how substrate and ions are released from the inward-facing state of the transporter. Our simulations reveal that the order of release is stochastic. Functional experiments were designed to test this prediction on the human homolog, hSGLT1, and we also found that cytoplasmic release is not ordered, but we confirmed that substrate and ion binding from the extracellular space is ordered. Our findings unify conflicting published results concerning cytoplasmic release of ions and substrate and hint at the possibility that other transporters in the superfamily may lack coordination between ions and substrate in the inward-facing state. PMID:27325773

Stochastic steps in secondary active sugar transport.

PubMed

Adelman, Joshua L; Ghezzi, Chiara; Bisignano, Paola; Loo, Donald D F; Choe, Seungho; Abramson, Jeff; Rosenberg, John M; Wright, Ernest M; Grabe, Michael

2016-07-05

Secondary active transporters, such as those that adopt the leucine-transporter fold, are found in all domains of life, and they have the unique capability of harnessing the energy stored in ion gradients to accumulate small molecules essential for life as well as expel toxic and harmful compounds. How these proteins couple ion binding and transport to the concomitant flow of substrates is a fundamental structural and biophysical question that is beginning to be answered at the atomistic level with the advent of high-resolution structures of transporters in different structural states. Nonetheless, the dynamic character of the transporters, such as ion/substrate binding order and how binding triggers conformational change, is not revealed from static structures, yet it is critical to understanding their function. Here, we report a series of molecular simulations carried out on the sugar transporter vSGLT that lend insight into how substrate and ions are released from the inward-facing state of the transporter. Our simulations reveal that the order of release is stochastic. Functional experiments were designed to test this prediction on the human homolog, hSGLT1, and we also found that cytoplasmic release is not ordered, but we confirmed that substrate and ion binding from the extracellular space is ordered. Our findings unify conflicting published results concerning cytoplasmic release of ions and substrate and hint at the possibility that other transporters in the superfamily may lack coordination between ions and substrate in the inward-facing state.

Tripartite ATP-independent Periplasmic (TRAP) Transporters Use an Arginine-mediated Selectivity Filter for High Affinity Substrate Binding*

PubMed Central

Fischer, Marcus; Hopkins, Adam P.; Severi, Emmanuele; Hawkhead, Judith; Bawdon, Daniel; Watts, Andrew G.; Hubbard, Roderick E.; Thomas, Gavin H.

2015-01-01

Tripartite ATP-independent periplasmic (TRAP) transporters are secondary transporters that have evolved an obligate dependence on a substrate-binding protein (SBP) to confer unidirectional transport. Different members of the DctP family of TRAP SBPs have binding sites that recognize a diverse range of organic acid ligands but appear to only share a common electrostatic interaction between a conserved arginine and a carboxylate group in the ligand. We investigated the significance of this interaction using the sialic acid-specific SBP, SiaP, from the Haemophilus influenzae virulence-related SiaPQM TRAP transporter. Using in vitro, in vivo, and structural methods applied to SiaP, we demonstrate that the coordination of the acidic ligand moiety of sialic acid by the conserved arginine (Arg-147) is essential for the function of the transporter as a high affinity scavenging system. However, at high substrate concentrations, the transporter can function in the absence of Arg-147 suggesting that this bi-molecular interaction is not involved in further stages of the transport cycle. As well as being required for high affinity binding, we also demonstrate that the Arg-147 is a strong selectivity filter for carboxylate-containing substrates in TRAP transporters by engineering the SBP to recognize a non-carboxylate-containing substrate, sialylamide, through water-mediated interactions. Together, these data provide biochemical and structural support that TRAP transporters function predominantly as high affinity transporters for carboxylate-containing substrates. PMID:26342690

Identification of ribozymes within a ribozyme library that efficiently cleave a long substrate RNA.

PubMed Central

Campbell, T B; Cech, T R

1995-01-01

Positions 2-6 of the substrate-binding internal guide sequence (IGS) of the L-21 Sca I form of the Tetrahymena thermophila intron were mutagenized to produce a GN5 IGS library. Ribozymes within the GN5 library capable of efficient cleavage of an 818-nt human immunodeficiency virus type 1 vif-vpr RNA, at 37 degrees C, were identified by ribozyme-catalyzed guanosine addition to the 3' cleavage product. Three ribozymes (IGS = GGGGCU, GGCUCC, and GUGGCU) within the GN5 library that actively cleaved the long substrate were characterized kinetically and compared to the wild-type ribozyme (GGAGGG) and two control ribozymes (GGAGUC and GGAGAU). The two control ribozymes have specific sites within the long substrate, but were not identified during screening of the library. Under single-turnover conditions, ribozymes GGGGCU, GGCUCC, and GUGGCU cleaved the 818-nt substrate 4- to 200-fold faster than control ribozymes. Short cognate substrates, which should be structureless and therefore accessible to ribozyme binding, were cleaved at similar rates by all ribozymes except GGGGCU, which showed a fourfold rate enhancement. The rate of cleavage of long relative to short substrate under single-turnover conditions suggests that GGCUCC and GUGGCU were identified because of accessibility to their specific cleavage sites within the long substrate (substrate-specific effects), whereas GGGGCU was identified because of an enhanced rate of substrate binding despite a less accessible site in the long substrate. Even though screening was performed with 100-fold excess substrate (relative to total ribozyme), the rate of multiple-turnover catalysis did not contribute to identification of trans-cleaving ribozymes in the GN5 library. PMID:7489519

Multiple binding modes of substrate to the catalytic RNA subunit of RNase P from Escherichia coli.

PubMed Central

Pomeranz Krummel, D A; Altman, S

1999-01-01

M1 RNA that contained 4'-thiouridine was photochemically cross-linked to different substrates and to a product of the reaction it governs. The locations of the cross-links in these photochemically induced complexes were identified. The cross-links indicated that different substrates share some contacts but have distinct binding modes to M1 RNA. The binding of some substrates also results in a substrate-dependent conformational change in the enzymatic RNA, as evidenced by the appearance of an M1 RNA intramolecular cross-link. The identification of the cross-links between M1 RNA and product indicate that they are shared with only one of the three cross-linked E-S complexes that were identified, an indication of noncompetitive inhibition by the product. We also examined whether the cross-linked complexes between M1 RNA and substrate(s) or product are altered in the presence of the enzyme's protein cofactor (C5 protein) and in the presence of different concentrations of divalent metal ions. C5 protein enhanced the yield of certain M1 RNA-substrate cross-linked complexes for both wild-type M1 RNA and a deletion mutant of M1 RNA (delta[273-281]), but not for the M1 RNA-product complex. High concentrations of Mg2+ increased the yield of all M1 RNA-substrate complexes but not the M1 RNA-product complex. PMID:10445877

Insights into Hsp70 Chaperone Activity from a Crystal Structure of the Yeast Hsp110 Sse1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu,Q.; Hendrickson, W.

2007-01-01

Classic Hsp70 chaperones assist in diverse processes of protein folding and translocation, and Hsp110s had seemed by sequence to be distant relatives within an Hsp70 superfamily. The 2.4 Angstroms resolution structure of Sse1 with ATP shows that Hsp110s are indeed Hsp70 relatives, and it provides insight into allosteric coupling between sites for ATP and polypeptide-substrate binding in Hsp70s. Subdomain structures are similar in intact Sse1(ATP) and in the separate Hsp70 domains, but conformational dispositions are radically different. Interfaces between Sse1 domains are extensive, intimate, and conservative in sequence with Hsp70s. We propose that Sse1(ATP) may be an evolutionary vestige ofmore » the Hsp70(ATP) state, and an analysis of 64 mutant variants in Sse1 and three Hsp70 homologs supports this hypothesis. An atomic-level understanding of Hsp70 communication between ATP and substrate-binding domains follows. Requirements on Sse1 for yeast viability are in keeping with the distinct function of Hsp110s as nucleotide exchange factors.« less

Clustering Molecular Dynamics Trajectories for Optimizing Docking Experiments

PubMed Central

De Paris, Renata; Quevedo, Christian V.; Ruiz, Duncan D.; Norberto de Souza, Osmar; Barros, Rodrigo C.

2015-01-01

Molecular dynamics simulations of protein receptors have become an attractive tool for rational drug discovery. However, the high computational cost of employing molecular dynamics trajectories in virtual screening of large repositories threats the feasibility of this task. Computational intelligence techniques have been applied in this context, with the ultimate goal of reducing the overall computational cost so the task can become feasible. Particularly, clustering algorithms have been widely used as a means to reduce the dimensionality of molecular dynamics trajectories. In this paper, we develop a novel methodology for clustering entire trajectories using structural features from the substrate-binding cavity of the receptor in order to optimize docking experiments on a cloud-based environment. The resulting partition was selected based on three clustering validity criteria, and it was further validated by analyzing the interactions between 20 ligands and a fully flexible receptor (FFR) model containing a 20 ns molecular dynamics simulation trajectory. Our proposed methodology shows that taking into account features of the substrate-binding cavity as input for the k-means algorithm is a promising technique for accurately selecting ensembles of representative structures tailored to a specific ligand. PMID:25873944

Non-ionic detergents facilitate non-specific binding of M13 bacteriophage to polystyrene surfaces.

PubMed

Hakami, Abdulrahim R; Ball, Jonathan K; Tarr, Alexander W

2015-09-01

Phage-displayed random peptide libraries are widely used for identifying peptide interactions with proteins and other substrates. Selection of peptide ligands involves iterative rounds of affinity enrichment. The binding properties of the selected phage clones are routinely tested using immunoassay after propagation to high titre in a bacterial host and precipitation using polyethylene glycol (PEG) and high salt concentration. These immunoassays can suffer from low sensitivity and high background signals. Polysorbate 20 (Tween(®) 20) is a non-ionic detergent commonly used in immunoassay washing buffers to reduce non-specific binding, and is also used as a blocking reagent. We have observed that Tween 20 enhances non-specific M13 library phage binding in a peptide-independent manner. Other non-ionic detergents were also found to promote significant, dose-dependent non-specific phage binding in ELISA. This effect was not observed for assays using phage concentrated by ultracentrifugation, suggesting that interactions occur between detergents and the PEG-precipitated phage, irrespective of the displayed peptide motif. This artefact may impact on successful affinity selection of peptides from phage-display libraries. We propose alternative methods for screening phage libraries for identifying binding interactions with target ligands. Copyright © 2015 Elsevier B.V. All rights reserved.

Complex coacervation in charge complementary biopolymers: Electrostatic versus surface patch binding.

PubMed

Pathak, Jyotsana; Priyadarshini, Eepsita; Rawat, Kamla; Bohidar, H B

2017-12-01

In this review, a number of systems are described to demonstrate the effect of polyelectrolyte chain stiffness (persistence length) on the coacervation phenomena, after we briefly review the field. We consider two specific types of complexation/coacervation: in the first type, DNA is used as a fixed substrate binding to flexible polyions such as gelatin A, bovine serum albumin and chitosan (large persistence length polyelectrolyte binding to low persistence length biopolymer), and in the second case, different substrates such as gelatin A, bovine serum albumin, and chitosan were made to bind to a polyion gelatin B (low persistence length substrate binding to comparable persistence length polyion). Polyelectrolyte chain flexibility was found to have remarkable effect on the polyelectrolyte-protein complex coacervation. The competitive interplay of electrostatic versus surface patch binding (SPB) leading to associative interaction followed by complex coacervation between these biopolymers is elucidated. We modelled the SPB interaction in terms of linear combination of attractive and repulsive Coulombic forces with respect to the solution ionic strength. The aforesaid interactions were established via a universal phase diagram, considering the persistence length of polyion as the sole independent variable. Copyright © 2017 Elsevier B.V. All rights reserved.

Identification and binding mechanism of phage displayed peptides with specific affinity to acid-alkali treated titanium.

PubMed

Sun, Yuhua; Tan, Jing; Wu, Baohua; Wang, Jianxin; Qu, Shuxin; Weng, Jie; Feng, Bo

2016-10-01

Acid-alkali treatment is one of means widely used for preparing bioactive titanium surfaces. Peptides with specific affinity to titanium surface modified by acid-alkali two-steps treatment were obtained via phage display technology. Out of the eight new unique peptides, titanium-binding peptide 54 displayed by monoclonal M13 phage at its pIII coat protein (TBP54-M13 phage) was proved to have higher binding affinity to the substrate. The binding interaction occurred at the domain from phenylalanine at position 1 to arginine at position 6 in the sequences of TBP54 (FAETHRGFHFSF) mainly via the reaction of these residues with the Ti surface. Together the coordination and electrostatic interactions controlled the specific binding of the phage to the substrate. The binding affinity was dependent on the surface basic hydroxyl group content. In addition, the phage showed a different interaction way with the Ti surface without acid-alkali treatment along with an impaired affinity. This study could provide more understanding of the interaction mechanism between the selected peptide and its specific substrate, and develop a promising method for the biofunctionalization of titanium. Copyright © 2016 Elsevier B.V. All rights reserved.

The inhibition of hemicellulosic sugars on cellulose hydrolysis are highly dependant on the cellulase productive binding, processivity, and substrate surface charges.

PubMed

Zhai, Rui; Hu, Jinguang; Saddler, Jack N

2018-06-01

In this study, the influence of major hemicellulosic sugars (mannose and xylose) on cellulose hydrolysis and major enzyme activities were evaluated by using both commercial enzyme cocktail and purified cellulase monocomponents over a "library" of cellulosic substrates. Surprisingly, the results showed that unlike glucose, mannose/xylose did not inhibit individual cellulase activities but significantly decreased their hydrolytic performance on cellulose substrates. When various enzyme-substrate interactions (e.g. adsorption/desorption, productive binding, and processive moving) were evaluated, it appeared that these hemicellulosic sugars significantly reduced the productive binding and processivity of Cel7A, which in turn limited cellulase hydrolytic efficacy. Among a range of major cellulose characteristics (e.g. crystallinity, degree of polymerization, accessibility, and surface charges), the acid group content of the cellulosic substrates seemed to be the main driver that determined the extent of hemicellulosic sugar inhibition. Our results provided new insights for better understanding the sugar inhibition mechanisms of cellulose hydrolysis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Maltase protein of Ogataea (Hansenula) polymorpha is a counterpart to the resurrected ancestor protein ancMALS of yeast maltases and isomaltases

PubMed Central

Viigand, Katrin; Visnapuu, Triinu; Mardo, Karin; Aasamets, Anneli

2016-01-01

Abstract Saccharomyces cerevisiae maltases use maltose, maltulose, turanose and maltotriose as substrates, isomaltases use isomaltose, α‐methylglucoside and palatinose and both use sucrose. These enzymes are hypothesized to have evolved from a promiscuous α‐glucosidase ancMALS through duplication and mutation of the genes. We studied substrate specificity of the maltase protein MAL1 from an earlier diverged yeast, Ogataea polymorpha (Op), in the light of this hypothesis. MAL1 has extended substrate specificity and its properties are strikingly similar to those of resurrected ancMALS. Moreover, amino acids considered to determine selective substrate binding are highly conserved between Op MAL1 and ancMALS. Op MAL1 represents an α‐glucosidase in which both maltase and isomaltase activities are well optimized in a single enzyme. Substitution of Thr200 (corresponds to Val216 in S. cerevisiae isomaltase IMA1) with Val in MAL1 drastically reduced the hydrolysis of maltose‐like substrates (α‐1,4‐glucosides), confirming the requirement of Thr at the respective position for this function. Differential scanning fluorimetry (DSF) of the catalytically inactive mutant Asp199Ala of MAL1 in the presence of its substrates and selected monosaccharides suggested that the substrate‐binding pocket of MAL1 has three subsites (–1, +1 and +2) and that binding is strongest at the –1 subsite. The DSF assay results were in good accordance with affinity (K m) and inhibition (K i) data of the enzyme for tested substrates, indicating the power of the method to predict substrate binding. Deletion of either the maltase (MAL1) or α‐glucoside permease (MAL2) gene in Op abolished the growth of yeast on MAL1 substrates, confirming the requirement of both proteins for usage of these sugars. © 2016 The Authors. Yeast published by John Wiley & Sons, Ltd. PMID:26919272

The interaction of representative members from two classes of antimycotics--the azoles and the allylamines--with cytochromes P-450 in steroidogenic tissues and liver.

PubMed

Schuster, I

1985-06-01

Spectrophotometric studies with ketoconazole, clotrimazole and miconazole show strong type-II interactions with several cytochromes P-450, particularly (Ks greater than 10(7)M-1; pH7.4; 25 degrees C) with the 11 beta-hydroxylase of adrenal mitochondria, with the 17 alpha/20 lyase of testis microsomes and with some forms of cytochromes P-450 of liver. A tight binding of the azoles also occurs to the reduced cytochromes, giving rise to an impeded CO binding to the haem iron. The binding of the azoles to 11 beta-hydroxylase and 17 alpha/20 lyase is much tighter than the binding of endogenous substrates, and consequently inhibition of steroidogenesis will occur at these sites. The metabolism of xenobiotic substrates by the cytochromes P-450 of liver will also be severely impeded. In contrast, the allylamines naftifine and SF 86-327 are type-I substrates for a small portion of cytochromes P-450 of liver microsomes only and there is no spectral evidence for binding to the cytochromes P-450 involved in steroid biosynthesis.

Structure and substrate-binding mechanism of human Ap4A hydrolase.

PubMed

Swarbrick, James D; Buyya, Smrithi; Gunawardana, Dilantha; Gayler, Kenwyn R; McLennan, Alexander G; Gooley, Paul R

2005-03-04

Asymmetric diadenosine 5',5'''-P(1),P(4)-tetraphosphate (Ap(4)A) hydrolases play a major role in maintaining homeostasis by cleaving the metabolite diadenosine tetraphosphate (Ap(4)A) back into ATP and AMP. The NMR solution structures of the 17-kDa human asymmetric Ap(4)A hydrolase have been solved in both the presence and absence of the product ATP. The adenine moiety of the nucleotide predominantly binds in a ring stacking arrangement equivalent to that observed in the x-ray structure of the homologue from Caenorhabditis elegans. The binding site is, however, markedly divergent to that observed in the plant/pathogenic bacteria class of enzymes, opening avenues for the exploration of specific therapeutics. Binding of ATP induces substantial conformational and dynamic changes that were not observed in the C. elegans structure. In contrast to the C. elegans homologue, important side chains that play a major role in substrate binding do not have to reorient to accommodate the ligand. This may have important implications in the mechanism of substrate recognition in this class of enzymes.

Protein Allostery and Conformational Dynamics.

PubMed

Guo, Jingjing; Zhou, Huan-Xiang

2016-06-08

The functions of many proteins are regulated through allostery, whereby effector binding at a distal site changes the functional activity (e.g., substrate binding affinity or catalytic efficiency) at the active site. Most allosteric studies have focused on thermodynamic properties, in particular, substrate binding affinity. Changes in substrate binding affinity by allosteric effectors have generally been thought to be mediated by conformational transitions of the proteins or, alternatively, by changes in the broadness of the free energy basin of the protein conformational state without shifting the basin minimum position. When effector binding changes the free energy landscape of a protein in conformational space, the change affects not only thermodynamic properties but also dynamic properties, including the amplitudes of motions on different time scales and rates of conformational transitions. Here we assess the roles of conformational dynamics in allosteric regulation. Two cases are highlighted where NMR spectroscopy and molecular dynamics simulation have been used as complementary approaches to identify residues possibly involved in allosteric communication. Perspectives on contentious issues, for example, the relationship between picosecond-nanosecond local and microsecond-millisecond conformational exchange dynamics, are presented.

Discovery of Potent Human Glutaminyl Cyclase Inhibitors as Anti-Alzheimer's Agents Based on Rational Design.

PubMed

Hoang, Van-Hai; Tran, Phuong-Thao; Cui, Minghua; Ngo, Van T H; Ann, Jihyae; Park, Jongmi; Lee, Jiyoun; Choi, Kwanghyun; Cho, Hanyang; Kim, Hee; Ha, Hee-Jin; Hong, Hyun-Seok; Choi, Sun; Kim, Young-Ho; Lee, Jeewoo

2017-03-23

Glutaminyl cyclase (QC) has been implicated in the formation of toxic amyloid plaques by generating the N-terminal pyroglutamate of β-amyloid peptides (pGlu-Aβ) and thus may participate in the pathogenesis of Alzheimer's disease (AD). We designed a library of glutamyl cyclase (QC) inhibitors based on the proposed binding mode of the preferred substrate, Aβ 3E-42 . An in vitro structure-activity relationship study identified several excellent QC inhibitors demonstrating 5- to 40-fold increases in potency compared to a known QC inhibitor. When tested in mouse models of AD, compound 212 significantly reduced the brain concentrations of pyroform Aβ and total Aβ and restored cognitive functions. This potent Aβ-lowering effect was achieved by incorporating an additional binding region into our previously established pharmacophoric model, resulting in strong interactions with the carboxylate group of Glu327 in the QC binding site. Our study offers useful insights in designing novel QC inhibitors as a potential treatment option for AD.

Super Spy variants implicate flexibility in chaperone action.

PubMed

Quan, Shu; Wang, Lili; Petrotchenko, Evgeniy V; Makepeace, Karl At; Horowitz, Scott; Yang, Jianyi; Zhang, Yang; Borchers, Christoph H; Bardwell, James Ca

2014-01-01

Experimental study of the role of disorder in protein function is challenging. It has been proposed that proteins utilize disordered regions in the adaptive recognition of their various binding partners. However apart from a few exceptions, defining the importance of disorder in promiscuous binding interactions has proven to be difficult. In this paper, we have utilized a genetic selection that links protein stability to antibiotic resistance to isolate variants of the newly discovered chaperone Spy that show an up to 7 fold improved chaperone activity against a variety of substrates. These "Super Spy" variants show tighter binding to client proteins and are generally more unstable than is wild type Spy and show increases in apparent flexibility. We establish a good relationship between the degree of their instability and the improvement they show in their chaperone activity. Our results provide evidence for the importance of disorder and flexibility in chaperone function. DOI: http://dx.doi.org/10.7554/eLife.01584.001.

Super Spy variants implicate flexibility in chaperone action

PubMed Central

Quan, Shu; Wang, Lili; Petrotchenko, Evgeniy V; Makepeace, Karl AT; Horowitz, Scott; Yang, Jianyi; Zhang, Yang; Borchers, Christoph H; Bardwell, James CA

2014-01-01

Experimental study of the role of disorder in protein function is challenging. It has been proposed that proteins utilize disordered regions in the adaptive recognition of their various binding partners. However apart from a few exceptions, defining the importance of disorder in promiscuous binding interactions has proven to be difficult. In this paper, we have utilized a genetic selection that links protein stability to antibiotic resistance to isolate variants of the newly discovered chaperone Spy that show an up to 7 fold improved chaperone activity against a variety of substrates. These “Super Spy” variants show tighter binding to client proteins and are generally more unstable than is wild type Spy and show increases in apparent flexibility. We establish a good relationship between the degree of their instability and the improvement they show in their chaperone activity. Our results provide evidence for the importance of disorder and flexibility in chaperone function. DOI: http://dx.doi.org/10.7554/eLife.01584.001 PMID:24497545

Charged/Polar-Residue Scanning of the Hydrophobic Face of Transmembrane Domain 9 of the Yeast Glutathione Transporter, Hgt1p, Reveals a Conformationally Critical Region for Substrate Transport

PubMed Central

Thakur, Anil; Bachhawat, Anand K.

2015-01-01

Unraveling the mechanistic workings of membrane transporters has remained a challenging task. We describe a novel strategy that involves subjecting the residues of the hydrophobic face of a transmembrane helix to a charged/polar scanning mutagenesis. TMD9 of the yeast glutathione transporter, Hgt1p, has been identified as being important in substrate binding, and two residues, F523 and Q526, are expected to line the substrate translocation channel while the other face is hydrophobic. The hydrophobic face of TMD9 helix consists of residues A509, V513, L517, L520, I524, and I528, and these were mutated to lysine, glutamine, and glutamic acid. Among the 16 charged mutants created, six were nonfunctional, revealing a surprising tolerance of charged residues in the hydrophobic part of TM helices. Furthermore, the only position that did not tolerate any charged residue was I524, proximal to the substrate binding residues. However, P525, also proximal to the substrate binding residues, did tolerate charged/polar residues, suggesting that mere proximity to the substrate binding residues was not the only factor. The I524K/E/Q mutants expressed well and localized correctly despite lacking any glutathione uptake capability. Isolation of suppressors for all nonfunctional mutants yielded second-site suppressors only for I524K and I524Q, and suppressors for these mutations appeared at G202K/I and G202K/Q, respectively. G202 is in the hydrophilic loop between TMD3 and TMD4. The results suggest that I524 in the hydrophobic face interacts with this region and is also in a conformationally critical region for substrate translocation. PMID:25784163

An intermolecular binding mechanism involving multiple LysM domains mediates carbohydrate recognition by an endopeptidase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wong, Jaslyn E. M. M.; Midtgaard, Søren Roi; Gysel, Kira

The crystal and solution structures of the T. thermophilus NlpC/P60 d, l-endopeptidase as well as the co-crystal structure of its N-terminal LysM domains bound to chitohexaose allow a proposal to be made regarding how the enzyme recognizes peptidoglycan. LysM domains, which are frequently present as repetitive entities in both bacterial and plant proteins, are known to interact with carbohydrates containing N-acetylglucosamine (GlcNAc) moieties, such as chitin and peptidoglycan. In bacteria, the functional significance of the involvement of multiple LysM domains in substrate binding has so far lacked support from high-resolution structures of ligand-bound complexes. Here, a structural study of themore » Thermus thermophilus NlpC/P60 endopeptidase containing two LysM domains is presented. The crystal structure and small-angle X-ray scattering solution studies of this endopeptidase revealed the presence of a homodimer. The structure of the two LysM domains co-crystallized with N-acetyl-chitohexaose revealed a new intermolecular binding mode that may explain the differential interaction between LysM domains and short or long chitin oligomers. By combining the structural information with the three-dimensional model of peptidoglycan, a model suggesting how protein dimerization enhances the recognition of peptidoglycan is proposed.« less

An Experiment Illustrating the Change in Ligand p"K"[subscript a] upon Protein Binding

ERIC Educational Resources Information Center

Chenprakhon, Pirom; Panijpan, Bhinyo; Chaiyen, Pimchai

2012-01-01

The modulation of ligand p"K"[subscript a] due to its surrounding environment is a crucial feature that controls many biological phenomena. For example, the shift in the p"K"[subscript a] of substrates or catalytic residues at enzyme active sites upon substrate binding often triggers and controls enzymatic reactions. In this work, we developed an…

Kinetics and binding sites for interaction of the prefoldin with a group II chaperonin: contiguous non-native substrate and chaperonin binding sites in the archaeal prefoldin.

PubMed

Okochi, Mina; Nomura, Tomoko; Zako, Tamotsu; Arakawa, Takatoshi; Iizuka, Ryo; Ueda, Hiroshi; Funatsu, Takashi; Leroux, Michel; Yohda, Masafumi

2004-07-23

Prefoldin is a jellyfish-shaped hexameric co-chaperone of the group II chaperonins. It captures a protein folding intermediate and transfers it to a group II chaperonin for completion of folding. The manner in which prefoldin interacts with its substrates and cooperates with the chaperonin is poorly understood. In this study, we have examined the interaction between a prefoldin and a chaperonin from hyperthermophilic archaea by immunoprecipitation, single molecule observation, and surface plasmon resonance. We demonstrate that Pyrococcus prefoldin interacts most tightly with its cognate chaperonin, and vice versa, suggesting species specificity in the interaction. Using truncation mutants, we uncovered by kinetic analyses that this interaction is multivalent in nature, consistent with multiple binding sites between the two chaperones. We present evidence that both N- and C-terminal regions of the prefoldin beta sub-unit are important for molecular chaperone activity and for the interaction with a chaperonin. Our data are consistent with substrate and chaperonin binding sites on prefoldin that are different but in close proximity, which suggests a possible handover mechanism of prefoldin substrates to the chaperonin.

Comparison of mechanistic transport cycle models of ABC exporters.

PubMed

Szöllősi, Dániel; Rose-Sperling, Dania; Hellmich, Ute A; Stockner, Thomas

2018-04-01

ABC (ATP binding cassette) transporters, ubiquitous in all kingdoms of life, carry out essential substrate transport reactions across cell membranes. Their transmembrane domains bind and translocate substrates and are connected to a pair of nucleotide binding domains, which bind and hydrolyze ATP to energize import or export of substrates. Over four decades of investigations into ABC transporters have revealed numerous details from atomic-level structural insights to their functional and physiological roles. Despite all these advances, a comprehensive understanding of the mechanistic principles of ABC transporter function remains elusive. The human multidrug resistance transporter ABCB1, also referred to as P-glycoprotein (P-gp), is one of the most intensively studied ABC exporters. Using ABCB1 as the reference point, we aim to compare the dominating mechanistic models of substrate transport and ATP hydrolysis for ABC exporters and to highlight the experimental and computational evidence in their support. In particular, we point out in silico studies that enhance and complement available biochemical data. "This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain." Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

The Structural Basis for Allosteric Inhibition of a Threonine-sensitive Aspartokinase*

PubMed Central

Liu, Xuying; Pavlovsky, Alexander G.; Viola, Ronald E.

2008-01-01

The commitment step to the aspartate pathway of amino acid biosynthesis is the phosphorylation of aspartic acid catalyzed by aspartokinase (AK). Most microorganisms and plants have multiple forms of this enzyme, and many of these isofunctional enzymes are subject to feedback regulation by the end products of the pathway. However, the archeal species Methanococcus jannaschii has only a single, monofunctional form of AK. The substrate l-aspartate binds to this recombinant enzyme in two different orientations, providing the first structural evidence supporting the relaxed regiospecificity previously observed with several alternative substrates of Escherichia coli AK (Angeles, T. S., Hunsley, J. R., and Viola, R. E. (1992) Biochemistry31 ,799 -8051731937). Binding of the nucleotide substrate triggers significant domain movements that result in a more compact quaternary structure. In contrast, the highly cooperative binding of the allosteric regulator l-threonine to multiple sites on this dimer of dimers leads to an open enzyme structure. A comparison of these structures supports a mechanism for allosteric regulation in which the domain movements induced by threonine binding causes displacement of the substrates from the enzyme, resulting in a relaxed, inactive conformation. PMID:18334478

Mutational analysis of microbial hydroxycinnamoyl-CoA hydratase-lyase (HCHL) towards enhancement of binding affinity: A computational approach.

PubMed

Kumar, Pravin; Ghosh Sachan, Shashwati; Poddar, Raju

2017-10-01

Improving the industrial enzyme for better yield of the product is important and a challenging task. One of such important industrial enzymes is microbial Hydroxycinnamoyl-CoA hydratase-lyase (HCHL). It converts feruloyl-CoA to vanillin. We place our efforts towards the improvement of its catalytic activity with comprehensive computational investigation. Catalytic core of the HCHL was explored with molecular modeling and docking approaches. Site-directed mutations were introduced in the catalytic site of HCHL in a sequential manner to generate different mutants of HCHL. Basis of mutation is to increase the interaction between HCHL and substrate feruloyl-CoA through interatomic forces and hydrogen bond formation. A rigorous molecular dynamics (MD) simulation was performed to check the stability of mutant's structure. Root mean square deviation (RMSD), root mean square fluctuation (RMSF), dynamic cross correlation (DCCM) and principal component analysis (PCA) were also performed to analyze flexibility and stability of structures. Docking studies were carried out between different mutants of HCHL and feruloyl-CoA. Investigation of the different binding sites and the interactions with mutant HCHLs and substrate allowed us to highlight the improved performance of mutants than wild type HCHL. This was further validated with MD simulation of complex consisting of different mutants and substrate. It further confirms all the structures are stable. However, mutant-2 showed better affinity towards substrate by forming hydrogen bond between active site and feruloyl-CoA. We propose that increase in hydrogen bond formation might facilitate in dissociation of vanillin from feruloyl-CoA. The current work may be useful for the future development of 'tailor-made' enzymes for better yield of vanillin. Copyright © 2017 Elsevier Inc. All rights reserved.

The Structure and Specificity of the Type III Secretion System Effector NleC Suggest a DNA Mimicry Mechanism of Substrate Recognition

PubMed Central

2015-01-01

Many pathogenic bacteria utilize the type III secretion system (T3SS) to translocate effector proteins directly into host cells, facilitating colonization. In enterohemmorhagic Escherichia coli (EHEC), a subset of T3SS effectors is essential for suppression of the inflammatory response in hosts, including humans. Identified as a zinc protease that cleaves NF-κB transcription factors, NleC is one such effector. Here, we investigate NleC substrate specificity, showing that four residues around the cleavage site in the DNA-binding loop of the NF-κB subunit RelA strongly influence the cleavage rate. Class I NF-κB subunit p50 is cleaved at a reduced rate consistent with conservation of only three of these four residues. However, peptides containing 10 residues on each side of the scissile bond were not efficiently cleaved by NleC, indicating that elements distal from the cleavage site are also important for substrate recognition. We present the crystal structure of NleC and show that it mimics DNA structurally and electrostatically. Consistent with this model, mutation of phosphate-mimicking residues in NleC reduces the level of RelA cleavage. We propose that global recognition of NF-κB subunits by DNA mimicry combined with a high sequence selectivity for the cleavage site results in exquisite NleC substrate specificity. The structure also shows that despite undetectable similarity of its sequence to those of other Zn2+ proteases beyond its conserved HExxH Zn2+-binding motif, NleC is a member of the Zincin protease superfamily, albeit divergent from its structural homologues. In particular, NleC displays a modified Ψ-loop motif that may be important for folding and refolding requirements implicit in T3SS translocation. PMID:25040221

On the structural and electronic properties of Ir-silicide nanowires on Si(001) surface

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fatima,; Hossain, Sehtab; Mohottige, Rasika

Iridium (Ir) modified Silicon (Si) (001) surface is studied with Scanning Tunneling Microscopy/Spectroscopy (STM/STS) and Density Functional Theory (DFT). A model for Ir-silicide nanowires based on STM images and ab-initio calculations is proposed. According to our model, the Ir adatom is on the top of the substrate dimer row and directly binds to the dimer atoms. I-V curves measured at 77 K shows that the nanowires are metallic. DFT calculations confirm strong metallic nature of the nanowires.

BiPPred: Combined sequence- and structure-based prediction of peptide binding to the Hsp70 chaperone BiP.

PubMed

Schneider, Markus; Rosam, Mathias; Glaser, Manuel; Patronov, Atanas; Shah, Harpreet; Back, Katrin Christiane; Daake, Marina Angelika; Buchner, Johannes; Antes, Iris

2016-10-01

Substrate binding to Hsp70 chaperones is involved in many biological processes, and the identification of potential substrates is important for a comprehensive understanding of these events. We present a multi-scale pipeline for an accurate, yet efficient prediction of peptides binding to the Hsp70 chaperone BiP by combining sequence-based prediction with molecular docking and MMPBSA calculations. First, we measured the binding of 15mer peptides from known substrate proteins of BiP by peptide array (PA) experiments and performed an accuracy assessment of the PA data by fluorescence anisotropy studies. Several sequence-based prediction models were fitted using this and other peptide binding data. A structure-based position-specific scoring matrix (SB-PSSM) derived solely from structural modeling data forms the core of all models. The matrix elements are based on a combination of binding energy estimations, molecular dynamics simulations, and analysis of the BiP binding site, which led to new insights into the peptide binding specificities of the chaperone. Using this SB-PSSM, peptide binders could be predicted with high selectivity even without training of the model on experimental data. Additional training further increased the prediction accuracies. Subsequent molecular docking (DynaDock) and MMGBSA/MMPBSA-based binding affinity estimations for predicted binders allowed the identification of the correct binding mode of the peptides as well as the calculation of nearly quantitative binding affinities. The general concept behind the developed multi-scale pipeline can readily be applied to other protein-peptide complexes with linearly bound peptides, for which sufficient experimental binding data for the training of classical sequence-based prediction models is not available. Proteins 2016; 84:1390-1407. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Insights into the molecular basis for substrate binding and specificity of the wild-type L-arginine/agmatine antiporter AdiC.

PubMed

Ilgü, Hüseyin; Jeckelmann, Jean-Marc; Gapsys, Vytautas; Ucurum, Zöhre; de Groot, Bert L; Fotiadis, Dimitrios

2016-09-13

Pathogenic enterobacteria need to survive the extreme acidity of the stomach to successfully colonize the human gut. Enteric bacteria circumvent the gastric acid barrier by activating extreme acid-resistance responses, such as the arginine-dependent acid resistance system. In this response, l-arginine is decarboxylated to agmatine, thereby consuming one proton from the cytoplasm. In Escherichia coli, the l-arginine/agmatine antiporter AdiC facilitates the export of agmatine in exchange of l-arginine, thus providing substrates for further removal of protons from the cytoplasm and balancing the intracellular pH. We have solved the crystal structures of wild-type AdiC in the presence and absence of the substrate agmatine at 2.6-Å and 2.2-Å resolution, respectively. The high-resolution structures made possible the identification of crucial water molecules in the substrate-binding sites, unveiling their functional roles for agmatine release and structure stabilization, which was further corroborated by molecular dynamics simulations. Structural analysis combined with site-directed mutagenesis and the scintillation proximity radioligand binding assay improved our understanding of substrate binding and specificity of the wild-type l-arginine/agmatine antiporter AdiC. Finally, we present a potential mechanism for conformational changes of the AdiC transport cycle involved in the release of agmatine into the periplasmic space of E. coli.

Ligand binding phenomena that pertain to the metabolic function of renalase.

PubMed

Beaupre, Brett A; Roman, Joseph V; Hoag, Matthew R; Meneely, Kathleen M; Silvaggi, Nicholas R; Lamb, Audrey L; Moran, Graham R

2016-12-15

Renalase catalyzes the oxidation of isomers of β-NAD(P)H that carry the hydride in the 2 or 6 positions of the nicotinamide base to form β-NAD(P) + . This activity is thought to alleviate inhibition of multiple β-NAD(P)-dependent enzymes of primary and secondary metabolism by these isomers. Here we present evidence for a variety of ligand binding phenomena relevant to the function of renalase. We offer evidence of the potential for primary metabolism inhibition with structures of malate dehydrogenase and lactate dehydrogenase bound to the 6-dihydroNAD isomer. The previously observed preference of renalase from Pseudomonas for NAD-derived substrates over those derived from NADP is accounted for by the structure of the enzyme in complex with NADPH. We also show that nicotinamide nucleosides and mononucleotides reduced in the 2- and 6-positions are renalase substrates, but bind weakly. A seven-fold enhancement of acquisition (k red /K d ) for 6-dihydronicotinamide riboside was observed for human renalase in the presence of ADP. However, generally the addition of complement ligands, AMP for mononucleotide or ADP for nucleoside substrates, did not enhance the reductive half-reaction. Non-substrate nicotinamide nucleosides or nucleotides bind weakly suggesting that only β-NADH and β-NADPH compete with dinucleotide substrates for access to the active site. Copyright © 2016 Elsevier Inc. All rights reserved.

NMR Mapping of Protein Conformational Landscapes using Coordinated Behavior of Chemical Shifts upon Ligand Binding

PubMed Central

Cembran, Alessandro; Kim, Jonggul; Gao, Jiali; Veglia, Gianluigi

2014-01-01

Proteins exist as an ensemble of conformers that are distributed on free energy landscapes resembling folding funnels. While the most stable conformers populate low energy basins, protein function is often carried out through low-populated conformational states that occupy high energy basins. Ligand binding shifts the populations of these states, changing the distribution of these conformers. Understanding how the equilibrium among the states is altered upon ligand binding, interaction with other binding partners, and/or mutations and post-translational modifications is of critical importance for explaining allosteric signaling in proteins. Here, we propose a statistical analysis of the chemical shifts (CONCISE, COordiNated ChemIcal Shifts bEhavior) for the interpretation of protein conformational equilibria following linear trajectories of NMR chemical shifts. CONCISE enables one to quantitatively measure the population shifts associated with ligand titrations and estimate the degree of collectiveness of the protein residues’ response to ligand binding, giving a concise view of the structural transitions. The combination of CONCISE with thermocalorimetric and kinetic data allows one to depict a protein’s approximate conformational energy landscape. We tested this method with the catalytic subunit of cAMP-dependent protein kinase A, a ubiquitous enzyme that undergoes conformational transitions upon both nucleotide and pseudo-substrate binding. When complemented with chemical shift covariance analysis (CHESCA), this new method offers both collective response and residue-specific correlations for ligand binding to proteins. PMID:24604024

Catalytic site interactions in yeast OMP synthase.

PubMed

Hansen, Michael Riis; Barr, Eric W; Jensen, Kaj Frank; Willemoës, Martin; Grubmeyer, Charles; Winther, Jakob R

2014-01-15

The enigmatic kinetics, half-of-the-sites binding, and structural asymmetry of the homodimeric microbial OMP synthases (orotate phosphoribosyltransferase, EC 2.4.2.10) have been proposed to result from an alternating site mechanism in these domain-swapped enzymes [R.W. McClard et al., Biochemistry 45 (2006) 5330-5342]. This behavior was investigated in the yeast enzyme by mutations in the conserved catalytic loop and 5-phosphoribosyl-1-diphosphate (PRPP) binding motif. Although the reaction is mechanistically sequential, the wild-type (WT) enzyme shows parallel lines in double reciprocal initial velocity plots. Replacement of Lys106, the postulated intersubunit communication device, produced intersecting lines in kinetic plots with a 2-fold reduction of kcat. Loop (R105G K109S H111G) and PRPP-binding motif (D131N D132N) mutant proteins, each without detectable enzymatic activity and ablated ability to bind PRPP, complemented to produce a heterodimer with a single fully functional active site showing intersecting initial velocity plots. Equilibrium binding of PRPP and orotidine 5'-monophosphate showed a single class of two binding sites per dimer in WT and K106S enzymes. Evidence here shows that the enzyme does not follow half-of-the-sites cooperativity; that interplay between catalytic sites is not an essential feature of the catalytic mechanism; and that parallel lines in steady-state kinetics probably arise from tight substrate binding. Copyright © 2013. Published by Elsevier Inc.

Functional role of R462 in the degradation of hyaluronan catalyzed by hyaluronate lyase from Streptococcus pneumoniae.

PubMed

Li, Fengxue; Xu, Dingguo

2015-08-01

Hyaluronan lyase from Streptococcus pneumoniae can degrade hyaluronic acid, which is one of the major components in the extracellular matrix. Hyaluronan can regulate water balance, osmotic pressure, and act as an ion exchange resin. Followed by our recent work on the catalytic reaction mechanism and substrate binding mode, we in this work further investigate the functional role of active site arginine residue, R462, in the degradation of hyaluronan. The site directed mutagenesis simulation of R462A and R462Q were modeled using a combined quantum mechanical and molecular mechanical method. The overall substrate binding features upon mutations do not have significant changes. The energetic profiles for the reaction processes are essentially the same as that in wild type enzyme, but significant activation barrier height changes can be observed. Both mutants were shown to accelerate the overall enzymatic activity, e.g., R462A can reduce the barrier height by about 2.8 kcal mol(-1), while R462Q reduces the activation energy by about 2.9 kcal mol(-1). Consistent with the active site model calculated using density functional theory, our results can support that the positive charge on R462 guanidino side chain group plays a negative role in the catalysis. Finally, the functional role of R462 was proposed to facilitate the formation of initial enzyme-substrate complex, but not in the subsequent catalytic degradation reaction. Graphical Abstract Degradation of hyaluronan catalyzed by hyaluronate lyase from Streptococcus pneumoniae.

Active site and laminarin binding in glycoside hydrolase family 55

DOE PAGES

Bianchetti, Christopher M.; Takasuka, Taichi E.; Deutsch, Sam; ...

2015-03-09

The Carbohydrate Active Enzyme (CAZy) database indicates that glycoside hydrolase family 55 (GH55) contains both endo- and exo-β-1,3-glucanases. The founding structure in the GH55 is PcLam55A from the white rot fungus Phanerochaete chrysosporium. Here, we present high resolution crystal structures of bacterial SacteLam55A from the highly cellulolytic Streptomyces sp. SirexAA-E with bound substrates and product. These structures, along with mutagenesis and kinetic studies, implicate Glu-502 as the catalytic acid (as proposed earlier for Glu-663 in PcLam55A) and a proton relay network of four residues in activating water as the nucleophile. Further, a set of conserved aromatic residues that define themore » active site apparently enforce an exo-glucanase reactivity as demonstrated by exhaustive hydrolysis reactions with purified laminarioligosaccharides. Two additional aromatic residues that line the substrate-binding channel show substrate-dependent conformational flexibility that may promote processive reactivity of the bound oligosaccharide in the bacterial enzymes. Gene synthesis carried out on ~30% of the GH55 family gave 34 active enzymes (19% functional coverage of the nonredundant members of GH55). These active enzymes reacted with only laminarin from a panel of 10 different soluble and insoluble polysaccharides and displayed a broad range of specific activities and optima for pH and temperature. Furthermore, application of this experimental method provides a new, systematic way to annotate glycoside hydrolase phylogenetic space for functional properties.« less

Hydration structure of the α-chymotrypsin substrate binding pocket: the impact of constrained geometry

NASA Astrophysics Data System (ADS)

Carey, Christina; Cheng, Yuen-Kit; Rossky, Peter J.

2000-08-01

The concave substrate binding pocket of α-chymotrypsin binds specifically hydrophobic side chains. In order to understand the hydration structure present in the absence of substrate, and elucidate the character of the solvent displaced on binding, molecular dynamics computer simulation of the solvent in a fully hydrated protein has been carried out and analyzed. The pocket is found to be characterized in terms of a mixed polar and apolar macromolecular surface. It is shown that the simulated solvent structure within it is spatially consistent with that seen via crystallography. The solvent structure is energetically characterized by large losses in hydrogen bonding among solvent molecules except at the mouth of the pocket where exposure to bulk-like solvent is possible. The loss in hydrogen bonding is attributed to the highly constrained geometry available to the solvent, preventing formation of a hydrogen bonding network, with only partial compensation by interactions with the macromolecular surface. The solvent displacement concomitant with substrate binding will therefore be associated with a large enthalpic driving force. This result is at the extreme of a continuum of variable cases of "hydrophobic" hydration, which differ most basically in surface curvature. These range from convex solute surfaces, inducing clathrate-like structures, with negligible hydrogen bond loss, to flat surfaces with significant interfacial loss, to the present concave case with hydrogen bonding losses exceeding 50%.

Spectroscopy on the wing: naturally inspired SERS substrates for biochemical analysis.

PubMed

Garrett, Natalie L; Vukusic, Peter; Ogrin, Feodor; Sirotkin, Evgeny; Winlove, C Peter; Moger, Julian

2009-03-01

We show that naturally occurring chitinous nanostructures found on the wings of the Graphium butterfly can be used as substrates for surface-enhanced Raman scattering when coated with a thin film of gold or silver. The substrates were found to exhibit excellent biocompatibility and sensitivity, making them ideal for protein assaying. An assay using avidin/biotin binding showed that the substrates could be used to quantify protein binding directly from changes in the surface-enhanced Raman scattering (SERS) spectra and were sensitive over a concentration range comparable with a typical enzyme-linked immunosorbent assays (ELISA) assay. A biomimetic version of the wing nanostructures produced using a highly reproducible, large-scale fabrication process, yielded comparable enhancement factors and biocompatibility. The excellent biocompatibility of the wings and biomimetic substrates is unparalleled by other lithographically produced substrates, and this could pave the way for widespread application of ultrasensitive SERS-based bioassays.

How Conformational Dynamics of DNA Polymerase Select Correct Substrates: Experiments and Simulations

PubMed Central

Kirmizialtin, Serdal; Nguyen, Virginia; Johnson, Kenneth A.; Elber, Ron

2012-01-01

Summary Nearly every enzyme undergoes a significant change in structure after binding it’s substrate. New experimental and theoretical analyses of the role of changes in HIV reverse transcriptase structure in selecting a correct substrate are presented. Atomically detailed simulations using the Milestoning method predict a rate and free energy profile of the conformational change commensurate with experimental data. A large conformational change occurring on a ms timescale locks the correct nucleotide at the active site, but promotes release of a mismatched nucleotide. The positions along the reaction coordinate that decide the yield of the reaction are not determined by the chemical step. Rather, the initial steps of weak substrate binding and protein conformational transition significantly enrich the yield of a reaction with a correct substrate, while the same steps diminish the reaction probability of an incorrect substrate. PMID:22483109

Crystal structure and enzymatic properties of chalcone isomerase from the Antarctic vascular plant Deschampsia antarctica Desv.

PubMed

Park, Sun-Ha; Lee, Chang Woo; Cho, Sung Mi; Lee, Hyoungseok; Park, Hyun; Lee, Jungeun; Lee, Jun Hyuck

2018-01-01

Chalcone isomerase (CHI) is an important enzyme for flavonoid biosynthesis that catalyzes the intramolecular cyclization of chalcones into (S)-flavanones. CHIs have been classified into two types based on their substrate specificity. Type I CHIs use naringenin chalcone as a substrate and are found in most of plants besides legumes, whereas type II CHIs in leguminous plants can also utilize isoliquiritigenin. In this study, we found that the CHI from the Antarctic plant Deschampsia antarctica (DaCHI1) is of type I based on sequence homology but can use type II CHI substrates. To clarify the enzymatic mechanism of DaCHI1 at the molecular level, the crystal structures of unliganded DaCHI1 and isoliquiritigenin-bound DaCHI1 were determined at 2.7 and 2.1 Å resolutions, respectively. The structures revealed that isoliquiritigenin binds to the active site of DaCHI1 and induces conformational changes. Additionally, the activity assay showed that while DaCHI1 exhibits substrate preference for naringenin chalcone, it can also utilize isoliquiritigenin although the catalytic activity was relatively low. Based on these results, we propose that DaCHI1 uses various substrates to produce antioxidant flavonoids as an adaptation to oxidative stresses associated with harsh environmental conditions.

Crystal structure and enzymatic properties of chalcone isomerase from the Antarctic vascular plant Deschampsia antarctica Desv.

PubMed Central

Lee, Hyoungseok; Park, Hyun; Lee, Jungeun

2018-01-01

Chalcone isomerase (CHI) is an important enzyme for flavonoid biosynthesis that catalyzes the intramolecular cyclization of chalcones into (S)-flavanones. CHIs have been classified into two types based on their substrate specificity. Type I CHIs use naringenin chalcone as a substrate and are found in most of plants besides legumes, whereas type II CHIs in leguminous plants can also utilize isoliquiritigenin. In this study, we found that the CHI from the Antarctic plant Deschampsia antarctica (DaCHI1) is of type I based on sequence homology but can use type II CHI substrates. To clarify the enzymatic mechanism of DaCHI1 at the molecular level, the crystal structures of unliganded DaCHI1 and isoliquiritigenin-bound DaCHI1 were determined at 2.7 and 2.1 Å resolutions, respectively. The structures revealed that isoliquiritigenin binds to the active site of DaCHI1 and induces conformational changes. Additionally, the activity assay showed that while DaCHI1 exhibits substrate preference for naringenin chalcone, it can also utilize isoliquiritigenin although the catalytic activity was relatively low. Based on these results, we propose that DaCHI1 uses various substrates to produce antioxidant flavonoids as an adaptation to oxidative stresses associated with harsh environmental conditions. PMID:29394293

Classification of a Haemophilus influenzae ABC Transporter HI1470/71 through Its Cognate Molybdate Periplasmic Binding Protein, MolA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tirado-Lee, Leidamarie; Lee, Allen; Rees, Douglas C.

2014-10-02

molA (HI1472) from H. influenzae encodes a periplasmic binding protein (PBP) that delivers substrate to the ABC transporter MolB{sub 2}C{sub 2} (formerly HI1470/71). The structures of MolA with molybdate and tungstate in the binding pocket were solved to 1.6 and 1.7 {angstrom} resolution, respectively. The MolA-binding protein binds molybdate and tungstate, but not other oxyanions such as sulfate and phosphate, making it the first class III molybdate-binding protein structurally solved. The {approx}100 {mu}M binding affinity for tungstate and molybdate is significantly lower than observed for the class II ModA molybdate-binding proteins that have nanomolar to low micromolar affinity for molybdate.more » The presence of two molybdate loci in H. influenzae suggests multiple transport systems for one substrate, with molABC constituting a low-affinity molybdate locus.« less

Crystal structure of tabtoxin resistance protein complexed with acetyl coenzyme A reveals the mechanism for {beta}-lactam acetylation.

DOE Office of Scientific and Technical Information (OSTI.GOV)

He, H.; Ding, Y.; Bartlam, M.

2003-01-31

Tabtoxin resistance protein (TTR) is an enzyme that renders tabtoxin-producing pathogens, such as Pseudomonas syringae, tolerant to their own phytotoxins. Here, we report the crystal structure of TTR complexed with its natural cofactor, acetyl coenzyme A (AcCoA), to 1.55 {angstrom} resolution. The binary complex forms a characteristic 'V' shape for substrate binding and contains the four motifs conserved in the GCN5-related N-acetyltransferase (GNAT) superfamily, which also includes the histone acetyltransferases (HATs). A single-step mechanism is proposed to explain the function of three conserved residues, Glu92, Asp130 and Tyr141, in catalyzing the acetyl group transfer to its substrate. We also reportmore » that TTR possesses HAT activity and suggest an evolutionary relationship between TTR and other GNAT members.« less

Crystal structure of tabtoxin resistance protein complexed with acetyl coenzyme A reveals the mechanism for beta-lactam acetylation.

PubMed

He, Hongzhen; Ding, Yi; Bartlam, Mark; Sun, Fei; Le, Yi; Qin, Xincheng; Tang, Hong; Zhang, Rongguang; Joachimiak, Andrzej; Liu, Jinyuan; Zhao, Nanming; Rao, Zihe

2003-01-31

Tabtoxin resistance protein (TTR) is an enzyme that renders tabtoxin-producing pathogens, such as Pseudomonas syringae, tolerant to their own phytotoxins. Here, we report the crystal structure of TTR complexed with its natural cofactor, acetyl coenzyme A (AcCoA), to 1.55A resolution. The binary complex forms a characteristic "V" shape for substrate binding and contains the four motifs conserved in the GCN5-related N-acetyltransferase (GNAT) superfamily, which also includes the histone acetyltransferases (HATs). A single-step mechanism is proposed to explain the function of three conserved residues, Glu92, Asp130 and Tyr141, in catalyzing the acetyl group transfer to its substrate. We also report that TTR possesses HAT activity and suggest an evolutionary relationship between TTR and other GNAT members.

Biochemistry Students' Ideas about How an Enzyme Interacts with a Substrate

ERIC Educational Resources Information Center

Linenberger, Kimberly J.; Bretz, Stacey Lowery

2015-01-01

Enzyme-substrate interactions are a fundamental concept of biochemistry that is built upon throughout multiple biochemistry courses. Central to understanding enzyme-substrate interactions is specific knowledge of exactly how an enzyme and substrate interact. Within this narrower topic, students must understand the various binding sites on an…

Structural insights into substrate and inhibitor binding sites in human indoleamine 2,3-dioxygenase 1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lewis-Ballester, Ariel; Pham, Khoa N.; Batabyal, Dipanwita

Human indoleamine 2,3-dioxygenase 1 (hIDO1) is an attractive cancer immunotherapeutic target owing to its role in promoting tumoral immune escape. However, drug development has been hindered by limited structural information. Here, we report the crystal structures of hIDO1 in complex with its substrate, Trp, an inhibitor, epacadostat, and/or an effector, indole ethanol (IDE). The data reveal structural features of the active site (Sa) critical for substrate activation; in addition, they disclose a new inhibitor-binding mode and a distinct small molecule binding site (Si). Structure-guided mutation of a critical residue, F270, to glycine perturbs the Si site, allowing structural determination ofmore » an inhibitory complex, where both the Sa and Si sites are occupied by Trp. The Si site offers a novel target site for allosteric inhibitors and a molecular explanation for the previously baffling substrate-inhibition behavior of the enzyme. Taken together, the data open exciting new avenues for structure-based drug design.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aich, Sanjukta; Prasad, Lata; Delbaere, Louis T.J.

GTP-dependent phosphoenolpyruvate carboxykinase (PCK) is the key enzyme that controls the blood glucose level during fasting in higher animals. Here we report the first substrate-free structure of a GTP-dependent phosphoenolpyruvate (PEP) carboxykinase from a bacterium, Corynebacterium glutamicum (CgPCK). The protein crystallizes in space group P2{sub 1} with four molecules per asymmetric unit. The 2.3 {angstrom} resolution structure was solved by molecular replacement using the human cytosolic PCK (hcPCK) structure (PDB ID: 1KHF) as the starting model. The four molecules in the asymmetric unit pack as two dimers, and is an artifact of crystal packing. However, the P-loop and the guaninemore » binding loop of the substrate-free CgPCK structure have different conformations from the other published GTP-specific PCK structures, which all have bound substrates and/or metal ions. It appears that a change in the P-loop and guanine binding loop conformation is necessary for substrate binding in GTP-specific PCKs, as opposed to overall domain movement in ATP-specific PCKs.« less

Kinetic Analysis and Probing with Substrate Analogues of the Reaction Pathway of the Nitrile Reductase QueF from Escherichia coli*

PubMed Central

Jung, Jihye; Czabany, Tibor; Wilding, Birgit; Klempier, Norbert; Nidetzky, Bernd

2016-01-01

The enzyme QueF catalyzes a four-electron reduction of a nitrile group into an amine, the only reaction of this kind known in biology. In nature, QueF converts 7-cyano-7-deazaguanine (preQ0) into 7-aminomethyl-7-deazaguanine (preQ1) for the biosynthesis of the tRNA-inserted nucleoside queuosine. The proposed QueF mechanism involves a covalent thioimide adduct between preQ0 and a cysteine nucleophile in the enzyme, and this adduct is subsequently converted into preQ1 in two NADPH-dependent reduction steps. Here, we show that the Escherichia coli QueF binds preQ0 in a strongly exothermic process (ΔH = −80.3 kJ/mol; −TΔS = 37.9 kJ/mol, Kd = 39 nm) whereby the thioimide adduct is formed with half-of-the-sites reactivity in the homodimeric enzyme. Both steps of preQ0 reduction involve transfer of the 4-pro-R-hydrogen from NADPH. They proceed about 4–7-fold more slowly than trapping of the enzyme-bound preQ0 as covalent thioimide (1.63 s−1) and are thus mainly rate-limiting for the enzyme's kcat (=0.12 s−1). Kinetic studies combined with simulation reveal a large primary deuterium kinetic isotope effect of 3.3 on the covalent thioimide reduction and a smaller kinetic isotope effect of 1.8 on the imine reduction to preQ1. 7-Formyl-7-deazaguanine, a carbonyl analogue of the imine intermediate, was synthesized chemically and is shown to be recognized by QueF as weak ligand for binding (ΔH = −2.3 kJ/mol; −TΔS = −19.5 kJ/mol) but not as substrate for reduction or oxidation. A model of QueF substrate recognition and a catalytic pathway for the enzyme are proposed based on these data. PMID:27754868

Structural Basis for the Ubiquitin-Linkage Specificity and deISGylating Activity of SARS-CoV Papain-Like Protease

PubMed Central

Ratia, Kiira; Kilianski, Andrew; Baez-Santos, Yahira M.; Baker, Susan C.; Mesecar, Andrew

2014-01-01

Severe acute respiratory syndrome coronavirus (SARS-CoV) encodes a papain-like protease (PLpro) with both deubiquitinating (DUB) and deISGylating activities that are proposed to counteract the post-translational modification of signaling molecules that activate the innate immune response. Here we examine the structural basis for PLpro's ubiquitin chain and interferon stimulated gene 15 (ISG15) specificity. We present the X-ray crystal structure of PLpro in complex with ubiquitin-aldehyde and model the interaction of PLpro with other ubiquitin-chain and ISG15 substrates. We show that PLpro greatly prefers K48- to K63-linked ubiquitin chains, and ISG15-based substrates to those that are mono-ubiquitinated. We propose that PLpro's higher affinity for K48-linked ubiquitin chains and ISG15 stems from a bivalent mechanism of binding, where two ubiquitin-like domains prefer to bind in the palm domain of PLpro with the most distal ubiquitin domain interacting with a “ridge” region of the thumb domain. Mutagenesis of residues within this ridge region revealed that these mutants retain viral protease activity and the ability to catalyze hydrolysis of mono-ubiquitin. However, a select number of these mutants have a significantly reduced ability to hydrolyze the substrate ISG15-AMC, or be inhibited by K48-linked diubuiquitin. For these latter residues, we found that PLpro antagonism of the nuclear factor kappa-light-chain-enhancer of activated B-cells (NFκB) signaling pathway is abrogated. This identification of key and unique sites in PLpro required for recognition and processing of diubiquitin and ISG15 versus mono-ubiquitin and protease activity provides new insight into ubiquitin-chain and ISG15 recognition and highlights a role for PLpro DUB and deISGylase activity in antagonism of the innate immune response. PMID:24854014

Crystal structures of the psychrophilic alpha-amylase from Alteromonas haloplanctis in its native form and complexed with an inhibitor.

PubMed Central

Aghajari, N.; Feller, G.; Gerday, C.; Haser, R.

1998-01-01

Alteromonas haloplanctis is a bacterium that flourishes in Antarctic sea-water and it is considered as an extreme psychrophile. We have determined the crystal structures of the alpha-amylase (AHA) secreted by this bacterium, in its native state to 2.0 angstroms resolution as well as in complex with Tris to 1.85 angstroms resolution. The structure of AHA, which is the first experimentally determined three-dimensional structure of a psychrophilic enzyme, resembles those of other known alpha-amylases of various origins with a surprisingly greatest similarity to mammalian alpha-amylases. AHA contains a chloride ion which activates the hydrolytic cleavage of substrate alpha-1,4-glycosidic bonds. The chloride binding site is situated approximately 5 angstroms from the active site which is characterized by a triad of acid residues (Asp 174, Glu 200, Asp 264). These are all involved in firm binding of the Tris moiety. A reaction mechanism for substrate hydrolysis is proposed on the basis of the Tris inhibitor binding and the chloride activation. A trio of residues (Ser 303, His 337, Glu 19) having a striking spatial resemblance with serine-protease like catalytic triads was found approximately 22 angstroms from the active site. We found that this triad is equally present in other chloride dependent alpha-amylases, and suggest that it could be responsible for autoproteolytic events observed in solution for this cold adapted alpha-amylase. PMID:9541387

Hsp70 Is a Novel Posttranscriptional Regulator of Gene Expression That Binds and Stabilizes Selected mRNAs Containing AU-Rich Elements

PubMed Central

Kishor, Aparna; Tandukar, Bishal; Ly, Yann V.; Toth, Eric A.; Suarez, Yvelisse; Brewer, Gary

2013-01-01

The AU-rich elements (AREs) encoded within many mRNA 3′ untranslated regions (3′UTRs) are targets for factors that control transcript longevity and translational efficiency. Hsp70, best known as a protein chaperone with well-defined peptide-refolding properties, is known to interact with ARE-like RNA substrates in vitro. Here, we show that cofactor-free preparations of Hsp70 form direct, high-affinity complexes with ARE substrates based on specific recognition of U-rich sequences by both the ATP- and peptide-binding domains. Suppressing Hsp70 in HeLa cells destabilized an ARE reporter mRNA, indicating a novel ARE-directed mRNA-stabilizing role for this protein. Hsp70 also bound and stabilized endogenous ARE-containing mRNAs encoding vascular endothelial growth factor (VEGF) and Cox-2, which involved a mechanism that was unaffected by an inhibitor of its protein chaperone function. Hsp70 recognition and stabilization of VEGF mRNA was mediated by an ARE-like sequence in the proximal 3′UTR. Finally, stabilization of VEGF mRNA coincided with the accumulation of Hsp70 protein in HL60 promyelocytic leukemia cells recovering from acute thermal stress. We propose that the binding and stabilization of selected ARE-containing mRNAs may contribute to the cytoprotective effects of Hsp70 following cellular stress but may also provide a novel mechanism linking constitutively elevated Hsp70 expression to the development of aggressive neoplastic phenotypes. PMID:23109422

Biochemical profiling in silico--predicting substrate specificities of large enzyme families.

PubMed

Tyagi, Sadhna; Pleiss, Juergen

2006-06-25

A general high-throughput method for in silico biochemical profiling of enzyme families has been developed based on covalent docking of potential substrates into the binding sites of target enzymes. The method has been tested by systematically docking transition state--analogous intermediates of 12 substrates into the binding sites of 20 alpha/beta hydrolases from 15 homologous families. To evaluate the effect of side chain orientations to the docking results, 137 crystal structures were included in the analysis. A good substrate must fulfil two criteria: it must bind in a productive geometry with four hydrogen bonds between the substrate and the catalytic histidine and the oxyanion hole, and a high affinity of the enzyme-substrate complex as predicted by a high docking score. The modelling results in general reproduce experimental data on substrate specificity and stereoselectivity: the differences in substrate specificity of cholinesterases toward acetyl- and butyrylcholine, the changes of activity of lipases and esterases upon the size of the acid moieties, activity of lipases and esterases toward tertiary alcohols, and the stereopreference of lipases and esterases toward chiral secondary alcohols. Rigidity of the docking procedure was the major reason for false positive and false negative predictions, as the geometry of the complex and docking score may sensitively depend on the orientation of individual side chains. Therefore, appropriate structures have to be identified. In silico biochemical profiling provides a time efficient and cost saving protocol for virtual screening to identify the potential substrates of the members of large enzyme family from a library of molecules.

A sensory complex consisting of an ATP-binding cassette transporter and a two-component regulatory system controls bacitracin resistance in Bacillus subtilis.

PubMed

Dintner, Sebastian; Heermann, Ralf; Fang, Chong; Jung, Kirsten; Gebhard, Susanne

2014-10-03

Resistance against antimicrobial peptides in many Firmicutes bacteria is mediated by detoxification systems that are composed of a two-component regulatory system (TCS) and an ATP-binding cassette (ABC) transporter. The histidine kinases of these systems depend entirely on the transporter for sensing of antimicrobial peptides, suggesting a novel mode of signal transduction where the transporter constitutes the actual sensor. The aim of this study was to investigate the molecular mechanisms of this unusual signaling pathway in more detail, using the bacitracin resistance system BceRS-BceAB of Bacillus subtilis as an example. To analyze the proposed communication between TCS and the ABC transporter, we characterized their interactions by bacterial two-hybrid analyses and could show that the permease BceB and the histidine kinase BceS interact directly. In vitro pulldown assays confirmed this interaction, which was found to be independent of bacitracin. Because it was unknown whether BceAB-type transporters could detect their substrate peptides directly or instead recognized the peptide-target complex in the cell envelope, we next analyzed substrate binding by the transport permease, BceB. Direct and specific binding of bacitracin by BceB was demonstrated by surface plasmon resonance spectroscopy. Finally, in vitro signal transduction assays indicated that complex formation with the transporter influenced the autophosphorylation activity of the histidine kinase. Taken together, our findings clearly show the existence of a sensory complex composed of TCS and ABC transporters and provide the first functional insights into the mechanisms of stimulus perception, signal transduction, and antimicrobial resistance employed by Bce-like detoxification systems. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

A Sensory Complex Consisting of an ATP-binding Cassette Transporter and a Two-component Regulatory System Controls Bacitracin Resistance in Bacillus subtilis*

PubMed Central

Dintner, Sebastian; Heermann, Ralf; Fang, Chong; Jung, Kirsten; Gebhard, Susanne

2014-01-01

Resistance against antimicrobial peptides in many Firmicutes bacteria is mediated by detoxification systems that are composed of a two-component regulatory system (TCS) and an ATP-binding cassette (ABC) transporter. The histidine kinases of these systems depend entirely on the transporter for sensing of antimicrobial peptides, suggesting a novel mode of signal transduction where the transporter constitutes the actual sensor. The aim of this study was to investigate the molecular mechanisms of this unusual signaling pathway in more detail, using the bacitracin resistance system BceRS-BceAB of Bacillus subtilis as an example. To analyze the proposed communication between TCS and the ABC transporter, we characterized their interactions by bacterial two-hybrid analyses and could show that the permease BceB and the histidine kinase BceS interact directly. In vitro pulldown assays confirmed this interaction, which was found to be independent of bacitracin. Because it was unknown whether BceAB-type transporters could detect their substrate peptides directly or instead recognized the peptide-target complex in the cell envelope, we next analyzed substrate binding by the transport permease, BceB. Direct and specific binding of bacitracin by BceB was demonstrated by surface plasmon resonance spectroscopy. Finally, in vitro signal transduction assays indicated that complex formation with the transporter influenced the autophosphorylation activity of the histidine kinase. Taken together, our findings clearly show the existence of a sensory complex composed of TCS and ABC transporters and provide the first functional insights into the mechanisms of stimulus perception, signal transduction, and antimicrobial resistance employed by Bce-like detoxification systems. PMID:25118291

Substrate-binding specificity of chitinase and chitosanase as revealed by active-site architecture analysis.

PubMed

Liu, Shijia; Shao, Shangjin; Li, Linlin; Cheng, Zhi; Tian, Li; Gao, Peiji; Wang, Lushan

2015-12-11

Chitinases and chitosanases, referred to as chitinolytic enzymes, are two important categories of glycoside hydrolases (GH) that play a key role in degrading chitin and chitosan, two naturally abundant polysaccharides. Here, we investigate the active site architecture of the major chitosanase (GH8, GH46) and chitinase families (GH18, GH19). Both charged (Glu, His, Arg, Asp) and aromatic amino acids (Tyr, Trp, Phe) are observed with higher frequency within chitinolytic active sites as compared to elsewhere in the enzyme structure, indicating significant roles related to enzyme function. Hydrogen bonds between chitinolytic enzymes and the substrate C2 functional groups, i.e. amino groups and N-acetyl groups, drive substrate recognition, while non-specific CH-π interactions between aromatic residues and substrate mainly contribute to tighter binding and enhanced processivity evident in GH8 and GH18 enzymes. For different families of chitinolytic enzymes, the number, type, and position of substrate atoms bound in the active site vary, resulting in different substrate-binding specificities. The data presented here explain the synergistic action of multiple enzyme families at a molecular level and provide a more reasonable method for functional annotation, which can be further applied toward the practical engineering of chitinases and chitosanases. Copyright © 2015 Elsevier Ltd. All rights reserved.

Structural Basis of J Cochaperone Binding and Regulation of Hsp70

PubMed Central

Jiang, Jianwen; Maes, E. Guy; Taylor, Alex B; Wang, Liping; Hinck, Andrew P; Lafer, Eileen M; Sousa, Rui

2007-01-01

The many protein processing reactions of the ATP-hydrolyzing Hsp70s are regulated by J cochaperones, which contain J domains that stimulate Hsp70 ATPase activity and accessory domains that present protein substrates to Hsp70s. We report the structure of a J domain complexed with a J responsive portion of a mammalian Hsp70. The J domain activates ATPase activity by directing the linker that connects the Hsp70 nucleotide binding domain (NBD) and substrate binding domain (SBD) towards a hydrophobic patch on the NBD surface. Binding of the J domain to Hsp70 displaces the SBD from the NBD, which may allow the SBD flexibility to capture diverse substrates. Unlike prokaryotic Hsp70, the SBD and NBD of the mammalian chaperone interact in the ADP state. Thus, while both nucleotides and J cochaperones modulate Hsp70 NBD:linker and NBD:SBD interactions, the intrinsic persistence of those interactions differs in different Hsp70s and this may optimize their activities for different cellular roles. PMID:17996706

Evidence of the direct involvement of the substrate TCP radical in functional switching from oxyferrous O2 carrier to ferric peroxidase in the dual-function hemoglobin/dehaloperoxidase from Amphitrite ornata.

PubMed

Sun, Shengfang; Sono, Masanori; Du, Jing; Dawson, John H

2014-08-05

The coelomic O2-binding hemoglobin dehaloperoxidase (DHP) from the sea worm Amphitrite ornata is a dual-function heme protein that also possesses a peroxidase activity. Two different starting oxidation states are required for reversible O2 binding (ferrous) and peroxidase (ferric) activity, bringing into question how DHP manages the two functions. In our previous study, the copresence of substrate 2,4,6-trichlorophenol (TCP) and H2O2 was found to be essential for the conversion of oxy-DHP to enzymatically active ferric DHP. On the basis of that study, a functional switching mechanism involving substrate radicals (TCP(•)) was proposed. To further support this mechanism, herein we report details of our investigations into the H2O2-mediated conversion of oxy-DHP to the ferric or ferryl ([TCP] < [H2O2]) state triggered by both biologically relevant [TCP and 4-bromophenol (4-BP)] and nonrelevant (ferrocyanide) compounds. At <50 μM H2O2, all of these conversion reactions are completely inhibited by ferric heme ligands (KCN and imidazole), indicating the involvement of ferric DHP. Furthermore, the spin-trapping reagent 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) effectively inhibits the TCP/4-BP (but not ferrocyanide)-triggered conversion of oxy-DHP to ferric DHP. These results and O2 concentration-dependent conversion rates observed in this study demonstrate that substrate TCP triggers the conversion of oxy-DHP to a peroxidase by TCP(•) oxidation of the deoxyferrous state. TCP(•) is progressively generated, by increasingly produced amounts of ferric DHP, upon H2O2 oxidation of TCP catalyzed initially by trace amounts of ferric enzyme present in the oxy-DHP sample. The data presented herein further address the mechanism of how the halophenolic substrate triggers the conversion of hemoglobin DHP into a peroxidase.

N ,N'-dimethylperylene-3,4,9,10-bis(dicarboximide) on alkali halide (001) surfaces

NASA Astrophysics Data System (ADS)

Fendrich, Markus; Lange, Manfred; Weiss, Christian; Kunstmann, Tobias; Möller, Rolf

2009-05-01

The growth of N ,N'-dimethylperylene-3,4,9,10-bis(dicarboximide) (DiMe-PTCDI) on KBr(001) and NaCl(001) surfaces has been studied. Experimental results have been achieved using frequency modulation atomic force microscopy at room temperature under ultrahigh vacuum conditions. On both substrates, DiMe-PTCDI forms molecular wires with a width of 10nm, typically, and a length of up to 600nm at low coverages. All wires grow along either the [110] direction (or [11¯0] direction, respectively) of the alkali halide (001) substrates. There is no wetting layer of molecules: atomic resolution of the substrates can be achieved between the wires. The wires are mobile on KBr but substantially more stable on NaCl. A p(2×2) superstructure in a brickwall arrangement on the ionic crystal surfaces is proposed based on electrostatic considerations. Calculations and Monte Carlo simulations using empirical potentials reveal possible growth mechanisms for molecules within the first layer for both substrates, also showing a significantly higher binding energy for NaCl(001). For KBr, the p(2×2) superstructure is confirmed by the simulations; for NaCl, a less dense, incommensurate superstructure is predicted.

Pharmacophore, QSAR, and binding mode studies of substrates of human cytochrome P450 2D6 (CYP2D6) using molecular docking and virtual mutations and an application to chinese herbal medicine screening.

PubMed

Mo, Sui-Lin; Liu, Wei-Feng; Li, Chun-Guang; Zhou, Zhi-Wei; Luo, Hai-Bin; Chew, Helen; Liang, Jun; Zhou, Shu-Feng

2012-07-01

The highly polymorphic human cytochrome P450 2D6 (CYP2D6) metabolizes about 25% of currently used drugs. In this study, we have explored the interaction of a large number of substrates (n = 120) with wild-type and mutated CYP2D6 by molecular docking using the CDOCKER module. Before we conducted the molecular docking and virtual mutations, the pharmacophore and QSAR models of CYP2D6 substrates were developed and validated. Finally, we explored the interaction of a traditional Chinese herbal formula, Fangjifuling decoction, with CYP2D6 by virtual screening. The optimized pharmacophore model derived from 20 substrates of CYP2D6 contained two hydrophobic features and one hydrogen bond acceptor feature, giving a relevance ratio of 76% when a validation set of substrates were tested. However, our QSAR models gave poor prediction of the binding affinity of substrates. Our docking study demonstrated that 117 out of 120 substrates could be docked into the active site of CYP2D6. Forty one out of 117 substrates (35.04%) formed hydrogen bonds with various active site residues of CYP2D6 and 53 (45.30%) substrates formed a strong π-π interaction with Phe120 (53/54), with only carvedilol showing π-π interaction with Phe483. The active site residues involving hydrogen bond formation with substrates included Leu213, Lys214, Glu216, Ser217, Gln244, Asp301, Ser304, Ala305, Phe483, and Phe484. Furthermore, the CDOCKER algorithm was further applied to study the impact of mutations of 28 active site residues (mostly non-conserved) of CYP2D6 on substrate binding modes using five probe substrates including bufuralol, debrisoquine, dextromethorphan, sparteine, and tramadol. All mutations of the residues examined altered the hydrogen bond formation and/or aromatic interactions, depending on the probe used in molecular docking. Apparent changes of the binding modes have been observed with the Glu216Asp and Asp301Glu mutants. Overall, 60 compounds out of 130 from Fangjifuling decoction matched our pharmacophore model for CYP2D6 substrates. Fifty four out of these 60 compounds could be docked into the active site of CYP2D6 and 24 of 54 compounds formed hydrogen bonds with Glu216, Asp301, Ser304, and Ala305 in CYP2D6. These results have provided further insights into the factors that determining the binding modes of substrates to CYP2D6. Screening of high-affinity ligands for CYP2D6 from herbal formula using computational models is a useful approach to identify potential herb-drug interactions.

A histone-mimicking interdomain linker in a multidomain protein modulates multivalent histone binding

PubMed Central

Kostrhon, Sebastian; Kontaxis, Georg; Kaufmann, Tanja; Schirghuber, Erika; Kubicek, Stefan; Konrat, Robert

2017-01-01

N-terminal histone tails are subject to many posttranslational modifications that are recognized by and interact with designated reader domains in histone-binding proteins. BROMO domain adjacent to zinc finger 2B (BAZ2B) is a multidomain histone-binding protein that contains two histone reader modules, a plant homeodomain (PHD) and a bromodomain (BRD), linked by a largely disordered linker. Although previous studies have reported specificity of the PHD domain for the unmodified N terminus of histone H3 and of the BRD domain for H3 acetylated at Lys14 (H3K14ac), the exact mode of H3 binding by BAZ2B and its regulation are underexplored. Here, using isothermal titration calorimetry and NMR spectroscopy, we report that acidic residues in the BAZ2B PHD domain are essential for H3 binding and that BAZ2B PHD–BRD establishes a polyvalent interaction with H3K14ac. Furthermore, we provide evidence that the disordered interdomain linker modulates the histone-binding affinity by interacting with the PHD domain. In particular, lysine-rich stretches in the linker, which resemble the positively charged N terminus of histone H3, reduce the binding affinity of the PHD finger toward the histone substrate. Phosphorylation, acetylation, or poly(ADP-ribosyl)ation of the linker residues may therefore act as a cellular mechanism to transiently tune BAZ2B histone-binding affinity. Our findings further support the concept of interdomain linkers serving a dual role in substrate binding by appropriately positioning the adjacent domains and by electrostatically modulating substrate binding. Moreover, inhibition of histone binding by a histone-mimicking interdomain linker represents another example of regulation of protein–protein interactions by intramolecular mimicry. PMID:28864776

The role of chloride in the mechanism of O(2) activation at the mononuclear nonheme Fe(II) center of the halogenase HctB.

PubMed

Pratter, Sarah M; Light, Kenneth M; Solomon, Edward I; Straganz, Grit D

2014-07-02

Mononuclear nonheme Fe(II) (MNH) and α-ketoglutarate (α-KG) dependent halogenases activate O2 to perform oxidative halogenations of activated and nonactivated carbon centers. While the mechanism of halide incorporation into a substrate has been investigated, the mechanism by which halogenases prevent oxidations in the absence of chloride is still obscure. Here, we characterize the impact of chloride on the metal center coordination and reactivity of the fatty acyl-halogenase HctB. Stopped-flow kinetic studies show that the oxidative transformation of the Fe(II)-α-KG-enzyme complex is >200-fold accelerated by saturating concentrations of chloride in both the absence and presence of a covalently bound substrate. By contrast, the presence of substrate, which generally brings about O2 activation at enzymatic MNH centers, only has an ∼10-fold effect in the absence of chloride. Circular dichroism (CD) and magnetic CD (MCD) studies demonstrate that chloride binding triggers changes in the metal center ligation: chloride binding induces the proper binding of the substrate as shown by variable-temperature, variable-field (VTVH) MCD studies of non-α-KG-containing forms and the conversion from six-coordinate (6C) to 5C/6C mixtures when α-KG is bound. In the presence of substrate, a site with square pyramidal five-coordinate (5C) geometry is observed, which is required for O2 activation at enzymatic MNH centers. In the absence of substrate an unusual trigonal bipyramidal site is formed, which accounts for the observed slow, uncoupled reactivity. Molecular dynamics simulations suggest that the binding of chloride to the metal center of HctB leads to a conformational change in the enzyme that makes the active site more accessible to the substrate and thus facilitates the formation of the catalytically competent enzyme-substrate complex. Results are discussed in relation to other MNH dependent halogenases.

X-ray structures of the Pseudomonas cichorii D-tagatose 3-epimerase mutant form C66S recognizing deoxy sugars as substrates.

PubMed

Yoshida, Hiromi; Yoshihara, Akihide; Ishii, Tomohiko; Izumori, Ken; Kamitori, Shigehiro

2016-12-01

Pseudomonas cichorii D-tagatose 3-epimerase (PcDTE), which has a broad substrate specificity, efficiently catalyzes the epimerization of not only D-tagatose to D-sorbose but also D-fructose to D-psicose (D-allulose) and also recognizes the deoxy sugars as substrates. In an attempt to elucidate the substrate recognition and catalytic reaction mechanisms of PcDTE for deoxy sugars, the X-ray structures of the PcDTE mutant form with the replacement of Cys66 by Ser (PcDTE_C66S) in complexes with deoxy sugars were determined. These X-ray structures showed that substrate recognition by the enzyme at the 1-, 2-, and 3-positions is responsible for enzymatic activity and that substrate-enzyme interactions at the 4-, 5-, and 6-positions are not essential for the catalytic reaction of the enzyme leading to the broad substrate specificity of PcDTE. They also showed that the epimerization site of 1-deoxy 3-keto D-galactitol is shifted from C3 to C4 and that 1-deoxy sugars may bind to the catalytic site in the inhibitor-binding mode. The hydrophobic groove that acts as an accessible surface for substrate binding is formed through the dimerization of PcDTE. In PcDTE_C66S/deoxy sugar complex structures, bound ligand molecules in both the linear and ring forms were detected in the hydrophobic groove, while bound ligand molecules in the catalytic site were in the linear form. This result suggests that the sugar-ring opening of a substrate may occur in the hydrophobic groove and also that the narrow channel of the passageway to the catalytic site allows a substrate in the linear form to pass through.

The Fanconi anemia associated protein FAAP24 uses two substrate specific binding surfaces for DNA recognition

PubMed Central

Wienk, Hans; Slootweg, Jack C.; Speerstra, Sietske; Kaptein, Robert; Boelens, Rolf; Folkers, Gert E.

2013-01-01

To maintain the integrity of the genome, multiple DNA repair systems exist to repair damaged DNA. Recognition of altered DNA, including bulky adducts, pyrimidine dimers and interstrand crosslinks (ICL), partially depends on proteins containing helix-hairpin-helix (HhH) domains. To understand how ICL is specifically recognized by the Fanconi anemia proteins FANCM and FAAP24, we determined the structure of the HhH domain of FAAP24. Although it resembles other HhH domains, the FAAP24 domain contains a canonical hairpin motif followed by distorted motif. The HhH domain can bind various DNA substrates; using nuclear magnetic resonance titration experiments, we demonstrate that the canonical HhH motif is required for double-stranded DNA (dsDNA) binding, whereas the unstructured N-terminus can interact with single-stranded DNA. Both DNA binding surfaces are used for binding to ICL-like single/double-strand junction-containing DNA substrates. A structural model for FAAP24 bound to dsDNA has been made based on homology with the translesion polymerase iota. Site-directed mutagenesis, sequence conservation and charge distribution support the dsDNA-binding model. Analogous to other HhH domain-containing proteins, we suggest that multiple FAAP24 regions together contribute to binding to single/double-strand junction, which could contribute to specificity in ICL DNA recognition. PMID:23661679

Effect of solid surface charge on the binding behaviour of a metal-binding peptide

PubMed Central

Donatan, Senem; Sarikaya, Mehmet; Tamerler, Candan; Urgen, Mustafa

2012-01-01

Over the last decade, solid-binding peptides have been increasingly used as molecular building blocks coupling bio- and nanotechnology. Despite considerable research being invested in this field, the effects of many surface-related parameters that define the binding of peptide to solids are still unknown. In the quest to control biological molecules at solid interfaces and, thereby, tailoring the binding characteristics of the peptides, the use of surface charge of the solid surface may probably play an important role, which then can be used as a potential tuning parameter of peptide adsorption. Here, we report quantitative investigation on the viscoelastic properties and binding kinetics of an engineered gold-binding peptide, 3RGBP1, adsorbed onto the gold surface at different surface charge densities. The experiments were performed in aqueous solutions using an electrochemical dissipative quartz crystal microbalance system. Hydrodynamic mass, hydration state and surface coverage of the adsorbed peptide films were determined as a function of surface charge density of the gold metal substrate. Under each charged condition, binding of 3rGBP1 displayed quantitative differences in terms of adsorbed peptide amount, surface coverage ratio and hydration state. Based on the intrinsically disordered structure of the peptide, we propose a possible mechanism for binding of the peptide that can be used for tuning surface adsorption in further studies. Controlled alteration of peptide binding on solid surfaces, as shown here, may provide novel methods for surface functionalization used for bioenabled processing and fabrication of future micro- and nanodevices. PMID:22491974

Altered Enthalpy-Entropy Compensation in Picomolar Transition State Analogues of Human Purine Nucleoside Phosphorylase†

PubMed Central

Edwards, Achelle A.; Mason, Jennifer M.; Clinch, Keith; Tyler, Peter C.; Evans, Gary B.; Schramm, Vern L.

2009-01-01

Human purine nucleoside phosphorylase (PNP) belongs to the trimeric class of PNPs and is essential for catabolism of deoxyguanosine. Genetic deficiency of PNP in humans causes a specific T-cell immune deficiency and transition state analogue inhibitors of PNP are in development for treatment of T-cell cancers and autoimmune disorders. Four generations of Immucillins have been developed, each of which contains inhibitors binding with picomolar affinity to human PNP. Full inhibition of PNP occurs upon binding to the first of three subunits and binding to subsequent sites occurs with negative cooperativity. In contrast, substrate analogue and product bind without cooperativity. Titrations of human PNP using isothermal calorimetery indicate that binding of a structurally rigid first-generation Immucillin (K d = 56 pM) is driven by large negative enthalpy values (ΔH = −21.2 kcal/mol) with a substantial entropic (-TΔS) penalty. The tightest-binding inhibitors (K d = 5 to 9 pM) have increased conformational flexibility. Despite their conformational freedom in solution, flexible inhibitors bind with high affinity because of reduced entropic penalties. Entropic penalties are proposed to arise from conformational freezing of the PNP·inhibitor complex with the entropy term dominated by protein dynamics. The conformationally flexible Immucillins reduce the system entropic penalty. Disrupting the ribosyl 5’-hydroxyl interaction of transition state analogues with PNP causes favorable entropy of binding. Tight binding of the seventeen Immucillins is characterized by large enthalpic contributions, emphasizing their similarity to the transition state. By introducing flexibility into the inhibitor structure, the enthalpy-entropy compensation pattern is altered to permit tighter binding. PMID:19425594

Porous light-emitting compositions

DOEpatents

Burrell, Anthony K [Los Alamos, NM; McCleskey, Thomas Mark [Los Alamos, NM; Jia, Quanxi [Los Alamos, NM; Bauer, Eve [Los Alamos, NM; Mueller, Alexander H [Los Alamos, NM

2012-04-17

Light-emitting devices are prepared by coating a porous substrate using a polymer-assisted deposition process. Solutions of metal precursor and soluble polymers having binding properties for metal precursor were coated onto porous substrates. The coated substrates were heated at high temperatures under a suitable atmosphere. The result was a substrate with a conformal coating that did not substantially block the pores of the substrate.

Novel characteristics of a carbohydrate-binding module 20 from hyperthermophilic bacterium.

PubMed

Oh, Il-Nam; Jane, Jay-Lin; Wang, Kan; Park, Jong-Tae; Park, Kwan-Hwa

2015-03-01

In this study, a gene fragment coding carbohydrate-binding module 20 (CBM20) in the amylopullulanase (APU) gene was cloned from the hyperthermophilic bacteria Thermoanaerobacter pseudoethanolicus 39E and expressed in Escherichia coli. The protein, hereafter Tp39E, possesses very low sequence similarity with the CBM20s previously reported and has no starch binding site 2. Tp39E did not demonstrate thermal denaturation at 50 °C; however, thermal unfolding of the protein was observed at 59.5 °C. A binding assay with Tp39E was conducted using various soluble and insoluble substrates, and starch was the best binding polysaccharide. Intriguingly, Tp39E bound, to a lesser extent, to soluble and insoluble xylan as well. The dissociation constant (K d) and the maximum specific binding (B max) of Tp39E to corn starch granules were 0.537 μM and 5.79 μM/g, respectively, at pH 5.5 and 20 °C. 99APU1357 with a Tp39E domain exhibited 2.2-fold greater activity than a CBM20-truncation mutant when starch granules were the substrate. Tp39E was an independently thermostable CBM and had a considerable effect on APU activity in the hydrolysis of insoluble substrates.

Coelenterazine-binding protein of Renilla muelleri: cDNA cloning, overexpression, and characterization as a substrate of luciferase.

PubMed

Titushin, Maxim S; Markova, Svetlana V; Frank, Ludmila A; Malikova, Natalia P; Stepanyuk, Galina A; Lee, John; Vysotski, Eugene S

2008-02-01

The Renilla bioluminescent system in vivo is comprised of three proteins--the luciferase, green-fluorescent protein, and coelenterazine-binding protein (CBP), previously called luciferin-binding protein (LBP). This work reports the cloning of the full-size cDNA encoding CBP from soft coral Renilla muelleri, its overexpression and properties of the recombinant protein. The apo-CBP was quantitatively converted to CBP by simple incubation with coelenterazine. The physicochemical properties of this recombinant CBP are determined to be practically the same as those reported for the CBP (LBP) of R. reniformis. CBP is a member of the four-EF-hand Ca(2+)-binding superfamily of proteins with only three of the EF-hand loops having the Ca(2+)-binding consensus sequences. There is weak sequence homology with the Ca(2+)-regulated photoproteins but only as a result of the necessary Ca(2+)-binding loop structure. In combination with Renilla luciferase, addition of only one Ca(2+) is sufficient to release the coelenterazine as a substrate for the luciferase for bioluminescence. This combination of the two proteins generates bioluminescence with higher reaction efficiency than using free coelenterazine alone as the substrate for luciferase. This increased quantum yield, a difference of bioluminescence spectra, and markedly different kinetics, implicate that a CBP-luciferase complex might be involved.

NMR study of complexes between low molecular mass inhibitors and the West Nile virus NS2B-NS3 protease.

PubMed

Su, Xun-Cheng; Ozawa, Kiyoshi; Yagi, Hiromasa; Lim, Siew P; Wen, Daying; Ekonomiuk, Dariusz; Huang, Danzhi; Keller, Thomas H; Sonntag, Sebastian; Caflisch, Amedeo; Vasudevan, Subhash G; Otting, Gottfried

2009-08-01

The two-component NS2B-NS3 protease of West Nile virus is essential for its replication and presents an attractive target for drug development. Here, we describe protocols for the high-yield expression of stable isotope-labelled samples in vivo and in vitro. We also describe the use of NMR spectroscopy to determine the binding mode of new low molecular mass inhibitors of the West Nile virus NS2B-NS3 protease which were discovered using high-throughput in vitro screening. Binding to the substrate-binding sites S1 and S3 is confirmed by intermolecular NOEs and comparison with the binding mode of a previously identified low molecular mass inhibitor. Our results show that all these inhibitors act by occupying the substrate-binding site of the protease rather than by an allosteric mechanism. In addition, the NS2B polypeptide chain was found to be positioned near the substrate-binding site, as observed previously in crystal structures of the protease in complex with peptide inhibitors or bovine pancreatic trypsin inhibitor. This indicates that the new low molecular mass compounds, although inhibiting the protease, also promote the proteolytically active conformation of NS2B, which is very different from the crystal structure of the protein without inhibitor.

Predominant nonproductive substrate binding by fungal cellobiohydrolase I and implications for activity improvement.

PubMed

Rabinovich, Mikhail L; Melnik, Maria S; Herner, Mikhail L; Voznyi, Yakov V; Vasilchenko, Lilia G

2018-05-21

Enzymatic conversion of the most abundant renewable source of organic compounds, cellulose to fermentable sugars is attractive for production of green fuels and chemicals. The major component of industrial enzyme systems, cellobiohydrolase I from Hypocrea jecorina (Trichoderma reesei) (HjCel7A) processively splits disaccharide units from the reducing ends of tightly packed cellulose chains. HjCel7A consists of a catalytic domain (CD) and a carbohydrate-binding module (CBM) separated by a linker peptide. A tunnel-shaped substrate-binding site in the CD includes 9 subsites for β-D-glucose units, 7 of which (-7 to -1) precede the catalytic center. Low catalytic activity of Cel7A is the bottleneck and the primary target for improvement. Here it is shown for the first time that, in spite of much lower apparent k cat of HjCel7A at the hydrolysis of β-1,4-glucosidic linkages in the fluorogenic cellotetra- and -pentaose compared to the structurally related endoglucanase I (HjCel7B), the specificity constants (catalytic efficiency) k cat /K m for both enzymes are almost equal in these reactions. The observed activity difference appears from strong nonproductive substrate binding by HjCel7A, particularly significant for MU-β-cellotetraose (MUG 4 ). Interaction of substrates with the subsites -6 and -5 proximal to the non-conserved Gln101 residue in HjCel7A decreases K m,ap by >1500 times. HjCel7A can be nonproductively bound onto cellulose surface with K d ∼2-9 nM via CBM and CD that captures 6 terminal glucose units of cellulose chain. Decomposition of this nonproductive complex can determine the rate of cellulose conversion. MUG 4 is a promising substrate to select active cellobiohydrolase I variants with reduced nonproductive substrate binding. This article is protected by copyright. All rights reserved.

The naphthoquinones, vitamin K3 and its structural analogue plumbagin, are substrates of the multidrug resistance linked ATP binding cassette drug transporter ABCG2.

PubMed

Shukla, Suneet; Wu, Chung-Pu; Nandigama, Krishnamachary; Ambudkar, Suresh V

2007-12-01

Vitamin K3 (menadione; 2-methyl-1,4-naphthoquinone) is a structural precursor of vitamins K1 and K2, which are essential for blood clotting. The naturally occurring structural analogue of this vitamin, plumbagin (5-hydroxy-menadione), is known to modulate cellular proliferation, apoptosis, carcinogenesis, and radioresistance. We here report that both vitamin K3 and plumbagin are substrates of the multidrug resistance-linked ATP binding cassette drug transporter, ABCG2. Vitamin K3 and plumbagin specifically inhibited the ABCG2-mediated efflux of mitoxantrone but did not have any effect on the ABCB1-mediated efflux of rhodamine 123. This inhibition of ABCG2 function was due to their interaction at the substrate-binding site(s). Vitamin K3 and plumbagin inhibited the binding of [(125)I]iodoarylazidoprazosin, a substrate of ABCG2, to this transporter in a concentration-dependent manner with IC(50) values of 7.3 and 22.6 micromol/L, respectively, but had no effect on the binding of the photoaffinity analogue to ABCB1. Both compounds stimulated ABCG2-mediated ATP hydrolysis and also inhibited the mitoxantrone-stimulated ATPase activity of the ABCG2 transporter, but did not have any significant effect on the ATPase activity of ABCB1. In a cytotoxicity assay, ABCG2-expressing HEK cells were 2.8- and 2.3-fold resistant to plumbagin and vitamin K3, respectively, compared with the control cells, suggesting that they are substrates of this transporter. Collectively, these data show for the first time that vitamin K3 is a substrate of the ABCG2 transporter. Thus, ABCG2 may have a role in the regulation of vitamin K3 levels in the body. In addition, vitamin K3 and its structural derivative, plumbagin, could potentially be used to modulate ABCG2 function.

Deciphering the Arginine-Binding Preferences at the Substrate-Binding Groove of Ser/Thr Kinases by Computational Surface Mapping

PubMed Central

Ben-Shimon, Avraham; Niv, Masha Y.

2011-01-01

Protein kinases are key signaling enzymes that catalyze the transfer of γ-phosphate from an ATP molecule to a phospho-accepting residue in the substrate. Unraveling the molecular features that govern the preference of kinases for particular residues flanking the phosphoacceptor is important for understanding kinase specificities toward their substrates and for designing substrate-like peptidic inhibitors. We applied ANCHORSmap, a new fragment-based computational approach for mapping amino acid side chains on protein surfaces, to predict and characterize the preference of kinases toward Arginine binding. We focus on positions P−2 and P−5, commonly occupied by Arginine (Arg) in substrates of basophilic Ser/Thr kinases. The method accurately identified all the P−2/P−5 Arg binding sites previously determined by X-ray crystallography and produced Arg preferences that corresponded to those experimentally found by peptide arrays. The predicted Arg-binding positions and their associated pockets were analyzed in terms of shape, physicochemical properties, amino acid composition, and in-silico mutagenesis, providing structural rationalization for previously unexplained trends in kinase preferences toward Arg moieties. This methodology sheds light on several kinases that were described in the literature as having non-trivial preferences for Arg, and provides some surprising departures from the prevailing views regarding residues that determine kinase specificity toward Arg. In particular, we found that the preference for a P−5 Arg is not necessarily governed by the 170/230 acidic pair, as was previously assumed, but by several different pairs of acidic residues, selected from positions 133, 169, and 230 (PKA numbering). The acidic residue at position 230 serves as a pivotal element in recognizing Arg from both the P−2 and P−5 positions. PMID:22125489

Family 46 Carbohydrate-binding Modules Contribute to the Enzymatic Hydrolysis of Xyloglucan and β-1,3-1,4-Glucans through Distinct Mechanisms.

PubMed

Venditto, Immacolata; Najmudin, Shabir; Luís, Ana S; Ferreira, Luís M A; Sakka, Kazuo; Knox, J Paul; Gilbert, Harry J; Fontes, Carlos M G A

2015-04-24

Structural carbohydrates comprise an extraordinary source of energy that remains poorly utilized by the biofuel sector as enzymes have restricted access to their substrates within the intricacy of plant cell walls. Carbohydrate active enzymes (CAZYmes) that target recalcitrant polysaccharides are modular enzymes containing noncatalytic carbohydrate-binding modules (CBMs) that direct enzymes to their cognate substrate, thus potentiating catalysis. In general, CBMs are functionally and structurally autonomous from their associated catalytic domains from which they are separated through flexible linker sequences. Here, we show that a C-terminal CBM46 derived from BhCel5B, a Bacillus halodurans endoglucanase, does not interact with β-glucans independently but, uniquely, acts cooperatively with the catalytic domain of the enzyme in substrate recognition. The structure of BhCBM46 revealed a β-sandwich fold that abuts onto the region of the substrate binding cleft upstream of the active site. BhCBM46 as a discrete entity is unable to bind to β-glucans. Removal of BhCBM46 from BhCel5B, however, abrogates binding to β-1,3-1,4-glucans while substantially decreasing the affinity for decorated β-1,4-glucan homopolymers such as xyloglucan. The CBM46 was shown to contribute to xyloglucan hydrolysis only in the context of intact plant cell walls, but it potentiates enzymatic activity against purified β-1,3-1,4-glucans in solution or within the cell wall. This report reveals the mechanism by which a CBM can promote enzyme activity through direct interaction with the substrate or by targeting regions of the plant cell wall where the target glucan is abundant. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Mechanistic Insights into Archaeal and Human Argonaute Substrate Binding and Cleavage Properties

PubMed Central

Willkomm, Sarah; Zander, Adrian; Grohmann, Dina; Restle, Tobias

2016-01-01

Argonaute (Ago) proteins from all three domains of life are key players in processes that specifically regulate cellular nucleic acid levels. Some of these Ago proteins, among them human Argonaute2 (hAgo2) and Ago from the archaeal organism Methanocaldococcus jannaschii (MjAgo), are able to cleave nucleic acid target strands that are recognised via an Ago-associated complementary guide strand. Here we present an in-depth kinetic side-by-side analysis of hAgo2 and MjAgo guide and target substrate binding as well as target strand cleavage, which enabled us to disclose similarities and differences in the mechanistic pathways as a function of the chemical nature of the substrate. Testing all possible guide-target combinations (i.e. RNA/RNA, RNA/DNA, DNA/RNA and DNA/DNA) with both Ago variants we demonstrate that the molecular mechanism of substrate association is highly conserved among archaeal-eukaryotic Argonautes. Furthermore, we show that hAgo2 binds RNA and DNA guide strands in the same fashion. On the other hand, despite striking homology between the two Ago variants, MjAgo cannot orientate guide RNA substrates in a way that allows interaction with the target DNA in a cleavage-compatible orientation. PMID:27741323

Concurrent Cooperativity and Substrate Inhibition in the Epoxidation of Carbamazepine by Cytochrome P450 3A4 Active Site Mutants Inspired by Molecular Dynamics Simulations

PubMed Central

2015-01-01

Cytochrome P450 3A4 (CYP3A4) is the major human P450 responsible for the metabolism of carbamazepine (CBZ). To explore the mechanisms of interactions of CYP3A4 with this anticonvulsive drug, we carried out multiple molecular dynamics (MD) simulations, starting with the complex of CYP3A4 manually docked with CBZ. On the basis of these simulations, we engineered CYP3A4 mutants I369F, I369L, A370V, and A370L, in which the productive binding orientation was expected to be stabilized, thus leading to increased turnover of CBZ to the 10,11-epoxide product. In addition, we generated CYP3A4 mutant S119A as a control construct with putative destabilization of the productive binding pose. Evaluation of the kinetics profiles of CBZ epoxidation demonstrate that CYP3A4-containing bacterial membranes (bactosomes) as well as purified CYP3A4 (wild-type and mutants I369L/F) exhibit substrate inhibition in reconstituted systems. In contrast, mutants S119A and A370V/L exhibit S-shaped profiles that are indicative of homotropic cooperativity. MD simulations with two to four CBZ molecules provide evidence that the substrate-binding pocket of CYP3A4 can accommodate more than one molecule of CBZ. Analysis of the kinetics profiles of CBZ metabolism with a model that combines the formalism of the Hill equation with an allowance for substrate inhibition demonstrates that the mechanism of interactions of CBZ with CYP3A4 involves multiple substrate-binding events (most likely three). Despite the retention of the multisite binding mechanism in the mutants, functional manifestations reveal an exquisite sensitivity to even minor structural changes in the binding pocket that are introduced by conservative substitutions such as I369F, I369L, and A370V. PMID:25545162

Concurrent cooperativity and substrate inhibition in the epoxidation of carbamazepine by cytochrome P450 3A4 active site mutants inspired by molecular dynamics simulations.

PubMed

Müller, Christian S; Knehans, Tim; Davydov, Dmitri R; Bounds, Patricia L; von Mandach, Ursula; Halpert, James R; Caflisch, Amedeo; Koppenol, Willem H

2015-01-27

Cytochrome P450 3A4 (CYP3A4) is the major human P450 responsible for the metabolism of carbamazepine (CBZ). To explore the mechanisms of interactions of CYP3A4 with this anticonvulsive drug, we carried out multiple molecular dynamics (MD) simulations, starting with the complex of CYP3A4 manually docked with CBZ. On the basis of these simulations, we engineered CYP3A4 mutants I369F, I369L, A370V, and A370L, in which the productive binding orientation was expected to be stabilized, thus leading to increased turnover of CBZ to the 10,11-epoxide product. In addition, we generated CYP3A4 mutant S119A as a control construct with putative destabilization of the productive binding pose. Evaluation of the kinetics profiles of CBZ epoxidation demonstrate that CYP3A4-containing bacterial membranes (bactosomes) as well as purified CYP3A4 (wild-type and mutants I369L/F) exhibit substrate inhibition in reconstituted systems. In contrast, mutants S119A and A370V/L exhibit S-shaped profiles that are indicative of homotropic cooperativity. MD simulations with two to four CBZ molecules provide evidence that the substrate-binding pocket of CYP3A4 can accommodate more than one molecule of CBZ. Analysis of the kinetics profiles of CBZ metabolism with a model that combines the formalism of the Hill equation with an allowance for substrate inhibition demonstrates that the mechanism of interactions of CBZ with CYP3A4 involves multiple substrate-binding events (most likely three). Despite the retention of the multisite binding mechanism in the mutants, functional manifestations reveal an exquisite sensitivity to even minor structural changes in the binding pocket that are introduced by conservative substitutions such as I369F, I369L, and A370V.

The tight binding model study of the role of band filling on the charge gap in graphene-on-substrate in paramagnetic state

NASA Astrophysics Data System (ADS)

Panda, Rudrashish; Sahu, Sivabrata; Rout, G. C.

2017-05-01

We communicate here a tight binding theoretical model study of the band filling effect on the charge gap in graphene-on-substrate. The Hamiltonian consists of nearest neighbor electron hopping and substrate induced gap. Besides this the Coulomb interaction is considered here within mean-field approximation in the paramagnetic limit. The electron occupancies at two sublattices are calculated by Green's function technique and are solved self consistently. Finally the charge gap i.e. Δ ¯=U [ < na > -< nb > ] is calculated and computed numerically. The results are reported.

Hydroxynitrile Lyases with α/β-Hydrolase Fold: Two Enzymes with Almost Identical 3D Structures but Opposite Enantioselectivities and Different Reaction Mechanisms

PubMed Central

Andexer, Jennifer N; Staunig, Nicole; Eggert, Thorsten; Kratky, Christoph; Pohl, Martina; Gruber, Karl

2012-01-01

Hydroxynitrile lyases (HNLs) catalyze the cleavage of cyanohydrins to yield hydrocyanic acid (HCN) and the respective carbonyl compound and are key enzymes in the process of cyanogenesis in plants. In organic syntheses, HNLs are used as biocatalysts for the formation of enantiopure cyanohydrins. We determined the structure of the recently identified, R-selective HNL from Arabidopsis thaliana (AtHNL) at a crystallographic resolution of 2.5 Å. The structure exhibits an α/β-hydrolase fold, very similar to the homologous, but S-selective, HNL from Hevea brasiliensis (HbHNL). The similarities also extend to the active sites of these enzymes, with a Ser-His-Asp catalytic triad present in all three cases. In order to elucidate the mode of substrate binding and to understand the unexpected opposite enantioselectivity of AtHNL, complexes of the enzyme with both (R)- and (S)-mandelonitrile were modeled using molecular docking simulations. Compared to the complex of HbHNL with (S)-mandelonitrile, the calculations produced an approximate mirror image binding mode of the substrate with the phenyl rings located at very similar positions, but with the cyano groups pointing in opposite directions. A catalytic mechanism for AtHNL is proposed, in which His236 from the catalytic triad acts as a general base and the emerging negative charge on the cyano group is stabilized by main-chain amide groups and an α-helix dipole very similar to α/β-hydrolases. This mechanistic proposal is additionally supported by mutagenesis studies. PMID:22851196

Non-Natural Linker Configuration in 2,6-Dipeptidyl-Anthraquinones Enhances the Inhibition of TAR RNA Binding/Annealing Activities by HIV-1 NC and Tat Proteins.

PubMed

Sosic, Alice; Saccone, Irene; Carraro, Caterina; Kenderdine, Thomas; Gamba, Elia; Caliendo, Giuseppe; Corvino, Angela; Di Vaio, Paola; Fiorino, Ferdinando; Magli, Elisa; Perissutti, Elisa; Santagada, Vincenzo; Severino, Beatrice; Spada, Valentina; Fabris, Dan; Frecentese, Francesco; Gatto, Barbara

2018-06-12

The HIV-1 nucleocapsid (NC) protein represents an excellent molecular target for the development of anti-retrovirals by virtue of its well-characterized chaperone activities, which play pivotal roles in essential steps of the viral life cycle. Our ongoing search for candidates able to impair NC binding/annealing activities led to the identification of peptidyl-anthraquinones as a promising class of nucleic acid ligands. Seeking to elucidate the inhibition determinants and increase the potency of this class of compounds, we have now explored the effects of chirality in the linker connecting the planar nucleus to the basic side chains. We show here that the non-natural linker configuration imparted unexpected TAR RNA targeting properties to the 2,6-peptidyl-anthraquinones and significantly enhanced their potency. Even if the new compounds were able to interact directly with the NC protein, they manifested a consistently higher affinity for the TAR RNA substrate and their TAR-binding properties mirrored their ability to interfere with NC-TAR interactions. Based on these findings, we propose that the viral Tat protein, sharing the same RNA substrate but acting in distinct phases of the viral life cycle, constitutes an additional druggable target for this class of peptidyl-anthraquinones. The inhibition of Tat-TAR interaction for the test compounds correlated again with their TAR-binding properties, while simultaneously failing to demonstrate any direct Tat-binding capabilities. These considerations highlighted the importance of TAR RNA in the elucidation of their inhibition mechanism, rather than direct protein inhibition. We have therefore identified anti-TAR compounds with dual in vitro inhibitory activity on different viral proteins, demonstrating that it is possible to develop multitarget compounds capable of interfering with processes mediated by the interactions of this essential RNA domain of HIV-1 genome with NC and Tat proteins.

Synthesis of rigidified flavin–guanidinium ion conjugates and investigation of their photocatalytic properties

PubMed Central

Schmaderer, Harald; Bhuyan, Mouchumi

2009-01-01

Summary Flavin chromophores can mediate redox reactions upon irradiation by blue light. In an attempt to increase their catalytic efficacy, flavin derivatives bearing a guanidinium ion as oxoanion binding site were prepared. Chromophore and substrate binding site are linked by a rigid Kemp’s acid structure. The molecular structure of the new flavins was confirmed by an X-ray structure analysis and their photocatalytic activity was investigated in benzyl ester cleavage, nitroarene reduction and a Diels–Alder reaction. The modified flavins photocatalyze the reactions, but the introduced substrate binding site does not enhance their performance. PMID:19590745

Synthesis of rigidified flavin-guanidinium ion conjugates and investigation of their photocatalytic properties.

PubMed

Schmaderer, Harald; Bhuyan, Mouchumi; König, Burkhard

2009-05-28

Flavin chromophores can mediate redox reactions upon irradiation by blue light. In an attempt to increase their catalytic efficacy, flavin derivatives bearing a guanidinium ion as oxoanion binding site were prepared. Chromophore and substrate binding site are linked by a rigid Kemp's acid structure. The molecular structure of the new flavins was confirmed by an X-ray structure analysis and their photocatalytic activity was investigated in benzyl ester cleavage, nitroarene reduction and a Diels-Alder reaction. The modified flavins photocatalyze the reactions, but the introduced substrate binding site does not enhance their performance.

Substrate-Induced Conformational Changes Occur in All Cleaved Forms of Caspase-6

DOE Office of Scientific and Technical Information (OSTI.GOV)

S Vaidya; E Velazquez-Delgado; G Abbruzzese

2011-12-31

Caspase-6 is an apoptotic cysteine protease that also governs disease progression in Huntington's and Alzheimer's diseases. Caspase-6 is of great interest as a target for treatment of these neurodegenerative diseases; however, the molecular basis of caspase-6 function and regulation remains poorly understood. In the recently reported structure of caspase-6, the 60's and 130's helices at the base of the substrate-binding groove extend upward, in a conformation entirely different from that of any other caspase. Presently, the central question about caspase-6 structure and function is whether the extended conformation is the catalytically competent conformation or whether the extended helices must undergomore » a large conformational rearrangement in order to bind substrate. We have generated a series of caspase-6 cleavage variants, including a novel constitutively two-chain form, and determined crystal structures of caspase-6 with and without the intersubunit linker. This series allows evaluation of the role of the prodomain and intersubunit linker on caspase-6 structure and function before and after substrate binding. Caspase-6 is inherently more stable than closely related caspases. Cleaved caspase-6 with both the prodomain and the linker present is the most stable, indicating that these two regions act in concert to increase stability, but maintain the extended conformation in the unliganded state. Moreover, these data suggest that caspase-6 undergoes a significant conformational change upon substrate binding, adopting a structure that is more like canonical caspases.« less

Structures of Prostacyclin Synthase and Its Complexes with Substrate Analog and Inhibitor Reveal a Ligand-specific Heme Conformation Change*s

PubMed Central

Li, Yi-Ching; Chiang, Chia-Wang; Yeh, Hui-Chun; Hsu, Pei-Yung; Whitby, Frank G.; Wang, Lee-Ho; Chan, Nei-Li

2008-01-01

Prostacyclin synthase (PGIS) is a cytochrome P450 (P450) enzyme that catalyzes production of prostacyclin from prostaglandin H2. PGIS is unusual in that it catalyzes an isomerization rather than a monooxygenation, which is typical of P450 enzymes. To understand the structural basis for prostacyclin biosynthesis in greater detail, we have determined the crystal structures of ligand-free, inhibitor (minoxidil)-bound and substrate analog U51605-bound PGIS. These structures demonstrate a stereo-specific substrate binding and suggest features of the enzyme that facilitate isomerization. Unlike most microsomal P450s, where large substrate-induced conformational changes take place at the distal side of the heme, conformational changes in PGIS are observed at the proximal side and in the heme itself. The conserved and extensive heme propionate-protein interactions seen in all other P450s, which are largely absent in the ligand-free PGIS, are recovered upon U51605 binding accompanied by water exclusion from the active site. In contrast, when minoxidil binds, the propionate-protein interactions are not recovered and water molecules are largely retained. These findings suggest that PGIS represents a divergent evolution of the P450 family, in which a heme barrier has evolved to ensure strict binding specificity for prostaglandin H2, leading to a radical-mediated isomerization with high product fidelity. The U51605-bound structure also provides a view of the substrate entrance and product exit channels. PMID:18032380

Structural basis for ligand-dependent dimerization of phenylalanine hydroxylase regulatory domain

PubMed Central

Patel, Dipali; Kopec, Jolanta; Fitzpatrick, Fiona; McCorvie, Thomas J.; Yue, Wyatt W.

2016-01-01

The multi-domain enzyme phenylalanine hydroxylase (PAH) catalyzes the hydroxylation of dietary I-phenylalanine (Phe) to I-tyrosine. Inherited mutations that result in PAH enzyme deficiency are the genetic cause of the autosomal recessive disorder phenylketonuria. Phe is the substrate for the PAH active site, but also an allosteric ligand that increases enzyme activity. Phe has been proposed to bind, in addition to the catalytic domain, a site at the PAH N-terminal regulatory domain (PAH-RD), to activate the enzyme via an unclear mechanism. Here we report the crystal structure of human PAH-RD bound with Phe at 1.8 Å resolution, revealing a homodimer of ACT folds with Phe bound at the dimer interface. This work delivers the structural evidence to support previous solution studies that a binding site exists in the RD for Phe, and that Phe binding results in dimerization of PAH-RD. Consistent with our structural observation, a disease-associated PAH mutant impaired in Phe binding disrupts the monomer:dimer equilibrium of PAH-RD. Our data therefore support an emerging model of PAH allosteric regulation, whereby Phe binds to PAH-RD and mediates the dimerization of regulatory modules that would bring about conformational changes to activate the enzyme. PMID:27049649

Structure and Ligand Binding Properties of the Epoxidase Component of Styrene Monooxygenase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ukaegbu, Uchechi E.; Kantz, Auric; Beaton, Michelle

2010-07-23

Styrene monooxygenase (SMO) is a two-component flavoprotein monooxygenase that transforms styrene to styrene oxide in the first step of the styrene catabolic and detoxification pathway of Pseudomonas putida S12. The crystal structure of the N-terminally histidine-tagged epoxidase component of this system, NSMOA, determined to 2.3 {angstrom} resolution, indicates the enzyme exists as a homodimer in which each monomer forms two distinct domains. The overall architecture is most similar to that of p-hydroxybenzoate hydroxylase (PHBH), although there are some significant differences in secondary structure. Structural comparisons suggest that a large cavity open to the surface forms the FAD binding site. Atmore » the base of this pocket is another cavity that likely represents the styrene binding site. Flavin binding and redox equilibria are tightly coupled such that reduced FAD binds apo NSMOA {approx}8000 times more tightly than the oxidized coenzyme. Equilibrium fluorescence and isothermal titration calorimetry data using benzene as a substrate analogue indicate that the oxidized flavin and substrate analogue binding equilibria of NSMOA are linked such that the binding affinity of each is increased by 60-fold when the enzyme is saturated with the other. A much weaker {approx}2-fold positive cooperative interaction is observed for the linked binding equilibria of benzene and reduced FAD. The low affinity of the substrate analogue for the reduced FAD complex of NSMOA is consistent with a preferred reaction order in which flavin reduction and reaction with oxygen precede the binding of styrene, identifying the apoenzyme structure as the key catalytic resting state of NSMOA poised to bind reduced FAD and initiate the oxygen reaction.« less

Enhancing the efficiency of sortase-mediated ligations through nickel-peptide complex formation.

PubMed

David Row, R; Roark, Travis J; Philip, Marina C; Perkins, Lorena L; Antos, John M

2015-08-14

A modified sortase A recognition motif containing a masked Ni(2+)-binding peptide was employed to boost the efficiency of sortase-catalyzed ligation reactions. Deactivation of the Ni(2+)-binding peptide using a Ni(2+) additive improved reaction performance at low to equimolar ratios of the glycine amine nucleophile and sortase substrate. The success of this approach was demonstrated with both peptide and protein substrates.

Structural Basis of Interdomain Communication in the Hsc70 Chaperone

PubMed Central

Jiang, Jianwen; Prasad, Kondury; Lafer, Eileen M.; Sousa, Rui

2015-01-01

Summary Hsp70 family proteins are highly conserved chaperones involved in protein folding, degradation, targeting and translocation, and protein complex remodeling. They are comprised of an N-terminal nucleotide binding domain (NBD) and a C-terminal protein substrate binding domain (SBD). ATP binding to the NBD alters SBD conformation and substrate binding kinetics, but an understanding of the mechanism of interdomain communication has been hampered by the lack of a crystal structure of an intact chaperone. Were-port here the 2.6 Å structure of a functionally intact bovine Hsc70 (bHsc70) and a mutational analysis of the observed interdomain interface and the immediately adjacent interdomain linker. This analysis identifies interdomain interactions critical for chaperone function and supports an allosteric mechanism in which the interdomain linker invades and disrupts the interdomain interface when ATP binds. PMID:16307916

Mapping specificity landscapes of RNA-protein interactions by high throughput sequencing.

PubMed

Jankowsky, Eckhard; Harris, Michael E

2017-04-15

To function in a biological setting, RNA binding proteins (RBPs) have to discriminate between alternative binding sites in RNAs. This discrimination can occur in the ground state of an RNA-protein binding reaction, in its transition state, or in both. The extent by which RBPs discriminate at these reaction states defines RBP specificity landscapes. Here, we describe the HiTS-Kin and HiTS-EQ techniques, which combine kinetic and equilibrium binding experiments with high throughput sequencing to quantitatively assess substrate discrimination for large numbers of substrate variants at ground and transition states of RNA-protein binding reactions. We discuss experimental design, practical considerations and data analysis and outline how a combination of HiTS-Kin and HiTS-EQ allows the mapping of RBP specificity landscapes. Copyright © 2017 Elsevier Inc. All rights reserved.

Enzyme architecture: optimization of transition state stabilization from a cation-phosphodianion pair.

PubMed

Reyes, Archie C; Koudelka, Astrid P; Amyes, Tina L; Richard, John P

2015-04-29

The side chain cation of R269 lies at the surface of l-glycerol 3-phosphate dehydrogenase (GPDH) and forms an ion pair to the phosphodianion of substrate dihydroxyacetone phosphate (DHAP), which is buried at the nonpolar protein interior. The R269A mutation of GPDH results in a 110-fold increase in K(m) (2.8 kcal/mol effect) and a 41,000-fold decrease in k(cat) (6.3 kcal/mol effect), which corresponds to a 9.1 kcal/mol destabilization of the transition state for GPDH-catalyzed reduction of DHAP by NADH. There is a 6.7 kcal/mol stabilization of the transition state for the R269A mutant GPDH-catalyzed reaction by 1.0 M guanidinium ion, and the transition state for the reaction of the substrate pieces is stabilized by an additional 2.4 kcal/mol by their covalent attachment at wildtype GPDH. These results provide strong support for the proposal that GPDH invests the 11 kcal/mol intrinsic phosphodianion binding energy of DHAP in trapping the substrate at a nonpolar active site, where strong electrostatic interactions are favored, and obtains a 9 kcal/mol return from stabilizing interactions between the side chain cation and transition state trianion. We propose a wide propagation for the catalytic motif examined in this work, which enables strong transition state stabilization from enzyme-phosphodianion pairs.

Molecular basis of P450 OleTJE: an investigation of substrate binding mechanism and major pathways

NASA Astrophysics Data System (ADS)

Du, Juan; Liu, Lin; Guo, Li Zhong; Yao, Xiao Jun; Yang, Jian Ming

2017-05-01

Cytochrome P450 OleTJE has attracted much attention for its ability to catalyze the decarboxylation of long chain fatty acids to generate alkenes, which are not only biofuel molecule, but also can be used broadly for making lubricants, polymers and detergents. In this study, the molecular basis of the binding mechanism of P450 OleTJE for arachidic acid, myristic acid, and caprylic acid was investigated by utilizing conventional molecular dynamics simulation and binding free energy calculations. Moreover, random acceleration molecular dynamics (RAMD) simulations were performed to uncover the most probable access/egress channels for different fatty acids. The predicted binding free energy shows an order of arachidic acid < myristic acid < caprylic acid. Key residues interacting with three substrates and residues specifically binding to one of them were identified. The RAMD results suggest the most likely channel for arachidic acid, myristic acid, and caprylic acid are 2e/2b, 2a and 2f/2a, respectively. It is suggested that the reaction is easier to carry out in myristic acid bound system than those in arachidic acid and caprylic acid bound system based on the distance of Hβ atom of substrate relative to P450 OleTJE Compound I states. This study provided novel insight to understand the substrate preference mechanism of P450 OleTJE and valuable information for rational enzyme design for short chain fatty acid decarboxylation.

In Vitro Reassembly of the Ribose ATP-binding Cassette Transporter Reveals a Distinct Set of Transport Complexes*

PubMed Central

Clifton, Matthew C.; Simon, Michael J.; Erramilli, Satchal K.; Zhang, Huide; Zaitseva, Jelena; Hermodson, Mark A.; Stauffacher, Cynthia V.

2015-01-01

Bacterial ATP-binding cassette (ABC) importers are primary active transporters that are critical for nutrient uptake. Based on structural and functional studies, ABC importers can be divided into two distinct classes, type I and type II. Type I importers follow a strict alternating access mechanism that is driven by the presence of the substrate. Type II importers accept substrates in a nucleotide-free state, with hydrolysis driving an inward facing conformation. The ribose transporter in Escherichia coli is a tripartite complex consisting of a cytoplasmic ATP-binding cassette protein, RbsA, with fused nucleotide binding domains; a transmembrane domain homodimer, RbsC2; and a periplasmic substrate binding protein, RbsB. To investigate the transport mechanism of the complex RbsABC2, we probed intersubunit interactions by varying the presence of the substrate ribose and the hydrolysis cofactors, ATP/ADP and Mg2+. We were able to purify a full complex, RbsABC2, in the presence of stable, transition state mimics (ATP, Mg2+, and VO4); a RbsAC complex in the presence of ADP and Mg2+; and a heretofore unobserved RbsBC complex in the absence of cofactors. The presence of excess ribose also destabilized complex formation between RbsB and RbsC. These observations suggest that RbsABC2 shares functional traits with both type I and type II importers, as well as possessing unique features, and employs a distinct mechanism relative to other ABC transporters. PMID:25533465

Fluconazole Binding and Sterol Demethylation in Three CYP51 Isoforms Indicate Differences in Active Site Topology

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bellamine, A.; Lepesheva, Galina I.; Waterman, Mike

2010-11-16

14{alpha}-Demethylase (CYP51) is a key enzyme in all sterol biosynthetic pathways (animals, fungi, plants, protists, and some bacteria), catalyzing the removal of the C-14 methyl group following cyclization of squalene. Based on mutations found in CYP51 genes from Candida albicans azole-resistant isolates obtained after fluconazole treatment of fungal infections, and using site-directed mutagenesis, we have found that fluconazole binding and substrate metabolism vary among three different CYP51 isoforms: human, fungal, and mycobacterial. In C. albicans, the Y132H mutant from isolates shows no effect on fluconazole binding, whereas the F145L mutant results in a 5-fold increase in its IC{sub 50} formore » fluconazole, suggesting that F145 (conserved only in fungal 14{alpha}-demethylases) interacts with this azole. In C. albicans, F145L accounts, in part, for the difference in fluconazole sensitivity reported between mammals and fungi, providing a basis for treatment of fungal infections. The C. albicans Y132H and human Y145H CYP51 mutants show essentially no effect on substrate metabolism, but the Mycobacterium tuberculosis F89H CYP51 mutant loses both its substrate binding and metabolism. Because these three residues align in the three isoforms, the results indicate that their active sites contain important structural differences, and further emphasize that fluconazole and substrate binding are uncoupled properties.« less

Molecular basis of HHQ biosynthesis: molecular dynamics simulations, enzyme kinetic and surface plasmon resonance studies

PubMed Central

2013-01-01

Background PQS (PseudomonasQuinolone Signal) and its precursor HHQ are signal molecules of the P. aeruginosa quorum sensing system. They explicate their role in mammalian pathogenicity by binding to the receptor PqsR that induces virulence factor production and biofilm formation. The enzyme PqsD catalyses the biosynthesis of HHQ. Results Enzyme kinetic analysis and surface plasmon resonance (SPR) biosensor experiments were used to determine mechanism and substrate order of the biosynthesis. Comparative analysis led to the identification of domains involved in functionality of PqsD. A kinetic cycle was set up and molecular dynamics (MD) simulations were used to study the molecular bases of the kinetics of PqsD. Trajectory analysis, pocket volume measurements, binding energy estimations and decompositions ensured insights into the binding mode of the substrates anthraniloyl-CoA and β-ketodecanoic acid. Conclusions Enzyme kinetics and SPR experiments hint at a ping-pong mechanism for PqsD with ACoA as first substrate. Trajectory analysis of different PqsD complexes evidenced ligand-dependent induced-fit motions affecting the modified ACoA funnel access to the exposure of a secondary channel. A tunnel-network is formed in which Ser317 plays an important role by binding to both substrates. Mutagenesis experiments resulting in the inactive S317F mutant confirmed the importance of this residue. Two binding modes for β-ketodecanoic acid were identified with distinct catalytic mechanism preferences. PMID:23916145

Redox process is crucial for inhibitory properties of aurintricarboxylic acid against activity of YopH: virulence factor of Yersinia pestis

PubMed Central

Kuban-Jankowska, Alicja; Gorska, Magdalena; Tuszynski, Jack A; Ossowski, Tadeusz; Wozniak, Michal

2015-01-01

YopH is a bacterial protein tyrosine phosphatase, which is essential for the viability and pathogenic virulence of the plague-causing Yersinia sp. bacteria. Inactivation of YopH activity would lead to the loss of bacterial pathogenicity. We have studied the inhibitory properties of aurintricarboxylic acid (ATA) against YopH phosphatase and found that at nanomolar concentrations ATA reversibly decreases the activity of YopH. Computational docking studies indicated that in all binding poses ATA binds in the YopH active site. Molecular dynamics simulations showed that in the predicted binding pose, ATA binds to the essential Cys403 and Arg409 residues in the active site and has a stronger binding affinity than the natural substrate (pTyr). The cyclic voltammetry experiments suggest that ATA reacts remarkably strongly with molecular oxygen. Additionally, the electrochemical reduction of ATA in the presence of a negative potential from −2.0 to 2.5 V generates a current signal, which is observed for hydrogen peroxide. Here we showed that ATA indicates a unique mechanism of YopH inactivation due to a redox process. We proposed that the potent inhibitory properties of ATA are a result of its strong binding in the YopH active site and in situ generation of hydrogen peroxide near catalytic cysteine residue. PMID:26286963

Structural basis of regulation and substrate specificity of protein kinase CK2 deduced from the modeling of protein-protein interactions

PubMed Central

Rekha, Nambudiry; Srinivasan, N

2003-01-01

Background Protein Kinase Casein Kinase 2 (PKCK2) is an ubiquitous Ser/Thr kinase expressed in all eukaryotes. It phosphorylates a number of proteins involved in various cellular processes. PKCK2 holoenzyme is catalytically active tetramer, composed of two homologous or identical and constitutively active catalytic (α) and two identical regulatory (β) subunits. The tetramer cannot phosphorylate some substrates that can be phosphorylated by PKCK2α in isolation. The present work explores the structural basis of this feature using computational analysis and modeling. Results We have initially built a model of PKCK2α bound to a substrate peptide with a conformation identical to that of the substrates in the available crystal structures of other kinases complexed with the substrates/ pseudosubstrates. In this model however, the fourth acidic residue in the consensus pattern of the substrate, S/T-X-X-D/E where S/T is the phosphorylation site, did not result in interaction with the active form of PKCK2α and is highly solvent exposed. Interaction of the acidic residue is observed if the substrate peptide adopts conformations as seen in β turn, α helix, or 310 helices. This type of conformation is observed and accommodated well by PKCK2α in calmodulin where the phosphorylation site is at the central helix. PP2A carries sequence patterns for PKCK2α phosphorylation. While the possibility of PP2A being phosphorylated by PKCK2 has been raised in the literature we use the model of PP2A to generate a model of PP2A-PKCK2α complex. PKCK2β undergoes phosphorylation by holoenzyme at the N-terminal region, and is accommodated very well in the limited space available at the substrate-binding site of the holoenzyme while the space is insufficient to accommodate the binding of PP2A or calmodulin in the holoenzyme. Conclusion Charge and shape complimentarity seems to play a role in substrate recognition and binding to PKCK2α, along with the consensus pattern. The detailed conformation of the substrate peptide binding to PKCK2 differs from the conformation of the substrate/pseudo substrate peptide that is bound to other kinases in the crystal structures reported. The ability of holoenzyme to phosphorylate substrate proteins seems to depend on the accessibility of the P-site in limited space available in holoenzyme. PMID:12740046

Multiple allosteric sites are involved in the modulation of insulin-degrading-enzyme activity by somatostatin.

PubMed

Tundo, Grazia R; Di Muzio, Elena; Ciaccio, Chiara; Sbardella, Diego; Di Pierro, Donato; Polticelli, Fabio; Coletta, Massimo; Marini, Stefano

2016-10-01

Somatostatin is a cyclic peptide, released in the gastrointestinal system and the central nervous system, where it is involved in the regulation of cognitive and sensory functions, motor activity and sleep. It is a substrate of insulin-degrading enzyme (IDE), as well as a modulator of its activity and expression. In the present study, we have investigated the modulatory role of somatostatin on IDE activity at 37 °C and pH 7.3 for various substrates [i.e. insulin, β-amyloid (Aβ) 1-40 and bradykinin], aiming to quantitatively characterize the correlation between the specific features of the substrates and the regulatory mechanism. Functional data indicate that somatostatin, in addition to the catalytic site of IDE (being a substrate), is also able to bind to two additional exosites, which play different roles according to the size of the substrate and its binding mode to the IDE catalytic cleft. In particular, one exosite, which displays high affinity for somatostatin, regulates only the interaction of IDE with larger substrates (such as insulin and Aβ 1-40 ) in a differing fashion according to their various modes of binding to the enzyme. A second exosite, which is involved in the regulation of enzymatic processing by IDE of all substrates investigated (including a 10-25 amino acid long amyloid-like peptide, bradykinin and somatostatin itself, which had been studied previously), probably acts through the alteration of an 'open-closed' equilibrium. © 2016 Federation of European Biochemical Societies.

Hydrophobic fluorescent probes introduce artifacts into single molecule tracking experiments due to non-specific binding.

PubMed

Zanetti-Domingues, Laura C; Tynan, Christopher J; Rolfe, Daniel J; Clarke, David T; Martin-Fernandez, Marisa

2013-01-01

Single-molecule techniques are powerful tools to investigate the structure and dynamics of macromolecular complexes; however, data quality can suffer because of weak specific signal, background noise and dye bleaching and blinking. It is less well-known, but equally important, that non-specific binding of probe to substrates results in a large number of immobile fluorescent molecules, introducing significant artifacts in live cell experiments. Following from our previous work in which we investigated glass coating substrates and demonstrated that the main contribution to this non-specific probe adhesion comes from the dye, we carried out a systematic investigation of how different dye chemistries influence the behaviour of spectrally similar fluorescent probes. Single-molecule brightness, bleaching and probe mobility on the surface of live breast cancer cells cultured on a non-adhesive substrate were assessed for anti-EGFR affibody conjugates with 14 different dyes from 5 different manufacturers, belonging to 3 spectrally homogeneous bands (491 nm, 561 nm and 638 nm laser lines excitation). Our results indicate that, as well as influencing their photophysical properties, dye chemistry has a strong influence on the propensity of dye-protein conjugates to adhere non-specifically to the substrate. In particular, hydrophobicity has a strong influence on interactions with the substrate, with hydrophobic dyes showing much greater levels of binding. Crucially, high levels of non-specific substrate binding result in calculated diffusion coefficients significantly lower than the true values. We conclude that the physic-chemical properties of the dyes should be considered carefully when planning single-molecule experiments. Favourable dye characteristics such as photostability and brightness can be offset by the propensity of a conjugate for non-specific adhesion.

Hydrophobic Fluorescent Probes Introduce Artifacts into Single Molecule Tracking Experiments Due to Non-Specific Binding

PubMed Central

Rolfe, Daniel J.; Clarke, David T.; Martin-Fernandez, Marisa

2013-01-01

Single-molecule techniques are powerful tools to investigate the structure and dynamics of macromolecular complexes; however, data quality can suffer because of weak specific signal, background noise and dye bleaching and blinking. It is less well-known, but equally important, that non-specific binding of probe to substrates results in a large number of immobile fluorescent molecules, introducing significant artifacts in live cell experiments. Following from our previous work in which we investigated glass coating substrates and demonstrated that the main contribution to this non-specific probe adhesion comes from the dye, we carried out a systematic investigation of how different dye chemistries influence the behaviour of spectrally similar fluorescent probes. Single-molecule brightness, bleaching and probe mobility on the surface of live breast cancer cells cultured on a non-adhesive substrate were assessed for anti-EGFR affibody conjugates with 14 different dyes from 5 different manufacturers, belonging to 3 spectrally homogeneous bands (491 nm, 561 nm and 638 nm laser lines excitation). Our results indicate that, as well as influencing their photophysical properties, dye chemistry has a strong influence on the propensity of dye-protein conjugates to adhere non-specifically to the substrate. In particular, hydrophobicity has a strong influence on interactions with the substrate, with hydrophobic dyes showing much greater levels of binding. Crucially, high levels of non-specific substrate binding result in calculated diffusion coefficients significantly lower than the true values. We conclude that the physic-chemical properties of the dyes should be considered carefully when planning single-molecule experiments. Favourable dye characteristics such as photostability and brightness can be offset by the propensity of a conjugate for non-specific adhesion. PMID:24066121

Conformational and chemical selection by a trans-acting editing domain

PubMed Central

Danhart, Eric M.; Bakhtina, Marina; Cantara, William A.; Kuzmishin, Alexandra B.; Ma, Xiao; Sanford, Brianne L.; Vargas-Rodriguez, Oscar; Košutić, Marija; Goto, Yuki; Suga, Hiroaki; Nakanishi, Kotaro; Micura, Ronald; Musier-Forsyth, Karin

2017-01-01

Molecular sieves ensure proper pairing of tRNAs and amino acids during aminoacyl-tRNA biosynthesis, thereby avoiding detrimental effects of mistranslation on cell growth and viability. Mischarging errors are often corrected through the activity of specialized editing domains present in some aminoacyl-tRNA synthetases or via single-domain trans-editing proteins. ProXp-ala is a ubiquitous trans-editing enzyme that edits Ala-tRNAPro, the product of Ala mischarging by prolyl-tRNA synthetase, although the structural basis for discrimination between correctly charged Pro-tRNAPro and mischarged Ala-tRNAAla is unclear. Deacylation assays using substrate analogs reveal that size discrimination is only one component of selectivity. We used NMR spectroscopy and sequence conservation to guide extensive site-directed mutagenesis of Caulobacter crescentus ProXp-ala, along with binding and deacylation assays to map specificity determinants. Chemical shift perturbations induced by an uncharged tRNAPro acceptor stem mimic, microhelixPro, or a nonhydrolyzable mischarged Ala-microhelixPro substrate analog identified residues important for binding and deacylation. Backbone 15N NMR relaxation experiments revealed dynamics for a helix flanking the substrate binding site in free ProXp-ala, likely reflecting sampling of open and closed conformations. Dynamics persist on binding to the uncharged microhelix, but are attenuated when the stably mischarged analog is bound. Computational docking and molecular dynamics simulations provide structural context for these findings and predict a role for the substrate primary α-amine group in substrate recognition. Overall, our results illuminate strategies used by a trans-editing domain to ensure acceptance of only mischarged Ala-tRNAPro, including conformational selection by a dynamic helix, size-based exclusion, and optimal positioning of substrate chemical groups. PMID:28768811

Modeling of Arylamide Helix Mimetics in the p53 Peptide Binding Site of hDM2 Suggests Parallel and Anti-Parallel Conformations Are Both Stable

PubMed Central

Fuller, Jonathan C.; Jackson, Richard M.; Edwards, Thomas A.; Wilson, Andrew J.; Shirts, Michael R.

2012-01-01

The design of novel α-helix mimetic inhibitors of protein-protein interactions is of interest to pharmaceuticals and chemical genetics researchers as these inhibitors provide a chemical scaffold presenting side chains in the same geometry as an α-helix. This conformational arrangement allows the design of high affinity inhibitors mimicking known peptide sequences binding specific protein substrates. We show that GAFF and AutoDock potentials do not properly capture the conformational preferences of α-helix mimetics based on arylamide oligomers and identify alternate parameters matching solution NMR data and suitable for molecular dynamics simulation of arylamide compounds. Results from both docking and molecular dynamics simulations are consistent with the arylamides binding in the p53 peptide binding pocket. Simulations of arylamides in the p53 binding pocket of hDM2 are consistent with binding, exhibiting similar structural dynamics in the pocket as simulations of known hDM2 binders Nutlin-2 and a benzodiazepinedione compound. Arylamide conformations converge towards the same region of the binding pocket on the 20 ns time scale, and most, though not all dihedrals in the binding pocket are well sampled on this timescale. We show that there are two putative classes of binding modes for arylamide compounds supported equally by the modeling evidence. In the first, the arylamide compound lies parallel to the observed p53 helix. In the second class, not previously identified or proposed, the arylamide compound lies anti-parallel to the p53 helix. PMID:22916232

The anchorage function of CipA (CelL), a scaffolding protein of the Clostridium thermocellum cellulosome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kruus, K.; Wu, J.H.D.; Lua, A.C.

1995-09-26

Enzymatic cellulose degradation is a heterogeneous reaction requiring binding of soluble cellulase molecules to the solid substrate. Based on our studies of the cellulase complex of Clostridium thermocellum (the cellulosome), we have previously proposed that such binding can be brought about by a special {open_quotes}anchorage subunit.{close_quotes} In this {open_quotes}anchor-enzyme{close_quotes} model, CipA (a major subunit of the cellulosome) enhances the activity of CelS (the most abundant catalytic subunit of the cellulosome) by anchoring it to the cellulose surface. We have subsequently reported that CelS contains a conserved duplicated sequence at its C terminus and the CipA contains nine repeated sequences withmore » a cellulose binding domain (CBD) in between the second and third repeats. In this work, we reexamined the anchor-enzyme mechanism by using recombinant CelS (rCelS) and various CipA domains, CBD, R3 (the repeat next to CBD), and CBD/R3, expressed in Escherichia coli. As analyzed by non-denaturing gel electrophoresis, rCelS, through its conserved duplicated sequence, formed a stable complex with R3 or CBD/R3 but not with CBD. Although R3 or CBD alone did not affect the binding of rCelS to cellulose, such binding was dependent on CBD/R3, indicating the anchorage role of CBD/R3. Such anchorage apparently increased the rCelS activity toward crystalline cellulose. These results substantiate the proposed anchor-enzyme model and the expected roles of individual CipA domains and the conserved duplicated sequence of CelS.« less

The substrate binding interface of alkylpurine DNA glycosylase AlkD.

PubMed

Mullins, Elwood A; Rubinson, Emily H; Eichman, Brandt F

2014-01-01

Tandem helical repeats have emerged as an important DNA binding architecture. DNA glycosylase AlkD, which excises N3- and N7-alkylated nucleobases, uses repeating helical motifs to bind duplex DNA and to selectively pause at non-Watson-Crick base pairs. Remodeling of the DNA backbone promotes nucleotide flipping of the lesion and the complementary base into the solvent and toward the protein surface, respectively. The important features of this new DNA binding architecture that allow AlkD to distinguish between damaged and normal DNA without contacting the lesion are poorly understood. Here, we show through extensive mutational analysis that DNA binding and N3-methyladenine (3mA) and N7-methylguanine (7mG) excision are dependent upon each residue lining the DNA binding interface. Disrupting electrostatic or hydrophobic interactions with the DNA backbone substantially reduced binding affinity and catalytic activity. These results demonstrate that residues seemingly only involved in general DNA binding are important for catalytic activity and imply that base excision is driven by binding energy provided by the entire substrate interface of this novel DNA binding architecture. Copyright © 2013 Elsevier B.V. All rights reserved.

New perspective on glycoside hydrolase binding to lignin from pretreated corn stover

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yarbrough, John M.; Mittal, Ashutosh; Mansfield, Elisabeth

Background: Non-specific binding of cellulases to lignin has been implicated as a major factor in the loss of cellulase activity during biomass conversion to sugars. It is believed that this binding may strongly impact process economics through loss of enzyme activities during hydrolysis and enzyme recycling scenarios. The current model suggests glycoside hydrolase activities are lost though non-specific/non-productive binding of carbohydrate-binding domains to lignin, limiting catalytic site access to the carbohydrate components of the cell wall. Results: In this study, we compared component enzyme affinities of a commercial Trichoderma reesei cellulase formulation, Cellic CTec2, towards extracted corn stover lignin usingmore » sodium dodecyl sulfate-polyacrylamide gel electrophoresis and p-nitrophenyl substrate activities to monitor component binding, activity loss, and total protein binding. Protein binding was strongly affected by pH and ionic strength. β-D-glucosidases and xylanases, which do not have carbohydrate-binding modules (CBMs) and are basic proteins, demonstrated the strongest binding at low ionic strength, suggesting that CBMs are not the dominant factor in enzyme adsorption to lignin. Despite strong adsorption to insoluble lignin, β-D-glucosidase and xylanase activities remained high, with process yields decreasing only 4–15 % depending on lignin concentration. Conclusion: We propose that specific enzyme adsorption to lignin from a mixture of biomass-hydrolyzing enzymes is a competitive affinity where β-D-glucosidases and xylanases can displace CBM interactions with lignin. Process parameters, such as temperature, pH, and salt concentration influence the individual enzymes’ affinity for lignin, and both hydrophobic and electrostatic interactions are responsible for this binding phenomenon. Moreover, our results suggest that concern regarding loss of critical cell wall degrading enzymes to lignin adsorption may be unwarranted when complex enzyme mixtures are used to digest biomass.« less

New perspective on glycoside hydrolase binding to lignin from pretreated corn stover

DOE PAGES

Yarbrough, John M.; Mittal, Ashutosh; Mansfield, Elisabeth; ...

2015-12-18

Background: Non-specific binding of cellulases to lignin has been implicated as a major factor in the loss of cellulase activity during biomass conversion to sugars. It is believed that this binding may strongly impact process economics through loss of enzyme activities during hydrolysis and enzyme recycling scenarios. The current model suggests glycoside hydrolase activities are lost though non-specific/non-productive binding of carbohydrate-binding domains to lignin, limiting catalytic site access to the carbohydrate components of the cell wall. Results: In this study, we compared component enzyme affinities of a commercial Trichoderma reesei cellulase formulation, Cellic CTec2, towards extracted corn stover lignin usingmore » sodium dodecyl sulfate-polyacrylamide gel electrophoresis and p-nitrophenyl substrate activities to monitor component binding, activity loss, and total protein binding. Protein binding was strongly affected by pH and ionic strength. β-D-glucosidases and xylanases, which do not have carbohydrate-binding modules (CBMs) and are basic proteins, demonstrated the strongest binding at low ionic strength, suggesting that CBMs are not the dominant factor in enzyme adsorption to lignin. Despite strong adsorption to insoluble lignin, β-D-glucosidase and xylanase activities remained high, with process yields decreasing only 4–15 % depending on lignin concentration. Conclusion: We propose that specific enzyme adsorption to lignin from a mixture of biomass-hydrolyzing enzymes is a competitive affinity where β-D-glucosidases and xylanases can displace CBM interactions with lignin. Process parameters, such as temperature, pH, and salt concentration influence the individual enzymes’ affinity for lignin, and both hydrophobic and electrostatic interactions are responsible for this binding phenomenon. Moreover, our results suggest that concern regarding loss of critical cell wall degrading enzymes to lignin adsorption may be unwarranted when complex enzyme mixtures are used to digest biomass.« less

Decipher the mechanisms of protein conformational changes induced by nucleotide binding through free-energy landscape analysis: ATP binding to Hsp70.

PubMed

Nicolaï, Adrien; Delarue, Patrice; Senet, Patrick

2013-01-01

ATP regulates the function of many proteins in the cell by transducing its binding and hydrolysis energies into protein conformational changes by mechanisms which are challenging to identify at the atomic scale. Based on molecular dynamics (MD) simulations, a method is proposed to analyze the structural changes induced by ATP binding to a protein by computing the effective free-energy landscape (FEL) of a subset of its coordinates along its amino-acid sequence. The method is applied to characterize the mechanism by which the binding of ATP to the nucleotide-binding domain (NBD) of Hsp70 propagates a signal to its substrate-binding domain (SBD). Unbiased MD simulations were performed for Hsp70-DnaK chaperone in nucleotide-free, ADP-bound and ATP-bound states. The simulations revealed that the SBD does not interact with the NBD for DnaK in its nucleotide-free and ADP-bound states whereas the docking of the SBD was found in the ATP-bound state. The docked state induced by ATP binding found in MD is an intermediate state between the initial nucleotide-free and final ATP-bound states of Hsp70. The analysis of the FEL projected along the amino-acid sequence permitted to identify a subset of 27 protein internal coordinates corresponding to a network of 91 key residues involved in the conformational change induced by ATP binding. Among the 91 residues, 26 are identified for the first time, whereas the others were shown relevant for the allosteric communication of Hsp70 s in several experiments and bioinformatics analysis. The FEL analysis revealed also the origin of the ATP-induced structural modifications of the SBD recently measured by Electron Paramagnetic Resonance. The pathway between the nucleotide-free and the intermediate state of DnaK was extracted by applying principal component analysis to the subset of internal coordinates describing the transition. The methodology proposed is general and could be applied to analyze allosteric communication in other proteins.

Decipher the Mechanisms of Protein Conformational Changes Induced by Nucleotide Binding through Free-Energy Landscape Analysis: ATP Binding to Hsp70

PubMed Central

Nicolaï, Adrien; Delarue, Patrice; Senet, Patrick

2013-01-01

ATP regulates the function of many proteins in the cell by transducing its binding and hydrolysis energies into protein conformational changes by mechanisms which are challenging to identify at the atomic scale. Based on molecular dynamics (MD) simulations, a method is proposed to analyze the structural changes induced by ATP binding to a protein by computing the effective free-energy landscape (FEL) of a subset of its coordinates along its amino-acid sequence. The method is applied to characterize the mechanism by which the binding of ATP to the nucleotide-binding domain (NBD) of Hsp70 propagates a signal to its substrate-binding domain (SBD). Unbiased MD simulations were performed for Hsp70-DnaK chaperone in nucleotide-free, ADP-bound and ATP-bound states. The simulations revealed that the SBD does not interact with the NBD for DnaK in its nucleotide-free and ADP-bound states whereas the docking of the SBD was found in the ATP-bound state. The docked state induced by ATP binding found in MD is an intermediate state between the initial nucleotide-free and final ATP-bound states of Hsp70. The analysis of the FEL projected along the amino-acid sequence permitted to identify a subset of 27 protein internal coordinates corresponding to a network of 91 key residues involved in the conformational change induced by ATP binding. Among the 91 residues, 26 are identified for the first time, whereas the others were shown relevant for the allosteric communication of Hsp70 s in several experiments and bioinformatics analysis. The FEL analysis revealed also the origin of the ATP-induced structural modifications of the SBD recently measured by Electron Paramagnetic Resonance. The pathway between the nucleotide-free and the intermediate state of DnaK was extracted by applying principal component analysis to the subset of internal coordinates describing the transition. The methodology proposed is general and could be applied to analyze allosteric communication in other proteins. PMID:24348227

GroEL stimulates protein folding through forced unfolding

PubMed Central

Lin, Zong; Madan, Damian; Rye, Hays S

2013-01-01

Many proteins cannot fold without the assistance of chaperonin machines like GroEL and GroES. The nature of this assistance, however, remains poorly understood. Here we demonstrate that unfolding of a substrate protein by GroEL enhances protein folding. We first show that capture of a protein on the open ring of a GroEL–ADP–GroES complex, GroEL’s physiological acceptor state for non-native proteins in vivo, leaves the substrate protein in an unexpectedly compact state. Subsequent binding of ATP to the same GroEL ring causes rapid, forced unfolding of the substrate protein. Notably, the fraction of the substrate protein that commits to the native state following GroES binding and protein release into the GroEL–GroES cavity is proportional to the extent of substrate-protein unfolding. Forced protein unfolding is thus a central component of the multilayered stimulatory mechanism used by GroEL to drive protein folding. PMID:18311152

Reactive ion etched substrates and methods of making and using

DOEpatents

Rucker, Victor C [San Francisco, CA; Shediac, Rene [Oakland, CA; Simmons, Blake A [San Francisco, CA; Havenstrite, Karen L [New York, NY

2007-08-07

Disclosed herein are substrates comprising reactive ion etched surfaces and specific binding agents immobilized thereon. The substrates may be used in methods and devices for assaying or isolating analytes in a sample. Also disclosed are methods of making the reactive ion etched surfaces.

Designing a Binding Interface for Control of Cancer Cell Adhesion via 3D Topography and Metabolic Oligosaccharide Engineering

PubMed Central

Du, Jian; Che, Pao-Lin; Wang, Zhi-Yun; Aich, Udayanath; Yarema, Kevin J.

2011-01-01

This study combines metabolic oligosaccharide engineering (MOE), a technology where the glycocalyx of living cells is endowed with chemical features not normally found in sugars, with custom-designed three dimensional biomaterial substrates to enhance the adhesion of cancer cells and control their morphology and gene expression. Specifically, Ac5ManNTGc, a thiol-bearing analogue of N-acetyl-d-mannosamine (ManNAc) was used to introduce thiolated sialic acids into the glycocalyx of human Jurkat T-lymphoma derived cells. In parallel 2D films and 3D electrospun nanofibrous scaffolds were prepared from polyethersulfone (PES) and (as controls) left unmodified or aminated. Alternately, the materials were malemided or gold-coated to provide bioorthogonal binding partners for the thiol groups newly expressed on the cell surface. Cell attachment was modulated by both the topography of the substrate surface and by the chemical compatibility of the binding interface between the cell and the substrate; a substantial increase in binding for normally non-adhesive Jurkat line for 3D scaffold compared to 2D surfaces with an added degree of adhesion resulting from chemoselective binding to malemidede-derivatived or gold-coated surfaces. In addition, the morphology of the cells attached to the 3D scaffolds via MOE-mediated adhesion was dramatically altered and the expression of genes involved in cell adhesion changed in a time-dependent manner. This study showed that cell adhesion could be enhanced, gene expression modulated, and cell fate controlled by introducing the 3D topograhical cues into the growth substrate and by creating a glycoengineered binding interface where the chemistry of both the cell surface and biomaterials scaffold was controlled to facilitate a new mode of carbohydrate-mediated adhesion. PMID:21549424

The Nedd4-binding partner 1 (N4BP1) protein is an inhibitor of the E3 ligase Itch.

PubMed

Oberst, Andrew; Malatesta, Martina; Aqeilan, Rami I; Rossi, Mario; Salomoni, Paolo; Murillas, Rodolfo; Sharma, Prashant; Kuehn, Michael R; Oren, Moshe; Croce, Carlo M; Bernassola, Francesca; Melino, Gerry

2007-07-03

Nedd4-binding partner-1 (N4BP1) has been identified as a protein interactor and a substrate of the homologous to E6AP C terminus (HECT) domain-containing E3 ubiquitin-protein ligase (E3), Nedd4. Here, we describe a previously unrecognized functional interaction between N4BP1 and Itch, a Nedd4 structurally related E3, which contains four WW domains, conferring substrate-binding activity. We show that N4BP1 association with the second WW domain (WW2) of Itch interferes with E3 binding to its substrates. In particular, we found that N4BP1 and p73 alpha, a target of Itch-mediated ubiquitin/proteasome proteolysis, share the same binding site. By competing with p73 alpha for binding to the WW2 domain, N4BP1 reduces the ability of Itch to recruit and ubiquitylate p73 alpha and inhibits Itch autoubiquitylation activity both in in vitro and in vivo ubiquitylation assays. Similarly, both c-Jun and p63 polyubiquitylation by Itch are inhibited by N4BP1. As a consequence, genetic and RNAi knockdown of N4BP1 diminish the steady-state protein levels and significantly impair the transcriptional activity of Itch substrates. Notably, stress-induced induction of c-Jun was impaired in N4BP1(-/-) cells. These results demonstrate that N4BP1 functions as a negative regulator of Itch. In addition, because inhibition of Itch by N4BP1 results in the stabilization of crucial cell death regulators such as p73 alpha and c-Jun, it is conceivable that N4BP1 may have a role in regulating tumor progression and the response of cancer cells to chemotherapy.

USP7 small-molecule inhibitors interfere with ubiquitin binding.

PubMed

Kategaya, Lorna; Di Lello, Paola; Rougé, Lionel; Pastor, Richard; Clark, Kevin R; Drummond, Jason; Kleinheinz, Tracy; Lin, Eva; Upton, John-Paul; Prakash, Sumit; Heideker, Johanna; McCleland, Mark; Ritorto, Maria Stella; Alessi, Dario R; Trost, Matthias; Bainbridge, Travis W; Kwok, Michael C M; Ma, Taylur P; Stiffler, Zachary; Brasher, Bradley; Tang, Yinyan; Jaishankar, Priyadarshini; Hearn, Brian R; Renslo, Adam R; Arkin, Michelle R; Cohen, Frederick; Yu, Kebing; Peale, Frank; Gnad, Florian; Chang, Matthew T; Klijn, Christiaan; Blackwood, Elizabeth; Martin, Scott E; Forrest, William F; Ernst, James A; Ndubaku, Chudi; Wang, Xiaojing; Beresini, Maureen H; Tsui, Vickie; Schwerdtfeger, Carsten; Blake, Robert A; Murray, Jeremy; Maurer, Till; Wertz, Ingrid E

2017-10-26

The ubiquitin system regulates essential cellular processes in eukaryotes. Ubiquitin is ligated to substrate proteins as monomers or chains and the topology of ubiquitin modifications regulates substrate interactions with specific proteins. Thus ubiquitination directs a variety of substrate fates including proteasomal degradation. Deubiquitinase enzymes cleave ubiquitin from substrates and are implicated in disease; for example, ubiquitin-specific protease-7 (USP7) regulates stability of the p53 tumour suppressor and other proteins critical for tumour cell survival. However, developing selective deubiquitinase inhibitors has been challenging and no co-crystal structures have been solved with small-molecule inhibitors. Here, using nuclear magnetic resonance-based screening and structure-based design, we describe the development of selective USP7 inhibitors GNE-6640 and GNE-6776. These compounds induce tumour cell death and enhance cytotoxicity with chemotherapeutic agents and targeted compounds, including PIM kinase inhibitors. Structural studies reveal that GNE-6640 and GNE-6776 non-covalently target USP7 12 Å distant from the catalytic cysteine. The compounds attenuate ubiquitin binding and thus inhibit USP7 deubiquitinase activity. GNE-6640 and GNE-6776 interact with acidic residues that mediate hydrogen-bond interactions with the ubiquitin Lys48 side chain, suggesting that USP7 preferentially interacts with and cleaves ubiquitin moieties that have free Lys48 side chains. We investigated this idea by engineering di-ubiquitin chains containing differential proximal and distal isotopic labels and measuring USP7 binding by nuclear magnetic resonance. This preferential binding protracted the depolymerization kinetics of Lys48-linked ubiquitin chains relative to Lys63-linked chains. In summary, engineering compounds that inhibit USP7 activity by attenuating ubiquitin binding suggests opportunities for developing other deubiquitinase inhibitors and may be a strategy more broadly applicable to inhibiting proteins that require ubiquitin binding for full functional activity.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bosserman, Mary A.; Downey, Theresa; Noinaj, Nicholas

Baeyer–Villiger monooxygenases (BVMOs) have been shown to play key roles for the biosynthesis of important natural products. MtmOIV, a homodimeric FAD- and NADPH-dependent BVMO, catalyzes the key frame-modifying steps of the mithramycin biosynthetic pathway, including an oxidative C–C bond cleavage, by converting its natural substrate premithramycin B into mithramycin DK, the immediate precursor of mithramycin. The drastically improved protein structure of MtmOIV along with the high-resolution structure of MtmOIV in complex with its natural substrate premithramycin B are reported here, revealing previously undetected key residues that are important for substrate recognition and catalysis. Kinetic analyses of selected mutants allowed usmore » to probe the substrate binding pocket of MtmOIV and also to discover the putative NADPH binding site. This is the first substrate-bound structure of MtmOIV providing new insights into substrate recognition and catalysis, which paves the way for the future design of a tailored enzyme for the chemo-enzymatic preparation of novel mithramycin analogues.« less

Structural insights into the cofactor-assisted substrate recognition of yeast methylglyoxal/isovaleraldehyde reductase Gre2.

PubMed

Guo, Peng-Chao; Bao, Zhang-Zhi; Ma, Xiao-Xiao; Xia, Qingyou; Li, Wei-Fang

2014-09-01

Saccharomyces cerevisiae Gre2 (EC1.1.1.283) serves as a versatile enzyme that catalyzes the stereoselective reduction of a broad range of substrates including aliphatic and aromatic ketones, diketones, as well as aldehydes, using NADPH as the cofactor. Here we present the crystal structures of Gre2 from S. cerevisiae in an apo-form at 2.00Å and NADPH-complexed form at 2.40Å resolution. Gre2 forms a homodimer, each subunit of which contains an N-terminal Rossmann-fold domain and a variable C-terminal domain, which participates in substrate recognition. The induced fit upon binding to the cofactor NADPH makes the two domains shift toward each other, producing an interdomain cleft that better fits the substrate. Computational simulation combined with site-directed mutagenesis and enzymatic activity analysis enabled us to define a potential substrate-binding pocket that determines the stringent substrate stereoselectivity for catalysis. Copyright © 2014 Elsevier B.V. All rights reserved.

Resveratrol serves as a protein-substrate interaction stabilizer in human SIRT1 activation.

PubMed

Hou, Xuben; Rooklin, David; Fang, Hao; Zhang, Yingkai

2016-11-30

Resveratrol is a natural compound found in red wine that has been suggested to exert its potential health benefit through the activation of SIRT1, a crucial member of the mammalian NAD + -dependent deacetylases. SIRT1 has emerged as an attractive therapeutic target for many aging related diseases, however, how its activity can only be activated toward some specific substrates by resveratrol has been poorly understood. Herein, by employing extensive molecular dynamics simulations as well as fragment-centric topographical mapping of binding interfaces, we have clarified current controversies in the literature and elucidated that resveratrol plays an important activation role by stabilizing SIRT1/peptide interactions in a substrate-specific manner. This new mechanism highlights the importance of the N-terminal domain in substrate recognition, explains the activity restoration role of resveratrol toward some "loose-binding" substrates of SIRT1, and has significant implications for the rational design of new substrate-specific SIRT1 modulators.

Ternary structure reveals mechanism of a membrane diacylglycerol kinase

PubMed Central

Li, Dianfan; Stansfeld, Phillip J.; Sansom, Mark S. P.; Keogh, Aaron; Vogeley, Lutz; Howe, Nicole; Lyons, Joseph A.; Aragao, David; Fromme, Petra; Fromme, Raimund; Basu, Shibom; Grotjohann, Ingo; Kupitz, Christopher; Rendek, Kimberley; Weierstall, Uwe; Zatsepin, Nadia A.; Cherezov, Vadim; Liu, Wei; Bandaru, Sateesh; English, Niall J.; Gati, Cornelius; Barty, Anton; Yefanov, Oleksandr; Chapman, Henry N.; Diederichs, Kay; Messerschmidt, Marc; Boutet, Sébastien; Williams, Garth J.; Marvin Seibert, M.; Caffrey, Martin

2015-01-01

Diacylglycerol kinase catalyses the ATP-dependent conversion of diacylglycerol to phosphatidic acid in the plasma membrane of Escherichia coli. The small size of this integral membrane trimer, which has 121 residues per subunit, means that available protein must be used economically to craft three catalytic and substrate-binding sites centred about the membrane/cytosol interface. How nature has accomplished this extraordinary feat is revealed here in a crystal structure of the kinase captured as a ternary complex with bound lipid substrate and an ATP analogue. Residues, identified as essential for activity by mutagenesis, decorate the active site and are rationalized by the ternary structure. The γ-phosphate of the ATP analogue is positioned for direct transfer to the primary hydroxyl of the lipid whose acyl chain is in the membrane. A catalytic mechanism for this unique enzyme is proposed. The active site architecture shows clear evidence of having arisen by convergent evolution. PMID:26673816

Ternary structure reveals mechanism of a membrane diacylglycerol kinase

NASA Astrophysics Data System (ADS)

Li, Dianfan; Stansfeld, Phillip J.; Sansom, Mark S. P.; Keogh, Aaron; Vogeley, Lutz; Howe, Nicole; Lyons, Joseph A.; Aragao, David; Fromme, Petra; Fromme, Raimund; Basu, Shibom; Grotjohann, Ingo; Kupitz, Christopher; Rendek, Kimberley; Weierstall, Uwe; Zatsepin, Nadia A.; Cherezov, Vadim; Liu, Wei; Bandaru, Sateesh; English, Niall J.; Gati, Cornelius; Barty, Anton; Yefanov, Oleksandr; Chapman, Henry N.; Diederichs, Kay; Messerschmidt, Marc; Boutet, Sébastien; Williams, Garth J.; Marvin Seibert, M.; Caffrey, Martin

2015-12-01

Diacylglycerol kinase catalyses the ATP-dependent conversion of diacylglycerol to phosphatidic acid in the plasma membrane of Escherichia coli. The small size of this integral membrane trimer, which has 121 residues per subunit, means that available protein must be used economically to craft three catalytic and substrate-binding sites centred about the membrane/cytosol interface. How nature has accomplished this extraordinary feat is revealed here in a crystal structure of the kinase captured as a ternary complex with bound lipid substrate and an ATP analogue. Residues, identified as essential for activity by mutagenesis, decorate the active site and are rationalized by the ternary structure. The γ-phosphate of the ATP analogue is positioned for direct transfer to the primary hydroxyl of the lipid whose acyl chain is in the membrane. A catalytic mechanism for this unique enzyme is proposed. The active site architecture shows clear evidence of having arisen by convergent evolution.

Platinum-Catalyzed, Terminal-Selective C(sp(3))-H Oxidation of Aliphatic Amines.

PubMed

Lee, Melissa; Sanford, Melanie S

2015-10-14

This Communication describes the terminal-selective, Pt-catalyzed C(sp(3))-H oxidation of aliphatic amines without the requirement for directing groups. CuCl2 is employed as a stoichiometric oxidant, and the reactions proceed in high yield at Pt loadings as low as 1 mol%. These transformations are conducted in the presence of sulfuric acid, which reacts with the amine substrates in situ to form ammonium salts. We propose that protonation of the amine serves at least three important roles: (i) it renders the substrates soluble in the aqueous reaction medium; (ii) it limits binding of the amine nitrogen to Pt or Cu; and (iii) it electronically deactivates the C-H bonds proximal to the nitrogen center. We demonstrate that this strategy is effective for the terminal-selective C(sp(3))-H oxidation of a variety of primary, secondary, and tertiary amines.

Platinum-Catalyzed Terminal-Selective C(sp3)–H Oxidation of Aliphatic Amines

PubMed Central

Lee, Melissa; Sanford, Melanie S.

2016-01-01

This paper describes the terminal-selective Pt-catalyzed C(sp3)–H oxidation of aliphatic amines without the requirement for directing groups. CuCl2 is employed as a stoichiometric oxidant, and the reactions proceed in high yield at Pt loadings as low as 1 mol %. These transformations are conducted in the presence of sulfuric acid, which reacts with the amine substrates in situ to form ammonium salts. We propose that protonation of the amine serves at least three important roles: (i) it renders the substrates soluble in the aqueous reaction medium; (ii) it limits binding of the amine nitrogen to Pt or Cu; and (ii) it electronically deactivates the C–H bonds proximal to the nitrogen center. We demonstrate that this strategy is effective for the terminal-selective C(sp3)–H oxidation of a variety of primary, secondary and tertiary amines. PMID:26439251

Mechanistic study of manganese-substituted glycerol dehydrogenase using a kinetic and thermodynamic analysis.

PubMed

Fang, Baishan; Niu, Jin; Ren, Hong; Guo, Yingxia; Wang, Shizhen

2014-01-01

Mechanistic insights regarding the activity enhancement of dehydrogenase by metal ion substitution were investigated by a simple method using a kinetic and thermodynamic analysis. By profiling the binding energy of both the substrate and product, the metal ion's role in catalysis enhancement was revealed. Glycerol dehydrogenase (GDH) from Klebsiella pneumoniae sp., which demonstrated an improvement in activity by the substitution of a zinc ion with a manganese ion, was used as a model for the mechanistic study of metal ion substitution. A kinetic model based on an ordered Bi-Bi mechanism was proposed considering the noncompetitive product inhibition of dihydroxyacetone (DHA) and the competitive product inhibition of NADH. By obtaining preliminary kinetic parameters of substrate and product inhibition, the number of estimated parameters was reduced from 10 to 4 for a nonlinear regression-based kinetic parameter estimation. The simulated values of time-concentration curves fit the experimental values well, with an average relative error of 11.5% and 12.7% for Mn-GDH and GDH, respectively. A comparison of the binding energy of enzyme ternary complex for Mn-GDH and GDH derived from kinetic parameters indicated that metal ion substitution accelerated the release of dioxyacetone. The metal ion's role in catalysis enhancement was explicated.

Human Cytochrome P450 21A2, the Major Steroid 21-Hydroxylase

PubMed Central

Pallan, Pradeep S.; Wang, Chunxue; Lei, Li; Yoshimoto, Francis K.; Auchus, Richard J.; Waterman, Michael R.; Guengerich, F. Peter; Egli, Martin

2015-01-01

Cytochrome P450 (P450) 21A2 is the major steroid 21-hydroxylase, and deficiency of this enzyme is involved in ∼95% of cases of human congenital adrenal hyperplasia, a disorder of adrenal steroidogenesis. A structure of the bovine enzyme that we published previously (Zhao, B., Lei, L., Kagawa, N., Sundaramoorthy, M., Banerjee, S., Nagy, L. D., Guengerich, F. P., and Waterman, M. R. (2012) Three-dimensional structure of steroid 21-hydroxylase (cytochrome P450 21A2) with two substrates reveals locations of disease-associated variants. J. Biol. Chem. 287, 10613–10622), containing two molecules of the substrate 17α-hydroxyprogesterone, has been used as a template for understanding genetic deficiencies. We have now obtained a crystal structure of human P450 21A2 in complex with progesterone, a substrate in adrenal 21-hydroxylation. Substrate binding and release were fast for human P450 21A2 with both substrates, and pre-steady-state kinetics showed a partial burst but only with progesterone as substrate and not 17α-hydroxyprogesterone. High intermolecular non-competitive kinetic deuterium isotope effects on both kcat and kcat/Km, from 5 to 11, were observed with both substrates, indicative of rate-limiting C–H bond cleavage and suggesting that the juxtaposition of the C21 carbon in the active site is critical for efficient oxidation. The estimated rate of binding of the substrate progesterone (kon 2.4 × 107 m−1 s−1) is only ∼2-fold greater than the catalytic efficiency (kcat/Km = 1.3 × 107 m−1 s−1) with this substrate, suggesting that the rate of substrate binding may also be partially rate-limiting. The structure of the human P450 21A2-substrate complex provides direct insight into mechanistic effects of genetic variants. PMID:25855791

Synthesis and evaluation of conformationally restricted inhibitors of aspartate semialdehyde dehydrogenase.

PubMed

Evitt, Andrew S; Cox, Russell J

2011-05-01

Inhibitors of the enzyme aspartate semialdehyde dehydrogenase, a key biological target for the generation of a new class of antibiotic compounds, have been developed. To investigate improvements to binding within an inhibitor series, the lowering of the entropic barrier to binding through conformational restriction was investigated. A library of linear and cyclic substrate analogues was generated and computational docking used to aid in structure selection. The cyclic phosphonate inhibitor 18 was thus identified as complimentary to the enzyme active-site. Synthesis and in vitro inhibition assay revealed a K(i) of 3.8 mM against natural substrate, where the linear analogue of 18, compound 15, had previously shown no inhibitory activity. Two further inhibitors, phosphate analogue diastereoisomers 17a and 17b, were synthesised and also found to have low millimolar K(i) values. As a result of the computational docking investigations, a novel substrate binding interaction was discovered: hydrogen bonding between the substrate (phosphate hydroxy-group as the hydrogen bond donor) and the NADPH cofactor (2'-oxygen as the hydrogen bond acceptor).

Structure of the substrate-binding b′ domain of the Protein disulfide isomerase-like protein of the testis

PubMed Central

Bastos-Aristizabal, Sara; Kozlov, Guennadi; Gehring, Kalle

2014-01-01

Protein Disulfide Isomerase-Like protein of the Testis (PDILT) is a testis-specific member of the PDI family. PDILT displays similar domain architecture to PDIA1, the founding member of this protein family, but lacks catalytic cysteines needed for oxidoreduction reactions. This suggests special importance of chaperone activity of PDILT, but how it recognizes misfolded protein substrates is unknown. Here, we report the high-resolution crystal structure of the b′ domain of human PDILT. The structure reveals a conserved hydrophobic pocket, which is likely a principal substrate-binding site in PDILT. In the crystal, this pocket is occupied by side chains of tyrosine and tryptophan residues from another PDILT molecule, suggesting a preference for binding exposed aromatic residues in protein substrates. The lack of interaction of the b′ domain with the P-domains of calreticulin-3 and calmegin hints at a novel way of interaction between testis-specific lectin chaperones and PDILT. Further studies of this recently discovered PDI member would help to understand the important role that PDILT plays in the differentiation and maturation of spermatozoids. PMID:24662985

The ATP-binding cassette transporter Cbc (choline/betaine/carnitine) recruits multiple substrate-binding proteins with strong specificity for distinct quaternary ammonium compounds

PubMed Central

Chen, Chiliang; Malek, Adel A.; Wargo, Matthew J.; Hogan, Deborah A.; Beattie, Gwyn A.

2017-01-01

Summary We identified a choline, betaine and carnitine transporter, designated Cbc, from Pseudomonas syringae and Pseudomonas aeruginosa that is unusual among members of the ATP-binding cassette (ABC) transporter family in its use of multiple periplasmic substrate-binding proteins (SBPs) that are highly specific for their substrates. The SBP encoded by the cbcXWV operon, CbcX, binds choline with a high affinity (Km, 2.6 μM) and, although it also binds betaine (Km, 24.2 μM), CbcXWV-mediated betaine uptake did not occur in the presence of choline. The CbcX orthologue ChoX from Sinorhizobium meliloti was similar to CbcX in these binding properties. The core transporter CbcWV also interacts with the carnitine-specific SBP CaiX (Km, 24 μM) and the betaine-specific SBP BetX (Km, 0.6 μM). Unlike most ABC transporter loci, caiX, betX and cbcXWV are separated in the genome. CaiX-mediated carnitine uptake was reduced by CbcX and BetX only when they were bound by their individual ligands, providing the first in vivo evidence for a higher affinity for ligand-bound than ligand-free SBPs by an ABC transporter. These studies demonstrate not only that the Cbc transporter serves as a useful model for exploring ABC transporter component interactions, but also that the orphan SBP genes common to bacterial genomes can encode functional SBPs. PMID:19919675

The ATP-binding cassette transporter Cbc (choline/betaine/carnitine) recruits multiple substrate-binding proteins with strong specificity for distinct quaternary ammonium compounds.

PubMed

Chen, Chiliang; Malek, Adel A; Wargo, Matthew J; Hogan, Deborah A; Beattie, Gwyn A

2010-01-01

We identified a choline, betaine and carnitine transporter, designated Cbc, from Pseudomonas syringae and Pseudomonas aeruginosa that is unusual among members of the ATP-binding cassette (ABC) transporter family in its use of multiple periplasmic substrate-binding proteins (SBPs) that are highly specific for their substrates. The SBP encoded by the cbcXWV operon, CbcX, binds choline with a high affinity (K(m), 2.6 microM) and, although it also binds betaine (K(m), 24.2 microM), CbcXWV-mediated betaine uptake did not occur in the presence of choline. The CbcX orthologue ChoX from Sinorhizobium meliloti was similar to CbcX in these binding properties. The core transporter CbcWV also interacts with the carnitine-specific SBP CaiX (K(m), 24 microM) and the betaine-specific SBP BetX (K(m), 0.6 microM). Unlike most ABC transporter loci, caiX, betX and cbcXWV are separated in the genome. CaiX-mediated carnitine uptake was reduced by CbcX and BetX only when they were bound by their individual ligands, providing the first in vivo evidence for a higher affinity for ligand-bound than ligand-free SBPs by an ABC transporter. These studies demonstrate not only that the Cbc transporter serves as a useful model for exploring ABC transporter component interactions, but also that the orphan SBP genes common to bacterial genomes can encode functional SBPs.

Effect of redox partner binding on CYP101D1 conformational dynamics.

PubMed

Batabyal, Dipanwita; Poulos, Thomas L

2018-06-01

We have compared the thermodynamics of substrate and redox partner binding of P450cam to its close homologue, CYP101D1, using isothermal titration calorimetry (ITC). CYP101D1 binds camphor about 10-fold more weakly than P450cam which is consistent with the inability of camphor to cause a complete low- to high-spin shift in CYP101D1. Even so molecular dynamics simulations show that camphor is very stable in the CYP101D1 active site similar to P450cam. ITC data on the binding of the CYP101D1 ferredoxin redox partner (abbreviated Arx) shows that the substrate-bound closed state of CYP101D1 binds Arx more tightly than the substrate-free open form. This is just the opposite to P450cam where Pdx (ferredoxin redox partner of P450cam) favors binding to the P450cam open state. In addition, CYP101D1-Arx binding has a large negative ΔS while the P450cam-Pdx has a much smaller ΔS indicating that interactions at the docking interface are different. The most obvious difference is that PDX D38 which forms an important ion pair with P450cam R112 at the center of the interface is Arx L39 in Arx. This suggests that Arx may adopt a different orientation than Pdx in order to optimize nonpolar interactions with Arx L39 . Copyright © 2018. Published by Elsevier Inc.

An additional substrate binding site in a bacterial phenylalanine hydroxylase

PubMed Central

Ronau, Judith A.; Paul, Lake N.; Fuchs, Julian E.; Corn, Isaac R.; Wagner, Kyle T.; Liedl, Klaus R.; Abu-Omar, Mahdi M.; Das, Chittaranjan

2014-01-01

Phenylalanine hydroxylase (PAH) is a non-heme iron enzyme that catalyzes phenylalanine oxidation to tyrosine, a reaction that must be kept under tight regulatory control. Mammalian PAH features a regulatory domain where binding of the substrate leads to allosteric activation of the enzyme. However, existence of PAH regulation in evolutionarily distant organisms, such as certain bacteria in which it occurs, has so far been underappreciated. In an attempt to crystallographically characterize substrate binding by PAH from Chromobacterium violaceum (cPAH), a single-domain monomeric enzyme, electron density for phenylalanine was observed at a distal site, 15.7Å from the active site. Isothermal titration calorimetry (ITC) experiments revealed a dissociation constant of 24 ± 1.1 µM for phenylalanine. Under the same conditions, no detectable binding was observed in ITC for alanine, tyrosine, or isoleucine, indicating the distal site may be selective for phenylalanine. Point mutations of residues in the distal site that contact phenylalanine (F258A, Y155A, T254A) lead to impaired binding, consistent with the presence of distal site binding in solution. Kinetic analysis reveals that the distal site mutants suffer a discernible loss in their catalytic activity. However, x-ray structures of Y155A and F258A, two of the mutants showing more noticeable defect in their activity, show no discernible change in their active site structure, suggesting that the effect of distal binding may transpire through protein dynamics in solution. PMID:23860686

The chitin-binding domain of a GH-18 chitinase from Vibrio harveyi is crucial for chitin-chitinase interactions.

PubMed

Suginta, Wipa; Sirimontree, Paknisa; Sritho, Natchanok; Ohnuma, Takayuki; Fukamizo, Tamo

2016-12-01

Vibrio harveyi chitinase A (VhChiA) is a GH-18 glycosyl hydrolase with a structure containing three distinct domains: i) the N-terminal chitin-binding domain; ii) the (α/β) 8 TIM barrel catalytic domain; and iii) the α+β insertion domain. In this study, we cloned the gene fragment encoding the chitin-binding domain of VhChiA, termed ChBD Vh ChiA . The recombinant ChBD Vh ChiA was heterologously expressed in E. coli BL21 strain Tuner(DE3)pLacI host cells, and purified to homogeneity. CD measurements suggested that ChBD Vh ChiA contained β-sheets as major structural components and fluorescence spectroscopy showed that the protein domain was folded correctly, and suitable for functional characterization. Chitin binding assays showed that ChBD Vh ChiA bound to both α- and β-chitins, with the greatest affinity for β-colloidal chitin, but barely bound to polymeric chitosan. These results identified the tandem N-acetamido functionality on chitin chains as the specific sites of enzyme-substrate interactions. The binding affinity of the isolated domain was significantly lower than that of intact VhChiA, suggesting that the catalytic domain works synergistically with the chitin-binding domain to guide the polymeric substrate into the substrate binding cleft. These data confirm the physiological role of the chitin-binding domain of the marine bacterial GH-18 chitinase A in chitin-chitinase interactions. Copyright © 2016 Elsevier B.V. All rights reserved.

AFRRI Reports, Second Quarter 1994

DTIC Science & Technology

1994-08-01

the antrum wete immediately placed in sterile 0.9% NaCl, kept on ice, coded, and then prepared for culture, smears, and urease assay by homogeniza...high urease specific activity (>1 |J.mol- min-1 ■ mg protein-1) plus high-affinity substrate binding (Mi- chaelis constant [K^\\ < 1 mmol/L),27 in at...031, respectively), and the characteristic bacterial growth with high-activity product.on of a urease with tight substrate binding " was found in

Real-time observation of the conformational dynamics of mitochondrial Hsp70 by spFRET.

PubMed

Sikor, Martin; Mapa, Koyeli; von Voithenberg, Lena Voith; Mokranjac, Dejana; Lamb, Don C

2013-05-29

The numerous functions of the important class of molecular chaperones, heat shock proteins 70 (Hsp70), rely on cycles of intricate conformational changes driven by ATP-hydrolysis and regulated by cochaperones and substrates. Here, we used Förster resonance energy transfer to study the conformational dynamics of individual molecules of Ssc1, a mitochondrial Hsp70, in real time. The intrinsic dynamics of the substrate-binding domain of Ssc1 was observed to be uncoupled from the dynamic interactions between substrate- and nucleotide-binding domains. Analysis of the fluctuations in the interdomain separation revealed frequent transitions to a nucleotide-free state. The nucleotide-exchange factor Mge1 did not induce ADP release, as expected, but rather facilitated binding of ATP. These results indicate that the conformational cycle of Ssc1 is more elaborate than previously thought and provide insight into how the Hsp70s can perform a wide variety of functions.

Rifampin phosphotransferase is an unusual antibiotic resistance kinase

PubMed Central

Stogios, Peter J.; Cox, Georgina; Spanogiannopoulos, Peter; Pillon, Monica C.; Waglechner, Nicholas; Skarina, Tatiana; Koteva, Kalinka; Guarné, Alba; Savchenko, Alexei; Wright, Gerard D.

2016-01-01

Rifampin (RIF) phosphotransferase (RPH) confers antibiotic resistance by conversion of RIF and ATP, to inactive phospho-RIF, AMP and Pi. Here we present the crystal structure of RPH from Listeria monocytogenes (RPH-Lm), which reveals that the enzyme is comprised of three domains: two substrate-binding domains (ATP-grasp and RIF-binding domains); and a smaller phosphate-carrying His swivel domain. Using solution small-angle X-ray scattering and mutagenesis, we reveal a mechanism where the swivel domain transits between the spatially distinct substrate-binding sites during catalysis. RPHs are previously uncharacterized dikinases that are widespread in environmental and pathogenic bacteria. These enzymes are members of a large unexplored group of bacterial enzymes with substrate affinities that have yet to be fully explored. Such an enzymatically complex mechanism of antibiotic resistance augments the spectrum of strategies used by bacteria to evade antimicrobial compounds. PMID:27103605

Electrostatic interactions guide the active site face of a structure-specific ribonuclease to its RNA substrate.

PubMed

Plantinga, Matthew J; Korennykh, Alexei V; Piccirilli, Joseph A; Correll, Carl C

2008-08-26

Restrictocin, a member of the alpha-sarcin family of site-specific endoribonucleases, uses electrostatic interactions to bind to the ribosome and to RNA oligonucleotides, including the minimal specific substrate, the sarcin/ricin loop (SRL) of 23S-28S rRNA. Restrictocin binds to the SRL by forming a ground-state E:S complex that is stabilized predominantly by Coulomb interactions and depends on neither the sequence nor structure of the RNA, suggesting a nonspecific complex. The 22 cationic residues of restrictocin are dispersed throughout this protein surface, complicating a priori identification of a Coulomb interacting surface. Structural studies have identified an enzyme-substrate interface, which is expected to overlap with the electrostatic E:S interface. Here, we identified restrictocin residues that contribute to binding in the E:S complex by determining the salt dependence [partial differential log(k 2/ K 1/2)/ partial differential log[KCl

Fungal chitinases: diversity, mechanistic properties and biotechnological potential.

PubMed

Hartl, Lukas; Zach, Simone; Seidl-Seiboth, Verena

2012-01-01

Chitin derivatives, chitosan and substituted chito-oligosaccharides have a wide spectrum of applications ranging from medicine to cosmetics and dietary supplements. With advancing knowledge about the substrate-binding properties of chitinases, enzyme-based production of these biotechnologically relevant sugars from biological resources is becoming increasingly interesting. Fungi have high numbers of glycoside hydrolase family 18 chitinases with different substrate-binding site architectures. As presented in this review, the large diversity of fungal chitinases is an interesting starting point for protein engineering. In this review, recent data about the architecture of the substrate-binding clefts of fungal chitinases, in connection with their hydrolytic and transglycolytic abilities, and the development of chitinase inhibitors are summarized. Furthermore, the biological functions of chitinases, chitin and chitosan utilization by fungi, and the effects of these aspects on biotechnological applications, including protein overexpression and autolysis during industrial processes, are discussed in this review.

Coelenterazine-v ligated to Ca2+-triggered coelenterazine-binding protein is a stable and efficient substrate of the red-shifted mutant of Renilla muelleri luciferase.

PubMed

Stepanyuk, Galina A; Unch, James; Malikova, Natalia P; Markova, Svetlana V; Lee, John; Vysotski, Eugene S

2010-10-01

It has been shown that the coelenterazine analog, coelenterazine-v, is an efficient substrate for a reaction catalyzed by Renilla luciferase. The resulting bioluminescence emission maximum is shifted to a longer wavelength up to 40 nm, which allows the use of some "yellow" Renilla luciferase mutants for in vivo imaging. However, the utility of coelenterazine-v in small-animal imaging has been hampered by its instability in solution and in biological tissues. To overcome this drawback, we ligated coelenterazine-v to Ca(2+)-triggered coelenterazine-binding protein from Renilla muelleri, which apparently functions in the organism for stabilizing and protecting coelenterazine from oxidation. The coelenterazine-v bound within coelenterazine-binding protein has revealed a greater long-term stability at both 4 and 37 °C. In addition, the coelenterazine-binding protein ligated by coelenterazine-v yields twice the total light over free coelenterazine-v as a substrate for the red-shifted R. muelleri luciferase. These findings suggest the possibility for effective application of coelenterazine-v in various in vitro assays.

High-affinity DNA-binding Domains of Replication Protein A (RPA) Direct SMARCAL1-dependent Replication Fork Remodeling*

PubMed Central

Bhat, Kamakoti P.; Bétous, Rémy; Cortez, David

2015-01-01

SMARCAL1 catalyzes replication fork remodeling to maintain genome stability. It is recruited to replication forks via an interaction with replication protein A (RPA), the major ssDNA-binding protein in eukaryotic cells. In addition to directing its localization, RPA also activates SMARCAL1 on some fork substrates but inhibits it on others, thereby conferring substrate specificity to SMARCAL1 fork-remodeling reactions. We investigated the mechanism by which RPA regulates SMARCAL1. Our results indicate that although an interaction between SMARCAL1 and RPA is essential for SMARCAL1 activation, the location of the interacting surface on RPA is not. Counterintuitively, high-affinity DNA binding of RPA DNA-binding domain (DBD) A and DBD-B near the fork junction makes it easier for SMARCAL1 to remodel the fork, which requires removing RPA. We also found that RPA DBD-C and DBD-D are not required for SMARCAL1 regulation. Thus, the orientation of the high-affinity RPA DBDs at forks dictates SMARCAL1 substrate specificity. PMID:25552480

High-affinity DNA-binding domains of replication protein A (RPA) direct SMARCAL1-dependent replication fork remodeling.

PubMed

Bhat, Kamakoti P; Bétous, Rémy; Cortez, David

2015-02-13

SMARCAL1 catalyzes replication fork remodeling to maintain genome stability. It is recruited to replication forks via an interaction with replication protein A (RPA), the major ssDNA-binding protein in eukaryotic cells. In addition to directing its localization, RPA also activates SMARCAL1 on some fork substrates but inhibits it on others, thereby conferring substrate specificity to SMARCAL1 fork-remodeling reactions. We investigated the mechanism by which RPA regulates SMARCAL1. Our results indicate that although an interaction between SMARCAL1 and RPA is essential for SMARCAL1 activation, the location of the interacting surface on RPA is not. Counterintuitively, high-affinity DNA binding of RPA DNA-binding domain (DBD) A and DBD-B near the fork junction makes it easier for SMARCAL1 to remodel the fork, which requires removing RPA. We also found that RPA DBD-C and DBD-D are not required for SMARCAL1 regulation. Thus, the orientation of the high-affinity RPA DBDs at forks dictates SMARCAL1 substrate specificity. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Local entropy difference upon a substrate binding of a psychrophilic α-amylase and a mesophilic homologue

NASA Astrophysics Data System (ADS)

Kosugi, Takahiro; Hayashi, Shigehiko

2011-01-01

Psychrophilic α-amylase from the antarctic bacterium pseudoalteromonashaloplanktis (AHA) and its mesophilic homologue, porcine pancreatic α-amylase (PPA) are theoretically investigated with molecular dynamics (MD) simulations. We carried out 240-ns MD simulations for four systems, AHA and PPA with/without the bound substrate, and examined protein conformational entropy changes upon the substrate binding. We developed an analysis that decomposes the entropy changes into contributions of individual amino acids, and successfully identified protein regions responsible for the entropy changes. The results provide a molecular insight into the structural flexibilities of those enzymes related to the temperature dependences of the enzymatic activity.

The active site of hydroxynitrile lyase from Prunus amygdalus: Modeling studies provide new insights into the mechanism of cyanogenesis

PubMed Central

Dreveny, Ingrid; Kratky, Christoph; Gruber, Karl

2002-01-01

The FAD-dependent hydroxynitrile lyase from almond (Prunus amygdalus, PaHNL) catalyzes the cleavage of R-mandelonitrile into benzaldehyde and hydrocyanic acid. Catalysis of the reverse reaction—the enantiospecific formation of α-hydroxynitriles—is now widely utilized in organic syntheses as one of the few industrially relevant examples of enzyme-mediated C–C bond formation. Starting from the recently determined X-ray crystal structure, systematic docking calculations with the natural substrate were used to locate the active site of the enzyme and to identify amino acid residues involved in substrate binding and catalysis. Analysis of the modeled substrate complexes supports an enzymatic mechanism that includes the flavin cofactor as a mere "spectator" of the reaction and relies on general acid/base catalysis by the conserved His-497. Stabilization of the negative charge of the cyanide ion is accomplished by a pronounced positive electrostatic potential at the binding site. PaHNL activity requires the FAD cofactor to be bound in its oxidized form, and calculations of the pKa of enzyme-bound HCN showed that the observed inactivation upon cofactor reduction is largely caused by the reversal of the electrostatic potential within the active site. The suggested mechanism closely resembles the one proposed for the FAD-independent, and structurally unrelated HNL from Hevea brasiliensis. Although the actual amino acid residues involved in the catalytic cycle are completely different in the two enzymes, a common motif for the mechanism of cyanogenesis (general acid/base catalysis plus electrostatic stabilization of the cyanide ion) becomes evident. PMID:11790839

Characterization of the interaction of yeast enolase with polynucleotides.

PubMed

al-Giery, A G; Brewer, J M

1992-09-23

Yeast enolase is inhibited under certain conditions by DNA. The enzyme binds to single-stranded DNA-cellulose. Inhibition was used for routine characterization of the interaction. The presence of the substrate 2-phospho-D-glycerate reduces inhibition and binding. Both yeast enolase isozymes behave similarly. Impure yeast enolase was purified by adsorption onto a single-stranded DNA-cellulose column followed by elution with substrate. Interaction with RNA, double-stranded DNA, or degraded DNA results in less inhibition, suggesting that yeast enolase preferentially binds single-stranded DNA. However, yeast enolase is not a DNA-unwinding protein. The enzyme is inhibited by the short synthetic oligodeoxynucleotides G6, G8 and G10 but not T8 or T6, suggesting some base specificity in the interaction. The interaction is stronger at more acid pH values, with an apparent pK of 5.6. The interaction is prevented by 0.3 M KCl, suggesting that electrostatic factors are important. Histidine or lysine reverse the inhibition at lower concentrations, while phosphate is still more effective. Binding of single-stranded DNA to enolase reduces the reaction of protein histidyl residues with diethylpyrocarbonate. The inhibition of yeast enolase by single-stranded DNA is not total, and suggests the active site is not directly involved in the interaction. Binding of substrate may induce a conformational change in the enzyme that interferes with DNA binding and vice versa.

Carbene footprinting accurately maps binding sites in protein-ligand and protein-protein interactions

NASA Astrophysics Data System (ADS)

Manzi, Lucio; Barrow, Andrew S.; Scott, Daniel; Layfield, Robert; Wright, Timothy G.; Moses, John E.; Oldham, Neil J.

2016-11-01

Specific interactions between proteins and their binding partners are fundamental to life processes. The ability to detect protein complexes, and map their sites of binding, is crucial to understanding basic biology at the molecular level. Methods that employ sensitive analytical techniques such as mass spectrometry have the potential to provide valuable insights with very little material and on short time scales. Here we present a differential protein footprinting technique employing an efficient photo-activated probe for use with mass spectrometry. Using this methodology the location of a carbohydrate substrate was accurately mapped to the binding cleft of lysozyme, and in a more complex example, the interactions between a 100 kDa, multi-domain deubiquitinating enzyme, USP5 and a diubiquitin substrate were located to different functional domains. The much improved properties of this probe make carbene footprinting a viable method for rapid and accurate identification of protein binding sites utilizing benign, near-UV photoactivation.

Snapshots of Dynamics in Synthesizing N6-isopentenyladenosine at tRNA Anticodon†,‡

PubMed Central

Chimnaronk, Sarin; Forouhar, Farhad; Sakai, Junichi; Yao, Min; Tron, Cecile M.; Atta, Mohamed; Fontecave, Marc; Hunt, John F.; Tanaka, Isao

2009-01-01

Bacterial and eukaryotic transfer RNAs that decode codons starting with uridine have a hydrophobically-hypermodified adenosine at the position 37 (A37) adjacent to the 3′-end of the anticodon, which is essential for efficient and highly accurate protein translation by the ribosome. However, it remains unclear how the corresponding tRNAs are selected to be modified by alkylation at the correct position of the adenosine base. We have determined a series of the crystal structures of bacterial tRNA isopentenyltransferase (MiaA) in apo- and tRNA-bound forms, which completely render snapshots of substrate selections during modification of RNA. A compact evolutionary inserted domain (herein ‘swinging domain’) in MiaA that exhibits as a highly mobile entity moves around the catalytic domain as likely to reach and trap the tRNA substrate. Thereby, MiaA clamps the anticodon stem loop of tRNA substrate between the catalytic and swinging domains, where the two conserved elongated residues from the swinging domain pinch the two flanking A36 and A38 together to squeeze out A37 into the reaction tunnel. The site-specific isopentenylation of RNA is thus ensured by a characteristic pinch-and-flip mechanism and by a reaction tunnel to confine the substrate selection. Furthermore, combining information from soaking experiments with structural comparisons, we propose a mechanism for the ordered substrate-binding of MiaA. PMID:19435325

Crystal Structure of the Heme d1 Biosynthesis Enzyme NirE in Complex with Its Substrate Reveals New Insights into the Catalytic Mechanism of S-Adenosyl-l-methionine-dependent Uroporphyrinogen III Methyltransferases*

PubMed Central

Storbeck, Sonja; Saha, Sayantan; Krausze, Joern; Klink, Björn U.; Heinz, Dirk W.; Layer, Gunhild

2011-01-01

During the biosynthesis of heme d1, the essential cofactor of cytochrome cd1 nitrite reductase, the NirE protein catalyzes the methylation of uroporphyrinogen III to precorrin-2 using S-adenosyl-l-methionine (SAM) as the methyl group donor. The crystal structure of Pseudomonas aeruginosa NirE in complex with its substrate uroporphyrinogen III and the reaction by-product S-adenosyl-l-homocysteine (SAH) was solved to 2.0 Å resolution. This represents the first enzyme-substrate complex structure for a SAM-dependent uroporphyrinogen III methyltransferase. The large substrate binds on top of the SAH in a “puckered” conformation in which the two pyrrole rings facing each other point into the same direction either upward or downward. Three arginine residues, a histidine, and a methionine are involved in the coordination of uroporphyrinogen III. Through site-directed mutagenesis of the nirE gene and biochemical characterization of the corresponding NirE variants the amino acid residues Arg-111, Glu-114, and Arg-149 were identified to be involved in NirE catalysis. Based on our structural and biochemical findings, we propose a potential catalytic mechanism for NirE in which the methyl transfer reaction is initiated by an arginine catalyzed proton abstraction from the C-20 position of the substrate. PMID:21632530

Application of the SSB biosensor to study in vitro transcription.

PubMed

Cook, Alexander; Hari-Gupta, Yukti; Toseland, Christopher P

2018-02-12

Gene expression, catalysed by RNA polymerases (RNAP), is one of the most fundamental processes in living cells. The majority of methods to quantify mRNA are based upon purification of the nucleic acid which leads to experimental inaccuracies and loss of product, or use of high cost dyes and sensitive spectrophotometers. Here, we describe the use of a fluorescent biosensor based upon the single stranded binding (SSB) protein. In this study, the SSB biosensor showed similar binding properties to mRNA, to that of its native substrate, single-stranded DNA (ssDNA). We found the biosensor to be reproducible with no associated loss of product through purification, or the requirement for expensive dyes. Therefore, we propose that the SSB biosensor is a useful tool for comparative measurement of mRNA yield following in vitro transcription. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Correlating single-molecule and ensemble-average measurements of peptide adsorption onto different inorganic materials.

PubMed

Kim, Seong-Oh; Jackman, Joshua A; Mochizuki, Masahito; Yoon, Bo Kyeong; Hayashi, Tomohiro; Cho, Nam-Joon

2016-06-07

The coating of solid-binding peptides (SBPs) on inorganic material surfaces holds significant potential for improved surface functionalization at nano-bio interfaces. In most related studies, the goal has been to engineer peptides with selective and high binding affinity for a target material. The role of the material substrate itself in modulating the adsorption behavior of a peptide molecule remains less explored and there are few studies that compare the interaction of one peptide with different inorganic substrates. Herein, using a combination of two experimental techniques, we investigated the adsorption of a 16 amino acid-long random coil peptide to various inorganic substrates - gold, silicon oxide, titanium oxide and aluminum oxide. Quartz crystal microbalance-dissipation (QCM-D) experiments were performed in order to measure the peptide binding affinity for inorganic solid supports at the ensemble average level, and atomic force microscopy (AFM) experiments were conducted in order to determine the adhesion force of a single peptide molecule. A positive trend was observed between the total mass uptake of attached peptide and the single-molecule adhesion force on each substrate. Peptide affinity for gold was appreciably greater than for the oxide substrates. Collectively, the results obtained in this study offer insight into the ways in which inorganic materials can differentially influence and modulate the adhesion of SBPs.

Structural complex of sterol 14α-demethylase (CYP51) with 14α-methylenecyclopropyl-Delta7-24, 25-dihydrolanosterol.

PubMed

Hargrove, Tatiana Y; Wawrzak, Zdzislaw; Liu, Jialin; Waterman, Michael R; Nes, W David; Lepesheva, Galina I

2012-02-01

Sterol 14α-demethylase (CYP51) that catalyzes the removal of the 14α-methyl group from the sterol nucleus is an essential enzyme in sterol biosynthesis, a primary target for clinical and agricultural antifungal azoles and an emerging target for antitrypanosomal chemotherapy. Here, we present the crystal structure of Trypanosoma (T) brucei CYP51 in complex with the substrate analog 14α-methylenecyclopropyl-Δ7-24,25-dihydrolanosterol (MCP). This sterol binds tightly to all protozoan CYP51s and acts as a competitive inhibitor of F105-containing (plant-like) T. brucei and Leishmania (L) infantum orthologs, but it has a much stronger, mechanism-based inhibitory effect on I105-containing (animal/fungi-like) T. cruzi CYP51. Depicting substrate orientation in the conserved CYP51 binding cavity, the complex specifies the roles of the contact amino acid residues and sheds new light on CYP51 substrate specificity. It also provides an explanation for the effect of MCP on T. cruzi CYP51. Comparison with the ligand-free and azole-bound structures supports the notion of structural rigidity as the characteristic feature of the CYP51 substrate binding cavity, confirming the enzyme as an excellent candidate for structure-directed design of new drugs, including mechanism-based substrate analog inhibitors.

A Camelid-derived Antibody Fragment Targeting the Active Site of a Serine Protease Balances between Inhibitor and Substrate Behavior*

PubMed Central

Kromann-Hansen, Tobias; Oldenburg, Emil; Yung, Kristen Wing Yu; Ghassabeh, Gholamreza H.; Muyldermans, Serge; Declerck, Paul J.; Huang, Mingdong; Andreasen, Peter A.; Ngo, Jacky Chi Ki

2016-01-01

A peptide segment that binds the active site of a serine protease in a substrate-like manner may behave like an inhibitor or a substrate. However, there is sparse information on which factors determine the behavior a particular peptide segment will exhibit. Here, we describe the first x-ray crystal structure of a nanobody in complex with a serine protease. The nanobody displays a new type of interaction between an antibody and a serine protease as it inserts its complementary determining region-H3 loop into the active site of the protease in a substrate-like manner. The unique binding mechanism causes the nanobody to behave as a strong inhibitor as well as a poor substrate. Intriguingly, its substrate behavior is incomplete, as 30–40% of the nanobody remained intact and inhibitory after prolonged incubation with the protease. Biochemical analysis reveals that an intra-loop interaction network within the complementary determining region-H3 of the nanobody balances its inhibitor versus substrate behavior. Collectively, our results unveil molecular factors, which may be a general mechanism to determine the substrate versus inhibitor behavior of other protease inhibitors. PMID:27226628

Crystal Structure of Thioesterase SgcE10 Supporting Common Polyene Intermediates in 9- and 10-Membered Enediyne Core Biosynthesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Annaval, Thibault; Rudolf, Jeffrey D.; Chang, Chin-Yuan

Enediynes are potent natural product anticancer antibiotics, and are classified as 9- or 10-membered according to the size of their enediyne core carbon skeleton. Both 9- and 10-membered enediyne cores are biosynthesized by the enediyne polyketide synthase (PKSE), thioesterase (TE), and PKSE-associated enzymes. Though the divergence between 9- and 10-membered enediyne core biosynthesis remains unclear, it has been observed that nascent polyketide intermediates, tethered to the acyl carrier protein (ACP) domain of PKSE, could be released by TE in the absence of the PKSE-associated enzymes. Here, we determined the crystal structure of SgcE10, the TE that participates in the biosynthesismore » of the 9-membered enediyne C-1027. Structural comparison of SgcE10 with CalE7 and DynE7, two TEs that participate in the biosynthesis of the 10-membered enediynes calicheamicin and dynemicin, respectively, revealed that they share a common α/β hot-dog fold. The amino acids involved in both substrate binding and catalysis are conserved among SgcE10, CalE7, and DynE7. The volume and the shape of the substrate-binding channel and active site in SgcE10, CalE7, and DynE7 confirm that TEs from both 9- and 10-membered enediyne biosynthetic machineries bind the linear form of similar ACP-tethered polyene intermediates. Taken together, our findings further support the proposal that the divergence between 9- and 10-membered enediyne core biosynthesis occurs beyond PKSE and TE catalysis.« less

Crystal Structure of Thioesterase SgcE10 Supporting Common Polyene Intermediates in 9- and 10-Membered Enediyne Core Biosynthesis

DOE PAGES

Annaval, Thibault; Rudolf, Jeffrey D.; Chang, Chin-Yuan; ...

2017-08-30

Enediynes are potent natural product anticancer antibiotics, and are classified as 9- or 10-membered according to the size of their enediyne core carbon skeleton. Both 9- and 10-membered enediyne cores are biosynthesized by the enediyne polyketide synthase (PKSE), thioesterase (TE), and PKSE-associated enzymes. Though the divergence between 9- and 10-membered enediyne core biosynthesis remains unclear, it has been observed that nascent polyketide intermediates, tethered to the acyl carrier protein (ACP) domain of PKSE, could be released by TE in the absence of the PKSE-associated enzymes. Here, we determined the crystal structure of SgcE10, the TE that participates in the biosynthesismore » of the 9-membered enediyne C-1027. Structural comparison of SgcE10 with CalE7 and DynE7, two TEs that participate in the biosynthesis of the 10-membered enediynes calicheamicin and dynemicin, respectively, revealed that they share a common α/β hot-dog fold. The amino acids involved in both substrate binding and catalysis are conserved among SgcE10, CalE7, and DynE7. The volume and the shape of the substrate-binding channel and active site in SgcE10, CalE7, and DynE7 confirm that TEs from both 9- and 10-membered enediyne biosynthetic machineries bind the linear form of similar ACP-tethered polyene intermediates. Taken together, our findings further support the proposal that the divergence between 9- and 10-membered enediyne core biosynthesis occurs beyond PKSE and TE catalysis.« less

Mechanism of N[superscript 10]-formyltetrahydrofolate synthetase derived from complexes with intermediates and inhibitors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Celeste, Lesa R.; Chai, Geqing; Bielak, Magdalena

N{sup 10}-formyltetrahydrofolate synthetase (FTHFS) is a folate enzyme that catalyzes the formylation of tetrahydrofolate (THF) in an ATP dependent manner. Structures of FTHFS from the thermophilic homoacetogen, Moorella thermoacetica, complexed with (1) a catalytic intermediate-formylphosphate (XPO) and product-ADP; (2) with an inhibitory substrate analog-folate; (3) with XPO and an inhibitory THF analog, ZD9331, were used to analyze the enzyme mechanism. Nucleophilic attack of the formate ion on the gamma phosphate of ATP leads to the formation of XPO and the first product ADP. A channel that leads to the putative formate binding pocket allows for the binding of ATP andmore » formate in random order. Formate binding is due to interactions with the gamma-phosphate moiety of ATP and additionally to two hydrogen bonds from the backbone nitrogen of Ala276 and the side chain of Arg97. Upon ADP dissociation, XPO reorients and moves to the position previously occupied by the beta-phosphate of ATP. Conformational changes that occur due to the XPO presence apparently allow for the recruitment of the third substrate, THF, with its pterin moiety positioned between Phe384 and Trp412. This position overlaps with that of the bound nucleoside, which is consistent with a catalytic mechanism hypothesis that FTHFS works via a sequential ping-pong mechanism. More specifically, a random bi uni uni bi ping-pong ter ter mechanism is proposed. Additionally, the native structure originally reported at a 2.5 {angstrom} resolution was redetermined at a 2.2 {angstrom} resolution.« less

Exploring the structure and function of Thermotoga maritima CorA reveals the mechanism of gating and ion selectivity in Co2+/Mg2+ transport

PubMed Central

Nordin, Nurhuda; Guskov, Albert; Phua, Terri; Sahaf, Newsha; Xia, Yu; Lu, Siyan; Eshaghi, Hojjat; Eshaghi, Said

2013-01-01

The CorA family of divalent cation transporters utilizes Mg2+ and Co2+ as primary substrates. The molecular mechanism of its function, including ion selectivity and gating, has not been fully characterized. Recently we reported a new structure of a CorA homologue from Methanocaldococcus jannaschii, which provided novel structural details that offered the conception of a unique gating mechanism involving conversion of an open hydrophilic gate into a closed hydrophobic one. In the present study we report functional evidence for this novel gating mechanism in the Thermotoga maritima CorA together with an improved crystal structure of this CorA to 2.7 Å (1 Å=0.1 nm) resolution. The latter reveals the organization of the selectivity filter to be similar to that of M. jannaschii CorA and also the previously unknown organization of the second signature motif of the CorA family. The proposed gating is achieved by a helical rotation upon the binding of a metal ion substrate to the regulatory binding sites. Additionally, our data suggest that the preference of this CorA for Co2+ over Mg2+ is controlled by the presence of threonine side chains in the channel. Finally, the roles of the intracellular metal-binding sites have been assigned to increased thermostability and regulation of the gating. These mechanisms most likely apply to the entire CorA family as they are regulated by the highly conserved amino acids. PMID:23425532

Some Surprising Implications of NMR-directed Simulations of Substrate Recognition and Binding by Cytochrome P450cam (CYP101A1).

PubMed

Asciutto, Eliana K; Pochapsky, Thomas C

2018-04-27

Cytochrome P450 cam (CYP101A1) catalyzes the stereospecific 5-exo hydroxylation of d-camphor by molecular oxygen. Previously, residual dipolar couplings measured for backbone amide 1 H- 15 N correlations in both substrate-free and bound forms of CYP101A1 were used as restraints in soft annealing molecular dynamic simulations in order to identify average conformations of the enzyme with and without substrate bound. Multiple substrate-dependent conformational changes remote from the enzyme active site were identified, and site-directed mutagenesis and activity assays confirmed the importance of these changes in substrate recognition. The current work makes use of perturbation response scanning (PRS) and umbrella sampling molecular dynamic of the residual dipolar coupling-derived CYP101A1 structures to probe the roles of remote structural features in enforcing the regio- and stereospecific nature of the hydroxylation reaction catalyzed by CYP101A1. An improper dihedral angle Ψ was defined and used to maintain substrate orientation in the CYP101A1 active site, and it was observed that different values of Ψ result in different PRS response maps. Umbrella sampling methods show that the free energy of the system is sensitive to Ψ, and bound substrate forms an important mechanical link in the transmission of mechanical coupling through the enzyme structure. Finally, a qualitative approach to interpreting PRS maps in terms of the roles of secondary structural features is proposed. Copyright © 2018 Elsevier Ltd. All rights reserved.

Electrostatic field of the large fragment of Escherichia coli DNA polymerase I.

PubMed

Warwicker, J; Ollis, D; Richards, F M; Steitz, T A

1985-12-05

The electrostatic field of the large fragment of Escherichia coli DNA polymerase I (Klenow fragment) has been calculated by the finite difference procedure on a 2 A grid. The potential field is substantially negative at physiological pH (reflecting the net negative charge at this pH). The largest regions of positive potential are in the deep crevice of the C-terminal domain, which is the proposed binding site for the DNA substrate. Within the crevice, the electrostatic potential has a partly helical form. If the DNA is positioned to fulfil stereochemical requirements, then the positive potential generally follows the major groove and (to a lesser extent) the negative potential is in the minor groove. Such an arrangement could stabilize DNA configurations related by screw symmetry. The histidine residues of the Klenow fragment give the positive field of the groove a sensitivity to relatively small pH changes around neutrality. We suggest that the histidine residues could change their ionization states in response to DNA binding, and that this effect could contribute to the protein-DNA binding energy.

Crystal Structures of Xanthomonas campestris OleA Reveal Features That Promote Head-to-Head Condensation of Two Long-Chain Fatty Acids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Goblirsch, BR; Frias, JA; Wackett, LP

2012-05-22

OleA is a thiolase superfamily enzyme that has been shown to catalyze the condensation of two long-chain fatty acylcoenzyme A (CoA) substrates. The enzyme is part of a larger gene cluster responsible for generating long-chain olefin products, a potential biofuel precursor. In thiolase superfamily enzymes, catalysis is achieved via a ping-pong mechanism. The first substrate forms a covalent intermediate with an active site cysteine that is followed by reaction with the second substrate. For OleA, this conjugation proceeds by a nondecarboxylative Claisen condensation. The OleA from Xanthomonas campestris has been crystallized and its structure determined, along with inhibitor-bound and xenon-derivatizedmore » structures, to improve our understanding of substrate positioning in the context of enzyme turnover. OleA is the first characterized thiolase superfamily member that has two long-chain alkyl substrates that need to be bound simultaneously and therefore uniquely requires an additional alkyl binding channel. The location of the fatty acid biosynthesis inhibitor, cerulenin, that possesses an alkyl chain length in the range of known OleA substrates, in conjunction with a single xenon binding site, leads to the putative assignment of this novel alkyl binding channel. Structural overlays between the OleA homologues, 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase and the fatty acid biosynthesis enzyme FabH, allow assignment of the two remaining channels: one for the thioester-containing pantetheinate arm and the second for the alkyl group of one substrate. A short beta-hairpin region is ordered in only one of the crystal forms, and that may suggest open and closed states relevant for substrate binding. Cys143 is the conserved catalytic cysteine within the superfamily, and the site of alkylation by cerulenin. The alkylated structure suggests that a glutamic acid residue (Glu117 beta) likely promotes Claisen condensation by acting as the catalytic base. Unexpectedly, Glu117 beta comes from the other monomer of the physiological dimer.« less

Crystal Structures of Xanthomonas campestris OleA Reveal Features That Promote Head-to-Head Condensation of Two Long-Chain Fatty Acids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Goblirsch, Brandon R.; Frias, Janice A.; Wackett, Lawrence P.

2012-10-25

OleA is a thiolase superfamily enzyme that has been shown to catalyze the condensation of two long-chain fatty acyl-coenzyme A (CoA) substrates. The enzyme is part of a larger gene cluster responsible for generating long-chain olefin products, a potential biofuel precursor. In thiolase superfamily enzymes, catalysis is achieved via a ping-pong mechanism. The first substrate forms a covalent intermediate with an active site cysteine that is followed by reaction with the second substrate. For OleA, this conjugation proceeds by a nondecarboxylative Claisen condensation. The OleA from Xanthomonas campestris has been crystallized and its structure determined, along with inhibitor-bound and xenon-derivatizedmore » structures, to improve our understanding of substrate positioning in the context of enzyme turnover. OleA is the first characterized thiolase superfamily member that has two long-chain alkyl substrates that need to be bound simultaneously and therefore uniquely requires an additional alkyl binding channel. The location of the fatty acid biosynthesis inhibitor, cerulenin, that possesses an alkyl chain length in the range of known OleA substrates, in conjunction with a single xenon binding site, leads to the putative assignment of this novel alkyl binding channel. Structural overlays between the OleA homologues, 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase and the fatty acid biosynthesis enzyme FabH, allow assignment of the two remaining channels: one for the thioester-containing pantetheinate arm and the second for the alkyl group of one substrate. A short {beta}-hairpin region is ordered in only one of the crystal forms, and that may suggest open and closed states relevant for substrate binding. Cys143 is the conserved catalytic cysteine within the superfamily, and the site of alkylation by cerulenin. The alkylated structure suggests that a glutamic acid residue (Glu117{beta}) likely promotes Claisen condensation by acting as the catalytic base. Unexpectedly, Glu117{beta} comes from the other monomer of the physiological dimer.« less

Growth- and Stress-Induced PASTA Kinase Phosphorylation in Enterococcus faecalis.

PubMed

Labbe, Benjamin D; Kristich, Christopher J

2017-11-01

Transmembrane Ser/Thr kinases containing extracellular PASTA domains are ubiquitous among Actinobacteria and Firmicutes Such PASTA kinases regulate critical processes, including antibiotic resistance, cell division, toxin production, and virulence, and are essential for viability in certain organisms. Based on in vitro studies with purified extracellular and intracellular fragments of PASTA kinases, a model for signaling has been proposed, in which the extracellular PASTA domains bind currently undefined ligands (typically thought to be peptidoglycan, or fragments thereof) to drive kinase dimerization, which leads to enhanced kinase autophosphorylation and enhanced phosphorylation of substrates. However, this model has not been rigorously tested in vivo Enterococcus faecalis is a Gram-positive intestinal commensal and major antibiotic-resistant opportunistic pathogen. In E. faecalis , the PASTA kinase IreK drives intrinsic resistance to cell wall-active antimicrobials, suggesting that such antimicrobials may trigger IreK signaling. Here we show that IreK responds to cell wall stress in vivo by enhancing its phosphorylation and that of a downstream substrate. This response requires both the extracellular PASTA domains and specific phosphorylatable residues in the kinase domain. Thus, our results provide in vivo evidence, with an intact full-length PASTA kinase in its native physiological environment, that supports the prevailing model of PASTA kinase signaling. In addition, we show that IreK responds to a signal associated with growth and/or cell division, in the absence of cell wall-active antimicrobials. Surprisingly, the ability of IreK to respond to growth and/or division does not require the extracellular PASTA domains, suggesting that IreK monitors multiple parameters for sensory input in vivo IMPORTANCE Transmembrane Ser/Thr kinases containing extracellular PASTA domains are ubiquitous among Actinobacteria and Firmicutes and regulate critical processes. The prevailing model for signaling by PASTA kinases proposes that the extracellular PASTA domains bind ligands to drive kinase dimerization, enhanced autophosphorylation, and enhanced phosphorylation of substrates. However, this model has not been rigorously tested in vivo We show that the PASTA kinase IreK of Enterococcus faecalis responds to cell wall stress in vivo by enhancing its phosphorylation and that of a downstream substrate. This response requires the PASTA domains and phosphorylatable residues in the kinase domain. Thus, our results provide in vivo evidence, with an intact full-length PASTA kinase in its native physiological environment, that supports the prevailing model of PASTA kinase signaling. Copyright © 2017 American Society for Microbiology.

Growth- and Stress-Induced PASTA Kinase Phosphorylation in Enterococcus faecalis

PubMed Central

Labbe, Benjamin D.

2017-01-01

ABSTRACT Transmembrane Ser/Thr kinases containing extracellular PASTA domains are ubiquitous among Actinobacteria and Firmicutes. Such PASTA kinases regulate critical processes, including antibiotic resistance, cell division, toxin production, and virulence, and are essential for viability in certain organisms. Based on in vitro studies with purified extracellular and intracellular fragments of PASTA kinases, a model for signaling has been proposed, in which the extracellular PASTA domains bind currently undefined ligands (typically thought to be peptidoglycan, or fragments thereof) to drive kinase dimerization, which leads to enhanced kinase autophosphorylation and enhanced phosphorylation of substrates. However, this model has not been rigorously tested in vivo. Enterococcus faecalis is a Gram-positive intestinal commensal and major antibiotic-resistant opportunistic pathogen. In E. faecalis, the PASTA kinase IreK drives intrinsic resistance to cell wall-active antimicrobials, suggesting that such antimicrobials may trigger IreK signaling. Here we show that IreK responds to cell wall stress in vivo by enhancing its phosphorylation and that of a downstream substrate. This response requires both the extracellular PASTA domains and specific phosphorylatable residues in the kinase domain. Thus, our results provide in vivo evidence, with an intact full-length PASTA kinase in its native physiological environment, that supports the prevailing model of PASTA kinase signaling. In addition, we show that IreK responds to a signal associated with growth and/or cell division, in the absence of cell wall-active antimicrobials. Surprisingly, the ability of IreK to respond to growth and/or division does not require the extracellular PASTA domains, suggesting that IreK monitors multiple parameters for sensory input in vivo. IMPORTANCE Transmembrane Ser/Thr kinases containing extracellular PASTA domains are ubiquitous among Actinobacteria and Firmicutes and regulate critical processes. The prevailing model for signaling by PASTA kinases proposes that the extracellular PASTA domains bind ligands to drive kinase dimerization, enhanced autophosphorylation, and enhanced phosphorylation of substrates. However, this model has not been rigorously tested in vivo. We show that the PASTA kinase IreK of Enterococcus faecalis responds to cell wall stress in vivo by enhancing its phosphorylation and that of a downstream substrate. This response requires the PASTA domains and phosphorylatable residues in the kinase domain. Thus, our results provide in vivo evidence, with an intact full-length PASTA kinase in its native physiological environment, that supports the prevailing model of PASTA kinase signaling. PMID:28808126

Dynamics of Preferential Substrate Recognition in HIV-1 Protease: Redefining the Substrate Envelope

PubMed Central

Özen, Ayşegül; Haliloğlu, Türkan; Schiffer, Celia A.

2011-01-01

HIV-1 protease (PR) permits viral maturation by processing the Gag and Gag-Pro-Pol polyproteins. Though HIV-1 PR inhibitors (PIs) are used in combination antiviral therapy, the emergence of drug resistance has limited their efficacy. The rapid evolution of HIV-1 necessitates the consideration of drug resistance in novel drug-design strategies. Drug-resistant HIV-1 PR variants, while no longer efficiently inhibited, continue to efficiently hydrolyze the natural viral substrates. Though highly diverse in sequence, the HIV-1 PR substrates bind in a conserved three-dimensional shape we defined as the “substrate envelope”. We previously showed that resistance mutations arise where PIs protrude beyond the substrate envelope, as these regions are crucial for drug binding but not for substrate recognition. Here, we extend this model by considering the role of protein dynamics in the interaction of HIV-1 PR with its substrates. Seven molecular dynamics simulations of PR-substrate complexes were performed to estimate the conformational flexibility of substrates in their complexes. Interdependency of the substrate-protease interactions may compensate for the variations in cleavage-site sequences, and explain how a diverse set of sequences can be recognized as substrates by the same enzyme. This diversity may be essential for regulating sequential processing of substrates. We also define a dynamic substrate envelope as a more accurate representation of PR-substrate interactions. This dynamic substrate envelope, described by a probability distribution function, is a powerful tool for drug design efforts targeting ensembles of resistant HIV-1 PR variants with the aim of developing drugs that are less susceptible to resistance. PMID:21762811

Nicotinamide Cofactors Suppress Active-Site Labeling of Aldehyde Dehydrogenases.

PubMed

Stiti, Naim; Chandrasekar, Balakumaran; Strubl, Laura; Mohammed, Shabaz; Bartels, Dorothea; van der Hoorn, Renier A L

2016-06-17

Active site labeling by (re)activity-based probes is a powerful chemical proteomic tool to globally map active sites in native proteomes without using substrates. Active site labeling is usually taken as a readout for the active state of the enzyme because labeling reflects the availability and reactivity of active sites, which are hallmarks for enzyme activities. Here, we show that this relationship holds tightly, but we also reveal an important exception to this rule. Labeling of Arabidopsis ALDH3H1 with a chloroacetamide probe occurs at the catalytic Cys, and labeling is suppressed upon nitrosylation and oxidation, and upon treatment with other Cys modifiers. These experiments display a consistent and strong correlation between active site labeling and enzymatic activity. Surprisingly, however, labeling is suppressed by the cofactor NAD(+), and this property is shared with other members of the ALDH superfamily and also detected for unrelated GAPDH enzymes with an unrelated hydantoin-based probe in crude extracts of plant cell cultures. Suppression requires cofactor binding to its binding pocket. Labeling is also suppressed by ALDH modulators that bind at the substrate entrance tunnel, confirming that labeling occurs through the substrate-binding cavity. Our data indicate that cofactor binding adjusts the catalytic Cys into a conformation that reduces the reactivity toward chloroacetamide probes.

Chlorella virus DNA ligase: nick recognition and mutational analysis.

PubMed

Sriskanda, V; Shuman, S

1998-01-15

Chlorella virus PBCV-1 DNA ligase seals nicked DNA substrates consisting of a 5'-phosphate-terminated strand and a 3'-hydroxyl-terminated strand annealed to a bridging DNA template strand. The enzyme discriminates at the DNA binding step between substrates containing a 5'-phosphate versus a 5'-hydroxyl at the nick. Mutational analysis of the active site motif KxDGxR (residues 27-32) illuminates essential roles for the conserved Lys, Asp and Arg moieties at different steps of the ligase reaction. Mutant K27A is unable to form the covalent ligase-(Lys-straightepsilonN-P)-adenylate intermediate and hence cannot activate a nicked DNA substrate via formation of the DNA-adenylate intermediate. Nonetheless, K27A catalyzes phosphodiester bond formation at a pre-adenylated nick. This shows that the active site lysine is not required for the strand closure reaction. K27A binds to nicked DNA-adenylate, but not to a standard DNA nick. This suggests that occupancy of the AMP binding pocket of DNA ligase is important for nick recognition. Mutant D29A is active in enzyme-adenylate formation and binds readily to nicked DNA, but is inert in DNA-adenylate formation. R32A is unable to catalyze any of the three reactions of the ligation pathway and does not bind to nicked DNA.

Monobromobimane occupies a distinct xenobiotic substrate site in glutathione S-transferase π

PubMed Central

Ralat, Luis A.; Colman, Roberta F.

2003-01-01

Monobromobimane (mBBr), functions as a substrate of porcine glutathione S-transferase π (GST π): The enzyme catalyzes the reaction of mBBr with glutathione. S-(Hydroxyethyl)bimane, a nonreactive analog of monobromobimane, acts as a competitive inhibitor with respect to mBBr as substrate but does not affect the reaction of GST π with another substrate, 1-chloro-2,4-dinitrobenzene (CDNB). In the absence of glutathione, monobromobimane inactivates GST π at pH 7.0 and 25°C as assayed using mBBr as substrate, with a lesser effect on the enzyme’s use of CDNB as substrate. These results indicate that the sites occupied by CDNB and mBBr are not identical. Inactivation is proportional to the incorporation of 2 moles of bimane/mole of subunit. Modification of GST π with mBBr does not interfere with its binding of 8-anilino-1-naphthalene sulfonate, indicating that this hydrophobic site is not the target of monobromobimane. S-Methylglutathione and S-(hydroxyethyl)bimane each yield partial protection against inactivation and decrease reagent incorporation, while glutathionyl-bimane protects completely against inactivation. Peptide analysis after trypsin digestion indicates that mBBr modifies Cys45 and Cys99 equally. Modification of Cys45 is reduced in the presence of S-methylglutathione, indicating that this residue is at or near the glutathione binding region. In contrast, modification of Cys99 is reduced in the presence of S-(hydroxyethyl)bimane, suggesting that this residue is at or near the mBBr xenobiotic substrate binding site. Modification of Cys99 can best be understood by reaction with monobromobimane while it is bound to its xenobiotic substrate site in an alternate orientation. These results support the concept that glutathione S-transferase accomplishes its ability to react with a diversity of substrates in part by harboring distinct xenobiotic substrate sites. PMID:14573868

Monobromobimane occupies a distinct xenobiotic substrate site in glutathione S-transferase pi.

PubMed

Ralat, Luis A; Colman, Roberta F

2003-11-01

Monobromobimane (mBBr), functions as a substrate of porcine glutathione S-transferase pi (GST pi): The enzyme catalyzes the reaction of mBBr with glutathione. S-(Hydroxyethyl)bimane, a nonreactive analog of monobromobimane, acts as a competitive inhibitor with respect to mBBr as substrate but does not affect the reaction of GST pi with another substrate, 1-chloro-2,4-dinitrobenzene (CDNB). In the absence of glutathione, monobromobimane inactivates GST pi at pH 7.0 and 25 degrees C as assayed using mBBr as substrate, with a lesser effect on the enzyme's use of CDNB as substrate. These results indicate that the sites occupied by CDNB and mBBr are not identical. Inactivation is proportional to the incorporation of 2 moles of bimane/mole of subunit. Modification of GST pi with mBBr does not interfere with its binding of 8-anilino-1-naphthalene sulfonate, indicating that this hydrophobic site is not the target of monobromobimane. S-Methylglutathione and S-(hydroxyethyl)bimane each yield partial protection against inactivation and decrease reagent incorporation, while glutathionyl-bimane protects completely against inactivation. Peptide analysis after trypsin digestion indicates that mBBr modifies Cys45 and Cys99 equally. Modification of Cys45 is reduced in the presence of S-methylglutathione, indicating that this residue is at or near the glutathione binding region. In contrast, modification of Cys99 is reduced in the presence of S-(hydroxyethyl)bimane, suggesting that this residue is at or near the mBBr xenobiotic substrate binding site. Modification of Cys99 can best be understood by reaction with monobromobimane while it is bound to its xenobiotic substrate site in an alternate orientation. These results support the concept that glutathione S-transferase accomplishes its ability to react with a diversity of substrates in part by harboring distinct xenobiotic substrate sites.

Modified Acyl-ACP desaturase

DOEpatents

Cahoon, Edgar B.; Shanklin, John; Lindqvist, Ylva; Schneider, Gunter

1999-03-30

Disclosed is a method for modifying the chain length and double bond positional specificities of a soluble plant fatty acid desaturase. More specifically, the method involves modifying amino acid contact residues in the substrate binding channel of the soluble fatty acid desaturase which contact the fatty acid. Specifically disclosed is the modification of an acyl-ACP desaturase. Amino acid contact residues which lie within the substrate binding channel are identified, and subsequently replaced with different residues to effect the modification of activity.

Modified acyl-ACP desaturase

DOEpatents

Cahoon, Edgar B.; Shanklin, John; Lindgvist, Ylva; Schneider, Gunter

1998-01-06

Disclosed is a methods for modifying the chain length and double bond positional specificities of a soluble plant fatty acid desaturase. More specifically, the method involves modifying amino acid contact residues in the substrate binding channel of the soluble fatty acid desaturase which contact the fatty acid. Specifically disclosed is the modification of an acyl-ACP desaturase. Amino acid contact residues which lie within the substrate binding channel are identified, and subsequently replaced with different residues to effect the modification of activity.

Inhibition of ligand exchange kinetics via active-site trapping with an antibody fragment.

PubMed

Oyen, David; Steyaert, Jan; Barlow, John N

2014-04-01

We describe the first example of an inhibitory antibody fragment (nanobody ca1697) that binds simultaneously to an enzyme (the enzyme dihydrofolate reductase from Escherichia coli) and its bound substrate (folate). Binding of the antibody to the substrate causes a 20-fold reduction in the rate of folate exchange kinetics. This work opens up the prospect of designing new types of antibody-based inhibitors of enzymes and receptors through suitable design of immunogens.

DNA Damage: Quantum Mechanics/Molecular Mechanics Study on the Oxygen Binding and Substrate Hydroxylation Step in AlkB Repair Enzymes

PubMed Central

Quesne, Matthew G; Latifi, Reza; Gonzalez-Ovalle, Luis E; Kumar, Devesh; de Visser, Sam P

2014-01-01

AlkB repair enzymes are important nonheme iron enzymes that catalyse the demethylation of alkylated DNA bases in humans, which is a vital reaction in the body that heals externally damaged DNA bases. Its mechanism is currently controversial and in order to resolve the catalytic mechanism of these enzymes, a quantum mechanics/molecular mechanics (QM/MM) study was performed on the demethylation of the N1-methyladenine fragment by AlkB repair enzymes. Firstly, the initial modelling identified the oxygen binding site of the enzyme. Secondly, the oxygen activation mechanism was investigated and a novel pathway was found, whereby the catalytically active iron(IV)–oxo intermediate in the catalytic cycle undergoes an initial isomerisation assisted by an Arg residue in the substrate binding pocket, which then brings the oxo group in close contact with the methyl group of the alkylated DNA base. This enables a subsequent rate-determining hydrogen-atom abstraction on competitive σ-and π-pathways on a quintet spin-state surface. These findings give evidence of different locations of the oxygen and substrate binding channels in the enzyme and the origin of the separation of the oxygen-bound intermediates in the catalytic cycle from substrate. Our studies are compared with small model complexes and the effect of protein and environment on the kinetics and mechanism is explained. PMID:24339041

Nicked-site substrates for a serine recombinase reveal enzyme-DNA communications and an essential tethering role of covalent enzyme-DNA linkages.

PubMed

Olorunniji, Femi J; McPherson, Arlene L; Pavlou, Hania J; McIlwraith, Michael J; Brazier, John A; Cosstick, Richard; Stark, W Marshall

2015-07-13

To analyse the mechanism and kinetics of DNA strand cleavages catalysed by the serine recombinase Tn3 resolvase, we made modified recombination sites with a single-strand nick in one of the two DNA strands. Resolvase acting on these sites cleaves the intact strand very rapidly, giving an abnormal half-site product which accumulates. We propose that these reactions mimic second-strand cleavage of an unmodified site. Cleavage occurs in a synapse of two sites, held together by a resolvase tetramer; cleavage at one site stimulates cleavage at the partner site. After cleavage of a nicked-site substrate, the half-site that is not covalently linked to a resolvase subunit dissociates rapidly from the synapse, destabilizing the entire complex. The covalent resolvase-DNA linkages in the natural reaction intermediate thus perform an essential DNA-tethering function. Chemical modifications of a nicked-site substrate at the positions of the scissile phosphodiesters result in abolition or inhibition of resolvase-mediated cleavage and effects on resolvase binding and synapsis, providing insight into the serine recombinase catalytic mechanism and how resolvase interacts with the substrate DNA. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Mechanisms of Enhanced Catalysis in Enzyme-DNA Nanostructures Revealed through Molecular Simulations and Experimental Analysis.

PubMed

Gao, Yingning; Roberts, Christopher C; Toop, Aaron; Chang, Chia-En A; Wheeldon, Ian

2016-08-03

Understanding and controlling the molecular interactions between enzyme substrates and DNA nanostructures has important implications in the advancement of enzyme-DNA technologies as solutions in biocatalysis. Such hybrid nanostructures can be used to create enzyme systems with enhanced catalysis by controlling the local chemical and physical environments and the spatial organization of enzymes. Here we have used molecular simulations with corresponding experiments to describe a mechanism of enhanced catalysis due to locally increased substrate concentrations. With a series of DNA nanostructures conjugated to horseradish peroxidase, we show that binding interactions between substrates and the DNA structures can increase local substrate concentrations. Increased local substrate concentrations in HRP(DNA) nanostructures resulted in 2.9- and 2.4-fold decreases in the apparent Michaelis constants of tetramethylbenzidine and 4-aminophenol, substrates of HRP with tunable binding interactions to DNA nanostructures with dissociation constants in the micromolar range. Molecular simulations and kinetic analysis also revealed that increased local substrate concentrations enhanced the rates of substrate association. Identification of the mechanism of increased local concentration of substrates in close proximity to enzymes and their active sites adds to our understanding of nanostructured biocatalysis from which we can develop guidelines for enhancing catalysis in rationally designed systems. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Rate and Equilibrium Constants for an Enzyme Conformational Change during Catalysis by Orotidine 5'-Monophosphate Decarboxylase.

PubMed

Goryanova, Bogdana; Goldman, Lawrence M; Ming, Shonoi; Amyes, Tina L; Gerlt, John A; Richard, John P

2015-07-28

The caged complex between orotidine 5'-monophosphate decarboxylase (ScOMPDC) and 5-fluoroorotidine 5'-monophosphate (FOMP) undergoes decarboxylation ∼300 times faster than the caged complex between ScOMPDC and the physiological substrate, orotidine 5'-monophosphate (OMP). Consequently, the enzyme conformational changes required to lock FOMP at a protein cage and release product 5-fluorouridine 5'-monophosphate (FUMP) are kinetically significant steps. The caged form of ScOMPDC is stabilized by interactions between the side chains from Gln215, Tyr217, and Arg235 and the substrate phosphodianion. The control of these interactions over the barrier to the binding of FOMP and the release of FUMP was probed by determining the effect of all combinations of single, double, and triple Q215A, Y217F, and R235A mutations on kcat/Km and kcat for turnover of FOMP by wild-type ScOMPDC; its values are limited by the rates of substrate binding and product release, respectively. The Q215A and Y217F mutations each result in an increase in kcat and a decrease in kcat/Km, due to a weakening of the protein-phosphodianion interactions that favor fast product release and slow substrate binding. The Q215A/R235A mutation causes a large decrease in the kinetic parameters for ScOMPDC-catalyzed decarboxylation of OMP, which are limited by the rate of the decarboxylation step, but much smaller decreases in the kinetic parameters for ScOMPDC-catalyzed decarboxylation of FOMP, which are limited by the rate of enzyme conformational changes. By contrast, the Y217A mutation results in large decreases in kcat/Km for ScOMPDC-catalyzed decarboxylation of both OMP and FOMP, because of the comparable effects of this mutation on rate-determining decarboxylation of enzyme-bound OMP and on the rate-determining enzyme conformational change for decarboxylation of FOMP. We propose that kcat = 8.2 s(-1) for decarboxylation of FOMP by the Y217A mutant is equal to the rate constant for cage formation from the complex between FOMP and the open enzyme, that the tyrosyl phenol group stabilizes the closed form of ScOMPDC by hydrogen bonding to the substrate phosphodianion, and that the phenyl group of Y217 and F217 facilitates formation of the transition state for the rate-limiting conformational change. An analysis of kinetic data for mutant enzyme-catalyzed decarboxylation of OMP and FOMP provides estimates for the rate and equilibrium constants for the conformational change that traps FOMP at the enzyme active site.

Chemistry and Molecular Dynamics Simulations of Heme b-HemQ and Coproheme-HemQ

PubMed Central

2016-01-01

Recently, a novel pathway for heme b biosynthesis in Gram-positive bacteria has been proposed. The final poorly understood step is catalyzed by an enzyme called HemQ and includes two decarboxylation reactions leading from coproheme to heme b. Coproheme has been suggested to act as both substrate and redox active cofactor in this reaction. In the study presented here, we focus on HemQs from Listeria monocytogenes (LmHemQ) and Staphylococcus aureus (SaHemQ) recombinantly produced as apoproteins in Escherichia coli. We demonstrate the rapid and two-phase uptake of coproheme by both apo forms and the significant differences in thermal stability of the apo forms, coproheme-HemQ and heme b-HemQ. Reduction of ferric high-spin coproheme-HemQ to the ferrous form is shown to be enthalpically favored but entropically disfavored with standard reduction potentials of −205 ± 3 mV for LmHemQ and −207 ± 3 mV for SaHemQ versus the standard hydrogen electrode at pH 7.0. Redox thermodynamics suggests the presence of a pronounced H-bonding network and restricted solvent mobility in the heme cavity. Binding of cyanide to the sixth coproheme position is monophasic but relatively slow (∼1 × 104 M–1 s–1). On the basis of the available structures of apo-HemQ and modeling of both loaded forms, molecular dynamics simulation allowed analysis of the interaction of coproheme and heme b with the protein as well as the role of the flexibility at the proximal heme cavity and the substrate access channel for coproheme binding and heme b release. Obtained data are discussed with respect to the proposed function of HemQ in monoderm bacteria. PMID:27599156

Structural analysis of group II chitinase (ChtII) catalysis completes the puzzle of chitin hydrolysis in insects.

PubMed

Chen, Wei; Qu, Mingbo; Zhou, Yong; Yang, Qing

2018-02-23

Chitin is a linear homopolymer of N -acetyl-β-d-glucosamines and a major structural component of insect cuticles. Chitin hydrolysis involves glycoside hydrolase family 18 (GH18) chitinases. In insects, chitin hydrolysis is essential for periodic shedding of the old cuticle ecdysis and proceeds via a pathway different from that in the well studied bacterial chitinolytic system. Group II chitinase (ChtII) is a widespread chitinolytic enzyme in insects and contains the greatest number of catalytic domains and chitin-binding domains among chitinases. In Lepidopterans, ChtII and two other chitinases, ChtI and Chi-h, are essential for chitin hydrolysis. Although ChtI and Chi-h have been well studied, the role of ChtII remains elusive. Here, we investigated the structure and enzymology of Of ChtII, a ChtII derived from the insect pest Ostrinia furnacalis We present the crystal structures of two catalytically active domains of Of ChtII, Of ChtII-C1 and Of ChtII-C2, both in unliganded form and complexed with chitooligosaccharide substrates. We found that Of ChtII-C1 and Of ChtII-C2 both possess long, deep substrate-binding clefts with endochitinase activities. Of ChtII exhibited structural characteristics within the substrate-binding cleft similar to those in Of Chi-h and Of ChtI. However, Of ChtII lacked structural elements favoring substrate binding beyond the active sites, including an extra wall structure present in Of Chi-h. Nevertheless, the numerous domains in Of ChtII may compensate for this difference; a truncation containing one catalytic domain and three chitin-binding modules ( Of ChtII-B4C1) displayed activity toward insoluble polymeric substrates that was higher than those of Of Chi-h and Of ChtI. Our observations provide the last piece of the puzzle of chitin hydrolysis in insects. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Computational Investigation of the Interplay of Substrate Positioning and Reactivity in Catechol O-Methyltransferase

PubMed Central

Patra, Niladri; Ioannidis, Efthymios I.

2016-01-01

Catechol O-methyltransferase (COMT) is a SAM- and Mg2+-dependent methyltransferase that regulates neurotransmitters through methylation. Simulations and experiments have identified divergent catecholamine substrate orientations in the COMT active site: molecular dynamics simulations have favored a monodentate coordination of catecholate substrates to the active site Mg2+, and crystal structures instead preserve bidentate coordination along with short (2.65 Å) methyl donor-acceptor distances. We carry out longer dynamics (up to 350 ns) to quantify interconversion between bidentate and monodentate binding poses. We provide a systematic determination of the relative free energy of the monodentate and bidentate structures in order to identify whether structural differences alter the nature of the methyl transfer mechanism and source of enzymatic rate enhancement. We demonstrate that the bidentate and monodentate binding modes are close in energy but separated by a 7 kcal/mol free energy barrier. Analysis of interactions in the two binding modes reveals that the driving force for monodentate catecholate orientations in classical molecular dynamics simulations is derived from stronger electrostatic stabilization afforded by alternate Mg2+ coordination with strongly charged active site carboxylates. Mixed semi-empirical-classical (SQM/MM) substrate C-O distances (2.7 Å) for the bidentate case are in excellent agreement with COMT X-ray crystal structures, as long as charge transfer between the substrates, Mg2+, and surrounding ligands is permitted. SQM/MM free energy barriers for methyl transfer from bidentate and monodentate catecholate configurations are comparable at around 21–22 kcal/mol, in good agreement with experiment (18–19 kcal/mol). Overall, the work suggests that both binding poses are viable for methyl transfer, and accurate descriptions of charge transfer and electrostatics are needed to provide balanced relative barriers when multiple binding poses are accessible, for example in other transferases. PMID:27564542

How Does (E)-2-(Acetamidomethylene)succinate Bind to Its Hydrolase? From the Binding Process to the Final Result

PubMed Central

Zhang, Ji-Long; Zheng, Qing-Chuan; Li, Zheng-Qiang; Zhang, Hong-Xing

2013-01-01

The binding of (E)-2-(acetamidomethylene)succinate (E-2AMS) to E-2AMS hydrolase is crucial for biological function of the enzyme and the last step reaction of vitamin B6 biological degradation. In the present study, several molecular simulation methods, including molecular docking, conventional molecular dynamics (MD), steered MD (SMD), and free energy calculation methods, were properly integrated to investigate the detailed binding process of E-2AMS to its hydrolase and to assign the optimal enzyme-substrate complex conformation. It was demonstrated that the substrate binding conformation with trans-form amide bond is energetically preferred conformation, in which E-2AMS's pose not only ensures hydrogen bond formation of its amide oxygen atom with the vicinal oxyanion hole but also provides probability of the hydrophobic interaction between its methyl moiety and the related enzyme's hydrophobic cavity. Several key residues, Arg146, Arg167, Tyr168, Arg179, and Tyr259, orientate the E-2AMS's pose and stabilize its conformation in the active site via the hydrogen bond interaction with E-2AMS. Sequentially, the binding process of E-2AMS to E-2AMS hydrolase was studied by SMD simulation, which shows the surprising conformational reversal of E-2AMS. Several important intermediate structures and some significant residues were identified in the simulation. It is stressed that Arg146 and Arg167 are two pivotal residues responsible for the conformational reversal of E-2AMS in the binding or unbinding. Our research has shed light onto the full binding process of the substrate to E-2AMS hydrolase, which could provide more penetrating insight into the interaction of E-2AMS with the enzyme and would help in the further exploration on the catalysis mechanism. PMID:23308285

Preferential Binding of Hot Spot Mutant p53 Proteins to Supercoiled DNA In Vitro and in Cells

PubMed Central

Brázdová, Marie; Navrátilová, Lucie; Tichý, Vlastimil; Němcová, Kateřina; Lexa, Matej; Hrstka, Roman; Pečinka, Petr; Adámik, Matej; Vojtesek, Borivoj; Paleček, Emil; Deppert, Wolfgang; Fojta, Miroslav

2013-01-01

Hot spot mutant p53 (mutp53) proteins exert oncogenic gain-of-function activities. Binding of mutp53 to DNA is assumed to be involved in mutp53-mediated repression or activation of several mutp53 target genes. To investigate the importance of DNA topology on mutp53-DNA recognition in vitro and in cells, we analyzed the interaction of seven hot spot mutp53 proteins with topologically different DNA substrates (supercoiled, linear and relaxed) containing and/or lacking mutp53 binding sites (mutp53BS) using a variety of electrophoresis and immunoprecipitation based techniques. All seven hot spot mutp53 proteins (R175H, G245S, R248W, R249S, R273C, R273H and R282W) were found to have retained the ability of wild-type p53 to preferentially bind circular DNA at native negative superhelix density, while linear or relaxed circular DNA was a poor substrate. The preference of mutp53 proteins for supercoiled DNA (supercoil-selective binding) was further substantiated by competition experiments with linear DNA or relaxed DNA in vitro and ex vivo. Using chromatin immunoprecipitation, the preferential binding of mutp53 to a sc mutp53BS was detected also in cells. Furthermore, we have shown by luciferase reporter assay that the DNA topology influences p53 regulation of BAX and MSP/MST1 promoters. Possible modes of mutp53 binding to topologically constrained DNA substrates and their biological consequences are discussed. PMID:23555710

Binding of Substrate Locks the Electrochemistry of CRY-DASH into DNA Repair.

PubMed

Gindt, Yvonne M; Messyasz, Adriana; Jumbo, Pamela I

2015-05-12

VcCry1, a member of the CRY-DASH family, may serve two diverse roles in vivo, including blue-light signaling and repair of UV-damaged DNA. We have discovered that the electrochemistry of the flavin adenine dinucleotide cofactor of VcCry1 is locked to cycle only between the hydroquinone and neutral semiquinone states when UV-damaged DNA is present. Other potential substrates, including undamaged DNA and ATP, have no discernible effect on the electrochemistry, and the kinetics of the reduction is unaffected by damaged DNA. Binding of the damaged DNA substrate determines the role of the protein and prevents the presumed photochemistry required for blue-light signaling.

Gold nanoclusters-Cu(2+) ensemble-based fluorescence turn-on and real-time assay for acetylcholinesterase activity and inhibitor screening.

PubMed

Sun, Jian; Yang, Xiurong

2015-12-15

Based on the specific binding of Cu(2+) ions to the 11-mercaptoundecanoic acid (11-MUA)-protected AuNCs with intense orange-red emission, we have proposed and constructed a novel fluorescent nanomaterials-metal ions ensemble at a nonfluorescence off-state. Subsequently, an AuNCs@11-MUA-Cu(2+) ensemble-based fluorescent chemosensor, which is amenable to convenient, sensitive, selective, turn-on and real-time assay of acetylcholinesterase (AChE), could be developed by using acetylthiocholine (ATCh) as the substrate. Herein, the sensing ensemble solution exhibits a marvelous fluorescent enhancement in the presence of AChE and ATCh, where AChE hydrolyzes its active substrate ATCh into thiocholine (TCh), and then TCh captures Cu(2+) from the ensemble, accompanied by the conversion from fluorescence off-state to on-state of the AuNCs. The AChE activity could be detected less than 0.05 mU/mL within a good linear range from 0.05 to 2.5 mU/mL. Our proposed fluorescence assay can be utilized to evaluate the AChE activity quantitatively in real biological sample, and furthermore to screen the inhibitor of AChE. As far as we know, the present study has reported the first analytical proposal for sensing AChE activity in real time by using a fluorescent nanomaterials-Cu(2+) ensemble or focusing on the Cu(2+)-triggered fluorescence quenching/recovery. This strategy paves a new avenue for exploring the biosensing applications of fluorescent AuNCs, and presents the prospect of AuNCs@11-MUA-Cu(2+) ensemble as versatile enzyme activity assay platforms by means of other appropriate substrates/analytes. Copyright © 2015 Elsevier B.V. All rights reserved.

High-Resolution Crystal Structures of Streptococcus pneumoniae Nicotinamidase with Trapped Intermediates Provide Insights into the Catalytic Mechanism and Inhibition by Aldehydes

DOE Office of Scientific and Technical Information (OSTI.GOV)

French, Jarrod B.; Cen, Yana; Sauve, Anthony A.

2010-11-11

Nicotinamidases are salvage enzymes that convert nicotinamide to nicotinic acid. These enzymes are essential for the recycling of nicotinamide into NAD{sup +} in most prokaryotes and most single-cell and multicellular eukaryotes, but not in mammals. The significance of these enzymes for nicotinamide salvage and for NAD{sup +} homeostasis has stimulated interest in nicotinamidases as possible antibiotic targets. Nicotinamidases are also regulators of intracellular nicotinamide concentrations, thereby regulating signaling of downstream NAD{sup +}-consuming enzymes, such as the NAD{sup +}-dependent deacetylases (sirtuins). Here, we report several high-resolution crystal structures of the nicotinamidase from Streptococcus pneumoniae (SpNic) in unliganded and ligand-bound forms. Themore » structure of the C136S mutant in complex with nicotinamide provides details about substrate binding, while a trapped nicotinoyl thioester in a complex with SpNic reveals the structure of the proposed thioester reaction intermediate. Examination of the active site of SpNic reveals several important features, including a metal ion that coordinates the substrate and the catalytically relevant water molecule and an oxyanion hole that both orients the substrate and offsets the negative charge that builds up during catalysis. Structures of this enzyme with bound nicotinaldehyde inhibitors elucidate the mechanism of inhibition and provide further details about the catalytic mechanism. In addition, we provide a biochemical analysis of the identity and role of the metal ion that orients the ligand in the active site and activates the water molecule responsible for hydrolysis of the substrate. These data provide structural evidence of several proposed reaction intermediates and allow for a more complete understanding of the catalytic mechanism of this enzyme.« less

Crystal Structures of Human SIRT[subscript 3] Displaying Substrate-induced Conformational Changes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jin, Lei; Wei, Wentao; Jiang, Yaobin

2009-11-04

SIRT3 is a major mitochondrial NAD{sup +}-dependent protein deacetylase playing important roles in regulating mitochondrial metabolism and energy production and has been linked to the beneficial effects of exercise and caloric restriction. SIRT3 is emerging as a potential therapeutic target to treat metabolic and neurological diseases. We report the first sets of crystal structures of human SIRT3, an apo-structure with no substrate, a structure with a peptide containing acetyl lysine of its natural substrate acetyl-CoA synthetase 2, a reaction intermediate structure trapped by a thioacetyl peptide, and a structure with the dethioacetylated peptide bound. These structures provide insights into themore » conformational changes induced by the two substrates required for the reaction, the acetylated substrate peptide and NAD+. In addition, the binding study by isothermal titration calorimetry suggests that the acetylated peptide is the first substrate to bind to SIRT3, before NAD{sup +}. These structures and biophysical studies provide key insight into the structural and functional relationship of the SIRT3 deacetylation activity.« less