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Sample records for atp catalytic domain

  1. Dynamic coupling between the LID and NMP domain motions in the catalytic conversion of ATP and AMP to ADP by adenylate kinase.

    PubMed

    Jana, Biman; Adkar, Bharat V; Biswas, Rajib; Bagchi, Biman

    2011-01-21

    The catalytic conversion of adenosine triphosphate (ATP) and adenosine monophosphate (AMP) to adenosine diphosphate (ADP) by adenylate kinase (ADK) involves large amplitude, ligand induced domain motions, involving the opening and the closing of ATP binding domain (LID) and AMP binding domain (NMP) domains, during the repeated catalytic cycle. We discover and analyze an interesting dynamical coupling between the motion of the two domains during the opening, using large scale atomistic molecular dynamics trajectory analysis, covariance analysis, and multidimensional free energy calculations with explicit water. Initially, the LID domain must open by a certain amount before the NMP domain can begin to open. Dynamical correlation map shows interesting cross-peak between LID and NMP domain which suggests the presence of correlated motion between them. This is also reflected in our calculated two-dimensional free energy surface contour diagram which has an interesting elliptic shape, revealing a strong correlation between the opening of the LID domain and that of the NMP domain. Our free energy surface of the LID domain motion is rugged due to interaction with water and the signature of ruggedness is evident in the observed root mean square deviation variation and its fluctuation time correlation functions. We develop a correlated dynamical disorder-type theoretical model to explain the observed dynamic coupling between the motion of the two domains in ADK. Our model correctly reproduces several features of the cross-correlation observed in simulations.

  2. Dynamics of the metal binding domains and regulation of the human copper transporters ATP7B and ATP7A.

    PubMed

    Yu, Corey H; Dolgova, Natalia V; Dmitriev, Oleg Y

    2017-04-01

    Copper transporters ATP7A and ATP7B regulate copper levels in the human cells and deliver copper to the biosynthetic pathways. ATP7A and ATP7B belong to the P-type ATPases and share much of the domain architecture and the mechanism of ATP hydrolysis with the other, well-studied, enzymes of this type. A unique structural feature of the copper ATPases is the chain of six cytosolic metal-binding domains (MBDs), which are believed to be involved in copper-dependent regulation of the activity and intracellular localization of these enzymes. Although the structures of all the MBDs have been solved, the mechanism of copper-dependent regulation of ATP7B and ATP7A, the roles of individual MBDs, and the relationship between the regulatory and catalytic copper binding are still unknown. We describe the structure and dynamics of the MBDs, review the current knowledge about their functional roles and propose a mechanism of regulation of ATP7B by copper-dependent changes in the dynamics and conformation of the MBD chain. Transient interactions between the MBDs, rather than transitions between distinct static conformations are likely to form the structural basis of regulation of the ATP-dependent copper transporters in human cells. © 2016 IUBMB Life, 69(4):226-235, 2017. © 2017 International Union of Biochemistry and Molecular Biology.

  3. Catalytic Mechanism of the Maltose Transporter Hydrolyzing ATP.

    PubMed

    Huang, Wenting; Liao, Jie-Lou

    2016-01-12

    We use quantum mechanical and molecular mechanical (QM/MM) simulations to study ATP hydrolysis catalyzed by the maltose transporter. This protein is a prototypical member of a large family that consists of ATP-binding cassette (ABC) transporters. The ABC proteins catalyze ATP hydrolysis to perform a variety of biological functions. Despite extensive research efforts, the precise molecular mechanism of ATP hydrolysis catalyzed by the ABC enzymes remains elusive. In this work, the reaction pathway for ATP hydrolysis in the maltose transporter is evaluated using a QM/MM implementation of the nudged elastic band method without presuming reaction coordinates. The potential of mean force along the reaction pathway is obtained with an activation free energy of 19.2 kcal/mol in agreement with experiments. The results demonstrate that the reaction proceeds via a dissociative-like pathway with a trigonal bipyramidal transition state in which the cleavage of the γ-phosphate P-O bond occurs and the O-H bond of the lytic water molecule is not yet broken. Our calculations clearly show that the Walker B glutamate as well as the switch histidine stabilizes the transition state via electrostatic interactions rather than serving as a catalytic base. The results are consistent with biochemical and structural experiments, providing novel insight into the molecular mechanism of ATP hydrolysis in the ABC proteins.

  4. Catalytic strategy used by the myosin motor to hydrolyze ATP.

    PubMed

    Kiani, Farooq Ahmad; Fischer, Stefan

    2014-07-22

    Myosin is a molecular motor responsible for biological motions such as muscle contraction and intracellular cargo transport, for which it hydrolyzes adenosine 5'-triphosphate (ATP). Early steps of the mechanism by which myosin catalyzes ATP hydrolysis have been investigated, but still missing are the structure of the final ADP·inorganic phosphate (Pi) product and the complete pathway leading to it. Here, a comprehensive description of the catalytic strategy of myosin is formulated, based on combined quantum-classical molecular mechanics calculations. A full exploration of catalytic pathways was performed and a final product structure was found that is consistent with all experiments. Molecular movies of the relevant pathways show the different reorganizations of the H-bond network that lead to the final product, whose γ-phosphate is not in the previously reported HPγO4(2-) state, but in the H2PγO4(-) state. The simulations reveal that the catalytic strategy of myosin employs a three-pronged tactic: (i) Stabilization of the γ-phosphate of ATP in a dissociated metaphosphate (PγO3(-)) state. (ii) Polarization of the attacking water molecule, to abstract a proton from that water. (iii) Formation of multiple proton wires in the active site, for efficient transfer of the abstracted proton to various product precursors. The specific role played in this strategy by each of the three loops enclosing ATP is identified unambiguously. It explains how the precise timing of the ATPase activation during the force generating cycle is achieved in myosin. The catalytic strategy described here for myosin is likely to be very similar in most nucleotide hydrolyzing enzymes.

  5. Comparing the catalytic strategy of ATP hydrolysis in biomolecular motors.

    PubMed

    Kiani, Farooq Ahmad; Fischer, Stefan

    2016-07-27

    ATP-driven biomolecular motors utilize the chemical energy obtained from the ATP hydrolysis to perform vital tasks in living cells. Understanding the mechanism of enzyme-catalyzed ATP hydrolysis reaction has substantially progressed lately thanks to combined quantum/classical molecular mechanics (QM/MM) simulations. Here, we present a comparative summary of the most recent QM/MM results for myosin, kinesin and F1-ATPase motors. These completely different motors achieve the acceleration of ATP hydrolysis through a very similar catalytic mechanism. ATP hydrolysis has high activation energy because it involves the breaking of two strong bonds, namely the Pγ-Oβγ bond of ATP and the H-O bond of lytic water. The key to the four-fold decrease in the activation barrier by the three enzymes is that the breaking of the Pγ-Oβγ bond precedes the deprotonation of the lytic water molecule, generating a metaphosphate hydrate complex. The resulting singly charged trigonal planar PγO3(-) metaphosphate is a better electrophilic target for attack by an OaH(-) hydroxyl group. The formation of this OaH(-) is promoted by a strong polarization of the lytic water: in all three proteins, this water is forming a hydrogen-bond with a backbone carbonyl group and interacts with the carboxylate group of glutamate (either directly or via an intercalated water molecule). This favors the shedding of one proton by the attacking water. The abstracted proton is transferred to the γ-phosphate via various proton wires, resulting in a H2PγO4(-)/ADP(3-) product state. This catalytic strategy is so effective that most other nucleotide hydrolyzing enzymes adopt a similar approach, as suggested by their very similar triphosphate binding sites.

  6. Structure of the ATP Synthase Catalytic Complex (F1) from Escherichia coli in an Autoinhibited conformation

    SciTech Connect

    G Cingolani; T Duncan

    2011-12-31

    ATP synthase is a membrane-bound rotary motor enzyme that is critical for cellular energy metabolism in all kingdoms of life. Despite conservation of its basic structure and function, autoinhibition by one of its rotary stalk subunits occurs in bacteria and chloroplasts but not in mitochondria. The crystal structure of the ATP synthase catalytic complex (F{sub 1}) from Escherichia coli described here reveals the structural basis for this inhibition. The C-terminal domain of subunit {var_epsilon} adopts a heretofore unknown, highly extended conformation that inserts deeply into the central cavity of the enzyme and engages both rotor and stator subunits in extensive contacts that are incompatible with functional rotation. As a result, the three catalytic subunits are stabilized in a set of conformations and rotational positions distinct from previous F{sub 1} structures.

  7. Human small cell lung cancer NYH cells selected for resistance to the bisdioxopiperazine topoisomerase II catalytic inhibitor ICRF-187 demonstrate a functional R162Q mutation in the Walker A consensus ATP binding domain of the alpha isoform.

    PubMed

    Wessel, I; Jensen, L H; Jensen, P B; Falck, J; Rose, A; Roerth, M; Nitiss, J L; Sehested, M

    1999-07-15

    Bisdioxopiperazine drugs such as ICRF-187 are catalytic inhibitors of DNA topoisomerase II, with at least two effects on the enzyme: namely, locking it in a closed-clamp form and inhibiting its ATPase activity. This is in contrast to topoisomerase II poisons as etoposide and amsacrine (m-AMSA), which act by stabilizing enzyme-DNA-drug complexes at a stage in which the DNA gate strand is cleaved and the protein is covalently attached to DNA. Human small cell lung cancer NYH cells selected for resistance to ICRF-187 (NYH/187) showed a 25% increase in topoisomerase IIalpha level and no change in expression of the beta isoform. Sequencing of the entire topoisomerase IIalpha cDNA from NYH/187 cells demonstrated a homozygous G-->A point mutation at nucleotide 485, leading to a R162Q conversion in the Walker A consensus ATP binding site (residues 161-165 in the alpha isoform), this being the first drug-selected mutation described at this site. Western blotting after incubation with ICRF-187 showed no depletion of the alpha isoform in NYH/187 cells in contrast to wild-type (wt) cells, whereas equal depletion of the beta isoform was observed in the two sublines. Alkaline elution assay demonstrated a lack of inhibition of etoposide-induced DNA single-stranded breaks in NYH/187 cells, whereas this inhibition was readily apparent in NYH cells. Site-directed mutagenesis in human topoisomerase IIalpha introduced into a yeast Saccharomyces cerevisiae strain with a temperature-conditional yeast TOP2 mutant demonstrated that R162Q conferred resistance to the bisdioxopiperazines ICRF-187 and -193 but not to etoposide or m-AMSA. Both etoposide and m-AMSA induced more DNA cleavage with purified R162Q enzyme than with the wt. The R162Q enzyme has a 20-25% decreased catalytic capacity compared to the wt and was almost inactive at <0.25 mM ATP compared to the wt. Kinetoplast DNA decatenation by the R162Q enzyme at 1 mM ATP was not resistant to ICRF-187 compared to wt, whereas it was

  8. Kinetic and stability properties of Penicillium chrysogenum ATP sulfurylase missing the C-terminal regulatory domain.

    PubMed

    Hanna, Eissa; Ng, Kit Fai; MacRae, Ian J; Bley, Christopher J; Fisher, Andrew J; Segel, Irwin H

    2004-02-06

    ATP sulfurylase from Penicillium chrysogenum is a homohexameric enzyme that is subject to allosteric inhibition by 3'-phosphoadenosine 5'-phosphosulfate. In contrast to the wild type enzyme, recombinant ATP sulfurylase lacking the C-terminal allosteric domain was monomeric and noncooperative. All kcat values were decreased (the adenosine 5'-phosphosulfate (adenylylsulfate) (APS) synthesis reaction to 17% of the wild type value). Additionally, the Michaelis constants for MgATP and sulfate (or molybdate), the dissociation constant of E.APS, and the monovalent oxyanion dissociation constants of dead end E.MgATP.oxyanion complexes were all increased. APS release (the k6 step) was rate-limiting in the wild type enzyme. Without the C-terminal domain, the composite k5 step (isomerization of the central complex and MgPPi release) became rate-limiting. The cumulative results indicate that besides (a) serving as a receptor for the allosteric inhibitor, the C-terminal domain (b) stabilizes the hexameric structure and indirectly, individual subunits. Additionally, (c) the domain interacts with and perfects the catalytic site such that one or more steps following the formation of the binary E.MgATP and E.SO4(2-) complexes and preceding the release of MgPPi are optimized. The more negative entropy of activation of the truncated enzyme for APS synthesis is consistent with a role of the C-terminal domain in promoting the effective orientation of MgATP and sulfate at the active site.

  9. Catalytic mechanism of bacteriophage T4 Rad50 ATP hydrolysis.

    PubMed

    Herdendorf, Timothy J; Nelson, Scott W

    2014-09-09

    Spontaneous double-strand breaks (DSBs) are one of the most deleterious forms of DNA damage, and their improper repair can lead to cellular dysfunction. The Mre11 and Rad50 proteins, a nuclease and an ATPase, respectively, form a well-conserved complex that is involved in the initial processing of DSBs. Here we examine the kinetic and catalytic mechanism of ATP hydrolysis by T4 Rad50 (gp46) in the presence and absence of Mre11 (gp47) and DNA. Single-turnover and pre-steady state kinetics on the wild-type protein indicate that the rate-limiting step for Rad50, the MR complex, and the MR-DNA complex is either chemistry or a conformational change prior to catalysis. Pre-steady state product release kinetics, coupled with viscosity steady state kinetics, also supports that the binding of DNA to the MR complex does not alter the rate-limiting step. The lack of a positive deuterium solvent isotope effect for the wild type and several active site mutants, combined with pH-rate profiles, implies that chemistry is rate-limiting and the ATPase mechanism proceeds via an asymmetric, dissociative-like transition state. Mutation of the Walker A/B and H-loop residues also affects the allosteric communication between Rad50 active sites, suggesting possible routes for cooperativity between the ATP active sites.

  10. Architecture and function of metallopeptidase catalytic domains

    PubMed Central

    Cerdà-Costa, Núria; Gomis-Rüth, Francesc Xavier

    2014-01-01

    The cleavage of peptide bonds by metallopeptidases (MPs) is essential for life. These ubiquitous enzymes participate in all major physiological processes, and so their deregulation leads to diseases ranging from cancer and metastasis, inflammation, and microbial infection to neurological insults and cardiovascular disorders. MPs cleave their substrates without a covalent intermediate in a single-step reaction involving a solvent molecule, a general base/acid, and a mono-or dinuclear catalytic metal site. Most monometallic MPs comprise a short metal-binding motif (HEXXH), which includes two metal-binding histidines and a general base/acid glutamate, and they are grouped into the zincin tribe of MPs. The latter divides mainly into the gluzincin and metzincin clans. Metzincins consist of globular ∼130–270-residue catalytic domains, which are usually preceded by N-terminal pro-segments, typically required for folding and latency maintenance. The catalytic domains are often followed by C-terminal domains for substrate recognition and other protein–protein interactions, anchoring to membranes, oligomerization, and compartmentalization. Metzincin catalytic domains consist of a structurally conserved N-terminal subdomain spanning a five-stranded β-sheet, a backing helix, and an active-site helix. The latter contains most of the metal-binding motif, which is here characteristically extended to HEXXHXXGXX(H,D). Downstream C-terminal subdomains are generally shorter, differ more among metzincins, and mainly share a conserved loop—the Met-turn—and a C-terminal helix. The accumulated structural data from more than 300 deposited structures of the 12 currently characterized metzincin families reviewed here provide detailed knowledge of the molecular features of their catalytic domains, help in our understanding of their working mechanisms, and form the basis for the design of novel drugs. PMID:24596965

  11. Effects of surface adsorption on catalytic activity of heavy meromyosin studied using a fluorescent ATP analogue.

    PubMed

    Balaz, Martina; Sundberg, Mark; Persson, Malin; Kvassman, Jan; Månsson, Alf

    2007-06-19

    Biochemical studies in solution and with myosin motor fragments adsorbed to surfaces (in vitro motility assays) are invaluable for elucidation of actomyosin function. However, there is limited understanding of how surface adsorption affects motor properties, e.g., catalytic activity. Here we address this issue by comparing the catalytic activity of heavy meromyosin (HMM) in solution and adsorbed to standard motility assay surfaces [derivatized with trimethylchlorosilane (TMCS)]. For these studies we first characterized the interaction of HMM and actomyosin with the fluorescent ATP analogue adenosine 5'-triphosphate Alexa Fluor 647 2'- (or 3'-) O-(N-(2-aminoethyl)urethane) hexa(triethylammonium) salt (Alexa-ATP). The data suggest that Alexa-ATP is hydrolyzed by HMM in solution at a slightly higher rate than ATP but with a generally similar mechanism. Furthermore, Alexa-ATP is effective as a fuel for HMM-propelled actin filament sliding. The catalytic activity of HMM on TMCS surfaces was studied using (1) Alexa-ATP in total internal reflection fluorescence (TIRF) spectroscopy experiments and (2) Alexa-ATP and ATP in HPLC-aided ATPase measurements. The results support the hypothesis of different HMM configurations on the surface. However, a dominant proportion of the myosin heads were catalytically active, and their average steady-state hydrolysis rate was slightly higher (with Alexa-ATP) or markedly higher (with ATP) on the surface than in solution. The results are discussed in relation to the use of TMCS surfaces and Alexa-ATP for in vitro motility assays and single molecule studies. Furthermore, we propose a novel TIRF microscopy method to accurately determine the surface density of catalytically active myosin motors.

  12. Structure of the ATP synthase catalytic complex (F1) from Escherichia coli in an auto-inhibited conformation

    PubMed Central

    Cingolani, Gino; Duncan, Thomas M.

    2011-01-01

    ATP synthase is a membrane-bound, rotary motor enzyme that is critical for cellular energy metabolism in all kingdoms of life. Despite conservation of its basic structure and function, auto-inhibition by one of its rotary stalk subunits occurs in bacteria and chloroplasts but not in mitochondria. The crystal structure of the ATP synthase catalytic complex (F1) from Escherichia coli described here reveals the structural basis for this inhibition. The C-terminal domain of subunit ε adopts a novel, highly extended conformation that inserts deeply into the central cavity of the enzyme and engages both rotor and stator subunits in extensive contacts that are incompatible with functional rotation. As a result, the three catalytic subunits are stabilized in a set of conformations and rotational positions distinct from previous F1 structures. PMID:21602818

  13. In situ structure of trypanosomal ATP synthase dimer reveals a unique arrangement of catalytic subunits

    PubMed Central

    Mühleip, Alexander W.; Dewar, Caroline E.; Schnaufer, Achim; Kühlbrandt, Werner; Davies, Karen M.

    2017-01-01

    We used electron cryotomography and subtomogram averaging to determine the in situ structures of mitochondrial ATP synthase dimers from two organisms belonging to the phylum euglenozoa: Trypanosoma brucei, a lethal human parasite, and Euglena gracilis, a photosynthetic protist. At a resolution of 32.5 Å and 27.5 Å, respectively, the two structures clearly exhibit a noncanonical F1 head, in which the catalytic (αβ)3 assembly forms a triangular pyramid rather than the pseudo-sixfold ring arrangement typical of all other ATP synthases investigated so far. Fitting of known X-ray structures reveals that this unusual geometry results from a phylum-specific cleavage of the α subunit, in which the C-terminal αC fragments are displaced by ∼20 Å and rotated by ∼30° from their expected positions. In this location, the αC fragment is unable to form the conserved catalytic interface that was thought to be essential for ATP synthesis, and cannot convert γ-subunit rotation into the conformational changes implicit in rotary catalysis. The new arrangement of catalytic subunits suggests that the mechanism of ATP generation by rotary ATPases is less strictly conserved than has been generally assumed. The ATP synthases of these organisms present a unique model system for discerning the individual contributions of the α and β subunits to the fundamental process of ATP synthesis. PMID:28096380

  14. In situ structure of trypanosomal ATP synthase dimer reveals a unique arrangement of catalytic subunits.

    PubMed

    Mühleip, Alexander W; Dewar, Caroline E; Schnaufer, Achim; Kühlbrandt, Werner; Davies, Karen M

    2017-01-31

    We used electron cryotomography and subtomogram averaging to determine the in situ structures of mitochondrial ATP synthase dimers from two organisms belonging to the phylum euglenozoa: Trypanosoma brucei, a lethal human parasite, and Euglena gracilis, a photosynthetic protist. At a resolution of 32.5 Å and 27.5 Å, respectively, the two structures clearly exhibit a noncanonical F1 head, in which the catalytic (αβ)3 assembly forms a triangular pyramid rather than the pseudo-sixfold ring arrangement typical of all other ATP synthases investigated so far. Fitting of known X-ray structures reveals that this unusual geometry results from a phylum-specific cleavage of the α subunit, in which the C-terminal αC fragments are displaced by ∼20 Å and rotated by ∼30° from their expected positions. In this location, the αC fragment is unable to form the conserved catalytic interface that was thought to be essential for ATP synthesis, and cannot convert γ-subunit rotation into the conformational changes implicit in rotary catalysis. The new arrangement of catalytic subunits suggests that the mechanism of ATP generation by rotary ATPases is less strictly conserved than has been generally assumed. The ATP synthases of these organisms present a unique model system for discerning the individual contributions of the α and β subunits to the fundamental process of ATP synthesis.

  15. An open conformation of the Thermus thermophilus gyrase B ATP-binding domain.

    PubMed

    Lamour, Valérie; Hoermann, Laurence; Jeltsch, Jean-Marc; Oudet, Pierre; Moras, Dino

    2002-05-24

    DNA gyrase forms an A(2)B(2) tetramer involved in DNA replication, repair, recombination, and transcription in which the B subunit catalyzes ATP hydrolysis. The Thermus thermophilus and Escherichia coli gyrases are homologues and present the same catalytic activity. When compared with that of the E. coli 43K-5'-adenylyl-beta,gamma-imidodiphosphate complex, the crystal structure of Gyrase B 43K ATPase domain in complex with novobiocin, one of the most potent inhibitors of gyrase shows large conformational changes of the subdomains within the dimer. The stabilization of loop 98-118 closing the active site through dimeric contacts and interaction with domain 2 allows to observe novobiocin-protein interactions that could not be seen in the 24K-inhibitor complexes. Furthermore, this loop adopts a position which defines an "open" conformation of the active site in absence of ATP, in contrast with the "closed" conformation adopted upon ATP binding. All together, these results indicate how the subdomains may propagate conformational changes from the active site and provide crucial information for the design of more specific inhibitors.

  16. An analysis of ATP binding with kinase catalytic subunit by molecular dynamics simulation of the CDK2 active kinase crystal lattice

    NASA Astrophysics Data System (ADS)

    Kretov, D. A.; Kholmurodov, Kh. T.; Koltovaya, N. A.

    2007-10-01

    Molecular dynamics simulation was used to analyze changes in the functionally significant structural elements of the crystal lattices of pT160-CDK2/cyclin and A/ATP-Mg2+/substrate complexes of the native (CDK2-G16) and mutant (CDK2-S16) active kinases at physiological temperatures (300 K). The structural rearrangement of ATP caused by changes in the kinase catalytic domain was studied. ATP was fixed by the ionic and H-bond interactions of several residues, including Lys33, Asp145, and side-chain amides of the G loop between β1 and β2. The binding of the kinases to complexes with cyclin and the phosphorylation of T160 in the active complex of the CDK2 kinase result in the ATP orientation more convenient for the transfer of the phosphate group to the substrate. An analysis of interatomic distances in the ATP active site region and Asp145, Asn132, Lys33 catalytic sites participating in the orientation of ATP phosphates revealed that the Asp 145 amino acid residue was situated noticeably closer to the ATP molecule in the native complex than in its mutant counterpart. The same is true of the arrangement of the Lys33 residue with respect to ATP.

  17. ErbB3/HER3 intracellular domain is competent to bind ATP and catalyze autophosphorylation

    SciTech Connect

    Shi, Fumin; Telesco, Shannon E.; Liu, Yingting; Radhakrishnan, Ravi; Lemmon, Mark A.

    2010-06-21

    ErbB3/HER3 is one of four members of the human epidermal growth factor receptor (EGFR/HER) or ErbB receptor tyrosine kinase family. ErbB3 binds neuregulins via its extracellular region and signals primarily by heterodimerizing with ErbB2/HER2/Neu. A recently appreciated role for ErbB3 in resistance of tumor cells to EGFR/ErbB2-targeted therapeutics has made it a focus of attention. However, efforts to inactivate ErbB3 therapeutically in parallel with other ErbB receptors are challenging because its intracellular kinase domain is thought to be an inactive pseudokinase that lacks several key conserved (and catalytically important) residues - including the catalytic base aspartate. We report here that, despite these sequence alterations, ErbB3 retains sufficient kinase activity to robustly trans-autophosphorylate its intracellular region - although it is substantially less active than EGFR and does not phosphorylate exogenous peptides. The ErbB3 kinase domain binds ATP with a K{sub d} of approximately 1.1 {micro}M. We describe a crystal structure of ErbB3 kinase bound to an ATP analogue, which resembles the inactive EGFR and ErbB4 kinase domains (but with a shortened {alpha}C-helix). Whereas mutations that destabilize this configuration activate EGFR and ErbB4 (and promote EGFR-dependent lung cancers), a similar mutation conversely inactivates ErbB3. Using quantum mechanics/molecular mechanics simulations, we delineate a reaction pathway for ErbB3-catalyzed phosphoryl transfer that does not require the conserved catalytic base and can be catalyzed by the 'inactive-like'configuration observed crystallographically. These findings suggest that ErbB3 kinase activity within receptor dimers may be crucial for signaling and could represent an important therapeutic target.

  18. ErbB3/HER3 intracellular domain is competent to bind ATP and catalyze autophosphorylation.

    PubMed

    Shi, Fumin; Telesco, Shannon E; Liu, Yingting; Radhakrishnan, Ravi; Lemmon, Mark A

    2010-04-27

    ErbB3/HER3 is one of four members of the human epidermal growth factor receptor (EGFR/HER) or ErbB receptor tyrosine kinase family. ErbB3 binds neuregulins via its extracellular region and signals primarily by heterodimerizing with ErbB2/HER2/Neu. A recently appreciated role for ErbB3 in resistance of tumor cells to EGFR/ErbB2-targeted therapeutics has made it a focus of attention. However, efforts to inactivate ErbB3 therapeutically in parallel with other ErbB receptors are challenging because its intracellular kinase domain is thought to be an inactive pseudokinase that lacks several key conserved (and catalytically important) residues-including the catalytic base aspartate. We report here that, despite these sequence alterations, ErbB3 retains sufficient kinase activity to robustly trans-autophosphorylate its intracellular region--although it is substantially less active than EGFR and does not phosphorylate exogenous peptides. The ErbB3 kinase domain binds ATP with a K(d) of approximately 1.1 microM. We describe a crystal structure of ErbB3 kinase bound to an ATP analogue, which resembles the inactive EGFR and ErbB4 kinase domains (but with a shortened alphaC-helix). Whereas mutations that destabilize this configuration activate EGFR and ErbB4 (and promote EGFR-dependent lung cancers), a similar mutation conversely inactivates ErbB3. Using quantum mechanics/molecular mechanics simulations, we delineate a reaction pathway for ErbB3-catalyzed phosphoryl transfer that does not require the conserved catalytic base and can be catalyzed by the "inactive-like" configuration observed crystallographically. These findings suggest that ErbB3 kinase activity within receptor dimers may be crucial for signaling and could represent an important therapeutic target.

  19. Catalytic Domain Architecture of Metzincin Metalloproteases*

    PubMed Central

    Gomis-Rüth, F. Xavier

    2009-01-01

    Metalloproteases cleave proteins and peptides, and deregulation of their function leads to pathology. An understanding of their structure and mechanisms of action is necessary to the development of strategies for their regulation. Among metallopeptidases are the metzincins, which are mostly multidomain proteins with ∼130–260-residue globular catalytic domains showing a common core architecture characterized by a long zinc-binding consensus motif, HEXXHXXGXX(H/D), and a methionine-containing Met-turn. Metzincins participate in unspecific protein degradation such as digestion of intake proteins and tissue development, maintenance, and remodeling, but they are also involved in highly specific cleavage events to activate or inactivate themselves or other (pro)enzymes and bioactive peptides. Metzincins are subdivided into families, and seven such families have been analyzed at the structural level: the astacins, ADAMs/adamalysins/reprolysins, serralysins, matrix metalloproteinases, snapalysins, leishmanolysins, and pappalysins. These families are reviewed from a structural point of view. PMID:19201757

  20. Trapped intermediate state of plant pyruvate phosphate dikinase indicates substeps in catalytic swiveling domain mechanism.

    PubMed

    Minges, Alexander; Höppner, Astrid; Groth, Georg

    2017-08-01

    Pyruvate phosphate dikinase (PPDK) is an essential enzyme of both the C4 photosynthetic pathway and cellular energy metabolism of some bacteria and unicellular protists. In C4 plants, it catalyzes the ATP- and Pi -dependent formation of phosphoenolpyruvate (PEP) while in bacteria and protozoa the ATP-forming direction is used. PPDK is composed out of three distinct domains and exhibits one of the largest single domain movements known today during its catalytic cycle. However, little information about potential intermediate steps of this movement was available. A recent study resolved a discrete intermediate step of PPDK's swiveling movement, shedding light on the details of this intriguing mechanism. Here we present an additional structural intermediate that possibly represents another crucial step in the catalytic cycle of PPDK, providing means to get a more detailed understanding of PPDK's mode of function. © 2017 The Protein Society.

  1. The role of metal binding and phosphorylation domains in the regulation of cisplatin-induced trafficking of ATP7B.

    PubMed

    Safaei, Roohangiz; Adams, Preston L; Mathews, Ryan A; Manorek, Gerald; Howell, Stephen B

    2013-08-01

    The copper (Cu) exporter ATP7B mediates cellular resistance to cisplatin (cDDP) by increasing drug efflux. ATP7B binds and sequesters cDDP in into secretory vesicles. Upon cDDP exposure ATP7B traffics from the trans-Golgi network (TGN) to the periphery of the cell in a manner that requires the cysteine residues in its metal binding domains (MBD). To elucidate the role of the various domains of ATP7B in its cDDP-induced trafficking we expressed a series of mCherry-tagged variants of ATP7B in HEK293T cells and analyzed their subcellular localization in basal media and after a 1 h exposure to 30 μM cDDP. The wild type ATP7B and a variant in which the cysteines in the CXXC motifs of MBD 1-5 were converted to serines trafficked out of the trans-Golgi (TGN) when exposed to cDDP. Conversion of the cysteines in all 6 of the CXXC motifs to serines, or in only the sixth MBD, rendered ATP7B incapable of trafficking on exposure to cDDP. Truncation of MBD1-5 or MBD1-6 resulted in the loss of TGN localization. Addition of the first 63 amino acids of ATP7B to these variants restored TGN localization to a great extent and enabled the MBD1-5 variant to undergo cDDP-induced trafficking. A variant of ATP7B in which the aspartate 1027 residue in the phosphorylation domain was converted to glutamine localized to the TGN but was incapable of cDDP-induced trafficking. These results demonstrate that the CXXC motif in the sixth MBD and the catalytic activity of ATP7B are required for cDDP-induced trafficking as they are for Cu-induced redistribution of ATP7B; this provides further evidence that cDDP mimics Cu with respect to the molecular mechanisms by they control the subcellular distribution of ATP7B.

  2. Catalytic and mechanical cycles in F-ATP synthases: Fourth in the Cycles Review Series

    PubMed Central

    Dimroth, Peter; von Ballmoos, Christoph; Meier, T

    2006-01-01

    Cycles have a profound role in cellular life at all levels of organization. Well-known cycles in cell metabolism include the tricarboxylic acid and the urea cycle, in which a specific carrier substrate undergoes a sequence of chemical transformations and is regenerated at the end. Other examples include the interconversions of cofactors, such as NADH or ATP, which are present in the cell in limiting amounts and have to be recycled effectively for metabolism to continue. Every living cell performs a rapid turnover of ATP to ADP to fulfil various energetic demands and effectively regenerates the ATP from ADP in an energy-consuming process. The turnover of the ATP cycle is impressive; a human uses about its body weight in ATP per day. Enzymes perform catalytic reaction cycles in which they undergo several chemical and physical transformations before they are converted back to their original states. The ubiquitous F1Fo ATP synthase is of particular interest not only because of its biological importance, but also owing to its unique rotational mechanism. Here, we give an overview of the membrane-embedded Fo sector, particularly with respect to the recent crystal structure of the c ring from Ilyobacter tartaricus, and summarize current hypotheses for the mechanism by which rotation of the c ring is generated. PMID:16607397

  3. Energy-dependent dissociation of ATP from high affinity catalytic sites of beef heart mitochondrial adenosine triphosphatase

    SciTech Connect

    Penefsky, H.S.

    1985-11-05

    Incubation of (gamma-TSP)ATP with a molar excess of the membrane-bound form of mitochondrial ATPase (F1) results in binding of the bulk of the radioactive nucleotide in high affinity catalytic sites (Ka = 10(12) M-1). Subsequent initiation of respiration by addition of succinate or NADH is accompanied by a profound decrease in the affinity for ATP. About one-third of the bound radioactive ATP appears to dissociate, that is, the (gamma-TSP)ATP becomes accessible to hexokinase. The NADH-stimulated dissociation of (gamma-TSP)ATP is energy-dependent since the stimulation is inhibited by uncouplers of oxidative phosphorylation and is prevented by respiratory chain inhibitors. The rate of the energy-dependent dissociation of ATP that occurs in the presence of NADH, ADP, and Pi is commensurate with the measured initial rate of ATP synthesis in NADH-supported oxidative phosphorylation catalyzed by the same submitochondrial particles. Thus, the rate of dissociation of ATP from the high affinity catalytic site of submitochondrial particles meets the criterion of kinetic competency under the conditions of oxidative phosphorylation. These experiments provide evidence in support of the argument that energy conserved during the oxidation of substrates by the respiratory chain can be utilized to reduce the very tight binding of product ATP in high affinity catalytic sites and to promote dissociation of the nucleotide.

  4. Comparison and correlation of binding mode of ATP in the kinase domains of Hexokinase family

    PubMed Central

    Kumar, Yellapu Nanda; Kumar, Pasupuleti Santhosh; Sowjenya, Gopal; Rao, Valasani Koteswara; Yeswanth, Sthanikam; Prasad, Uppu Venkateswara; Pradeepkiran, Jangampalli Adi; Sarma, PVGK; Bhaskar, Matcha

    2012-01-01

    Hexokinases (HKs) are the enzymes that catalyses the ATP dependent phosphorylation of Hexose sugars to Hexose-6-Phosphate (Hex-6-P). There exist four different forms of HKs namely HK-I, HK-II, HK-III and HK-IV and all of them share a common ATP binding site core surrounded by more variable sequence that determine substrate affinities. Although they share a common binding site but they differ in their kinetic functions, hence the present study is aimed to analyze the binding mode of ATP. The analysis revealed that the four ATP binding domains are showing 13 identical, 7 similar and 6 dissimilar residues with similar structural conformation. Molecular docking of ATP into the kinase domains using Molecular Operating Environment (MOE) soft ware tool clearly showed the variation in the binding mode of ATP with variable docking scores. This probably explains the variable phosphorylation rates among hexokinases family. PMID:22829728

  5. Hydrolysis at One of the Two Nucleotide-binding Sites Drives the Dissociation of ATP-binding Cassette Nucleotide-binding Domain Dimers

    SciTech Connect

    Zoghbi, M. E.; Altenberg, G. A.

    2013-10-15

    The functional unit of ATP-binding cassette (ABC) transporters consists of two transmembrane domains and two nucleotide-binding domains (NBDs). ATP binding elicits association of the two NBDs, forming a dimer in a head-to-tail arrangement, with two nucleotides “sandwiched” at the dimer interface. Each of the two nucleotide-binding sites is formed by residues from the two NBDs. We recently found that the prototypical NBD MJ0796 from Methanocaldococcus jannaschii dimerizes in response to ATP binding and dissociates completely following ATP hydrolysis. However, it is still unknown whether dissociation of NBD dimers follows ATP hydrolysis at one or both nucleotide-binding sites. Here, we used luminescence resonance energy transfer to study heterodimers formed by one active (donor-labeled) and one catalytically defective (acceptor-labeled) NBD. Rapid mixing experiments in a stop-flow chamber showed that NBD heterodimers with one functional and one inactive site dissociated at a rate indistinguishable from that of dimers with two hydrolysis-competent sites. Comparison of the rates of NBD dimer dissociation and ATP hydrolysis indicated that dissociation followed hydrolysis of one ATP. We conclude that ATP hydrolysis at one nucleotide-binding site drives NBD dimer dissociation.

  6. Hydrolysis at One of the Two Nucleotide-binding Sites Drives the Dissociation of ATP-binding Cassette Nucleotide-binding Domain Dimers*

    PubMed Central

    Zoghbi, Maria E.; Altenberg, Guillermo A.

    2013-01-01

    The functional unit of ATP-binding cassette (ABC) transporters consists of two transmembrane domains and two nucleotide-binding domains (NBDs). ATP binding elicits association of the two NBDs, forming a dimer in a head-to-tail arrangement, with two nucleotides “sandwiched” at the dimer interface. Each of the two nucleotide-binding sites is formed by residues from the two NBDs. We recently found that the prototypical NBD MJ0796 from Methanocaldococcus jannaschii dimerizes in response to ATP binding and dissociates completely following ATP hydrolysis. However, it is still unknown whether dissociation of NBD dimers follows ATP hydrolysis at one or both nucleotide-binding sites. Here, we used luminescence resonance energy transfer to study heterodimers formed by one active (donor-labeled) and one catalytically defective (acceptor-labeled) NBD. Rapid mixing experiments in a stop-flow chamber showed that NBD heterodimers with one functional and one inactive site dissociated at a rate indistinguishable from that of dimers with two hydrolysis-competent sites. Comparison of the rates of NBD dimer dissociation and ATP hydrolysis indicated that dissociation followed hydrolysis of one ATP. We conclude that ATP hydrolysis at one nucleotide-binding site drives NBD dimer dissociation. PMID:24129575

  7. Closer to nature: an ATP-driven bioinspired catalytic oxidation process.

    PubMed

    Stefan, Loic; Duret, Damien; Spinelli, Nicolas; Defrancq, Eric; Monchaud, David

    2013-02-21

    The capability of DNA to acquire enzyme-like properties has led to the emergence of the so-called DNAzyme field; herein, we take a further leap along this nature-inspired road, demonstrating that a template assembled synthetic G-quartet (TASQ) can act as a pre-catalyst for catalytic peroxidase-mimicking oxidation reactions, whatever its nature (guanine or guanosine-based G-quartets), in an ATP-dependent manner, thereby bringing this bioinspired TASQzyme process even closer to nature.

  8. ATP interacts with the CPVT mutation-associated central domain of the cardiac ryanodine receptor.

    PubMed

    Blayney, Lynda; Beck, Konrad; MacDonald, Ewan; D'Cruz, Leon; Nomikos, Michail; Griffiths, Julia; Thanassoulas, Angelos; Nounesis, George; Lai, F Anthony

    2013-10-01

    This study was designed to determine whether the cardiac ryanodine receptor (RyR2) central domain, a region associated with catecholamine polymorphic ventricular tachycardia (CPVT) mutations, interacts with the RyR2 regulators, ATP and the FK506-binding protein 12.6 (FKBP12.6). Wild-type (WT) RyR2 central domain constructs (G(2236)to G(2491)) and those containing the CPVT mutations P2328S and N2386I, were expressed as recombinant proteins. Folding and stability of the proteins were examined by circular dichroism (CD) spectroscopy and guanidine hydrochloride chemical denaturation. The far-UV CD spectra showed a soluble stably-folded protein with WT and mutant proteins exhibiting a similar secondary structure. Chemical denaturation analysis also confirmed a stable protein for both WT and mutant constructs with similar two-state unfolding. ATP and caffeine binding was measured by fluorescence spectroscopy. Both ATP and caffeine bound with an EC50 of ~200-400μM, and the affinity was the same for WT and mutant constructs. Sequence alignment with other ATP binding proteins indicated the RyR2 central domain contains the signature of an ATP binding pocket. Interaction of the central domain with FKBP12.6 was tested by glutaraldehyde cross-linking and no association was found. The RyR2 central domain, expressed as a 'correctly' folded recombinant protein, bound ATP in accord with bioinformatics evidence of conserved ATP binding sequence motifs. An interaction with FKBP12.6 was not evident. CPVT mutations did not disrupt the secondary structure nor binding to ATP. Part of the RyR2 central domain CPVT mutation cluster, can be expressed independently with retention of ATP binding. Copyright © 2013 Elsevier B.V. All rights reserved.

  9. Phospholipid binding to the FAK catalytic domain impacts function

    PubMed Central

    Schaller, Michael D.

    2017-01-01

    Focal adhesion kinase is an essential nonreceptor tyrosine kinase that plays an important role in development, in homeostasis and in the progression of human disease. Multiple stimuli activate FAK, which requires a change in structure from an autoinhibited to activated conformation. In the autoinhibited conformation the FERM domain associates with the catalytic domain of FAK and PI(4,5)P2 binding to the FERM domain plays a role in the release of autoinhibition, activating the enzyme. An in silico model of FAK/PI(4,5)P2 interaction suggests that residues on the catalytic domain interact with PI(4,5)P2, in addition to the known FERM domain PI(4,5)P2 binding site. This study was undertaken to test the significance of this in silico observation. Mutations designed to disrupt the putative PI(4,5)P2 binding site were engineered into FAK. These mutants exhibited defects in phosphorylation and failed to completely rescue the phenotype associated with fak -/- phenotype fibroblasts demonstrating the importance of these residues in FAK function. The catalytic domain of FAK exhibited PI(4,5)P2 binding in vitro and binding activity was lost upon mutation of putative PI(4,5)P2 binding site basic residues. However, binding was not selective for PI(4,5)P2, and the catalytic domain bound to several phosphatidylinositol phosphorylation variants. The mutant exhibiting the most severe biological defect was defective for phosphatidylinositol phosphate binding, supporting the model that catalytic domain phospholipid binding is important for biochemical and biological function. PMID:28222177

  10. The function of Glu338 in the catalytic triad of the carbamoyl phosphate synthetase amidotransferase domain.

    PubMed

    Hewagama, A; Guy, H I; Chaparian, M; Evans, D R

    1998-11-10

    The synthesis of carbamoyl phosphate by the mammalian multifunctional protein, CAD, involves the concerted action of the 40 kDa amidotransferase domain (GLN), that hydrolyzes glutamine and the 120 kDa synthetase (CPS) domain that uses the ammonia, thus produced, ATP and bicarbonate to make carbamoyl phosphate. The separately cloned GLN domain has very low activity due to a reduction in kcat and an increase in Km but forms a hybrid complex with the isolated Escherichia coli CPS subunit. The hybrid has full glutamine-dependent catalytic activity and a functional interdomain linkage. The mammalian-E. coli hybrid was used to investigate the functional consequence of replacing His336 and Glu338, two residues postulated to participate in catalysis as part of a catalytic triad. The mutant mammalian GLN domains formed stable complexes with the E. coli CPS subunit, but the catalytic activity was severely impaired. While the His336Asn mutant does not form measurable amounts of the gamma-glutamyl thioester, the steady state concentration of the intermediate with the Glu338Gly mutant was comparable to the wild type hybrid because both the rate of formation and breakdown of the thioester are reduced. This result is consistent with the postulated role of Glu338 in maintaining His336 in the optimal orientation for catalysis and suggests a mechanism for the GLN CPS functional linkage.

  11. Functional roles of ATP-binding residues in the catalytic site of human mitochondrial NAD(P)+-dependent malic enzyme.

    PubMed

    Hung, Hui-Chih; Chien, Yu-Ching; Hsieh, Ju-Yi; Chang, Gu-Gang; Liu, Guang-Yaw

    2005-09-27

    Human mitochondrial NAD(P)+-dependent malic enzyme is inhibited by ATP. The X-ray crystal structures have revealed that two ATP molecules occupy both the active and exo site of the enzyme, suggesting that ATP might act as an allosteric inhibitor of the enzyme. However, mutagenesis studies and kinetic evidences indicated that the catalytic activity of the enzyme is inhibited by ATP through a competitive inhibition mechanism in the active site and not in the exo site. Three amino acid residues, Arg165, Asn259, and Glu314, which are hydrogen-bonded with NAD+ or ATP, are chosen to characterize their possible roles on the inhibitory effect of ATP for the enzyme. Our kinetic data clearly demonstrate that Arg165 is essential for catalysis. The R165A enzyme had very low enzyme activity, and it was only slightly inhibited by ATP and not activated by fumarate. The values of K(m,NAD) and K(i,ATP) to both NAD+ and malate were elevated. Elimination of the guanidino side chain of R165 made the enzyme defective on the binding of NAD+ and ATP, and it caused the charge imbalance in the active site. These effects possibly caused the enzyme to malfunction on its catalytic power. The N259A enzyme was less inhibited by ATP but could be fully activated by fumarate at a similar extent compared with the wild-type enzyme. For the N259A enzyme, the value of K(i,ATP) to NAD+ but not to malate was elevated, indicating that the hydrogen bonding between ATP and the amide side chain of this residue is important for the binding stability of ATP. Removal of this side chain did not cause any harmful effect on the fumarate-induced activation of the enzyme. The E314A enzyme, however, was severely inhibited by ATP and only slightly activated by fumarate. The values of K(m,malate), K(m,NAD), and K(i,ATP) to both NAD+ and malate for E314A were reduced to about 2-7-folds compared with those of the wild-type enzyme. It can be concluded that mutation of Glu314 to Ala eliminated the repulsive effects

  12. Molecular Dynamics Studies on the HIV-1 Integrase Catalytic Domain

    SciTech Connect

    Lins, Roberto D.; Briggs, J. M.; Straatsma, TP; Carlson, Heather A.; Greenwald, Jason; Choe, Senyon; Mccammon, Andy

    1999-06-30

    The HIV-1 integrase, which is essential for viral replication, catalyzes the insertion of viral DNA into the host chromosome, thereby recruiting host cell machinery into making viral proteins. It represents the third main HIV enzyme target for inhibitor design, the first two being the reverse transcriptase and the protease. Two 1-ns molecular dynamics simulations have been carried out on completely hydrated models of the HIV-1 integrase catalytic domain, one with no metal ions and another with one magnesium ion in the catalytic site. The simulations predict that the region of the active site that is missing in the published crystal structures has (at the time of this work) more secondary structure than previously thought. The flexibility of this region has been discussed with respect to the mechanistic function of the enzyme. The results of these simulations will be used as part of inhibitor design projects directed against the catalytic domain of the enzyme.

  13. Photoaffinity labeling of ribulose-1,5-bisphosphate carboxylase/oxygenase activase with ATP gamma-benzophenone. Identification of the ATP gamma-phosphate binding domain.

    PubMed

    Salvucci, M E; Rajagopalan, K; Sievert, G; Haley, B E; Watt, D S

    1993-07-05

    The phosphate-binding domain of the ATP-binding site of tobacco Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) activase was elucidated by photo-affinity labeling with a monoanhydride of ADP with N-(4-(benzoyl)phenylmethyl)phosphoramide ([gamma-32P]ATP gamma BP). Covalent incorporation of [gamma-32P]ATP gamma BP into the 42-kDa Rubisco activase subunit was dependent upon irradiation with ultraviolet light. Photolabelling of Rubisco activase with ATP gamma BP exhibited saturation kinetics; the apparent Kd for photolabeling was 5 microM. Two lines of evidence showed that ATP gamma BP modified Rubisco activase at the ATP-binding domain. First, physiological concentrations of ATP and ADP afforded complete protection against photolabeling of Rubisco activase by ATP gamma BP. Second, photolysis of Rubisco activase in the presence of ATP gamma BP decreased both the ATPase and the Rubisco activating activities. Inactivation of enzyme activity was dependent on ATP gamma BP concentration and could be prevented by including ADP during photolabeling. The region of Rubisco activase that was modified by ATP gamma BP was identified by isolating photolabeled peptides. Sequence analysis showed that ATP gamma BP modified Rubisco activase in two distinct regions; one region, S117-A136, is adjacent to the P-loop and the other region, V223-T234, exhibits homology to a region of adenylate kinase that ligates the essential metal ion. Photolabeling of these two regions of Rubisco activase was consistent with modification of the ATP gamma-phosphate-binding domain of Rubisco activase with ATP gamma BP.

  14. Regulatory and Catalytic Domain Dynamics of Smooth Muscle Myosin Filaments†

    PubMed Central

    Li, Hui-Chun; Song, Likai; Salzameda, Bridget; Cremo, Christine R.; Fajer, Piotr G.

    2016-01-01

    Domain dynamics of the chicken gizzard smooth muscle myosin catalytic domain (heavy chain Cys-717) and regulatory domain (regulatory light chain Cys-108) were determined in the absence of nucleotides using saturation-transfer electron paramagnetic resonance. In unphosphorylated synthetic filaments, the effective rotational correlation times, τr, were 24 ± 6 μs and 441 ± 79 μs for the catalytic and regulatory domains, respectively. The corresponding amplitudes of motion were 42 ± 4° and 24 ± 9° as determined from steady-state phosphorescence anisotropy. These results suggest that the two domains have independent mobility due to a hinge between the two domains. Although a similar hinge was observed for skeletal myosin (Adhikari and Fajer (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 9643–9647. Brown et al. (2001) Biochemistry 40, 8283–8291), the latter displayed higher regulatory domain mobility, τr = 40 ± 3 μs, suggesting a smooth muscle specific mechanism of constraining regulatory domain dynamics. In the myosin monomers the correlation times for both domains were the same (~4 μs) for both smooth and skeletal myosin, suggesting that the motional difference between the two isoforms in the filaments was not due to intrinsic variation of hinge stiffness. Heavy chain/regulatory light chain chimeras of smooth and skeletal myosin pinpointed the origin of the restriction to the heavy chain and established correlation between the regulatory domain dynamics with the ability of myosin to switch off but not to switch on the ATPase and the actin sliding velocity. Phosphorylation of smooth muscle myosin filaments caused a small increase in the amplitude of motion of the regulatory domain (from 24 ± 4° to 36 ± 7°) but did not significantly affect the rotational correlation time of the regulatory domain (441 to 408 μs) or the catalytic domain (24 to 17 μs). These data are not consistent with a stable interaction between the two catalytic domains in

  15. Mitochondrial ATP synthases cluster as discrete domains that reorganize with the cellular demand for oxidative phosphorylation.

    PubMed

    Jimenez, Laure; Laporte, Damien; Duvezin-Caubet, Stephane; Courtout, Fabien; Sagot, Isabelle

    2014-02-15

    Mitochondria are double membrane-bounded organelles that form a dynamic tubular network. Mitochondria energetic functions depend on a complex internal architecture. Cristae, inner membrane invaginations that fold into the matrix space, are proposed to be the site of oxidative phosphorylation, reactions by which ATP synthase produces ATP. ATP synthase is also thought to have a role in crista morphogenesis. To date, the exploration of the processes regulating mitochondrial internal compartmentalization have been mostly limited to electron microscopy. Here, we describe ATP synthase localization in living yeast cells and show that it clusters as discrete inner membrane domains. These domains are dynamic within the mitochondrial network. They are impaired in mutants defective in crista morphology and partially overlap with the crista-associated MICOS-MINOS-MITOS complex. Finally, ATP synthase occupancy increases with the cellular demand for OXPHOS. Overall our data suggest that domains in which ATP synthases are clustered correspond to mitochondrial cristae. Being able to follow mitochondrial sub-compartments in living yeast cells opens new avenues to explore the mechanisms involved in inner membrane remodeling, an architectural feature crucial for mitochondrial activities.

  16. Spectroscopic investigation of the reaction mechanism of CopB-B, the catalytic fragment from an archaeal thermophilic ATP-driven heavy metal transporter.

    PubMed

    Völlmecke, Christian; Kötting, Carsten; Gerwert, Klaus; Lübben, Mathias

    2009-11-01

    The mechanism of ATP hydrolysis of a shortened variant of the heavy metal-translocating P-type ATPase CopB of Sulfolobus solfataricus was studied. The catalytic fragment, named CopB-B, comprises the nucleotide binding and phosphorylation domains. We demonstrated stoichiometric high-affinity binding of one nucleotide to the protein (K(diss) 1-20 microm). Mg is not necessary for nucleotide association but is essential for the phosphatase activity. Binding and hydrolysis of ATP released photolytically from the caged precursor nitrophenylethyl-ATP was measured at 30 degrees C by infrared spectroscopy, demonstrating that phosphate groups are not involved in nucleotide binding. The hydrolytic kinetics was biphasic, and provides evidence for at least one reaction intermediate. Modelling of the forward reaction gave rise to three kinetic states connected by two intrinsic rate constants. The lower kinetic constant (k(1) = 4.7 x 10(-3) s(-1) at 30 degrees C) represents the first and rate-limiting reaction, probably reflecting the transition between the open and closed conformations of the domain pair. The subsequent step has a faster rate (k(2) = 17 x 10(-3) s(-1) at 30 degrees C), leading to product formation. Although the latter appears to be a single step, it probably comprises several reactions with presently unresolved intermediates. Based on these data, we suggest a model of the hydrolytic mechanism.

  17. Sirt1 carboxyl-domain is an ATP-repressible domain that is transferrable to other proteins

    PubMed Central

    Kang, Hyeog; Oka, Shinichi; Lee, Duck-Yeon; Park, Junhong; Aponte, Angel M.; Jung, Young-Sang; Bitterman, Jacob; Zhai, Peiyong; He, Yi; Kooshapur, Hamed; Ghirlando, Rodolfo; Tjandra, Nico; Lee, Sean B.; Kim, Myung K.; Sadoshima, Junichi; Chung, Jay H.

    2017-01-01

    Sirt1 is an NAD+-dependent protein deacetylase that regulates many physiological functions, including stress resistance, adipogenesis, cell senescence and energy production. Sirt1 can be activated by energy deprivation, but the mechanism is poorly understood. Here, we report that Sirt1 is negatively regulated by ATP, which binds to the C-terminal domain (CTD) of Sirt1. ATP suppresses Sirt1 activity by impairing the CTD's ability to bind to the deacetylase domain as well as its ability to function as the substrate recruitment site. ATP, but not NAD+, causes a conformational shift to a less compact structure. Mutations that prevent ATP binding increase Sirt1's ability to promote stress resistance and inhibit adipogenesis under high-ATP conditions. Interestingly, the CTD can be attached to other proteins, thereby converting them into energy-regulated proteins. These discoveries provide insight into how extreme energy deprivation can impact Sirt1 activity and underscore the complex nature of Sirt1 structure and regulation. PMID:28504272

  18. Ligand-induced global transitions in the catalytic domain of protein kinase A

    PubMed Central

    Hyeon, Changbong; Jennings, Patricia A.; Adams, Joseph A.; Onuchic, José N.

    2009-01-01

    Conformational transitions play a central role in the phosphorylation mechanisms of protein kinase. To understand the nature of these transitions, we investigated the dynamics of nucleotide binding to the catalytic domain of PKA, a prototype for the protein kinase enzyme family. The open-to-closed transition in PKA was constructed as a function of ATP association by using available X-ray data and Brownian dynamics. Analyzing the multiple kinetic trajectories at the residue level, we find that the spatial rearrangement of the residues around the nucleotide-binding pocket, along with suppressed local fluctuations, controls the compaction of the entire molecule. In addition, to accommodate the stresses induced by ATP binding at the early transition stage, partial unfoldings (cracking) and reformations of several native contacts occur at the interfaces between the secondary structure motifs enveloping the binding pocket. This suggests that the enzyme experiences local structural deformations while reaching its functional, ATP-bound state. Our dynamical view of the ligand-induced transitions in PKA suggests that the kinetic hierarchy of local and global dynamics, the variable fluctuation of residues and the necessity of partial local unfolding may be fundamental components in other large scale allosteric transitions. PMID:19204278

  19. Myosin motor domain lever arm rotation is coupled to ATP hydrolysis.

    PubMed

    Highsmith, S; Polosukhina, K; Eden, D

    2000-10-10

    We have investigated coupling of lever arm rotation to the ATP binding and hydrolysis steps for the myosin motor domain. In several current hypotheses of the mechanism of force production by muscle, the primary mechanical feature is the rotation of a lever arm that is a subdomain of the myosin motor domain. In these models, the lever arm rotates while the myosin motor domain is free, and then reverses the rotation to produce force while it is bound to actin. These mechanical steps are coupled to steps in the ATP hydrolysis cycle. Our hypothesis is that ATP hydrolysis induces lever arm rotation to produce a more compact motor domain that has stored mechanical energy. Our approach is to use transient electric birefringence techniques to measure changes in hydrodynamic size that result from lever arm rotation when various ligands are bound to isolated skeletal muscle myosin motor domain in solution. Results for ATP and CTP, which do support force production by muscle fibers, are compared to those of ATPgammaS and GTP, which do not. Measurements are also made of conformational changes when the motor domain is bound to NDP's and PP(i) in the absence and presence of the phosphate analogue orthovanadate, to determine the roles the nucleoside moieties of the nucleotides have on lever arm rotation. The results indicate that for the substrates investigated, rotation does not occur upon substrate binding, but is coupled to the NTP hydrolysis step. The data are consistent with a model in which only substrates that produce a motor domain-NDP-P(i) complex as the steady-state intermediate make the motor domain more compact, and only those substrates support force production.

  20. The catalytically active domain in the A subunit of calcineurin.

    PubMed

    Xiang, Benqiong; Liu, Ping; Jiang, Guohua; Zou, Ke; Yi, Fang; Yang, Shujie; Wei, Qun

    2003-01-01

    Calcineurin (CaN) is a heterodimer composed of a catalytic subunit A (CaNA) and a regulatory subunit B (CaNB). We report here an active truncated mutation of the rat CaNAdelta that contains only the catalytic domain (residues 1-347, also known as a/CaNA). The p-nitrophenyl phosphatase activity and protein phosphatase activity of a/CaNA were higher than that of CaNA. Both p-nitrophenyl phosphatase activity and protein phosphatase activity of a/CaNA were unaffected by CaM and the B-subunit; the B-subunit and CaM have relatively little effect on p-nitrophenyl phosphatase activity and a crucial effect on protein phosphatase activity of CaNA. Mn2+ and Ni2+ ions effeciently activated CaNA. The Km of a/CaNA was about 16 mM, and the k(cat) of a/CaNA was 10.03 s(-1) using pNPP as substrate. With RII peptide as a substrate, the Km of a/CaNA was about 21 microM and the k(cat) of a/CaNA was 0.51 s(-1). The optimum reaction temperature was about 45 degrees C, and the optimum reaction pH was about 7.2. Our results indicate that a/CaNA is the catalytic core of CaNA, and CaN and the B-subunit binding domain itself might play roles in the negative regulation of the phosphatase activity of CaN. The results provide the basis for future studies on the catalytic domain of CaN.

  1. ATP binding to p97/VCP D1 domain regulates selective recruitment of adaptors to its proximal N-domain.

    PubMed

    Chia, Wei Sheng; Chia, Diana Xueqi; Rao, Feng; Bar Nun, Shoshana; Geifman Shochat, Susana

    2012-01-01

    p97/Valosin-containing protein (VCP) is a member of the AAA-ATPase family involved in many cellular processes including cell division, intracellular trafficking and extraction of misfolded proteins in endoplasmic reticulum-associated degradation (ERAD). It is a homohexamer with each subunit containing two tandem D1 and D2 ATPase domains and N- and C-terminal regions that function as adaptor protein binding domains. p97/VCP is directed to its many different functional pathways by associating with various adaptor proteins. The regulation of the recruitment of the adaptor proteins remains unclear. Two adaptor proteins, Ufd1/Npl4 and p47, which bind exclusively to the p97/VCP N-domain and direct p97/VCP to either ERAD-related processes or homotypic fusion of Golgi fragments, were studied here. Surface plasmon resonance biosensor-based assays allowed the study of binding kinetics in real time. In competition experiments, it was observed that in the presence of ATP, Ufd1/Npl4 was able to compete more effectively with p47 for binding to p97/VCP. By using non-hydrolysable ATP analogues and the hexameric truncated p97/N-D1 fragment, it was shown that binding rather than hydrolysis of ATP to the proximal D1 domain strengthened the Ufd1/Npl4 association with the N-domain, thus regulating the recruitment of either Ufd1/Npl4 or p47. This novel role of ATP and an assigned function to the D1 AAA-ATPase domain link the multiple functions of p97/VCP to the metabolic status of the cell.

  2. Catalytic properties of ADAM12 and its domain deletion mutants.

    PubMed

    Jacobsen, Jonas; Visse, Robert; Sørensen, Hans Peter; Enghild, Jan J; Brew, Keith; Wewer, Ulla M; Nagase, Hideaki

    2008-01-15

    Human ADAM12 (a disintegrin and metalloproteinase) is a multidomain zinc metalloproteinase expressed at high levels during development and in human tumors. ADAM12 exists as two splice variants: a classical type 1 membrane-anchored form (ADAM12-L) and a secreted splice variant (ADAM12-S) consisting of pro, catalytic, disintegrin, cysteine-rich, and EGF domains. Here we present a novel activity of recombinant ADAM12-S and its domain deletion mutants on S-carboxymethylated transferrin (Cm-Tf). Cleavage of Cm-Tf occurred at multiple sites, and N-terminal sequencing showed that the enzyme exhibits restricted specificity but a consensus sequence could not be defined as its subsite requirements are promiscuous. Kinetic analysis revealed that the noncatalytic C-terminal domains are important regulators of Cm-Tf activity and that ADAM12-PC consisting of the pro domain and catalytic domain is the most active on this substrate. It was also observed that NaCl inhibits ADAM12. Among the tissue inhibitors of metalloproteinases (TIMP) examined, the N-terminal domain of TIMP-3 (N-TIMP-3) inhibits ADAM12-S and ADAM12-PC with low nanomolar Ki(app) values while TIMP-2 inhibits them with a slightly lower affinity (9-44 nM). However, TIMP-1 is a much weaker inhibitor. N-TIMP-3 variants that lack MMP inhibitory activity but retained the ability to inhibit ADAM17/TACE failed to inhibit ADAM12. These results indicate unique enzymatic properties of ADAM12 among the members of the ADAM family of metalloproteinases.

  3. Time-resolved Fourier Transform Infrared Spectroscopy of the Nucleotide-binding Domain from the ATP-binding Cassette Transporter MsbA

    PubMed Central

    Syberg, Falk; Suveyzdis, Yan; Kötting, Carsten; Gerwert, Klaus; Hofmann, Eckhard

    2012-01-01

    MsbA is an essential Escherichia coli ATP-binding cassette (ABC) transporter involved in the flipping of lipid A across the cytoplasmic membrane. It is a close homologue of human P-glycoprotein involved in multidrug resistance, and it similarly accepts a variety of small hydrophobic xenobiotics as transport substrates. X-ray structures of three full-length ABC multidrug exporters (including MsbA) have been published recently and reveal large conformational changes during the transport cycle. However, how ATP hydrolysis couples to these conformational changes and finally the transport is still an open question. We employed time-resolved FTIR spectroscopy, a powerful method to elucidate molecular reaction mechanisms of soluble and membrane proteins, to address this question with high spatiotemporal resolution. Here, we monitored the hydrolysis reaction in the nucleotide-binding domain of MsbA at the atomic level. The isolated MsbA nucleotide-binding domain hydrolyzed ATP with Vmax = 45 nmol mg−1 min−1, similar to the full-length transporter. A Hill coefficient of 1.49 demonstrates positive cooperativity between the two catalytic sites formed upon dimerization. Global fit analysis of time-resolved FTIR data revealed two apparent rate constants of ∼1 and 0.01 s−1, which were assigned to formation of the catalytic site and hydrolysis, respectively. Using isotopically labeled ATP, we identified specific marker bands for protein-bound ATP (1245 cm−1), ADP (1101 and 1205 cm−1), and free phosphate (1078 cm−1). Cleavage of the β-phosphate–γ-phosphate bond was found to be the rate-limiting step; no protein-bound phosphate intermediate was resolved. PMID:22593573

  4. Topology of ATP-binding domain of adrenoleukodystrophy gene product in peroxisomes.

    PubMed

    Contreras, M; Sengupta, T K; Sheikh, F; Aubourg, P; Singh, I

    1996-10-15

    Adrenoleukodystrophy (X-ALD) is a demyelinating disorder characterized by the accumulation of saturated very-long-chain fatty acids (> C22:0) due to the impaired activity of lignoceroyl-CoA ligase. The gene responsible for the disease was found to code for a 84-kDa peroxisomal integral membrane protein. Its amino acid sequence has high homology with the ATP-binding cassette superfamily of transporters and it is predicted to have six membrane-spanning segments and a putative ATP-binding domain. To define the function of ALDP, we studied the topology of its ATP-binding domain by using antibodies (1D6) against a hydrophobic domain (amino acid residues 279 to 482) and antibodies (Abct) against the C-terminal 15-amino-acid hydrophilic domain (amino acid residues 731 to 745) of ALDP. The observation of punctate fluorescence in permeabilized ALD fibroblasts, using Abct antibodies but not with antibodies against catalase, suggests that the C-terminal segment of ALDP is projected toward the cytoplasm from the peroxisomal membrane. Trypsinization of intact peroxisomes under isotonic conditions abolishes the Abct antibody recognition site, whereas the 1D6 antibodies identify a degradation product of 43-kDa protein that has been protected and retained by the membrane. This again suggests that the C-terminal portion of the ALDP protein is located on the outside (cytoplasmic) face of the peroxisomal membrane. Additional support for this conclusion was obtained by purification of the ALDP C-terminal domain, released from purified rat liver peroxisomes incubated with the cytosolic fraction, using blue-Sepharose affinity chromatography. A 47-kDa peptide retained by the column was recognized by Western blot analysis with Abct antibodies against the C-terminal sequence of ALDP and this polypeptide on polyvinylidene difluoride membrane was able to bind [gamma-32P]ATP in vitro in the presence of Mg2+. These results demonstrate that the C-terminal peptide containing the ATP

  5. Mutagenesis and modeling of the peroxiredoxin (Prx) complex with the NMR structure of ATP-bound human sulfiredoxin implicate aspartate 187 of Prx I as the catalytic residue in ATP hydrolysis.

    PubMed

    Lee, Duck-Yeon; Park, Sung Jun; Jeong, Woojin; Sung, Ho Jin; Oho, Taena; Wu, Xiongwu; Rhee, Sue Goo; Gruschus, James M

    2006-12-26

    The catalytic cysteine of certain members of the peroxiredoxin (Prx) family can be hyperoxidized to cysteinesulfinic acid during reduction of peroxides. Sulfiredoxin is responsible for the ATP-dependent reduction of cysteinesulfinic acid (SO2H) of hyperoxidized Prx. Here we report the NMR solution structure of human sulfiredoxin (hSrx), both with and without bound ATP, and we model the complex of ATP-bound hSrx with Prx. Binding ATP causes only small changes in the NMR structure of hSrx, and the bound ATP conformation is quite similar to that seen for the previously reported X-ray structure of the ADP-hSrx complex. Although hSrx binds ATP, it does not catalyze hydrolysis by itself and has no catalytic acid residue typical of most ATPase and kinase family proteins. For modeling the complex, the ATP-bound hSrx was docked to hyperoxidized Prx II using EMAP of CHARMM. In the model complex, Asn186 of Prx II (Asp187 of Prx I) is in contact with the hSrx-bound ATP beta- and gamma-phosphate groups. Asp187 of Prx I was mutated to alanine and asparagine, and binding and activity of the mutants with hSrx were compared to those of the wild type. For the D187N mutant, both binding and hydrolysis and reduction activities were comparable to those of the wild type, whereas for D187A, binding was unimpaired but ATP hydrolysis and reduction did not occur. The modeling and mutagenesis analyses strongly implicate Asp187 of Prx I as the catalytic residue responsible for ATP hydrolysis in the cysteinesulfinic acid reduction of Prx by hSrx.

  6. MgATP-concentration dependence of protection of yeast vacuolar V-ATPase from inactivation by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole supports a bi-site catalytic mechanism of ATP hydrolysis

    SciTech Connect

    Milgrom, Elena M.; Milgrom, Yakov M.

    2012-06-29

    Highlights: Black-Right-Pointing-Pointer MgATP protects V-ATPase from inactivation by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. Black-Right-Pointing-Pointer V-ATPase activity saturation with MgATP is not sufficient for complete protection. Black-Right-Pointing-Pointer The results support a bi-site catalytic mechanism for V-ATPase. -- Abstract: Catalytic site occupancy of the yeast vacuolar V-ATPase during ATP hydrolysis in the presence of an ATP-regenerating system was probed using sensitivity of the enzyme to inhibition by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). The results show that, regardless of the presence or absence of the proton-motive force across the vacuolar membrane, saturation of V-ATPase activity at increasing MgATP concentrations is accompanied by only partial protection of the enzyme from inhibition by NBD-Cl. Both in the presence and absence of an uncoupler, complete protection of V-ATPase from inhibition by NBD-Cl requires MgATP concentrations that are significantly higher than those expected from the K{sub m} values for MgATP. The results are inconsistent with a tri-site model and support a bi-site model for a mechanism of ATP hydrolysis by V-ATPase.

  7. Translocation of the Catalytic Domain of Diphtheria Toxin across Planar Phospholipid Bilayers by Its Own T Domain

    NASA Astrophysics Data System (ADS)

    Oh, Kyoung Joon; Senzel, Lisa; Collier, R. John; Finkelstein, Alan

    1999-07-01

    The T domain of diphtheria toxin is known to participate in the pH-dependent translocation of the catalytic C domain of the toxin across the endosomal membrane, but how it does so, and whether cellular proteins are also required for this process, remain unknown. Here, we report results showing that the T domain alone is capable of translocating the entire C domain across model, planar phospholipid bilayers in the absence of other proteins. The T domain therefore contains the entire molecular machinery for mediating transfer of the catalytic domain of diphtheria toxin across membranes.

  8. ATP forms a stable complex with the essential histidine kinase WalK (YycG) domain

    SciTech Connect

    Celikel, Reha; Veldore, Vidya Harini; Mathews, Irimpan; Devine, Kevin M.; Varughese, Kottayil I.

    2012-07-01

    The histidine WalK (YycG) plays a crucial role in coordinating murein synthesis with cell division and the crystal structure of its ATP binding domain has been determined. Interestingly the bound ATP was not hydrolyzed during crystallization and remains intact in the crystal lattice. In Bacillus subtilis, the WalRK (YycFG) two-component system coordinates murein synthesis with cell division. It regulates the expression of autolysins that function in cell-wall remodeling and of proteins that modulate autolysin activity. The transcription factor WalR is activated upon phosphorylation by the histidine kinase WalK, a multi-domain homodimer. It autophosphorylates one of its histidine residues by transferring the γ-phosphate from ATP bound to its ATP-binding domain. Here, the high-resolution crystal structure of the ATP-binding domain of WalK in complex with ATP is presented at 1.61 Å resolution. The bound ATP remains intact in the crystal lattice. It appears that the strong binding interactions and the nature of the binding pocket contribute to its stability. The triphosphate moiety of ATP wraps around an Mg{sup 2+} ion, providing three O atoms for coordination in a near-ideal octahedral geometry. The ATP molecule also makes strong interactions with the protein. In addition, there is a short contact between the exocyclic O3′ of the sugar ring and O2B of the β-phosphate, implying an internal hydrogen bond. The stability of the WalK–ATP complex in the crystal lattice suggests that such a complex may exist in vivo poised for initiation of signal transmission. This feature may therefore be part of the sensing mechanism by which the WalRK two-component system is so rapidly activated when cells encounter conditions conducive for growth.

  9. [Peptide hydrolases with catalytic dyad Ser-Lys. Similarity and distinctions of the active centers of ATP-dependent Lon proteases, LexA repressors, signal peptidases and C-terminal processing proteases].

    PubMed

    Rotanova, T V

    2002-01-01

    It is established that ATP-dependent protease Lon family belongs to the serine-lysine peptide hydrolases clan. Significant similarity of amino acid sequences of proteases Lon and repressors LexA in the regions including the catalytic serine and lysine residues is revealed by comparing primary structures of different families of the enzymes with Ser-Lys catalytic dyad. The both Lon and LexA families are shown to be divided into two subfamilies in accordance with the nature of amino acids in the catalytically active serine environment. Putative DNA binding sites are revealed in proteolytic domains of Lon A subfamily. Similarities and distinctions of the all families peptide hydrolases of the clan in the regions of their active centers are discussed.

  10. Dye removal, catalytic activity and 2D crystallization of chloroplast H(+)-ATP synthase purified by blue native electrophoresis.

    PubMed

    Poetsch, A; Neff, D; Seelert, H; Schägger, H; Dencher, N A

    2000-06-01

    The proton-ATP synthase of thylakoid membranes from spinach chloroplasts (CF(O)F(1)) and its subcomplexes CF(O) and CF(1) were isolated by blue native electrophoresis (BN-PAGE) [Neff, D. and Dencher, N.A. (1999) Biochem. Biophys. Res. Commun. 259, 569-575] and subsequently electroeluted from the gel. A method was developed to remove most of the dye Coomassie G-250 (CBG) using gel filtration, a prerequisite for many biophysical investigations. The dye was removed from the electroeluted CF(O)F(1), CF(O) or CF(1) and exchanged with the detergent CHAPS. ATP hydrolysis activity of CF(1) and ATP synthesis activity of reconstituted CF(O)F(1) were determined before and after dye removal. The secondary structure of CF(O) was studied by CD spectroscopy in the presence and the absence of the dye. CBG neither abolishes the catalytic activity of the isolated CF(O)F(1) and CF(1) nor affects the subunit composition and the high alpha-helical content of CF(O). In crystallization attempts, 2D arrays of CF(O)F(1) and of CF(O) before and after dye removal were obtained. In the aggregates of CF(O), circular structures with a mean diameter of 6.7 nm were observed. Our results indicate that the combination of BN-PAGE and dye removal by gel filtration is a suitable approach to obtain catalytically active protein complexes for further functional and structural characterization.

  11. Evidence for a requirement for ATP hydrolysis at two distinct steps during a single turnover of the catalytic cycle of human P-glycoprotein

    PubMed Central

    Sauna, Zuben E.; Ambudkar, Suresh V.

    2000-01-01

    P-glycoprotein (Pgp) is an ATP-dependent hydrophobic natural product anticancer drug efflux pump whose overexpression confers multidrug resistance to tumor cells. The work reported here deals with the elucidation of the energy requirement for substrate interaction with Pgp during the catalytic cycle. We show that the Kd (412 nM) of the substrate analogue [125I]iodoarylazidoprazoin for Pgp is not altered by the presence of the nonhydrolyzable nucleotide 5′-adenylylimididiphosphate and vanadate (Kd = 403 nM). Though binding of nucleotide per se does not affect interactions with the substrate, ATP hydrolysis results in a dramatic conformational change where the affinity of [125I]iodoarylazidoprazoin for Pgp trapped in transition-state conformation (Pgp⋅ADP⋅vanadate) is reduced >30-fold. To transform Pgp from this intermediate state of low affinity for substrate to the next catalytic cycle, i.e., a conformation that binds substrate with high affinity, requires conditions that permit ATP hydrolysis. Additionally, there is an inverse correlation (R2 = 0.96) between 8AzidoADP (or ADP) release and the recovery of substrate binding. These results suggest that the release of nucleotide is necessary for reactivation but not sufficient. The hydrolysis of additional molecule(s) of ATP (or 8AzidoATP) is obligatory for the catalytic cycle to advance to completion. These data are consistent with the observed stoichiometry of two ATP molecules hydrolyzed for the transport of every substrate molecule. Our data demonstrate two distinct roles for ATP hydrolysis in a single turnover of the catalytic cycle of Pgp, one in the transport of substrate and the other in effecting conformational changes to reset the pump for the next catalytic cycle. PMID:10716986

  12. The Lid Domain of Caenorhabditis elegans Hsc70 Influences ATP Turnover, Cofactor Binding and Protein Folding Activity

    PubMed Central

    Papsdorf, Katharina; Gaiser, Andreas M.; Richter, Klaus

    2012-01-01

    Hsc70 is a conserved ATP-dependent molecular chaperone, which utilizes the energy of ATP hydrolysis to alter the folding state of its client proteins. In contrast to the Hsc70 systems of bacteria, yeast and humans, the Hsc70 system of C. elegans (CeHsc70) has not been studied to date. We find that CeHsc70 is characterized by a high ATP turnover rate and limited by post-hydrolysis nucleotide exchange. This rate-limiting step is defined by the helical lid domain at the C-terminus. A certain truncation in this domain (CeHsc70-Δ545) reduces the turnover rate and renders the hydrolysis step rate-limiting. The helical lid domain also affects cofactor affinities as the lidless mutant CeHsc70-Δ512 binds more strongly to DNJ-13, forming large protein complexes in the presence of ATP. Despite preserving the ability to hydrolyze ATP and interact with its cofactors DNJ-13 and BAG-1, the truncation of the helical lid domain leads to the loss of all protein folding activity, highlighting the requirement of this domain for the functionality of the nematode's Hsc70 protein. PMID:22479492

  13. Molecular Identification of Carnosine Synthase as ATP-grasp Domain-containing Protein 1 (ATPGD1)*

    PubMed Central

    Drozak, Jakub; Veiga-da-Cunha, Maria; Vertommen, Didier; Stroobant, Vincent; Van Schaftingen, Emile

    2010-01-01

    Carnosine (β-alanyl-l-histidine) and homocarnosine (γ-aminobutyryl-l-histidine) are abundant dipeptides in skeletal muscle and brain of most vertebrates and some invertebrates. The formation of both compounds is catalyzed by carnosine synthase, which is thought to convert ATP to AMP and inorganic pyrophosphate, and whose molecular identity is unknown. In the present work, we have purified carnosine synthase from chicken pectoral muscle about 1500-fold until only two major polypeptides of 100 and 90 kDa were present in the preparation. Mass spectrometry analysis of these polypeptides did not yield any meaningful candidate. Carnosine formation catalyzed by the purified enzyme was accompanied by a stoichiometric formation, not of AMP, but of ADP, suggesting that carnosine synthase belongs to the “ATP-grasp family” of ligases. A data base mining approach identified ATPGD1 as a likely candidate. As this protein was absent from chicken protein data bases, we reconstituted its sequence from a PCR-amplified cDNA and found it to fit with the 100-kDa polypeptide of the chicken carnosine synthase preparation. Mouse and human ATPGD1 were expressed in HEK293T cells, purified to homogeneity, and shown to catalyze the formation of carnosine, as confirmed by mass spectrometry, and of homocarnosine. Specificity studies carried out on all three enzymes were in agreement with published data. In particular, they acted with 15–25-fold higher catalytic efficiencies on β-alanine than on γ-aminobutyrate. The identification of the gene encoding carnosine synthase will help for a better understanding of the biological functions of carnosine and related dipeptides, which still remain largely unknown. PMID:20097752

  14. Mutations in the NB-ARC Domain of I-2 That Impair ATP Hydrolysis Cause Autoactivation1[OA

    PubMed Central

    Tameling, Wladimir I.L.; Vossen, Jack H.; Albrecht, Mario; Lengauer, Thomas; Berden, Jan A.; Haring, Michel A.; Cornelissen, Ben J.C.; Takken, Frank L.W.

    2006-01-01

    Resistance (R) proteins in plants confer specificity to the innate immune system. Most R proteins have a centrally located NB-ARC (nucleotide-binding adaptor shared by APAF-1, R proteins, and CED-4) domain. For two tomato (Lycopersicon esculentum) R proteins, I-2 and Mi-1, we have previously shown that this domain acts as an ATPase module that can hydrolyze ATP in vitro. To investigate the role of nucleotide binding and hydrolysis for the function of I-2 in planta, specific mutations were introduced in conserved motifs of the NB-ARC domain. Two mutations resulted in autoactivating proteins that induce a pathogen-independent hypersensitive response upon expression in planta. These mutant forms of I-2 were found to be impaired in ATP hydrolysis, but not in ATP binding, suggesting that the ATP- rather than the ADP-bound state of I-2 is the active form that triggers defense signaling. In addition, upon ADP binding, the protein displayed an increased affinity for ADP suggestive of a change of conformation. Based on these data, we propose that the NB-ARC domain of I-2, and likely of related R proteins, functions as a molecular switch whose state (on/off) depends on the nucleotide bound (ATP/ADP). PMID:16489136

  15. The CXXC motifs in the metal binding domains are required for ATP7B to mediate resistance to cisplatin☆

    PubMed Central

    Safaei, Roohangiz; Adams, Preston L.; Maktabi, Mohammad H.; Mathews, Ryan A.; Howell, Stephen B.

    2013-01-01

    The copper (Cu) exporter ATP7B mediates resistance to cisplatin (cDDP) but details of the mechanism are unknown. We explored the role of the CXXC motifs in the metal binding domains (MBDs) of ATP7B by investigating binding of cDDP to the sixth metal binding domain (MBD6) or a variant in which the CXXC motif was converted to SXXS. Platinum measurement showed that cDDP bound to wild type MBD6 but not to the SXXS variant. Wild type ATP7B rendered ovarian 2008 cells resistant to cDDP. In 2008 and in HEK293T cells, wild type ATP7B trafficked from TGN to peripheral locations in response to Cu or cDDP. A variant in which the CXXC motifs in all 6 MBDs were converted to SXXS localized correctly to the TGN but failed to traffic when exposed to either Cu or cDDP. Deletion of either the first 5 MBDs or all 6 MBDs resulted in failure to localize to the TGN. Neither the SXXS variant nor the deletion variant was able to mediate resistance to cDDP. We conclude that cDDP binds to the CXXC motifs of ATP7B and that this interaction is essential to the trafficking of ATP7B and to its ability to mediate resistance to cDDP. PMID:22459168

  16. The CXXC motifs in the metal binding domains are required for ATP7B to mediate resistance to cisplatin.

    PubMed

    Safaei, Roohangiz; Adams, Preston L; Maktabi, Mohammad H; Mathews, Ryan A; Howell, Stephen B

    2012-05-01

    The copper (Cu) exporter ATP7B mediates resistance to cisplatin (cDDP) but details of the mechanism are unknown. We explored the role of the CXXC motifs in the metal binding domains (MBDs) of ATP7B by investigating binding of cDDP to the sixth metal binding domain (MBD6) or a variant in which the CXXC motif was converted to SXXS. Platinum measurement showed that cDDP bound to wild type MBD6 but not to the SXXS variant. Wild type ATP7B rendered ovarian 2008 cells resistant to cDDP. In 2008 and in HEK293T cells, wild type ATP7B trafficked from TGN to peripheral locations in response to Cu or cDDP. A variant in which the CXXC motifs in all 6 MBDs were converted to SXXS localized correctly to the TGN but failed to traffic when exposed to either Cu or cDDP. Deletion of either the first 5 MBDs or all 6 MBDs resulted in failure to localize to the TGN. Neither the SXXS variant nor the deletion variant was able to mediate resistance to cDDP. We conclude that cDDP binds to the CXXC motifs of ATP7B and that this interaction is essential to the trafficking of ATP7B and to its ability to mediate resistance to cDDP. Copyright © 2012 Elsevier Inc. All rights reserved.

  17. Possible role of inter-domain salt bridges in oligopeptidase B from Trypanosoma brucei: critical role of Glu172 of non-catalytic β-propeller domain in catalytic activity and Glu490 of catalytic domain in stability of OPB.

    PubMed

    Fukumoto, Junki; Ismail, Nor Ismaliza Mohd; Kubo, Masaki; Kinoshita, Keita; Inoue, Masahiro; Yuasa, Keizo; Nishimoto, Makoto; Matsuki, Hitoshi; Tsuji, Akihiko

    2013-11-01

    Oligopeptidase B (OPB) is a member of the prolyl oligopeptidase (POP) family of serine proteases. OPB in trypanosomes is an important virulence factor and potential pharmaceutical target. Characteristic structural features of POP family members include lack of a propeptide and presence of a β-propeller domain (PD), although the role of the β-PD has yet to be fully understood. In this work, residues Glu(172), Glu(490), Glu(524) and Arg(689) in Trypanosoma brucei OPB (Tb OPB), which are predicted to form inter-domain salt bridges, were substituted for Gln and Ala, respectively. These mutants were evaluated in terms of catalytic properties and stability. A negative effect on kcat/Km was obtained following mutation of Glu(172) or Arg(689). In contrast, the E490Q mutant exhibited markedly decreased thermal stability, although this mutation had less effect on catalytic properties compared to the E172Q and R689A mutants. Trypsin digestion showed that the boundary regions between the β-PD and catalytic domains (CDs) of the E490Q mutant are unfolded with heat treatment. These results indicated that Glu(490) in the CD plays a role in stabilization of Tb OPB, whereas Glu(172) in the β-PD is critical for the catalytic activity of Tb OPB.

  18. Crystal structures of Cg1458 reveal a catalytic lid domain and a common catalytic mechanism for the FAH family.

    PubMed

    Ran, Tingting; Gao, Yanyan; Marsh, May; Zhu, Wenjun; Wang, Meitian; Mao, Xiang; Xu, Langlai; Xu, Dongqing; Wang, Weiwu

    2013-01-01

    Cg1458 was recently characterized as a novel soluble oxaloacetate decarboxylase. However, sequence alignment identified that Cg1458 has no similarity with other oxaloacetate decarboxylases and instead belongs to the FAH (fumarylacetoacetate hydrolase) family. Differences in the function of Cg1458 and other FAH proteins may suggest a different catalytic mechanism. To help elucidate the catalytic mechanism of Cg1458, crystal structures of Cg1458 in both the open and closed conformations have been determined for the first time up to a resolution of 1.9 Å (1 Å=0.1 nm) and 2.0 Å respectively. Comparison of both structures and detailed biochemical studies confirmed the presence of a catalytic lid domain which is missing in the native enzyme structure. In this lid domain, a glutamic acid-histidine dyad was found to be critical in mediating enzymatic catalysis. On the basis of structural modelling and comparison, as well as large-scale sequence alignment studies, we further determined that the catalytic mechanism of Cg1458 is actually through a glutamic acid-histidine-water triad, and this catalytic triad is common among FAH family proteins that catalyse the cleavage of the C-C bond of the substrate. Two sequence motifs, HxxE and Hxx…xxE have been identified as the basis for this mechanism.

  19. Calcium-induced conformational changes in the regulatory domain of the human mitochondrial ATP-Mg/Pi carrier

    PubMed Central

    Harborne, Steven P.D.; Ruprecht, Jonathan J.; Kunji, Edmund R.S.

    2015-01-01

    The mitochondrial ATP-Mg/Pi carrier imports adenine nucleotides from the cytosol into the mitochondrial matrix and exports phosphate. The carrier is regulated by the concentration of cytosolic calcium, altering the size of the adenine nucleotide pool in the mitochondrial matrix in response to energetic demands. The protein consists of three domains; (i) the N-terminal regulatory domain, which is formed of two pairs of fused calcium-binding EF-hands, (ii) the C-terminal mitochondrial carrier domain, which is involved in transport, and (iii) a linker region with an amphipathic α-helix of unknown function. The mechanism by which calcium binding to the regulatory domain modulates substrate transport in the carrier domain has not been resolved. Here, we present two new crystal structures of the regulatory domain of the human isoform 1. Careful analysis by SEC confirmed that although the regulatory domain crystallised as dimers, full-length ATP-Mg/Pi carrier is monomeric. Therefore, the ATP-Mg/Pi carrier must have a different mechanism of calcium regulation than the architecturally related aspartate/glutamate carrier, which is dimeric. The structure showed that an amphipathic α-helix is bound to the regulatory domain in a hydrophobic cleft of EF-hand 3/4. Detailed bioinformatics analyses of different EF-hand states indicate that upon release of calcium, EF-hands close, meaning that the regulatory domain would release the amphipathic α-helix. We propose a mechanism for ATP-Mg/Pi carriers in which the amphipathic α-helix becomes mobile upon release of calcium and could block the transport of substrates across the mitochondrial inner membrane. PMID:26164100

  20. Asymmetric Hsp90 N-domain SUMOylation recruits Aha1 and ATP-competitive inhibitors

    PubMed Central

    Mollapour, Mehdi; Bourboulia, Dimitra; Beebe, Kristin; Woodford, Mark R.; Polier, Sigrun; Hoang, Anthony; Chelluri, Raju; Li, Yu; Guo, Ailan; Lee, Min-Jung; Fotooh-Abadi, Elham; Khan, Sahar; Prince, Thomas; Miyajima, Naoto; Yoshida, Soichiro; Tsutsumi, Shinji; Xu, Wanping; Panaretou, Barry; Stetler-Stevenson, William G.; Bratslavsky, Gennady; Trepel, Jane B.; Prodromou, Chrisostomos; Neckers, Len

    2014-01-01

    Summary The stability and activity of numerous signaling proteins in both normal and cancer cells depends on the dimeric molecular chaperone Heat Shock Protein 90 (Hsp90). Hsp90 function is coupled to ATP binding and hydrolysis, and requires a series of conformational changes that are regulated by co-chaperones and numerous posttranslational modifications (PTMs). SUMOylation is one of the least understood Hsp90 PTMs. Here we show that asymmetric SUMOylation of a conserved lysine residue in the N-domain of both yeast (K178) and human (K191) Hsp90 facilitates both recruitment of the ATPase activating co-chaperone Aha1 and, unexpectedly, the binding of Hsp90 inhibitors, suggesting that these drugs associate preferentially with Hsp90 proteins that are actively engaged in the chaperone cycle. Importantly, cellular transformation is accompanied by elevated steady-state N-domain SUMOylation and increased Hsp90 SUMOylation sensitizes yeast and mammalian cells to Hsp90 inhibitors, providing a mechanism to explain the sensitivity of cancer cells to these drugs. PMID:24462205

  1. Characterization of the Catalytic and Nucleotide Binding Properties of the α-Kinase Domain of Dictyostelium Myosin-II Heavy Chain Kinase A*

    PubMed Central

    Yang, Yidai; Ye, Qilu; Jia, Zongchao; Côté, Graham P.

    2015-01-01

    The α-kinases are a widely expressed family of serine/threonine protein kinases that exhibit no sequence identity with conventional eukaryotic protein kinases. In this report, we provide new information on the catalytic properties of the α-kinase domain of Dictyostelium myosin-II heavy chain kinase-A (termed A-CAT). Crystallization of A-CAT in the presence of MgATP yielded structures with AMP or adenosine in the catalytic cleft together with a phosphorylated Asp-766 residue. The results show that the β- and α-phosphoryl groups are transferred either directly or indirectly to the catalytically essential Asp-766. Biochemical assays confirmed that A-CAT hydrolyzed ATP, ADP, and AMP with kcat values of 1.9, 0.6, and 0.32 min−1, respectively, and showed that A-CAT can use ADP to phosphorylate peptides and proteins. Binding assays using fluorescent 2′/3′-O-(N-methylanthraniloyl) analogs of ATP and ADP yielded Kd values for ATP, ADP, AMP, and adenosine of 20 ± 3, 60 ± 20, 160 ± 60, and 45 ± 15 μm, respectively. Site-directed mutagenesis showed that Glu-713, Leu-716, and Lys-645, all of which interact with the adenine base, were critical for nucleotide binding. Mutation of the highly conserved Gln-758, which chelates a nucleotide-associated Mg2+ ion, eliminated catalytic activity, whereas loss of the highly conserved Lys-722 and Arg-592 decreased kcat values for kinase and ATPase activities by 3–6-fold. Mutation of Asp-663 impaired kinase activity to a much greater extent than ATPase, indicating a specific role in peptide substrate binding, whereas mutation of Gln-768 doubled ATPase activity, suggesting that it may act to exclude water from the active site. PMID:26260792

  2. Processing of metacaspase into a cytoplasmic catalytic domain mediating cell death in Leishmania major.

    PubMed

    Zalila, Habib; González, Iveth J; El-Fadili, Amal Kuendig; Delgado, Maria Belen; Desponds, Chantal; Schaff, Cédric; Fasel, Nicolas

    2011-01-01

    Metacaspases are cysteine peptidases that could play a role similar to caspases in the cell death programme of plants, fungi and protozoa. The human protozoan parasite Leishmania major expresses a single metacaspase (LmjMCA) harbouring a central domain with the catalytic dyad histidine and cysteine as found in caspases. In this study, we investigated the processing sites important for the maturation of LmjMCA catalytic domain, the cellular localization of LmjMCA polypeptides, and the functional role of the catalytic domain in the cell death pathway of Leishmania parasites. Although LmjMCA polypeptide precursor form harbours a functional mitochondrial localization signal (MLS), we determined that LmjMCA polypeptides are mainly localized in the cytoplasm. In stress conditions, LmjMCA precursor forms were extensively processed into soluble forms containing the catalytic domain. This domain was sufficient to enhance sensitivity of parasites to hydrogen peroxide by impairing the mitochondrion. These data provide experimental evidences of the importance of LmjMCA processing into an active catalytic domain and of its role in disrupting mitochondria, which could be relevant in the design of new drugs to fight leishmaniasis and likely other protozoan parasitic diseases.

  3. Inhibition of soluble guanylyl cyclase by small molecules targeting the catalytic domain.

    PubMed

    Vijayaraghavan, Jagamya; Kramp, Kristopher; Harris, Michael E; van den Akker, Focco

    2016-10-01

    Soluble guanylyl cyclase (sGC) plays a crucial role in cyclic nucleotide signaling that regulates numerous important physiological processes. To identify new sGC inhibitors that may prevent the formation of the active catalytic domain conformation, we carried out an in silico docking screen targeting a 'backside pocket' of the inactive sGC catalytic domain structure. Compounds 1 and 2 were discovered to inhibit sGC even at high/saturating nitric oxide concentrations. Both compounds also inhibit the BAY 58-2667-activated sGC as well as BAY 41-2272-stimulated sGC activity. Additional biochemical analyses showed that compound 2 also inhibits the isolated catalytic domain, thus demonstrating functional binding to this domain. Both compounds have micromolar affinity for sGC and are potential leads to develop more potent sGC inhibitors. © 2016 Federation of European Biochemical Societies.

  4. Agrobacterium rhizogenes GALLS Protein Contains Domains for ATP Binding, Nuclear Localization, and Type IV Secretion▿

    PubMed Central

    Hodges, Larry D.; Vergunst, Annette C.; Neal-McKinney, Jason; den Dulk-Ras, Amke; Moyer, Deborah M.; Hooykaas, Paul J. J.; Ream, Walt

    2006-01-01

    Agrobacterium tumefaciens and Agrobacterium rhizogenes are closely related plant pathogens that cause different diseases, crown gall and hairy root. Both diseases result from transfer, integration, and expression of plasmid-encoded bacterial genes located on the transferred DNA (T-DNA) in the plant genome. Bacterial virulence (Vir) proteins necessary for infection are also translocated into plant cells. Transfer of single-stranded DNA (ssDNA) and Vir proteins requires a type IV secretion system, a protein complex spanning the bacterial envelope. A. tumefaciens translocates the ssDNA-binding protein VirE2 into plant cells, where it binds single-stranded T-DNA and helps target it to the nucleus. Although some strains of A. rhizogenes lack VirE2, they are pathogenic and transfer T-DNA efficiently. Instead, these bacteria express the GALLS protein, which is essential for their virulence. The GALLS protein can complement an A. tumefaciens virE2 mutant for tumor formation, indicating that GALLS can substitute for VirE2. Unlike VirE2, GALLS contains ATP-binding and helicase motifs similar to those in TraA, a strand transferase involved in conjugation. Both GALLS and VirE2 contain nuclear localization sequences and a C-terminal type IV secretion signal. Here we show that mutations in any of these domains abolished the ability of GALLS to substitute for VirE2. PMID:17012398

  5. Autoinhibition of a calmodulin-dependent calcium pump involves a structure in the stalk that connects the transmembrane domain to the ATPase catalytic domain

    NASA Technical Reports Server (NTRS)

    Curran, A. C.; Hwang, I.; Corbin, J.; Martinez, S.; Rayle, D.; Sze, H.; Harper, J. F.; Evans, M. L. (Principal Investigator)

    2000-01-01

    The regulation of Ca(2+)-pumps is important for controlling [Ca(2+)] in the cytosol and organelles of all eukaryotes. Here, we report a genetic strategy to identify residues that function in autoinhibition of a novel calmodulin-activated Ca(2+)-pump with an N-terminal regulatory domain (isoform ACA2 from Arabidopsis). Mutant pumps with constitutive activity were identified by complementation of a yeast (K616) deficient in two Ca(2+)-pumps. Fifteen mutations were found that disrupted a segment of the N-terminal autoinhibitor located between Lys(23) and Arg(54). Three mutations (E167K, D219N, and E341K) were found associated with the stalk that connects the ATPase catalytic domain (head) and with the transmembrane domain. Enzyme assays indicated that the stalk mutations resulted in calmodulin-independent activity, with V(max), K(mATP), and K(mCa(2+)) similar to that of a pump in which the N-terminal autoinhibitor had been deleted. A highly conservative substitution at Asp(219) (D219E) still produced a deregulated pump, indicating that the autoinhibitory structure in the stalk is highly sensitive to perturbation. In plasma membrane H(+)-ATPases from yeast and plants, similarly positioned mutations resulted in hyperactive pumps. Together, these results suggest that a structural feature of the stalk is of general importance in regulating diverse P-type ATPases.

  6. Autoinhibition of a calmodulin-dependent calcium pump involves a structure in the stalk that connects the transmembrane domain to the ATPase catalytic domain.

    PubMed

    Curran, A C; Hwang, I; Corbin, J; Martinez, S; Rayle, D; Sze, H; Harper, J F

    2000-09-29

    The regulation of Ca(2+)-pumps is important for controlling [Ca(2+)] in the cytosol and organelles of all eukaryotes. Here, we report a genetic strategy to identify residues that function in autoinhibition of a novel calmodulin-activated Ca(2+)-pump with an N-terminal regulatory domain (isoform ACA2 from Arabidopsis). Mutant pumps with constitutive activity were identified by complementation of a yeast (K616) deficient in two Ca(2+)-pumps. Fifteen mutations were found that disrupted a segment of the N-terminal autoinhibitor located between Lys(23) and Arg(54). Three mutations (E167K, D219N, and E341K) were found associated with the stalk that connects the ATPase catalytic domain (head) and with the transmembrane domain. Enzyme assays indicated that the stalk mutations resulted in calmodulin-independent activity, with V(max), K(mATP), and K(mCa(2+)) similar to that of a pump in which the N-terminal autoinhibitor had been deleted. A highly conservative substitution at Asp(219) (D219E) still produced a deregulated pump, indicating that the autoinhibitory structure in the stalk is highly sensitive to perturbation. In plasma membrane H(+)-ATPases from yeast and plants, similarly positioned mutations resulted in hyperactive pumps. Together, these results suggest that a structural feature of the stalk is of general importance in regulating diverse P-type ATPases.

  7. Autoinhibition of a calmodulin-dependent calcium pump involves a structure in the stalk that connects the transmembrane domain to the ATPase catalytic domain

    NASA Technical Reports Server (NTRS)

    Curran, A. C.; Hwang, I.; Corbin, J.; Martinez, S.; Rayle, D.; Sze, H.; Harper, J. F.; Evans, M. L. (Principal Investigator)

    2000-01-01

    The regulation of Ca(2+)-pumps is important for controlling [Ca(2+)] in the cytosol and organelles of all eukaryotes. Here, we report a genetic strategy to identify residues that function in autoinhibition of a novel calmodulin-activated Ca(2+)-pump with an N-terminal regulatory domain (isoform ACA2 from Arabidopsis). Mutant pumps with constitutive activity were identified by complementation of a yeast (K616) deficient in two Ca(2+)-pumps. Fifteen mutations were found that disrupted a segment of the N-terminal autoinhibitor located between Lys(23) and Arg(54). Three mutations (E167K, D219N, and E341K) were found associated with the stalk that connects the ATPase catalytic domain (head) and with the transmembrane domain. Enzyme assays indicated that the stalk mutations resulted in calmodulin-independent activity, with V(max), K(mATP), and K(mCa(2+)) similar to that of a pump in which the N-terminal autoinhibitor had been deleted. A highly conservative substitution at Asp(219) (D219E) still produced a deregulated pump, indicating that the autoinhibitory structure in the stalk is highly sensitive to perturbation. In plasma membrane H(+)-ATPases from yeast and plants, similarly positioned mutations resulted in hyperactive pumps. Together, these results suggest that a structural feature of the stalk is of general importance in regulating diverse P-type ATPases.

  8. Domain closure in the catalytic chains of Escherichia coli aspartate transcarbamoylase influences the kinetic mechanism.

    PubMed

    Lee, B H; Ley, B W; Kantrowitz, E R; O'Leary, M H; Wedler, F C

    1995-06-30

    The closure of the two domains of the catalytic chains of Escherichia coli aspartate transcarbamoylase, which is critical for completion of the T-->R transition, is stabilized by salt-bridges between Glu-50 and both Arg-167 and Arg-234. Mutation of Glu-50 to Ala shifts the enzyme toward a low activity, low affinity state (Newton, C. J., and Kantrowitz, E. R. (1990) Biochemistry, 29, 1444-1451). Kinetic isotope effects (KIE) and equilibrium isotope exchange kinetics (EIEK) have been used to probe the dynamic properties of the Glu-50-->Ala enzyme. Unlike the behavior of the wild-type enzyme, the observed kinetic isotope effect for 13C versus 12C at the carbonyl group of carbamoyl phosphate (CP) increased upon the binding of ligands which promote the formation of the R-state (Asp, N-phosphonacetyl-L-aspartate (PALA), or ATP). The maximum rate for the [14C]Asp<-->Carbamoyl aspartate (CAsp) exchange with the Glu-50-->Ala enzyme was 500-fold slower than for the wild-type enzyme; however, the rate for the [14C]CP<-->CAsp exchange was only 50-fold slower, reversing the relative rates observed with the wild-type enzyme. In addition, upon variation of substrate pairs involving Asp or CAsp, loss of inhibition effects in the CP<-->CAsp exchange indicated that the Glu-50-->Ala substitution caused the kinetic mechanism for the mutant enzyme to shift from ordered to random. Computer simulations of the EIEK data indicate that the Glu-50-->Ala mutation specifically causes strong decreases in the rates of catalysis and association-dissociation for Asp and CAsp, with minimal effects on the CP and Pi on-off rates. With substrates bound, the Glu-50-->Ala enzyme apparently does not attain a full R-state conformation. The PALA-activated Glu-50-->Ala enzyme, however, exhibits substrate affinities comparable to those for the wild-type enzyme, but fails to restore the preferred order substrate binding. Unlike the wild-type enzyme, both the T and R-states of the Glu-50-->Ala enzyme

  9. The structure of the catalytic domain of a plant cellulose synthase and its assembly into dimers.

    PubMed

    Olek, Anna T; Rayon, Catherine; Makowski, Lee; Kim, Hyung Rae; Ciesielski, Peter; Badger, John; Paul, Lake N; Ghosh, Subhangi; Kihara, Daisuke; Crowley, Michael; Himmel, Michael E; Bolin, Jeffrey T; Carpita, Nicholas C

    2014-07-01

    Cellulose microfibrils are para-crystalline arrays of several dozen linear (1→4)-β-d-glucan chains synthesized at the surface of the cell membrane by large, multimeric complexes of synthase proteins. Recombinant catalytic domains of rice (Oryza sativa) CesA8 cellulose synthase form dimers reversibly as the fundamental scaffold units of architecture in the synthase complex. Specificity of binding to UDP and UDP-Glc indicates a properly folded protein, and binding kinetics indicate that each monomer independently synthesizes single glucan chains of cellulose, i.e., two chains per dimer pair. In contrast to structure modeling predictions, solution x-ray scattering studies demonstrate that the monomer is a two-domain, elongated structure, with the smaller domain coupling two monomers into a dimer. The catalytic core of the monomer is accommodated only near its center, with the plant-specific sequences occupying the small domain and an extension distal to the catalytic domain. This configuration is in stark contrast to the domain organization obtained in predicted structures of plant CesA. The arrangement of the catalytic domain within the CesA monomer and dimer provides a foundation for constructing structural models of the synthase complex and defining the relationship between the rosette structure and the cellulose microfibrils they synthesize. © 2014 American Society of Plant Biologists. All rights reserved.

  10. The structure of the catalytic domain of a plant cellulose synthase and its assembly into dimers

    DOE PAGES

    Olek, Anna T.; Rayon, Catherine; Makowski, Lee; ...

    2014-07-10

    Cellulose microfibrils are para-crystalline arrays of several dozen linear (1→4)-β-d-glucan chains synthesized at the surface of the cell membrane by large, multimeric complexes of synthase proteins. Recombinant catalytic domains of rice (Oryza sativa) CesA8 cellulose synthase form dimers reversibly as the fundamental scaffold units of architecture in the synthase complex. Specificity of binding to UDP and UDP-Glc indicates a properly folded protein, and binding kinetics indicate that each monomer independently synthesizes single glucan chains of cellulose, i.e., two chains per dimer pair. In contrast to structure modeling predictions, solution x-ray scattering studies demonstrate that the monomer is a two-domain, elongatedmore » structure, with the smaller domain coupling two monomers into a dimer. The catalytic core of the monomer is accommodated only near its center, with the plant-specific sequences occupying the small domain and an extension distal to the catalytic domain. This configuration is in stark contrast to the domain organization obtained in predicted structures of plant CesA. As a result, the arrangement of the catalytic domain within the CesA monomer and dimer provides a foundation for constructing structural models of the synthase complex and defining the relationship between the rosette structure and the cellulose microfibrils they synthesize.« less

  11. The structure of the catalytic domain of a plant cellulose synthase and its assembly into dimers

    SciTech Connect

    Olek, Anna T.; Rayon, Catherine; Makowski, Lee; Kim, Hyung Rae; Ciesielski, Peter; Badger, John; Paul, Lake N.; Ghosh, Subhangi; Kihara, Daisuke; Crowley, Michael; Himmel, Michael E.; Bolin, Jeffrey T.; Carpita, Nicholas C.

    2014-07-10

    Cellulose microfibrils are para-crystalline arrays of several dozen linear (1→4)-β-d-glucan chains synthesized at the surface of the cell membrane by large, multimeric complexes of synthase proteins. Recombinant catalytic domains of rice (Oryza sativa) CesA8 cellulose synthase form dimers reversibly as the fundamental scaffold units of architecture in the synthase complex. Specificity of binding to UDP and UDP-Glc indicates a properly folded protein, and binding kinetics indicate that each monomer independently synthesizes single glucan chains of cellulose, i.e., two chains per dimer pair. In contrast to structure modeling predictions, solution x-ray scattering studies demonstrate that the monomer is a two-domain, elongated structure, with the smaller domain coupling two monomers into a dimer. The catalytic core of the monomer is accommodated only near its center, with the plant-specific sequences occupying the small domain and an extension distal to the catalytic domain. This configuration is in stark contrast to the domain organization obtained in predicted structures of plant CesA. As a result, the arrangement of the catalytic domain within the CesA monomer and dimer provides a foundation for constructing structural models of the synthase complex and defining the relationship between the rosette structure and the cellulose microfibrils they synthesize.

  12. The Ω-loop lid domain of phosphoenolpyruvate carboxykinase is essential for catalytic function

    PubMed Central

    Johnson, Troy A.; Holyoak, Todd

    2012-01-01

    Phosphoenolpyruvate carboxykinase (PEPCK) is an essential metabolic enzyme operating in the gluconeogenesis and glyceroneogenesis pathways. Recent studies have demonstrated that the enzyme contains a mobile active site lid domain that transitions between an open/disorded conformation to a closed/ordered conformation as the enzyme progresses through the catalytic cycle. The understanding of how this mobile domain functions in catalysis is incomplete. Previous studies show that the closure of the lid domain stabilizes the reaction intermediate and protects the reactive intermediate from spurious protonation and thus contributes to the fidelity of the enzyme. In order to more fully investigate the roles of the lid domain in PEPCK function we created three mutations that replaced the 11-residue lid domain with one, two or three glycine residues. Kinetic analysis of the mutant enzymes demonstrates that none of the enzyme constructs exhibit any measurable kinetic activity resulting in a decrease in the catalytic parameters by at least 106. Structural characterization of the mutants in complexes representing the catalytic cycle suggest that the inactivity is due to a role for the lid domain in the formation of the fully closed state of the enzyme that is required for catalytic function. In the absence of the lid domain, the enzyme is unable to achieve the fully closed state and is rendered inactive despite possessing all of the residues and substrates required for catalytic function. This work demonstrates how enzyme catalytic function can be abolished through the alteration of conformational equilibria despite all elements required for chemical conversion of substrates to products remaining intact. PMID:23127136

  13. The Ω-loop lid domain of phosphoenolpyruvate carboxykinase is essential for catalytic function.

    PubMed

    Johnson, Troy A; Holyoak, Todd

    2012-11-27

    Phosphoenolpyruvate carboxykinase (PEPCK) is an essential metabolic enzyme operating in the gluconeogenesis and glyceroneogenesis pathways. Recent studies have demonstrated that the enzyme contains a mobile active site lid domain that undergoes a transition between an open, disorded conformation and a closed, ordered conformation as the enzyme progresses through the catalytic cycle. The understanding of how this mobile domain functions in catalysis is incomplete. Previous studies showed that the closure of the lid domain stabilizes the reaction intermediate and protects the reactive intermediate from spurious protonation and thus contributes to the fidelity of the enzyme. To more fully investigate the roles of the lid domain in PEPCK function, we introduced three mutations that replaced the 11-residue lid domain with one, two, and three glycine residues. Kinetic analysis of the mutant enzymes demonstrates that none of the enzyme constructs exhibit any measurable kinetic activity, resulting in a decrease in the catalytic parameters of at least 10(6). Structural characterization of the mutants in complexes representing the catalytic cycle suggests that the inactivity is due to a role for the lid domain in the formation of the fully closed state of the enzyme that is required for catalytic function. In the absence of the lid domain, the enzyme is unable to achieve the fully closed state and is rendered inactive despite possessing all of the residues and substrates required for catalytic function. This work demonstrates how enzyme catalytic function can be abolished through the alteration of conformational equilibria despite all the elements required for chemical conversion of substrates to products remaining intact.

  14. The NMR structure of the inhibited catalytic domain of human stromelysin-1.

    PubMed

    Gooley, P R; O'Connell, J F; Marcy, A I; Cuca, G C; Salowe, S P; Bush, B L; Hermes, J D; Esser, C K; Hagmann, W K; Springer, J P

    1994-02-01

    The three-dimensional structure of the catalytic domain of stromelysin-1 complexed with an N-carboxyl alkyl inhibitor has been determined by NMR methods. The global fold consists of three helices, a five stranded beta-sheet and a methionine located in a turn near the catalytic histidines, classifying stromelysin-1 as a metzincin. Stromelysin-1 is unique in having two independent zinc binding sites: a catalytic site and a structural site. The inhibitor binds in an extended conformation. The S1' subsite is a deep hydrophobic pocket, whereas S2' appears shallow and S3' open.

  15. Reduction in water activity greatly retards the phosphoryl transfer from ATP to enzyme protein in the catalytic cycle of sarcoplasmic reticulum Ca2+-ATPase.

    PubMed

    Suzuki, H; Kanazawa, T

    1996-03-08

    Cys-674 of the sarcoplasmic reticulum Ca2+-ATPase was labeled with N-acetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine without a loss of the catalytic activity. The ATP-induced drop in the fluorescence of the label, which was shown in our previous studies to reflect the conformational change upon formation of the calcium.enzyme.ATP complex, was followed by the stopped-flow method. The subsequent phosphoenzyme formation was followed by the rapid quenching method. Effects of a partial substitution of organic solvents for water in the medium on the conformational change and phosphoenzyme formation were investigated in the presence of 100 microM CaCl2 at pH 7.5, 0 degrees C. The rate of the conformational change increased with increasing ATP concentration (0.1 100 microM) and was unaffected by 30% (v/v) dimethyl sulfoxide. In contrast, the rate of phosphoenzyme formation decreased sharply with increasing concentration of dimethyl sulfoxide (20-40% (v/v)), even when phosphoenzyme formation was saturated with ATP. N,N-Dimethylformamide and glycerol had essentially the same effects as dimethyl sulfoxide. These results show that the reduction in water activity does not affect the rate of the conformational change upon formation of the calcium.enzyme.ATP complex, but greatly retards the subsequent phosphoryl transfer from ATP to the enzyme protein. This strongly suggests that in this early stage of the catalytic cycle water plays a critical role in ensuring the rapid turnover of the enzyme.

  16. Simulation of the coupling between nucleotide binding and transmembrane domains in the ATP binding cassette transporter BtuCD.

    PubMed

    Sonne, Jacob; Kandt, Christian; Peters, Günther H; Hansen, Flemming Y; Jensen, Morten Ø; Tieleman, D Peter

    2007-04-15

    The nucleotide-induced structural rearrangements in ATP binding cassette (ABC) transporters, leading to substrate translocation, are largely unknown. We have modeled nucleotide binding and release in the vitamin B(12) importer BtuCD using perturbed elastic network calculations and biased molecular dynamics simulations. Both models predict that nucleotide release decreases the tilt between the two transmembrane domains and opens the cytoplasmic gate. Nucleotide binding has the opposite effect. The observed coupling may be relevant for all ABC transporters because of the conservation of nucleotide binding domains and the shared role of ATP in ABC transporters. The rearrangements in the cytoplasmic gate region do not provide enough space for B(12) to diffuse from the transporter pore into the cytoplasm, which could suggest that peristaltic forces are needed to exclude B(12) from the transporter pore.

  17. Molecular evolution of dinoflagellate luciferases, enzymes with three catalytic domains in a single polypeptide

    PubMed Central

    Liu, Liyun; Wilson, Thérèse; Hastings, J. Woodland

    2004-01-01

    Enzymes with multiple catalytic sites are rare, and their evolutionary significance remains to be established. This study of luciferases from seven dinoflagellate species examines the previously undescribed evolution of such proteins. All these enzymes have the same unique structure: three homologous domains, each with catalytic activity, preceded by an N-terminal region of unknown function. Both pairwise comparison and phylogenetic inference indicate that the similarity of the corresponding individual domains between species is greater than that between the three different domains of each polypeptide. Trees constructed from each of the three individual domains are congruent with the tree of the full-length coding sequence. Luciferase and ribosomal DNA trees both indicate that the Lingulodinium polyedrum luciferase diverged early from the other six. In all species, the amino acid sequence in the central regions of the three domains is strongly conserved, suggesting it as the catalytic site. Synonymous substitution rates also are greatly reduced in the central regions of two species but not in the other five. This lineage-specific difference in synonymous substitution rates in the central region of the domains correlates inversely with the content of GC3, which can be accounted for by the biased usage toward C-ending codons at the degenerate sites. RNA modeling of the central region of the L. polyedrum luciferase domain suggests a function of the constrained synonymous substitutions in the circadian-controlled protein synthesis. PMID:15545598

  18. Importance of the lid and cap domains for the catalytic activity of gastric lipases.

    PubMed

    Miled, N; Bussetta, C; De caro, A; Rivière, M; Berti, L; Canaan, S

    2003-09-01

    Human gastric lipase (HGL) is an enzyme secreted by the stomach, which is stable and active despite the highly acidic environment. It has been clearly established that this enzyme is responsible for 30% of the fat digestion processes occurring in human. This globular protein belongs to the alpha/beta hydrolase fold family and its catalytic serine is deeply buried under a domain called the extrusion domain, which is composed of a 'cap' domain and a segment consisting of 58 residues, which can be defined as a lid. The exact roles played by the cap and the lid domains during the catalytic step have not yet been elucidated. We have recently solved the crystal structure of the open form of the dog gastric lipase in complex with a covalent inhibitor. The detergent molecule and the inhibitor were mimicking a triglyceride substrate that would interact with residues belonging to both the cap and the lid domains. In this study, we have investigated the role of the cap and the lid domains, using site-directed mutagenesis procedures. We have produced truncated mutants lacking the lid and the cap. After expressing these mutants and purifying them, their activity was found to have decreased drastically in comparison with the wild type HGL. The lid and the cap domains play an important role in the catalytic reaction mechanism. Based on these results and the structural data (open form of DGL), we have pointed out the cap and the lid residues involved in the binding with the lipidic substrate.

  19. Structure of the two-domain hexameric APS kinase from Thiobacillus denitrificans: structural basis for the absence of ATP sulfurylase activity

    SciTech Connect

    Gay, Sean C.; Segel, Irwin H.; Fisher, Andrew J.

    2009-10-01

    APS kinase from Thiobacillus denitrificans contains an inactive N-terminal ATP sulfurylase domain. The structure presented unveils the first hexameric assembly for an APS kinase, and reveals that structural changes in the N-terminal domain disrupt the ATP sulfurylase active site thus prohibiting activity. The Tbd-0210 gene of the chemolithotrophic bacterium Thiobacillus denitrificans is annotated to encode a 60.5 kDa bifunctional enzyme with ATP sulfurylase and APS kinase activity. This putative bifunctional enzyme was cloned, expressed and structurally characterized. The 2.95 Å resolution X-ray crystal structure reported here revealed a hexameric assembly with D{sub 3} symmetry. Each subunit contains a large N-terminal sulfurylase-like domain and a C-terminal APS kinase domain reminiscent of the two-domain fungal ATP sulfurylases of Penicillium chrysogenum and Saccharomyces cerevisiae, which also exhibit a hexameric assembly. However, the T. denitrificans enzyme exhibits numerous structural and sequence differences in the N-terminal domain that render it inactive with respect to ATP sulfurylase activity. Surprisingly, the C-terminal domain does indeed display APS kinase activity, indicating that this gene product is a true APS kinase. Therefore, these results provide the first structural insights into a unique hexameric APS kinase that contains a nonfunctional ATP sulfurylase-like domain of unknown function.

  20. Crystal Structure of the Catalytic Domain of a Serine Threonine Protein Phosphatase

    NASA Technical Reports Server (NTRS)

    Swinglel, Mark; Honkanel, Richard; Ciszak, Ewa

    2003-01-01

    Reversible phosphorylation of serine and threonine residues is a well-recognized mechanism in eukaryotic cells for the regulation of cell-cycle progression, cell growth and metabolism. Human serine/threonine phosphatases can be placed into two major families, PPP and PPM. To date the structure on one PPP family member (PPl) has been determined. Here we present the structure of a 323-residue catalytic domain of a second phosphatase belonging to the PPP family of enzyme. catalytic domain of the enzyme has been determined to 1.60Angstrom resolution and refined to R=17.5 and Rfree = 20.8%. The catalytic domain possesses a unique fold consisting of a largely monolithic structure, divisible into closely-associated helical and sheet regions. The catalytic site contains two manganese ions that are involved in substrate binding and catalysis. The enzyme crystallizes as a dimer that completely buries catalytic surfaces of both monomers, Also, the structure shows evidence of some flexibility around the active site cleft that may be related to substrate specificity of this enzyme.

  1. Crystal Structure of the Catalytic Domain of a Serine Threonine Protein Phosphatase

    NASA Technical Reports Server (NTRS)

    Swinglel, Mark; Honkanel, Richard; Ciszak, Ewa

    2003-01-01

    Reversible phosphorylation of serine and threonine residues is a well-recognized mechanism in eukaryotic cells for the regulation of cell-cycle progression, cell growth and metabolism. Human serine/threonine phosphatases can be placed into two major families, PPP and PPM. To date the structure on one PPP family member (PPl) has been determined. Here we present the structure of a 323-residue catalytic domain of a second phosphatase belonging to the PPP family of enzyme. catalytic domain of the enzyme has been determined to 1.60Angstrom resolution and refined to R=17.5 and Rfree = 20.8%. The catalytic domain possesses a unique fold consisting of a largely monolithic structure, divisible into closely-associated helical and sheet regions. The catalytic site contains two manganese ions that are involved in substrate binding and catalysis. The enzyme crystallizes as a dimer that completely buries catalytic surfaces of both monomers, Also, the structure shows evidence of some flexibility around the active site cleft that may be related to substrate specificity of this enzyme.

  2. Evolution of Domain Architectures and Catalytic Functions of Enzymes in Metabolic Systems

    PubMed Central

    Suen, Summit; Lu, Henry Horng-Shing; Yeang, Chen-Hsiang

    2012-01-01

    Domain architectures and catalytic functions of enzymes constitute the centerpieces of a metabolic network. These types of information are formulated as a two-layered network consisting of domains, proteins, and reactions—a domain–protein–reaction (DPR) network. We propose an algorithm to reconstruct the evolutionary history of DPR networks across multiple species and categorize the mechanisms of metabolic systems evolution in terms of network changes. The reconstructed history reveals distinct patterns of evolutionary mechanisms between prokaryotic and eukaryotic networks. Although the evolutionary mechanisms in early ancestors of prokaryotes and eukaryotes are quite similar, more novel and duplicated domain compositions with identical catalytic functions arise along the eukaryotic lineage. In contrast, prokaryotic enzymes become more versatile by catalyzing multiple reactions with similar chemical operations. Moreover, different metabolic pathways are enriched with distinct network evolution mechanisms. For instance, although the pathways of steroid biosynthesis, protein kinases, and glycosaminoglycan biosynthesis all constitute prominent features of animal-specific physiology, their evolution of domain architectures and catalytic functions follows distinct patterns. Steroid biosynthesis is enriched with reaction creations but retains a relatively conserved repertoire of domain compositions and proteins. Protein kinases retain conserved reactions but possess many novel domains and proteins. In contrast, glycosaminoglycan biosynthesis has high rates of reaction/protein creations and domain recruitments. Finally, we elicit and validate two general principles underlying the evolution of DPR networks: 1) duplicated enzyme proteins possess similar catalytic functions and 2) the majority of novel domains arise to catalyze novel reactions. These results shed new lights on the evolution of metabolic systems. PMID:22936075

  3. Uncoupling nitrogenase: catalytic reduction of hydrazine to ammonia by a MoFe protein in the absence of Fe protein-ATP.

    PubMed

    Danyal, Karamatullah; Inglet, Boyd S; Vincent, Kylie A; Barney, Brett M; Hoffman, Brian M; Armstrong, Fraser A; Dean, Dennis R; Seefeldt, Lance C

    2010-09-29

    The catalytic reduction of hydrazine (N(2)H(4)) to ammonia by a β-98(Tyr→His) MoFe protein in the absence of the Fe protein or ATP is reported. The reduction of N(2) or other substrates (e.g., hydrazine, protons, acetylene) by nitrogenase normally requires the transient association of the two nitrogenase component proteins, the Fe protein and the MoFe protein. The Fe protein, with two bound MgATP molecules, transfers one electron to the MoFe protein during each association, coupled to the hydrolysis of two MgATP. All substrate reduction reactions catalyzed by nitrogenase require delivery of electrons by the Fe protein coupled to the hydrolysis of MgATP. We report that when a single amino acid within the MoFe protein (β-98(Tyr)) is substituted by His, the resulting MoFe protein supports catalytic reduction of the nitrogenous substrate hydrazine (N(2)H(4)) to two ammonia molecules when provided with a low potential reductant, polyaminocarboxylate ligated Eu(II) (E(m) -1.1 V vs NHE). The wild-type and a number of other MoFe proteins with amino acid substitutions do not show significant rates of hydrazine reduction under these conditions, whereas the β-98(His) MoFe protein catalyzes hydrazine reduction at rates up to 170 nmol NH(3)/min/mg MoFe protein. This rate of hydrazine reduction is 94% of the rate catalyzed by the β-98(His) or wild-type MoFe protein when combined with the Fe protein, ATP, and reductant under comparable conditions. The β-98(His) MoFe protein reduction of hydrazine in the absence of the Fe protein showed saturation kinetics for the concentration of reductant and substrate. The implications of these results in understanding the nitrogenase mechanism are discussed.

  4. Structure of the catalytic domain of glucuronoyl esterase Cip2 from Hypocrea jecorina

    USDA-ARS?s Scientific Manuscript database

    The structure of the catalytic domain of glucuronoyl esterase Cip2 from the fungus Hypocrea jecorina was determined at a resolution of 1.9 Angstroms. This is the first structure of the newly established carbohydrate esterase family 15. The structure has revealed the residues Ser278–His411–Glu301 pre...

  5. 4,6-Substituted-1,3,5-triazin-2(1H)-ones as monocyclic catalytic inhibitors of human DNA topoisomerase IIα targeting the ATP binding site.

    PubMed

    Pogorelčnik, Barbara; Janežič, Matej; Sosič, Izidor; Gobec, Stanislav; Solmajer, Tom; Perdih, Andrej

    2015-08-01

    Human DNA topoisomerase IIα (htIIα) is a validated target for the development of novel anticancer agents. Starting from our discovered 4-amino-1,3,5-triazine inhibitors of htIIα, we investigated a library of 2,4,6-trisubstituted-1,3,5-triazines for novel inhibitors that bind to the htIIα ATP binding site using a combination of structure-based and ligand-based pharmacophore models and molecular docking. 4,6-substituted-1,3,5-triazin-2(1H)-ones 8, 9 and 14 were identified as novel inhibitors with activity comparable to the established drug etoposide (1). Compound 8 inhibits the htIIα decatenation in a superior fashion to etoposide. Cleavage assays demonstrated that selected compounds 8 and 14 do not act as poisons and antagonize the poison effect of etoposide. Microscale thermophoresis (MST) confirmed binding of compound 8 to the htIIα ATPase domain and compound 14 effectively inhibits the htIIα mediated ATP hydrolysis. The molecular dynamics simulation study provides further insight into the molecular recognition. The 4,6-disubstituted-1,3,5-triazin-2(1H)-ones represent the first validated monocyclic class of catalytic inhibitors that bind to the to the htIIα ATPase domain.

  6. Structure of the Brachydanio Rerio Polo-Like Kinase 1 (Plk1) Catalytic Domain in Complex With An Extended Inhibitor Targeting the Adaptive Pocket of the Enzyme

    SciTech Connect

    Elling, R.A.; Fucini, R.V.; Hanan, E.J.; Barr, K.J.; Zhu, J.; Paulvannan, K.; Yang, W.; Romanowski, M.J.

    2009-05-18

    Polo-like kinase 1 (Plk1) is a member of the Polo-like kinase family of serine/threonine kinases involved in the regulation of cell-cycle progression and cytokinesis and is an attractive target for the development of anticancer therapeutics. The catalytic domain of this enzyme shares significant primary amino-acid homology and structural similarity with another mitotic kinase, Aurora A. While screening an Aurora A library of ATP-competitive compounds, a urea-containing inhibitor with low affinity for mouse Aurora A but with submicromolar potency for human and zebrafish Plk1 (hPlk1 and zPlk1, respectively) was identified. A crystal structure of the zebrafish Plk1 kinase domain-inhibitor complex reveals that the small molecule occupies the purine pocket and extends past the catalytic lysine into the adaptive region of the active site. Analysis of the structures of this protein-inhibitor complex and of similar small molecules cocrystallized with other kinases facilitates understanding of the specificity of the inhibitor for Plk1 and documents for the first time that Plk1 can accommodate extended ATP-competitive compounds that project toward the adaptive pocket and help the enzyme order its activation segment.

  7. The MLLE domain of the ubiquitin ligase UBR5 binds to its catalytic domain to regulate substrate binding.

    PubMed

    Muñoz-Escobar, Juliana; Matta-Camacho, Edna; Kozlov, Guennadi; Gehring, Kalle

    2015-09-11

    E3 ubiquitin ligases catalyze the transfer of ubiquitin from an E2-conjugating enzyme to a substrate. UBR5, homologous to the E6AP C terminus (HECT)-type E3 ligase, mediates the ubiquitination of proteins involved in translation regulation, DNA damage response, and gluconeogenesis. In addition, UBR5 functions in a ligase-independent manner by prompting protein/protein interactions without ubiquitination of the binding partner. Despite recent functional studies, the mechanisms involved in substrate recognition and selective ubiquitination of its binding partners remain elusive. The C terminus of UBR5 harbors the HECT catalytic domain and an adjacent MLLE domain. MLLE domains mediate protein/protein interactions through the binding of a conserved peptide motif, termed PAM2. Here, we characterize the binding properties of the UBR5 MLLE domain to PAM2 peptides from Paip1 and GW182. The crystal structure with a Paip1 PAM2 peptide reveals the network of hydrophobic and ionic interactions that drive binding. In addition, we identify a novel interaction of the MLLE domain with the adjacent HECT domain mediated by a PAM2-like sequence. Our results confirm the role of the MLLE domain of UBR5 in substrate recruitment and suggest a potential role in regulating UBR5 ligase activity. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Copper binding triggers compaction in N-terminal tail of human copper pump ATP7B.

    PubMed

    Mondol, Tanumoy; Åden, Jörgen; Wittung-Stafshede, Pernilla

    2016-02-12

    Protein conformational changes are fundamental to biological reactions. For copper ion transport, the multi-domain protein ATP7B in the Golgi network receives copper from the cytoplasmic copper chaperone Atox1 and, with energy from ATP hydrolysis, moves the metal to the lumen for loading of copper-dependent enzymes. Although anticipated, conformational changes involved in ATP7B's functional cycle remain elusive. Using spectroscopic methods we here demonstrate that the four most N-terminal metal-binding domains in ATP7B, upon stoichiometric copper addition, adopt a more compact arrangement which has a higher thermal stability than in the absence of copper. In contrast to previous reports, no stable complex was found in solution between the metal-binding domains and the nucleotide-binding domain of ATP7B. Metal-dependent movement of the first four metal-binding domains in ATP7B may be a trigger that initiates the overall catalytic cycle.

  9. Long-range coupling between the extracellular gates and the intracellular ATP binding domains of multidrug resistance protein pumps and cystic fibrosis transmembrane conductance regulator channels

    PubMed Central

    Wei, Shipeng; Roessler, Bryan C.; Icyuz, Mert; Chauvet, Sylvain; Tao, Binli; Hartman, John L.; Kirk, Kevin L.

    2015-01-01

    The ABCC transporter subfamily includes pumps, the long and short multidrug resistance proteins (MRPs), and an ATP-gated anion channel, the cystic fibrosis transmembrane conductance regulator (CFTR). We show that despite their thermodynamic differences, these ABCC transporter subtypes use broadly similar mechanisms to couple their extracellular gates to the ATP occupancies of their cytosolic nucleotide binding domains. A conserved extracellular phenylalanine at this gate was a prime location for producing gain of function (GOF) mutants of a long MRP in yeast (Ycf1p cadmium transporter), a short yeast MRP (Yor1p oligomycin exporter), and human CFTR channels. Extracellular gate mutations rescued ATP binding mutants of the yeast MRPs and CFTR by increasing ATP sensitivity. Control ATPase-defective MRP mutants could not be rescued by this mechanism. A CFTR double mutant with an extracellular gate mutation plus a cytosolic GOF mutation was highly active (single-channel open probability >0.3) in the absence of ATP and protein kinase A, each normally required for CFTR activity. We conclude that all 3 ABCC transporter subtypes use similar mechanisms to couple their extracellular gates to ATP occupancy, and highly active CFTR channels that bypass defects in ATP binding or phosphorylation can be produced.—Wei, S., Roessler, B. C., Icyuz, M., Chauvet, S., Tao, B., Hartman IV, J. L., Kirk, K. L. Long-range coupling between the extracellular gates and the intracellular ATP binding domains of multidrug resistance protein pumps and cystic fibrosis transmembrane conductance regulator channels. PMID:26606940

  10. Domain Interactions in the Yeast ATP Binding Cassette Transporter Ycf1p: Intragenic Suppressor Analysis of Mutations in the Nucleotide Binding Domains

    PubMed Central

    Falcón-Pérez, Juan M.; Martínez-Burgos, Mónica; Molano, Jesús; Mazón, María J.; Eraso, Pilar

    2001-01-01

    The yeast cadmium factor (Ycf1p) is a vacuolar ATP binding cassette (ABC) transporter required for heavy metal and drug detoxification. Cluster analysis shows that Ycf1p is strongly related to the human multidrug-associated protein (MRP1) and cystic fibrosis transmembrane conductance regulator and therefore may serve as an excellent model for the study of eukaryotic ABC transporter structure and function. Identifying intramolecular interactions in these transporters may help to elucidate energy transfer mechanisms during transport. To identify regions in Ycf1p that may interact to couple ATPase activity to substrate binding and/or movement across the membrane, we sought intragenic suppressors of ycf1 mutations that affect highly conserved residues presumably involved in ATP binding and/or hydrolysis. Thirteen intragenic second-site suppressors were identified for the D777N mutation which affects the invariant Asp residue in the Walker B motif of the first nucleotide binding domain (NBD1). Two of the suppressor mutations (V543I and F565L) are located in the first transmembrane domain (TMD1), nine (A1003V, A1021T, A1021V, N1027D, Q1107R, G1207D, G1207S, S1212L, and W1225C) are found within TMD2, one (S674L) is in NBD1, and another one (R1415G) is in NBD2, indicating either physical proximity or functional interactions between NBD1 and the other three domains. The original D777N mutant protein exhibits a strong defect in the apparent affinity for ATP and Vmax of transport. The phenotypic characterization of the suppressor mutants shows that suppression does not result from restoring these alterations but rather from a change in substrate specificity. We discuss the possible involvement of Asp777 in coupling ATPase activity to substrate binding and/or transport across the membrane. PMID:11466279

  11. The role of individual domains and the significance of shedding of ATP6AP2/(pro)renin receptor in vacuolar H(+)-ATPase biogenesis.

    PubMed

    Kinouchi, Kenichiro; Ichihara, Atsuhiro; Sano, Motoaki; Sun-Wada, Ge-Hong; Wada, Yoh; Ochi, Hiroki; Fukuda, Toru; Bokuda, Kanako; Kurosawa, Hideaki; Yoshida, Naohiro; Takeda, Shu; Fukuda, Keiichi; Itoh, Hiroshi

    2013-01-01

    The ATPase 6 accessory protein 2 (ATP6AP2)/(pro)renin receptor (PRR) is essential for the biogenesis of active vacuolar H(+)-ATPase (V-ATPase). Genetic deletion of ATP6AP2/PRR causes V-ATPase dysfunction and compromises vesicular acidification. Here, we characterized the domains of ATP6AP2/PRR involved in active V-ATPase biogenesis. Three forms of ATP6AP2/PRR were found intracellularly: full-length protein and the N- and C-terminal fragments of furin cleavage products, with the N-terminal fragment secreted extracellularly. Genetic deletion of ATP6AP2/PRR did not affect the protein stability of V-ATPase subunits. The extracellular domain (ECD) and transmembrane domain (TM) of ATP6AP2/PRR were indispensable for the biogenesis of active V-ATPase. A deletion mutant of ATP6AP2/PRR, which lacks exon 4-encoded amino acids inside the ECD (Δ4M) and causes X-linked mental retardation Hedera type (MRXSH) and X-linked parkinsonism with spasticity (XPDS) in humans, was defective as a V-ATPase-associated protein. Prorenin had no effect on the biogenesis of active V-ATPase. The cleavage of ATP6AP2/PRR by furin seemed also dispensable for the biogenesis of active V-ATPase. We conclude that the N-terminal ECD of ATP6AP2/PRR, which is also involved in binding to prorenin or renin, is required for the biogenesis of active V-ATPase. The V-ATPase assembly occurs prior to its delivery to the trans-Golgi network and hence shedding of ATP6AP2/PRR would not affect the biogenesis of active V-ATPase.

  12. The dual role of CHAPS in the crystallization of stromelysin-3 catalytic domain.

    PubMed

    Gall, Anne-Laure; Ruff, Marc; Moras, Dino

    2003-03-01

    CHAPS [3-[(3-cholamidopropyl) dimethylammonio]-1-propane sulfonate] is a non-denaturing detergent widely used for protein solubilization and stabilization. CHAPS was used to avoid protein aggregation during concentration of the recombinant stromelysin-3 (ST3) catalytic domain and was required to stabilize the protein, allowing its crystallization. The crystal structure of the complex between the ST3 catalytic domain and a phosphinic inhibitor shows two CHAPS molecules binding to ST3 in two different orientations. One CHAPS molecule is masking a hydrophobic surface of the protein, thus avoiding protein aggregation. This detergent molecule is also involved in packing interactions. The other detergent molecule is located in a pocket formed by the N- and C-terminal parts of the ST3 and stabilizes a loop that normally binds a Ca atom.

  13. Heterologous expression and purification of catalytic domain of CESA1 from Arabidopsis thaliana

    DOE PAGES

    Vandavasi, Venu Gopal; O'Neill, Hugh Michael

    2016-10-20

    Here, heterologous expression of plant cellulose synthase (CESA) and its purification has remained a challenge for decades impeding detailed biophysical, biochemical and structural characterization of this key enzyme. An in-depth knowledge of structure and function of CESA proteins would enable us to better understand the hierarchical structure of the plant cell wall. Here, we report a detailed, and reproducible method of purification of catalytic domain of CESA1 from Arabidopsis thaliana that was recombinantly expressed in Escherichia coli. The method relies on a two stage purification procedure to obtain the catalytic domain in monomer and trimer forms. The biochemical and biophysicalmore » data including low resolution structures of the protein have been published. Currently the crystallization studies of this protein are underway.« less

  14. Interactions of a Pop5/Rpp1 heterodimer with the catalytic domain of RNase MRP.

    PubMed

    Perederina, Anna; Khanova, Elena; Quan, Chao; Berezin, Igor; Esakova, Olga; Krasilnikov, Andrey S

    2011-10-01

    Ribonuclease (RNase) MRP is a multicomponent ribonucleoprotein complex closely related to RNase P. RNase MRP and eukaryotic RNase P share most of their protein components, as well as multiple features of their catalytic RNA moieties, but have distinct substrate specificities. While RNase P is practically universally found in all three domains of life, RNase MRP is essential in eukaryotes. The structural organizations of eukaryotic RNase P and RNase MRP are poorly understood. Here, we show that Pop5 and Rpp1, protein components found in both RNase P and RNase MRP, form a heterodimer that binds directly to the conserved area of the putative catalytic domain of RNase MRP RNA. The Pop5/Rpp1 binding site corresponds to the protein binding site in bacterial RNase P RNA. Structural and evolutionary roles of the Pop5/Rpp1 heterodimer in RNases P and MRP are discussed.

  15. Structural Models of Zebrafish (Danio rerio) NOD1 and NOD2 NACHT Domains Suggest Differential ATP Binding Orientations: Insights from Computational Modeling, Docking and Molecular Dynamics Simulations

    PubMed Central

    Maharana, Jitendra; Sahoo, Bikash Ranjan; Bej, Aritra; Sahoo, Jyoti Ranjan; Dehury, Budheswar; Patra, Mahesh Chandra; Martha, Sushma Rani; Balabantray, Sucharita; Pradhan, Sukanta Kumar; Behera, Bijay Kumar

    2015-01-01

    Nucleotide-binding oligomerization domain-containing protein 1 (NOD1) and NOD2 are cytosolic pattern recognition receptors playing pivotal roles in innate immune signaling. NOD1 and NOD2 recognize bacterial peptidoglycan derivatives iE-DAP and MDP, respectively and undergoes conformational alternation and ATP-dependent self-oligomerization of NACHT domain followed by downstream signaling. Lack of structural adequacy of NACHT domain confines our understanding about the NOD-mediated signaling mechanism. Here, we predicted the structure of NACHT domain of both NOD1 and NOD2 from model organism zebrafish (Danio rerio) using computational methods. Our study highlighted the differential ATP binding modes in NOD1 and NOD2. In NOD1, γ-phosphate of ATP faced toward the central nucleotide binding cavity like NLRC4, whereas in NOD2 the cavity was occupied by adenine moiety. The conserved ‘Lysine’ at Walker A formed hydrogen bonds (H-bonds) and Aspartic acid (Walker B) formed electrostatic interaction with ATP. At Sensor 1, Arg328 of NOD1 exhibited an H-bond with ATP, whereas corresponding Arg404 of NOD2 did not. ‘Proline’ of GxP motif (Pro386 of NOD1 and Pro464 of NOD2) interacted with adenine moiety and His511 at Sensor 2 of NOD1 interacted with γ-phosphate group of ATP. In contrast, His579 of NOD2 interacted with the adenine moiety having a relatively inverted orientation. Our findings are well supplemented with the molecular interaction of ATP with NLRC4, and consistent with mutagenesis data reported for human, which indicates evolutionary shared NOD signaling mechanism. Together, this study provides novel insights into ATP binding mechanism, and highlights the differential ATP binding modes in zebrafish NOD1 and NOD2. PMID:25811192

  16. Phosphorylation in the Catalytic Cleft Stabilizes and Attracts Domains of a Phosphohexomutase

    PubMed Central

    Xu, Jia; Lee, Yingying; Beamer, Lesa J.; Van Doren, Steven R.

    2015-01-01

    Phosphorylation can modulate the activities of enzymes. The phosphoryl donor in the catalytic cleft of α-D-phosphohexomutases is transiently dephosphorylated while the reaction intermediate completes a 180° reorientation within the cleft. The phosphorylated form of 52 kDa bacterial phosphomannomutase/phosphoglucomutase is less accessible to dye or protease, more stable to chemical denaturation, and widely stabilized against NMR-detected hydrogen exchange across the core of domain 3 to juxtaposed domain 4 (each by ≥1.3 kcal/mol) and parts of domains 1 and 2. However, phosphorylation accelerates hydrogen exchange in specific regions of domains 1 and 2, including a metal-binding residue in the active site. Electrostatic field lines reveal attraction across the catalytic cleft between phosphorylated Ser-108 and domain 4, but repulsion when Ser-108 is dephosphorylated. Molecular dynamics (MD) simulated the dephosphorylated form to be expanded due to enhanced rotational freedom of domain 4. The contacts and fluctuations of the MD trajectories enabled correct simulation of more than 80% of sites that undergo either protection or deprotection from hydrogen exchange due to phosphorylation. Electrostatic attraction in the phosphorylated enzyme accounts for 1) domain 4 drawing closer to domains 1 and 3; 2) decreased accessibility; and 3) increased stability within these domains. The electrostriction due to phosphorylation may help capture substrate, whereas the opening of the cleft upon transient dephosphorylation allows rotation of the intermediate. The long-range effects of phosphorylation on hydrogen exchange parallel reports on protein kinases, suggesting a conceptual link among these multidomain, phosphoryl transfer enzymes. PMID:25606681

  17. Structure of Trypanosoma brucei glutathione synthetase: Domain and loop alterations in the catalytic cycle of a highly conserved enzyme

    PubMed Central

    Fyfe, Paul K.; Alphey, Magnus S.; Hunter, William N.

    2010-01-01

    Glutathione synthetase catalyses the synthesis of the low molecular mass thiol glutathione from l-γ-glutamyl-l-cysteine and glycine. We report the crystal structure of the dimeric enzyme from Trypanosoma brucei in complex with the product glutathione. The enzyme belongs to the ATP-grasp family, a group of enzymes known to undergo conformational changes upon ligand binding. The T. brucei enzyme crystal structure presents two dimers in the asymmetric unit. The structure reveals variability in the order and position of a small domain, which forms a lid for the active site and serves to capture conformations likely to exist during the catalytic cycle. Comparisons with orthologous enzymes, in particular from Homo sapiens and Saccharomyces cerevisae, indicate a high degree of sequence and structure conservation in part of the active site. Structural differences that are observed between the orthologous enzymes are assigned to different ligand binding states since key residues are conserved. This suggests that the molecular determinants of ligand recognition and reactivity are highly conserved across species. We conclude that it would be difficult to target the parasite enzyme in preference to the host enzyme and therefore glutathione synthetase may not be a suitable target for antiparasitic drug discovery. PMID:20045436

  18. Complete determination of the Pin1 catalytic domain thermodynamic cycle by NMR lineshape analysis.

    PubMed

    Greenwood, Alexander I; Rogals, Monique J; De, Soumya; Lu, Kun Ping; Kovrigin, Evgenii L; Nicholson, Linda K

    2011-09-01

    The phosphorylation-specific peptidyl-prolyl isomerase Pin1 catalyzes the isomerization of the peptide bond preceding a proline residue between cis and trans isomers. To best understand the mechanisms of Pin1 regulation, rigorous enzymatic assays of isomerization are required. However, most measures of isomerase activity require significant constraints on substrate sequence and only yield rate constants for the cis isomer, [Formula: see text] and apparent Michaelis constants, [Formula: see text]. By contrast, NMR lineshape analysis is a powerful tool for determining microscopic rates and populations of each state in a complex binding scheme. The isolated catalytic domain of Pin1 was employed as a first step towards elucidating the reaction scheme of the full-length enzyme. A 24-residue phosphopeptide derived from the amyloid precurser protein intracellular domain (AICD) phosphorylated at Thr668 served as a biologically-relevant Pin1 substrate. Specific (13)C labeling at the Pin1-targeted proline residue provided multiple reporters sensitive to individual isomer binding and on-enzyme catalysis. We have performed titration experiments and employed lineshape analysis of phosphopeptide (13)C-(1)H constant time HSQC spectra to determine [Formula: see text], [Formula: see text], [Formula: see text], and [Formula: see text] for the catalytic domain of Pin1 acting on this AICD substrate. The on-enzyme equilibrium value of [E·trans]/[E·cis] = 3.9 suggests that the catalytic domain of Pin1 is optimized to operate on this substrate near equilibrium in the cellular context. This highlights the power of lineshape analysis for determining the microscopic parameters of enzyme catalysis, and demonstrates the feasibility of future studies of Pin1-PPIase mutants to gain insights on the catalytic mechanism of this important enzyme.

  19. Energy transduction in ATP synthase

    NASA Astrophysics Data System (ADS)

    Elston, Timothy; Wang, Hongyun; Oster, George

    1998-01-01

    Mitochondria, bacteria and chloroplasts use the free energy stored in transmembrane ion gradients to manufacture ATP by the action of ATP synthase. This enzyme consists of two principal domains. The asymmetric membrane-spanning Fo portion contains the proton channel, and the soluble F1 portion contains three catalytic sites which cooperate in the synthetic reactions. The flow of protons through Fo is thought to generate a torque which is transmitted to F1 by an asymmetric shaft, the coiled-coil γ-subunit. This acts as a rotating `cam' within F1, sequentially releasing ATPs from the three active sites. The free-energy difference across the inner membrane of mitochondria and bacteria is sufficient to produce three ATPs per twelve protons passing through the motor. It has been suggested that this protonmotive force biases the rotor's diffusion so that Fo constitutes a rotary motor turning the γ shaft. Here we show that biased diffusion, augmented by electrostatic forces, does indeed generate sufficient torque to account for ATP production. Moreover, the motor's reversibility - supplying torque from ATP hydrolysis in F1 converts the motor into an efficient proton pump - can also be explained by our model.

  20. Structural Stability of Human Protein Tyrosine Phosphatase ρ Catalytic Domain: Effect of Point Mutations

    PubMed Central

    Knapp, Stefan; Alfano, Ivan; Ardini, Matteo; Stefanini, Simonetta; Chiaraluce, Roberta

    2012-01-01

    Protein tyrosine phosphatase ρ (PTPρ) belongs to the classical receptor type IIB family of protein tyrosine phosphatase, the most frequently mutated tyrosine phosphatase in human cancer. There are evidences to suggest that PTPρ may act as a tumor suppressor gene and dysregulation of Tyr phosphorylation can be observed in diverse diseases, such as diabetes, immune deficiencies and cancer. PTPρ variants in the catalytic domain have been identified in cancer tissues. These natural variants are nonsynonymous single nucleotide polymorphisms, variations of a single nucleotide occurring in the coding region and leading to amino acid substitutions. In this study we investigated the effect of amino acid substitution on the structural stability and on the activity of the membrane-proximal catalytic domain of PTPρ. We expressed and purified as soluble recombinant proteins some of the mutants of the membrane-proximal catalytic domain of PTPρ identified in colorectal cancer and in the single nucleotide polymorphisms database. The mutants show a decreased thermal and thermodynamic stability and decreased activation energy relative to phosphatase activity, when compared to wild- type. All the variants show three-state equilibrium unfolding transitions similar to that of the wild- type, with the accumulation of a folding intermediate populated at ∼4.0 M urea. PMID:22389709

  1. Crystallization and preliminary crystallographic study of a trypsin-resistant catalytic domain of human calcineurin

    PubMed Central

    Jin, Lei; Roehrl, Michael H. A.; Xiao, Li; He, Xiuyun; Li, Haibin; Ge, Linhu; Shi, Bingyi

    2012-01-01

    Calcineurin, a Ca2+/calmodulin-dependent serine/threonine protein phosphatase, plays a key role in a number of cellular pathways, including T-cell activation, and is an important molecular target of the immunosuppressive drugs cyclosporin A and FK506. To understand the structural basis underlying the activation of calcineurin by calmodulin, X-ray crystallography was employed to solve the three-dimensional structure of the free calcineurin catalytic domain (residues 20–347 of the A subunit). To accomplish this, a bacterially expressed glutathione S-­transferase (GST) fusion protein of the human calcineurin catalytic domain was first purified by GST-affinity chromatography. After limited digestion by trypsin, the catalytic domain (Cncat) was purified using anion-exchange and size-exclusion chromatography. Crystallization of Cncat was achieved by the hanging-drop vapour-diffusion method at pH 6.5 using PEG 6000 as precipitant. The diffraction results showed that the Cncat crystal belonged to the orthorhombic space group P21212, with unit-cell parameters a = 161.6, b = 87.4, c = 112.0 Å. There are four Cncat molecules in the asymmetric unit, with 49.5% solvent content. An X-ray diffraction data set was collected to 2.87 Å resolution and a clear molecular-replacement solution was obtained. The active site of Cncat is open to the solvent channels in the crystal packing. PMID:22691791

  2. DNA binding residues in the RQC domain of Werner protein are critical for its catalytic activities.

    PubMed

    Tadokoro, Takashi; Kulikowicz, Tomasz; Dawut, Lale; Croteau, Deborah L; Bohr, Vilhelm A

    2012-06-01

    Werner protein (WRN), member of the RecQ helicase family, is a helicase and exonuclease, and participates in multiple DNA metabolic processes including DNA replication, recombination and DNA repair. Mutations in the WRN gene cause Werner syndrome, associated with premature aging, genome instability and cancer predisposition. The RecQ C-terminal (RQC) domain of WRN, containing α2-α3 loop and β-wing motifs, is important for DNA binding and for many protein interactions. To better understand the critical functions of this domain, we generated recombinant WRN proteins (using a novel purification scheme) with mutations in Arg-993 within the α2-α3 loop of the RQC domain and in Phe-1037 of the -wing motif. We then studied the catalytic activities and DNA binding of these mutant proteins as well as some important functional protein interactions. The mutant proteins were defective in DNA binding and helicase activity, and interestingly, they had deficient exonuclease activity and strand annealing function. The RQC domain of WRN has not previously been implicated in exonuclease or annealing activities. The mutant proteins could not stimulate NEIL1 incision activity as did the wild type. Thus, the Arg-993 and Phe-1037 in the RQC domain play essential roles in catalytic activity, and in functional interactions mediated by WRN.

  3. DNA binding residues in the RQC domain of Werner protein are critical for its catalytic activities

    PubMed Central

    Tadokoro, Takashi; Kulikowicz, Tomasz; Dawut, Lale; Croteau, Deborah L.; Bohr, Vilhelm A.

    2012-01-01

    Werner protein (WRN), member of the RecQ helicase family, is a helicase and exonuclease, and participates in multiple DNA metabolic processes including DNA replication, recombination and DNA repair. Mutations in the WRN gene cause Werner syndrome, associated with premature aging, genome instability and cancer predisposition. The RecQ C-terminal (RQC) domain of WRN, containing α2-α3 loop and β-wing motifs, is important for DNA binding and for many protein interactions. To better understand the critical functions of this domain, we generated recombinant WRN proteins (using a novel purification scheme) with mutations in Arg-993 within the α2-α3 loop of the RQC domain and in Phe-1037 of the μ-wing motif. We then studied the catalytic activities and DNA binding of these mutant proteins as well as some important functional protein interactions. The mutant proteins were defective in DNA binding and helicase activity, and interestingly, they had deficient exonuclease activity and strand annealing function. The RQC domain of WRN has not previously been implicated in exonuclease or annealing activities. The mutant proteins could not stimulate NEIL1 incision activity as did the wild type. Thus, the Arg-993 and Phe-1037 in the RQC domain play essential roles in catalytic activity, and in functional interactions mediated by WRN. PMID:22713343

  4. Domain movements during CCA-addition: a new function for motif C in the catalytic core of the human tRNA nucleotidyltransferases.

    PubMed

    Ernst, Felix G M; Rickert, Christian; Bluschke, Alexander; Betat, Heike; Steinhoff, Heinz-Jürgen; Mörl, Mario

    2015-01-01

    CCA-adding enzymes are highly specific RNA polymerases that synthesize and maintain the sequence CCA at the tRNA 3'-end. This nucleotide triplet is a prerequisite for tRNAs to be aminoacylated and to participate in protein biosynthesis. During CCA-addition, a set of highly conserved motifs in the catalytic core of these enzymes is responsible for accurate sequential nucleotide incorporation. In the nucleotide binding pocket, three amino acid residues form Watson-Crick-like base pairs to the incoming CTP and ATP. A reorientation of these templating amino acids switches the enzyme's specificity from CTP to ATP recognition. However, the mechanism underlying this essential structural rearrangement is not understood. Here, we show that motif C, whose actual function has not been identified yet, contributes to the switch in nucleotide specificity during polymerization. Biochemical characterization as well as EPR spectroscopy measurements of the human enzyme reveal that mutating the highly conserved amino acid position D139 in this motif interferes with AMP incorporation and affects interdomain movements in the enzyme. We propose a model of action, where motif C forms a flexible spring element modulating the relative orientation of the enzyme's head and body domains to accommodate the growing 3'-end of the tRNA. Furthermore, these conformational transitions initiate the rearranging of the templating amino acids to switch the specificity of the nucleotide binding pocket from CTP to ATP during CCA-synthesis.

  5. Domain movements during CCA-addition: A new function for motif C in the catalytic core of the human tRNA nucleotidyltransferases

    PubMed Central

    Ernst, Felix G M; Rickert, Christian; Bluschke, Alexander; Betat, Heike; Steinhoff, Heinz-Jürgen; Mörl, Mario

    2015-01-01

    CCA-adding enzymes are highly specific RNA polymerases that synthesize and maintain the sequence CCA at the tRNA 3′-end. This nucleotide triplet is a prerequisite for tRNAs to be aminoacylated and to participate in protein biosynthesis. During CCA-addition, a set of highly conserved motifs in the catalytic core of these enzymes is responsible for accurate sequential nucleotide incorporation. In the nucleotide binding pocket, three amino acid residues form Watson-Crick-like base pairs to the incoming CTP and ATP. A reorientation of these templating amino acids switches the enzyme's specificity from CTP to ATP recognition. However, the mechanism underlying this essential structural rearrangement is not understood. Here, we show that motif C, whose actual function has not been identified yet, contributes to the switch in nucleotide specificity during polymerization. Biochemical characterization as well as EPR spectroscopy measurements of the human enzyme reveal that mutating the highly conserved amino acid position D139 in this motif interferes with AMP incorporation and affects interdomain movements in the enzyme. We propose a model of action, where motif C forms a flexible spring element modulating the relative orientation of the enzyme's head and body domains to accommodate the growing 3′-end of the tRNA. Furthermore, these conformational transitions initiate the rearranging of the templating amino acids to switch the specificity of the nucleotide binding pocket from CTP to ATP during CCA-synthesis. PMID:25849199

  6. The tandemly repeated domains of a β-propeller phytase act synergistically to increase catalytic efficiency.

    PubMed

    Li, Zhongyuan; Huang, Huoqing; Yang, Peilong; Yuan, Tiezheng; Shi, Pengjun; Zhao, Junqi; Meng, Kun; Yao, Bin

    2011-09-01

    β-Propeller phytases (BPPs) with tandemly repeated domains are abundant in nature. Previous studies have shown that the intact domain is responsible for phytate hydrolysis, but the function of the other domain is relatively unknown. In this study, a new dual-domain BPP (PhyH) from Bacillus sp. HJB17 was identified to contain an incomplete N-terminal BPP domain (PhyH-DI, residues 41-318) and a typical BPP domain (PhyH-DII, residues 319-644) at the C-terminus. Purified recombinant PhyH and PhyH-DII required Ca(2+) for phytase activity, showed activity at low temperatures (0-35 °C) and pH 6.0-8.0, and remained active (at 37 °C) after incubation at 60 °C and pH 6.0-12.0. Compared with PhyH-DII, PhyH is catalytically more active against phytate (catalytic constant 27.72 versus 4.17 s(-1)), which indicates the importance of PhyH-DI in phytate degradation. PhyH-DI was found to hydrolyze phytate intermediate D-Ins(1,4,5,6) P(4), and to act synergistically (a 1.2-2.5-fold increase in phosphate release) with PhyH-DII, other BPPs (PhyP and 168PhyA) and a histidine acid phosphatase. Furthermore, fusion of PhyH-DI with PhyP or 168PhyA significantly enhanced their catalytic efficiencies. This is the first report to elucidate the substrate specificity of the incomplete domain and the functional relationship of tandemly repeated domains in BPPs. We conjecture that dual-domain BPPs have succeeded evolutionarily because they can increase the amount of available phosphate by interacting together. Additionally, fusing PhyH-DI to a single-domain phytase appears to be an efficient way to improve the activity of the latter.

  7. Functional Analysis of the Streptomyces coelicolor NrdR ATP-Cone Domain: Role in Nucleotide Binding, Oligomerization, and DNA Interactions▿ †

    PubMed Central

    Grinberg, Inna; Shteinberg, Tatyana; Hassan, A. Quamrul; Aharonowitz, Yair; Borovok, Ilya; Cohen, Gerald

    2009-01-01

    Ribonucleotide reductases (RNRs) are essential enzymes in all living cells, providing the only known de novo pathway for the biosynthesis of deoxyribonucleotides (dNTPs), the immediate precursors of DNA synthesis and repair. RNRs catalyze the controlled reduction of all four ribonucleotides to maintain a balanced pool of dNTPs during the cell cycle. Streptomyces species contain genes, nrdAB and nrdJ, coding for oxygen-dependent class I and oxygen-independent class II RNRs, either of which is sufficient for vegetative growth. Both sets of genes are transcriptionally repressed by NrdR. NrdR contains a zinc ribbon DNA-binding domain and an ATP-cone domain similar to that present in the allosteric activity site of many class I and class III RNRs. Purified NrdR contains up to 1 mol of tightly bound ATP or dATP per mol of protein and binds to tandem 16-bp sequences, termed NrdR-boxes, present in the upstream regulatory regions of bacterial RNR operons. Previously, we showed that the ATP-cone domain alone determines nucleotide binding and that an NrdR mutant defective in nucleotide binding was unable to bind to DNA probes containing NrdR-boxes. These observations led us to propose that when NrdR binds ATP/dATP it undergoes a conformational change that affects DNA binding and hence RNR gene expression. In this study, we analyzed a collection of ATP-cone mutant proteins containing changes in residues inferred to be implicated in nucleotide binding and show that they result in pleiotrophic effects on ATP/dATP binding, on protein oligomerization, and on DNA binding. A model is proposed to integrate these observations. PMID:19047342

  8. Conservation of an ATP-binding domain among RecA proteins from Proteus vulgaris, Erwinia carotovora, Shigella flexneri, and Escherichia coli K-12 and B/r.

    PubMed

    Knight, K L; Hess, R M; McEntee, K

    1988-06-01

    The purified RecA proteins encoded by the cloned genes from Proteus vulgaris, Erwinia carotovora, Shigella flexneri, and Escherichia coli B/r were compared with the RecA protein from E. coli K-12. Each of the proteins hydrolyzed ATP in the presence of single-stranded DNA, and each was covalently modified with the photoaffinity ATP analog 8-azidoadenosine 5'-triphosphate (8N3ATP). Two-dimensional tryptic maps of the four heterologous RecA proteins demonstrated considerable structural conservation among these bacterial genera. Moreover, when the [alpha-32P]8N3ATP-modified proteins were digested with trypsin and analyzed by high-performance liquid chromatography, a single peak of radioactivity was detected in each of the digests and these peptides eluted identically with the tryptic peptide T31 of the E. coli K-12 RecA protein, which was the unique site of 8N3ATP photolabeling. Each of the heterologous recA genes hybridized to oligonucleotide probes derived from the ATP-binding domain sequence of the E. coli K-12 gene. These last results demonstrate that the ATP-binding domain of the RecA protein has been strongly conserved for greater than 10(7) years.

  9. Conservation of an ATP-binding domain among recA proteins from Proteus vulgaris, erwinia carotovora, Shigella flexneri, and Escherichia coli K-12 and B/r

    SciTech Connect

    Knight, K.L.; Hess, R.M.; McEntee, K.

    1988-06-01

    The purified RecA proteins encoded by the cloned genes from Proteus vulgaris, Erwinia carotovora, Shigella flexneri, and Escherichia coli B/r were compared with the RecA protein from E. coli K-12. Each of the proteins hydrolyzed ATP in the presence of single-stranded DNA, and each was covalently modified with the photoaffinity ATP analog 8-azidoadenosine 5'-triphosphate (8N/sub 3/ATP). Two-dimensional tryptic maps of the four heterologous RecA proteins demonstrated considerable structural conservation among these bacterial genera. Moreover, when the (..cap alpha..-/sup 32/P)8N/sub 3/ATP-modified proteins were digested with trypsin and analyzed by high-performance liquid chromatography, a single peak of radioactivity was detected in each of the digests and these peptides eluted identically with the tryptic peptide T/sub 31/ of the E. coli K-12 RecA protein, which was the unique site of 8N/sub 3/ATP photolabeling. Each of the heterologous recA genes hybridized to oligonucleotide probes derived from the ATP-binding domain sequence of the E. coli K-12 gene. These last results demonstrate that the ATP-binding domain of the RecA protein has been strongly conserved for greater than 10/sup 7/ years.

  10. Ca2+ translocation and catalytic activity of the sarcoplasmic reticulum ATPase. Modulation by ATP, Ca2+, and Pi.

    PubMed

    Galina, A; de Meis, L

    1991-09-25

    The ratio between Ca2+ uptake and Ca(2+)-dependent ATP hydrolysis measured in sarcoplasmic reticulum vesicles of rabbit skeletal muscle was found to vary greatly depending on the concentrations of oxalate or Pi used. In the presence of 5 mM oxalate, 20 mM Pi, and 1 mM Pi, the ratios found were in the range of 1.4-2.3, 0.6-0.8, and 0.01-0.10, respectively. The rates of Ca2+ exchange and ATP synthesis were measured at the steady state by adding trace amounts of 45Ca and 32Pi, after the vesicles had been loaded with Ca2+. In the presence of 1 mM Pi, 10 mM MgCl2, and 0.2 mM CaCl2, the ratio between Ca2+ exchange and ATP synthesis varied from 9 to 14. This ratio approached two when Ca2+ in the medium was reduced to a very low level, or when in the presence of Ca2+, dimethyl sulfoxide was added to the assay medium, or when the Pi concentration was raised from 1 to 20 mM. A ratio of two was also measured when the steady state was attained using ITP instead of ATP. In all the conditions that led to a ratio close to two, there was an increase in the fraction of enzyme phosphorylated by Pi. It is proposed that the coupling between Ca2+ translocation and ATP hydrolysis or synthesis is modulated by the phosphorylation of the ATPase by Pi.

  11. Functional hot spots in human ATP-binding cassette transporter nucleotide binding domains

    PubMed Central

    Kelly, Libusha; Fukushima, Hisayo; Karchin, Rachel; Gow, Jason M; Chinn, Leslie W; Pieper, Ursula; Segal, Mark R; Kroetz, Deanna L; Sali, Andrej

    2010-01-01

    The human ATP-binding cassette (ABC) transporter superfamily consists of 48 integral membrane proteins that couple the action of ATP binding and hydrolysis to the transport of diverse substrates across cellular membranes. Defects in 18 transporters have been implicated in human disease. In hundreds of cases, disease phenotypes and defects in function can be traced to nonsynonymous single nucleotide polymorphisms (nsSNPs). The functional impact of the majority of ABC transporter nsSNPs has yet to be experimentally characterized. Here, we combine experimental mutational studies with sequence and structural analysis to describe the impact of nsSNPs in human ABC transporters. First, the disease associations of 39 nsSNPs in 10 transporters were rationalized by identifying two conserved loops and a small α-helical region that may be involved in interdomain communication necessary for transport of substrates. Second, an approach to discriminate between disease-associated and neutral nsSNPs was developed and tailored to this superfamily. Finally, the functional impact of 40 unannotated nsSNPs in seven ABC transporters identified in 247 ethnically diverse individuals studied by the Pharmacogenetics of Membrane Transporters consortium was predicted. Three predictions were experimentally tested using human embryonic kidney epithelial (HEK) 293 cells stably transfected with the reference multidrug resistance transporter 4 and its variants to examine functional differences in transport of the antiviral drug, tenofovir. The experimental results confirmed two predictions. Our analysis provides a structural and evolutionary framework for rationalizing and predicting the functional effects of nsSNPs in this clinically important membrane transporter superfamily. PMID:20799350

  12. Crystal structure of Deinococcus radiodurans RecQ helicase catalytic core domain: the interdomain flexibility.

    PubMed

    Chen, Sheng-Chia; Huang, Chi-Hung; Yang, Chia Shin; Way, Tzong-Der; Chang, Ming-Chung; Chen, Yeh

    2014-01-01

    RecQ DNA helicases are key enzymes in the maintenance of genome integrity, and they have functions in DNA replication, recombination, and repair. In contrast to most RecQs, RecQ from Deinococcus radiodurans (DrRecQ) possesses an unusual domain architecture that is crucial for its remarkable ability to repair DNA. Here, we determined the crystal structures of the DrRecQ helicase catalytic core and its ADP-bound form, revealing interdomain flexibility in its first RecA-like and winged-helix (WH) domains. Additionally, the WH domain of DrRecQ is positioned in a different orientation from that of the E. coli RecQ (EcRecQ). These results suggest that the orientation of the protein during DNA-binding is significantly different when comparing DrRecQ and EcRecQ.

  13. Structure of the catalytic domain of human polo-like kinase 1.

    PubMed

    Kothe, Michael; Kohls, Darcy; Low, Simon; Coli, Rocco; Cheng, Alan C; Jacques, Suzanne L; Johnson, Theresa L; Lewis, Cristina; Loh, Christine; Nonomiya, Jim; Sheils, Alissa L; Verdries, Kimberly A; Wynn, Thomas A; Kuhn, Cyrille; Ding, Yuan-Hua

    2007-05-22

    Polo-like kinase 1 (Plk1) is an attractive target for the development of anticancer agents due to its importance in regulating cell-cycle progression. Overexpression of Plk1 has been detected in a variety of cancers, and expression levels often correlate with poor prognosis. Despite high interest in Plk1-targeted therapeutics, there is currently no structure publicly available to guide structure-based drug design of specific inhibitors. We determined the crystal structures of the T210V mutant of the kinase domain of human Plk1 complexed with the nonhydrolyzable ATP analogue adenylylimidodiphosphate (AMPPNP) or the pyrrolo-pyrazole inhibitor PHA-680626 at 2.4 and 2.1 A resolution, respectively. Plk1 adopts the typical kinase domain fold and crystallized in a conformation resembling the active state of other kinases. Comparison of the kinetic parameters determined for the (unphosphorylated) wild-type enzyme, as well as the T210V and T210D mutants, shows that the mutations primarily affect the kcat of the reaction, with little change in the apparent Km for the protein or nucleotide substrates (kcat = 0.0094, 0.0376, and 0.0049 s-1 and Km(ATP) = 3.2, 4.0, and 3.0 microM for WT, T210D, and T210V, respectively). The structure highlights features of the active site that can be exploited to obtain Plk1-specific inhibitors with selectivity over other kinases and Plk isoforms. These include the presence of a phenylalanine at the bottom of the ATP pocket, combined with a cysteine (as opposed to the more commonly found leucine) in the roof of the binding site, a pocket created by Leu132 in the hinge region, and a cluster of positively charged residues in the solvent-exposed area outside of the adenine pocket adjacent to the hinge region.

  14. Cloning and Iron Transportation of Nucleotide Binding Domain of Cryptosporidium andersoni ATP-Binding Cassette (CaABC) Gene.

    PubMed

    Wang, Ju-Hua; Xue, Xiu-Heng; Zhou, Jie; Fan, Cai-Yun; Xie, Qian-Qian; Wang, Pan

    2015-06-01

    Cryptosporidium andersoni ATP-binding cassette (CaABC) is an important membrane protein involved in substrate transport across the membrane. In this research, the nucleotide binding domain (NBD) of CaABC gene was amplified by PCR, and the eukaryotic expression vector of pEGFP-C1-CaNBD was reconstructed. Then, the recombinant plasmid of pEGFP-C1-CaNBD was transformed into the mouse intestinal epithelial cells (IECs) to study the iron transportation function of CaABC. The results indicated that NBD region of CaABC gene can significantly elevate the transport efficiency of Ca(2+), Mg(2+), K(+), and HCO3 (-) in IECs (P<0.05). The significance of this study is to find the ATPase inhibitors for NBD region of CaABC gene and to inhibit ATP binding and nutrient transport of CaABC transporter. Thus, C. andersoni will be killed by inhibition of nutrient uptake. This will open up a new way for treatment of cryptosporidiosis.

  15. Insertion of endocellulase catalytic domains into thermostable consensus ankyrin scaffolds: effects on stability and cellulolytic activity.

    PubMed

    Cunha, Eva S; Hatem, Christine L; Barrick, Doug

    2013-11-01

    Degradation of cellulose for biofuels production holds promise in solving important environmental and economic problems. However, the low activities (and thus high enzyme-to-substrate ratios needed) of hydrolytic cellulase enzymes, which convert cellulose into simple sugars, remain a major barrier. As a potential strategy to stabilize cellulases and enhance their activities, we have embedded cellulases of extremophiles into hyperstable α-helical consensus ankyrin domain scaffolds. We found the catalytic domains CelA (CA, GH8; Clostridium thermocellum) and Cel12A (C12A, GH12; Thermotoga maritima) to be stable in the context of the ankyrin scaffold and to be active against both soluble and insoluble substrates. The ankyrin repeats in each fusion are folded, although it appears that for the C12A catalytic domain (CD; where the N and C termini are distant in the crystal structure), the two flanking ankyrin domains are independent, whereas for CA (where termini are close), the flanking ankyrin domains stabilize each other. Although the activity of CA is unchanged in the context of the ankyrin scaffold, the activity of C12A is increased between 2- and 6-fold (for regenerated amorphous cellulose and carboxymethyl cellulose substrates) at high temperatures. For C12A, activity increases with the number of flanking ankyrin repeats. These results showed ankyrin arrays to be a promising scaffold for constructing designer cellulosomes, preserving or enhancing enzymatic activity and retaining thermostability. This modular architecture will make it possible to arrange multiple cellulase domains at a precise spacing within a single polypeptide, allowing us to search for spacings that may optimize reactivity toward the repetitive cellulose lattice.

  16. Insertion of Endocellulase Catalytic Domains into Thermostable Consensus Ankyrin Scaffolds: Effects on Stability and Cellulolytic Activity

    PubMed Central

    Cunha, Eva S.; Hatem, Christine L.

    2013-01-01

    Degradation of cellulose for biofuels production holds promise in solving important environmental and economic problems. However, the low activities (and thus high enzyme-to-substrate ratios needed) of hydrolytic cellulase enzymes, which convert cellulose into simple sugars, remain a major barrier. As a potential strategy to stabilize cellulases and enhance their activities, we have embedded cellulases of extremophiles into hyperstable α-helical consensus ankyrin domain scaffolds. We found the catalytic domains CelA (CA, GH8; Clostridium thermocellum) and Cel12A (C12A, GH12; Thermotoga maritima) to be stable in the context of the ankyrin scaffold and to be active against both soluble and insoluble substrates. The ankyrin repeats in each fusion are folded, although it appears that for the C12A catalytic domain (CD; where the N and C termini are distant in the crystal structure), the two flanking ankyrin domains are independent, whereas for CA (where termini are close), the flanking ankyrin domains stabilize each other. Although the activity of CA is unchanged in the context of the ankyrin scaffold, the activity of C12A is increased between 2- and 6-fold (for regenerated amorphous cellulose and carboxymethyl cellulose substrates) at high temperatures. For C12A, activity increases with the number of flanking ankyrin repeats. These results showed ankyrin arrays to be a promising scaffold for constructing designer cellulosomes, preserving or enhancing enzymatic activity and retaining thermostability. This modular architecture will make it possible to arrange multiple cellulase domains at a precise spacing within a single polypeptide, allowing us to search for spacings that may optimize reactivity toward the repetitive cellulose lattice. PMID:23974146

  17. Three dimensional model of severe acute respiratory syndrome coronavirus helicase ATPase catalytic domain and molecular design of severe acute respiratory syndrome coronavirus helicase inhibitors

    NASA Astrophysics Data System (ADS)

    Hoffmann, Marcin; Eitner, Krystian; von Grotthuss, Marcin; Rychlewski, Leszek; Banachowicz, Ewa; Grabarkiewicz, Tomasz; Szkoda, Tomasz; Kolinski, Andrzej

    2006-05-01

    The modeling of the severe acute respiratory syndrome coronavirus helicase ATPase catalytic domain was performed using the protein structure prediction Meta Server and the 3D Jury method for model selection, which resulted in the identification of 1JPR, 1UAA and 1W36 PDB structures as suitable templates for creating a full atom 3D model. This model was further utilized to design small molecules that are expected to block an ATPase catalytic pocket thus inhibit the enzymatic activity. Binding sites for various functional groups were identified in a series of molecular dynamics calculation. Their positions in the catalytic pocket were used as constraints in the Cambridge structural database search for molecules having the pharmacophores that interacted most strongly with the enzyme in a desired position. The subsequent MD simulations followed by calculations of binding energies of the designed molecules were compared to ATP identifying the most successful candidates, for likely inhibitors—molecules possessing two phosphonic acid moieties at distal ends of the molecule.

  18. Crystal structure of the catalytic domain of human bile salt activated lipase.

    PubMed Central

    Terzyan, S.; Wang, C. S.; Downs, D.; Hunter, B.; Zhang, X. C.

    2000-01-01

    Bile-salt activated lipase (BAL) is a pancreatic enzyme that digests a variety of lipids in the small intestine. A distinct property of BAL is its dependency on bile salts in hydrolyzing substrates of long acyl chains or bulky alcoholic motifs. A crystal structure of the catalytic domain of human BAL (residues 1-538) with two surface mutations (N186D and A298D), which were introduced in attempting to facilitate crystallization, has been determined at 2.3 A resolution. The crystal form belongs to space group P2(1)2(1)2(1) with one monomer per asymmetric unit, and the protein shows an alpha/beta hydrolase fold. In the absence of bound bile salt molecules, the protein possesses a preformed catalytic triad and a functional oxyanion hole. Several surface loops around the active site are mobile, including two loops potentially involved in substrate binding (residues 115-125 and 270-285). PMID:11045623

  19. Comprehensive Characterization of AMP-Activated Protein Kinase Catalytic Domain by Top-Down Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Yu, Deyang; Peng, Ying; Ayaz-Guner, Serife; Gregorich, Zachery R.; Ge, Ying

    2016-02-01

    AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase that is essential in regulating energy metabolism in all eukaryotic cells. It is a heterotrimeric protein complex composed of a catalytic subunit (α) and two regulatory subunits (β and γ). C-terminal truncation of AMPKα at residue 312 yielded a protein that is active upon phosphorylation of Thr172 in the absence of β and γ subunits, which is refered to as the AMPK catalytic domain and commonly used to substitute for the AMPK heterotrimeric complex in in vitro kinase assays. However, a comprehensive characterization of the AMPK catalytic domain is lacking. Herein, we expressed a His-tagged human AMPK catalytic domin (denoted as AMPKΔ) in E. coli, comprehensively characterized AMPKΔ in its basal state and after in vitro phosphorylation using top-down mass spectrometry (MS), and assessed how phosphorylation of AMPKΔ affects its activity. Unexpectedly, we found that bacterially-expressed AMPKΔ was basally phosphorylated and localized the phosphorylation site to the His-tag. We found that AMPKΔ had noticeable basal activity and was capable of phosphorylating itself and its substrates without activating phosphorylation at Thr172. Moreover, our data suggested that Thr172 is the only site phosphorylated by its upstream kinase, liver kinase B1, and that this phosphorylation dramatically increases the kinase activity of AMPKΔ. Importantly, we demonstrated that top-down MS in conjunction with in vitro phosphorylation assay is a powerful approach for monitoring phosphorylation reaction and determining sequential order of phosphorylation events in kinase-substrate systems.

  20. Comprehensive Characterization of AMP-activated Protein Kinase Catalytic Domain by Top-down Mass Spectrometry

    PubMed Central

    Yu, Deyang; Peng, Ying; Ayaz-Guner, Serife; Gregorich, Zachery R.; Ge, Ying

    2015-01-01

    AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase that is essential in regulating energy metabolism in all eukaryotic cells. It is a heterotrimeric protein complex composed of a catalytic subunit (α) and two regulatory subunits (β and γ. C-terminal truncation of AMPKα at residue 312 yielded a protein that is active upon phosphorylation of Thr172 in the absence of β and γ subunits, which is refered to as the AMPK catalytic domain and commonly used to substitute for the AMPK heterotrimeric complex in in vitro kinase assays. However, a comprehensive characterization of the AMPK catalytic domain is lacking. Herein, we expressed a His-tagged human AMPK catalytic domin (denoted as AMPKΔ) in E. coli, comprehensively characterized AMPKΔ in its basal state and after in vitro phosphorylation using top-down mass spectrometry (MS), and assessed how phosphorylation of AMPKΔ affects its activity. Unexpectedly, we found that bacterially-expressed AMPKΔ was basally phosphorylated and localized the phosphorylation site to the His-tag. We found that AMPKΔ has noticeable basal activity and was capable of phosphorylating itself and its substrates without activating phosphorylation at Thr172. Moreover, our data suggested that Thr172 is the only site phosphorylated by its upstream kinase, liver kinase B1, and that this phosphorylation dramatically increases the kinase activity of AMPKΔ. Importantly, we demonstrated that top-down MS in conjunction with in vitro phosphorylation assay is a powerful approach for monitoring phosphorylation reaction and determining sequential order of phosphorylation events in kinase-substrate systems. PMID:26489410

  1. ATP-dependent DNA ligase from Thermococcus sp. 1519 displays a new arrangement of the OB-fold domain.

    PubMed

    Petrova, T; Bezsudnova, E Y; Boyko, K M; Mardanov, A V; Polyakov, K M; Volkov, V V; Kozin, M; Ravin, N V; Shabalin, I G; Skryabin, K G; Stekhanova, T N; Kovalchuk, M V; Popov, V O

    2012-12-01

    DNA ligases join single-strand breaks in double-stranded DNA by catalyzing the formation of a phosphodiester bond between adjacent 5'-phosphate and 3'-hydroxyl termini. Their function is essential for maintaining genome integrity in the replication, recombination and repair of DNA. High flexibility is important for the function of DNA ligase molecules. Two types of overall conformations of archaeal DNA ligase that depend on the relative position of the OB-fold domain have previously been revealed: closed and open extended conformations. The structure of ATP-dependent DNA ligase from Thermococcus sp. 1519 (LigTh1519) in the crystalline state determined at a resolution of 3.02 Å shows a new relative arrangement of the OB-fold domain which is intermediate between the positions of this domain in the closed and the open extended conformations of previously determined archaeal DNA ligases. However, small-angle X-ray scattering (SAXS) measurements indicate that in solution the LigTh1519 molecule adopts either an open extended conformation or both an intermediate and an open extended conformation with the open extended conformation being dominant.

  2. Structure of the catalytic domain of the colistin resistance enzyme MCR-1

    DOE PAGES

    Stojanoski, Vlatko; Sankaran, Banumathi; Prasad, B. V. Venkataram; ...

    2016-09-21

    Due to the paucity of novel antibiotics, colistin has become a last resort antibiotic for treating multidrug resistant bacteria. Colistin acts by binding the lipid A component of lipopolysaccharides and subsequently disrupting the bacterial membrane. The recently identified plasmid-encoded MCR-1 enzyme is the first transmissible colistin resistance determinant and is a cause for concern for the spread of this resistance trait. MCR-1 is a phosphoethanolamine transferase that catalyzes the addition of phosphoethanolamine to lipid A to decrease colistin affinity. The structure of the catalytic domain of MCR-1 at 1.32 Å reveals the active site is similar to that of relatedmore » phosphoethanolamine transferases. The putative nucleophile for catalysis, threonine 285, is phosphorylated in cMCR-1 and a zinc is present at a conserved site in addition to three zincs more peripherally located in the active site. As noted for catalytic domains of other phosphoethanolamine transferases, binding sites for the lipid A and phosphatidylethanolamine substrates are not apparent in the cMCR-1 structure, suggesting that they are present in the membrane domain.« less

  3. Structure of the catalytic domain of the colistin resistance enzyme MCR-1

    SciTech Connect

    Stojanoski, Vlatko; Sankaran, Banumathi; Prasad, B. V. Venkataram; Poirel, Laurent; Nordmann, Patrice; Palzkill, Timothy

    2016-09-21

    Due to the paucity of novel antibiotics, colistin has become a last resort antibiotic for treating multidrug resistant bacteria. Colistin acts by binding the lipid A component of lipopolysaccharides and subsequently disrupting the bacterial membrane. The recently identified plasmid-encoded MCR-1 enzyme is the first transmissible colistin resistance determinant and is a cause for concern for the spread of this resistance trait. MCR-1 is a phosphoethanolamine transferase that catalyzes the addition of phosphoethanolamine to lipid A to decrease colistin affinity. The structure of the catalytic domain of MCR-1 at 1.32 Å reveals the active site is similar to that of related phosphoethanolamine transferases. The putative nucleophile for catalysis, threonine 285, is phosphorylated in cMCR-1 and a zinc is present at a conserved site in addition to three zincs more peripherally located in the active site. As noted for catalytic domains of other phosphoethanolamine transferases, binding sites for the lipid A and phosphatidylethanolamine substrates are not apparent in the cMCR-1 structure, suggesting that they are present in the membrane domain.

  4. Structure of the catalytic domain of the colistin resistance enzyme MCR-1.

    PubMed

    Stojanoski, Vlatko; Sankaran, Banumathi; Prasad, B V Venkataram; Poirel, Laurent; Nordmann, Patrice; Palzkill, Timothy

    2016-09-21

    Due to the paucity of novel antibiotics, colistin has become a last resort antibiotic for treating multidrug resistant bacteria. Colistin acts by binding the lipid A component of lipopolysaccharides and subsequently disrupting the bacterial membrane. The recently identified plasmid-encoded MCR-1 enzyme is the first transmissible colistin resistance determinant and is a cause for concern for the spread of this resistance trait. MCR-1 is a phosphoethanolamine transferase that catalyzes the addition of phosphoethanolamine to lipid A to decrease colistin affinity. The structure of the catalytic domain of MCR-1 at 1.32 Å reveals the active site is similar to that of related phosphoethanolamine transferases. The putative nucleophile for catalysis, threonine 285, is phosphorylated in cMCR-1 and a zinc is present at a conserved site in addition to three zincs more peripherally located in the active site. As noted for catalytic domains of other phosphoethanolamine transferases, binding sites for the lipid A and phosphatidylethanolamine substrates are not apparent in the cMCR-1 structure, suggesting that they are present in the membrane domain.

  5. Solution structure of the catalytic domain of human stromelysin complexed with a hydrophobic inhibitor.

    PubMed Central

    Van Doren, S. R.; Kurochkin, A. V.; Hu, W.; Ye, Q. Z.; Johnson, L. L.; Hupe, D. J.; Zuiderweg, E. R.

    1995-01-01

    Stromelysin, a representative matrix metalloproteinase and target of drug development efforts, plays a prominent role in the pathological proteolysis associated with arthritis and secondarily in that of cancer metastasis and invasion. To provide a structural template to aid the development of therapeutic inhibitors, we have determined a medium-resolution structure of a 20-kDa complex of human stromelysin's catalytic domain with a hydrophobic peptidic inhibitor using multinuclear, multidimensional NMR spectroscopy. This domain of this zinc hydrolase contains a mixed beta-sheet comprising one antiparallel strand and four parallel strands, three helices, and a methionine-containing turn near the catalytic center. The ensemble of 20 structures was calculated using, on average, 8 interresidue NOE restraints per residue for the 166-residue protein fragment complexed with a 4-residue substrate analogue. The mean RMS deviation (RMSD) to the average structure for backbone heavy atoms is 0.91 A and for all heavy atoms is 1.42 A. The structure has good stereochemical properties, including its backbone torsion angles. The beta-sheet and alpha-helices of the catalytic domains of human stromelysin (NMR model) and human fibroblast collagenase (X-ray crystallographic model of Lovejoy B et al., 1994b, Biochemistry 33:8207-8217) superimpose well, having a pairwise RMSD for backbone heavy atoms of 2.28 A when three loop segments are disregarded. The hydroxamate-substituted inhibitor binds across the hydrophobic active site of stromelysin in an extended conformation. The first hydrophobic side chain is deeply buried in the principal S'1 subsite, the second hydrophobic side chain is located on the opposite side of the inhibitor backbone in the hydrophobic S'2 surface subsite, and a third hydrophobic side chain (P'3) lies at the surface. PMID:8580839

  6. Crystal structure of the catalytic domain of the tumor-associated human carbonic anhydrase IX.

    PubMed

    Alterio, Vincenzo; Hilvo, Mika; Di Fiore, Anna; Supuran, Claudiu T; Pan, Peiwen; Parkkila, Seppo; Scaloni, Andrea; Pastorek, Jaromir; Pastorekova, Silvia; Pedone, Carlo; Scozzafava, Andrea; Monti, Simona Maria; De Simone, Giuseppina

    2009-09-22

    Carbonic anhydrase (CA) IX is a plasma membrane-associated member of the alpha-CA enzyme family, which is involved in solid tumor acidification. It is a marker of tumor hypoxia and a prognostic factor in several human cancers. An aberrant increase in CA IX expression in chronic hypoxia and during development of various carcinomas contributes to tumorigenesis through at least two mechanisms: pH regulation and cell adhesion control. Here we report the X-ray structure of the catalytic domain of CA IX in complex with a classical, clinically used sulfonamide inhibitor, acetazolamide. The structure reveals a typical alpha-CA fold, which significantly differs from the other CA isozymes when the protein quaternary structure is considered. Thus, two catalytic domains of CA IX associate to form a dimer, which is stabilized by the formation of an intermolecular disulfide bond. The active site clefts and the PG domains are located on one face of the dimer, while the C-termini are located on the opposite face to facilitate protein anchoring to the cell membrane. A correlation between the three-dimensional structure and the physiological role of the enzyme is here suggested, based on the measurement of the pH profile of the catalytic activity for the physiological reaction, CO(2) hydration to bicarbonate and protons. On the basis of the structural differences observed between CA IX and the other membrane-associated alpha-CAs, further prospects for the rational drug design of isozyme-specific CA inhibitors are proposed, given that inhibition of this enzyme shows antitumor activity both in vitro and in vivo.

  7. Structure of USP7 catalytic domain and three Ubl-domains reveals a connector α-helix with regulatory role.

    PubMed

    Kim, Robbert Q; van Dijk, Willem J; Sixma, Titia K

    2016-07-01

    Ubiquitin conjugation is an important signal in cellular pathways, changing the fate of a target protein, by degradation, relocalisation or complex formation. These signals are balanced by deubiquitinating enzymes (DUBs), which antagonize ubiquitination of specific protein substrates. Because ubiquitination pathways are critically important, DUB activity is often carefully controlled. USP7 is a highly abundant DUB with numerous targets that plays complex roles in diverse pathways, including DNA regulation, p53 stress response and endosomal protein recycling. Full-length USP7 switches between an inactive and an active state, tuned by the positioning of 5 Ubl folds in the C-terminal HUBL domain. The active state requires interaction between the last two Ubls (USP7(45)) and the catalytic domain (USP7(CD)), and this can be promoted by allosteric interaction from the first 3 Ubl domains of USP7 (USP7(123)) interacting with GMPS. Here we study the transition between USP7 states. We provide a crystal structure of USP7(CD123) and show that CD and Ubl123 are connected via an extended charged alpha helix. Mutational analysis is used to determine whether the charge and rigidity of this 'connector helix' are important for full USP7 activity. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  8. Structure of the catalytic domain of the Salmonella virulence factor SseI.

    PubMed

    Bhaskaran, Shyam S; Stebbins, C Erec

    2012-12-01

    SseI is secreted into host cells by Salmonella and contributes to the establishment of systemic infections. The crystal structure of the C-terminal domain of SseI has been solved to 1.70 Å resolution, revealing it to be a member of the cysteine protease superfamily with a catalytic triad consisting of Cys178, His216 and Asp231 that is critical to its virulence activities. Structure-based analysis revealed that SseI is likely to possess either acyl hydrolase or acyltransferase activity, placing this virulence factor in the rapidly growing class of enzymes of this family utilized by bacterial pathogens inside eukaryotic cells.

  9. High Resolution Crystal Structure of the Catalytic Domain of ADAMTS-5 (Aggrecanase-2)

    SciTech Connect

    Shieh, Huey-Sheng; Mathis, Karl J.; Williams, Jennifer M.; Hills, Robert L.; Wiese, Joe F.; Benson, Timothy E.; Kiefer, James R.; Marino, Margaret H.; Carroll, Jeffery N.; Leone, Joseph W.; Malfait, Anne-Marie; Arner, Elizabeth C.; Tortorella, Micky D.; Tomasselli, Alfredo

    2008-06-30

    Aggrecanase-2 (a disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS-5)), a member of the ADAMTS protein family, is critically involved in arthritic diseases because of its direct role in cleaving the cartilage component aggrecan. The catalytic domain of aggrecanase-2 has been refolded, purified, and crystallized, and its three-dimensional structure determined to 1.4{angstrom} resolution in the presence of an inhibitor. A high resolution structure of an ADAMTS/aggrecanase protein provides an opportunity for the development of therapeutics to treat osteoarthritis.

  10. ATP-Driven Remodeling of the Linker Domain in the Dynein Motor

    PubMed Central

    Roberts, Anthony J.; Malkova, Bara; Walker, Matt L.; Sakakibara, Hitoshi; Numata, Naoki; Kon, Takahide; Ohkura, Reiko; Edwards, Thomas A.; Knight, Peter J.; Sutoh, Kazuo; Oiwa, Kazuhiro; Burgess, Stan A.

    2012-01-01

    Summary Dynein ATPases are the largest known cytoskeletal motors and perform critical functions in cells: carrying cargo along microtubules in the cytoplasm and powering flagellar beating. Dyneins are members of the AAA+ superfamily of ring-shaped enzymes, but how they harness this architecture to produce movement is poorly understood. Here, we have used cryo-EM to determine 3D maps of native flagellar dynein-c and a cytoplasmic dynein motor domain in different nucleotide states. The structures show key sites of conformational change within the AAA+ ring and a large rearrangement of the “linker” domain, involving a hinge near its middle. Analysis of a mutant in which the linker “undocks” from the ring indicates that linker remodeling requires energy that is supplied by interactions with the AAA+ modules. Fitting the dynein-c structures into flagellar tomograms suggests how this mechanism could drive sliding between microtubules, and also has implications for cytoplasmic cargo transport. PMID:22863569

  11. Mutation in cysteine bridge domain of the gamma-subunit affects light regulation of the ATP synthase in Arabidopsis

    USDA-ARS?s Scientific Manuscript database

    The chloroplast ATP synthase functions to synthesize ATP from ADP and free phosphate coupled by the electrochemical potential across the thylakoid membrane in the light. The light-dependent regulation of ATP synthase activity is carried out in part through redox modulation of a cysteine bridge in CF...

  12. Purification of catalytic domain of rat spleen p72syk kinase and its phosphorylation and activation by protein kinase C.

    PubMed Central

    Borowski, P; Heiland, M; Kornetzky, L; Medem, S; Laufs, R

    1998-01-01

    The catalytic domain of p72(syk) kinase (CDp72(syk)) was purified from a 30000 g particulate fraction of rat spleen. The purification procedure employed sequential chromatography on columns of DEAE-Sephacel and Superdex-200, and elution from HA-Ultrogel by chloride. The analysis of the final CDp72(syk) preparation by SDS/PAGE revealed a major silver-stained 40 kDa protein. The kinase was identified by covalent modification of its ATP-binding site with [14C]5'-fluorosulphonylbenzoyladenosine and by immunoblotting with a polyclonal antibody against the 'linker' region of p72(syk). By using poly(Glu4, Tyr1) as a substrate, the specific activity of the enzyme was determined as 18.5 nmol Pi/min per mg. Casein, histones H1 and H2B and myelin basic protein were efficiently phosphorylated by CDp72(syk). The kinase exhibited a limited ability to phosphorylate random polymers containing tyrosine residues. CDp72(syk) autophosphorylation activity was associated with an activation of the kinase towards exogenous substrates. The extent of activation was dependent on the substrates added. CDp72(syk) was phosphorylated by protein kinase C (PKC) on serine and threonine residues. With a newly developed assay method, we demonstrated that the PKC-mediated phosphorylation had a strong activating effect on the tyrosine kinase activity of CDp72(syk). Studies extended to conventional PKC isoforms revealed an isoform-dependent manner (alpha > betaI = betaII > gamma) of CDp72(syk) phosphorylation. The different phosphorylation efficiencies of the PKC isoforms closely correlated with the ability to enhance the tyrosine kinase activity. PMID:9531509

  13. Non-catalytic participation of the Pin1 peptidyl-prolyl isomerase domain in target binding

    PubMed Central

    Innes, Brendan T.; Bailey, Melanie L.; Brandl, Christopher J.; Shilton, Brian H.; Litchfield, David W.

    2012-01-01

    Pin1 is a phosphorylation-dependent peptidyl-prolyl isomerase (PPIase) that has the potential to add an additional level of regulation within protein kinase mediated signaling pathways. Furthermore, there is a mounting body of evidence implicating Pin1 in the emergence of pathological phenotypes in neurodegeneration and cancer through the isomerization of a wide variety of substrates at peptidyl-prolyl bonds where the residue preceding proline is a phosphorylated serine or threonine residue (i.e., pS/T-P motifs). A key step in this regulatory process is the interaction of Pin-1 with its substrates. This is a complex process since Pin1 is composed of two domains, the catalytic PPIase domain, and a type IV WW domain, both of which recognize pS/T-P motifs. The observation that the WW domain exhibits considerably higher binding affinity for pS/T-P motifs has led to predictions that the two domains may have distinct roles in mediating the actions of Pin1 on its substrates. To evaluate the participation of its individual domains in target binding, we performed GST pulldowns to monitor interactions between various forms of Pin1 and mitotic phospho-proteins that revealed two classes of Pin-1 interacting proteins, differing in their requirement for residues within the PPIase domain. From these observations, we consider models for Pin1-substrate interactions and the potential functions of the different classes of Pin1 interacting proteins. We also compare sequences that are recognized by Pin1 within its individual interaction partners to investigate the underlying basis for its different types of interactions. PMID:23407864

  14. A core catalytic domain of the TyrA protein family: arogenate dehydrogenase from Synechocystis

    PubMed Central

    2004-01-01

    The TyrA protein family includes prephenate dehydrogenases, cyclohexadienyl dehydrogenases and TyrAas (arogenate dehydrogenases). tyrAa from Synechocystis sp. PCC 6803, encoding a 30 kDa TyrAa protein, was cloned into an overexpression vector in Escherichia coli. TyrAa was then purified to apparent homogeneity and characterized. This protein is a model structure for a catalytic core domain in the TyrA superfamily, uncomplicated by allosteric or fused domains. Competitive inhibitors acting at the catalytic core of TyrA proteins are analogues of any accepted cyclohexadienyl substrate. The homodimeric enzyme was specific for L-arogenate (Km=331 μM) and NADP+ (Km=38 μM), being unable to substitute prephenate or NAD+ respectively. L-Tyrosine was a potent inhibitor of the enzyme (Ki=70 μM). NADPH had no detectable ability to inhibit the reaction. Although the mechanism is probably steady-state random order, properties of 2′,5′-ADP as an inhibitor suggest a high preference for L-arogenate binding first. Comparative enzymology established that both of the arogenate-pathway enzymes, prephenate aminotransferase and TyrAa, were present in many diverse cyanobacteria and in a variety of eukaryotic red and green algae. PMID:15171683

  15. Distribution and prediction of catalytic domains in 2-oxoglutarate dependent dioxygenases

    PubMed Central

    2012-01-01

    Background The 2-oxoglutarate dependent superfamily is a diverse group of non-haem dioxygenases, and is present in prokaryotes, eukaryotes, and archaea. The enzymes differ in substrate preference and reaction chemistry, a factor that precludes their classification by homology studies and electronic annotation schemes alone. In this work, I propose and explore the rationale of using substrates to classify structurally similar alpha-ketoglutarate dependent enzymes. Findings Differential catalysis in phylogenetic clades of 2-OG dependent enzymes, is determined by the interactions of a subset of active-site amino acids. Identifying these with existing computational methods is challenging and not feasible for all proteins. A clustering protocol based on validated mechanisms of catalysis of known molecules, in tandem with group specific hidden markov model profiles is able to differentiate and sequester these enzymes. Access to this repository is by a web server that compares user defined unknown sequences to these pre-defined profiles and outputs a list of predicted catalytic domains. The server is free and is accessible at the following URL ( http://comp-biol.theacms.in/H2OGpred.html). Conclusions The proposed stratification is a novel attempt at classifying and predicting 2-oxoglutarate dependent function. In addition, the server will provide researchers with a tool to compare their data to a comprehensive list of HMM profiles of catalytic domains. This work, will aid efforts by investigators to screen and characterize putative 2-OG dependent sequences. The profile database will be updated at regular intervals. PMID:22862831

  16. A non-catalytic N-terminal domain negatively influences the nucleotide exchange activity of translation elongation factor 1Bα.

    PubMed

    Trosiuk, Tetiana V; Shalak, Vyacheslav F; Szczepanowski, Roman H; Negrutskii, Boris S; El'skaya, Anna V

    2016-02-01

    Eukaryotic translation elongation factor 1Bα (eEF1Bα) is a functional homolog of the bacterial factor EF-Ts, and is a component of the macromolecular eEF1B complex. eEF1Bα functions as a catalyst of guanine nucleotide exchange on translation elongation factor 1A (eEF1A). The C-terminal domain of eEF1Bα is necessary and sufficient for its catalytic activity, whereas the N-terminal domain interacts with eukaryotic translation elongation factor 1Bγ (eEF1Bγ) to form a tight complex. However, eEF1Bγ has been shown to enhance the catalytic activity of eEF1Bα attributed to the C-terminal domain of eEF1Bα. This suggests that the N-terminal domain of eEF1Bα may in some way influence the guanine nucleotide exchange process. We have shown that full-length recombinant eEF1Bα and its truncated forms are non-globular proteins with elongated shapes. Truncation of the N-terminal domain of eEF1Bα, which is dispensable for catalytic activity, resulted in acceleration of the rate of guanine nucleotide exchange on eEF1A compared to full-length eEF1Bα. A similar effect on the catalytic activity of eEF1Bα was observed after its interaction with eEF1Bγ. We suggest that the non-catalytic N-terminal domain of eEF1Bα may interfere with eEF1A binding to the C-terminal catalytic domain, resulting in a decrease in the overall rate of the guanine nucleotide exchange reaction. Formation of a tight complex between the eEF1Bγ and eEF1Bα N-terminal domains abolishes this inhibitory effect.

  17. Plasma cholesteryl ester-triglyceride transfer protein. The catalytic domain is a low molecular weight proteolipid.

    PubMed

    Busch, S J; Stuart, W D; Hug, B; Mao, S J; Harmony, J A

    1987-12-25

    The lipid transfer protein complex (LTC) isolated from human plasma by immunoaffinity chromatography transfers cholesteryl esters (CE), triglycerides, and phosphatidylcholine (PC) between lipoproteins in vitro. The molecular weight of this lipid transfer catalyst in sodium dodecyl sulfate-polyacrylamide gels was 65,000. When resolved on a gel filtration column by high performance liquid chromatography (HPLC), LTC was composed of fractions of high (greater than 150,000) to low (18,000) molecular weight, although sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of each fraction revealed bands at Mr 65,000 (major) and 52,000 (minor). The CE and triglyceride transfer activity of the low Mr HPLC fraction (1049 nmol of triglyceride/mg/h and 244 nmol of CE/mg/h) was significantly greater than that of the high Mr HPLC fraction (15-27 nmol of triglyceride/mg/h and 20-30 nmol of CE/mg/h). The PC transfer activity of the HPLC fractions was not determined. LTC proteins were separated by dialysis in acidified chloroform:methanol solution into dialysand and dialysate proteins. The dialysate contained a low Mr proteolipid, designated the catalytic domain Cd, which catalyzed CE and triglyceride transfer at equivalent rates (11.0 versus 9.5 mumol/mg/h, respectively). PC transfer activity was approximately 10% of these levels (1.5 mumol/mg/h). The dialysand consisted of a protein, designated the transfer protein TP, which facilitated CE (3.4 mumol/mg/h) preferentially over triglyceride and PC (1.0 mumol/mg/h) transfer, and a catalytically inactive protein, designated the heparin-binding domain Hd. We propose a model of the LTC protein (based on catalytic activities, monoclonal antibody reactivities, and heparin-binding capacities of the isolated proteins) in which both Hd (approximately 13 kDa) and Cd (approximately 3 kDa) originate from a single lipid transfer protein, TP.

  18. Catalytic efficiency of chitinase-D on insoluble chitinous substrates was improved by fusing auxiliary domains.

    PubMed

    Madhuprakash, Jogi; El Gueddari, Nour Eddine; Moerschbacher, Bruno M; Podile, Appa Rao

    2015-01-01

    Chitin is an abundant renewable polysaccharide, next only to cellulose. Chitinases are important for effective utilization of this biopolymer. Chitinase D from Serratia proteamaculans (SpChiD) is a single domain chitinase with both hydrolytic and transglycosylation (TG) activities. SpChiD had less of hydrolytic activity on insoluble polymeric chitin substrates due to the absence of auxiliary binding domains. We improved catalytic efficiency of SpChiD in degradation of insoluble chitin substrates by fusing with auxiliary domains like polycystic kidney disease (PKD) domain and chitin binding protein 21 (CBP21). Of the six different SpChiD fusion chimeras, two C-terminal fusions viz. ChiD+PKD and ChiD+CBP resulted in improved hydrolytic activity on α- and β-chitin, respectively. Time-course degradation of colloidal chitin also confirmed that these two C-terminal SpChiD fusion chimeras were more active than other chimeras. More TG products were produced for a longer duration by the fusion chimeras ChiD+PKD and PKD+ChiD+CBP.

  19. Catalytic Efficiency of Chitinase-D on Insoluble Chitinous Substrates Was Improved by Fusing Auxiliary Domains

    PubMed Central

    Madhuprakash, Jogi; El Gueddari, Nour Eddine; Moerschbacher, Bruno M.; Podile, Appa Rao

    2015-01-01

    Chitin is an abundant renewable polysaccharide, next only to cellulose. Chitinases are important for effective utilization of this biopolymer. Chitinase D from Serratia proteamaculans (SpChiD) is a single domain chitinase with both hydrolytic and transglycosylation (TG) activities. SpChiD had less of hydrolytic activity on insoluble polymeric chitin substrates due to the absence of auxiliary binding domains. We improved catalytic efficiency of SpChiD in degradation of insoluble chitin substrates by fusing with auxiliary domains like polycystic kidney disease (PKD) domain and chitin binding protein 21 (CBP21). Of the six different SpChiD fusion chimeras, two C-terminal fusions viz. ChiD+PKD and ChiD+CBP resulted in improved hydrolytic activity on α- and β-chitin, respectively. Time-course degradation of colloidal chitin also confirmed that these two C-terminal SpChiD fusion chimeras were more active than other chimeras. More TG products were produced for a longer duration by the fusion chimeras ChiD+PKD and PKD+ChiD+CBP. PMID:25615694

  20. Visualizing Dealumination of a Single Zeolite Domain in a Real-Life Catalytic Cracking Particle.

    PubMed

    Kalirai, Sam; Paalanen, Pasi P; Wang, Jian; Meirer, Florian; Weckhuysen, Bert M

    2016-09-05

    Fluid catalytic cracking (FCC) catalysts play a central role in the chemical conversion of crude oil fractions. Using scanning transmission X-ray microscopy (STXM) we investigate the chemistry of one fresh and two industrially deactivated (ECAT) FCC catalysts at the single zeolite domain level. Spectro-microscopic data at the Fe L3 , La M5 , and Al K X-ray absorption edges reveal differing levels of deposited Fe on the ECAT catalysts corresponding with an overall loss in tetrahedral Al within the zeolite domains. Using La as a localization marker, we have developed a novel methodology to map the changing Al distribution of single zeolite domains within real-life FCC catalysts. It was found that significant changes in the zeolite domain size distributions as well as the loss of Al from the zeolite framework occur. Furthermore, inter- and intraparticle heterogeneities in the dealumination process were observed, revealing the complex interplay between metal-mediated pore accessibility loss and zeolite dealumination.

  1. Role of α-Subunit VISIT-DG Sequence Residues Ser-347 and Gly-351 in the Catalytic Sites of Escherichia coli ATP Synthase*

    PubMed Central

    Li, Wenzong; Brudecki, Laura E.; Senior, Alan E.; Ahmad, Zulfiqar

    2009-01-01

    This paper describes the role of α-subunit VISIT-DG sequence residues αSer-347 and αGly-351 in catalytic sites of Escherichia coli F1Fo ATP synthase. X-ray structures show the very highly conserved α-subunit VISIT-DG sequence in close proximity to the conserved phosphate-binding residues αArg-376, βArg-182, βLys-155, and βArg-246 in the phosphate-binding subdomain. Mutations αS347Q and αG351Q caused loss of oxidative phosphorylation and reduced ATPase activity of F1Fo in membranes by 100- and 150-fold, respectively, whereas αS347A mutation showed only a 13-fold loss of activity and also retained some oxidative phosphorylation activity. The ATPase of αS347Q mutant was not inhibited, and the αS347A mutant was slightly inhibited by MgADP-azide, MgADP-fluoroaluminate, or MgADP-fluoroscandium, in contrast to wild type and αG351Q mutant. Whereas 7-chloro-4-nitrobenzo-2-oxa-1, 3-diazole (NBD-Cl) inhibited wild type and αG351Q mutant ATPase essentially completely, ATPase in αS347A or αS347Q mutant was inhibited maximally by ∼80–90%, although reaction still occurred at residue βTyr-297, proximal to the α-subunit VISIT-DG sequence, near the phosphate-binding pocket. Inhibition characteristics supported the conclusion that NBD-Cl reacts inβE (empty) catalytic sites, as shown previously by x-ray structure analysis. Phosphate protected against NBD-Cl inhibition in wild type and αG351Q mutant but not in αS347Q or αS347A mutant. The results demonstrate that αSer-347 is an additional residue involved in phosphate-binding and transition state stabilization in ATP synthase catalytic sites. In contrast, αGly-351, although strongly conserved and clearly important for function, appears not to play a direct role. PMID:19240022

  2. A Novel Method of Production and Biophysical Characterization of the Catalytic Domain of Yeast Oligosaccharyl Transferase

    PubMed Central

    Huang, Chengdong; Mohanty, Smita; Banerjee, Monimoy

    2010-01-01

    Oligosaccharyl transferase (OT) is a multi-subunit enzyme that catalyzes N-linked glycosylation of nascent polypeptides in the lumen of the endoplasmic reticulum. In the case of Saccharomyces cerevisiae, OT is composed of nine integral membrane protein subunits. Defects in N-linked glycosylation cause a series of disorders known as congenital disorders of glycosylation (CDG). The C-terminal domain of Stt3p subunit has been reported to contain the acceptor protein recognition site and/or catalytic site. We report here the subcloning, overexpression, a robust but novel method of production of pure C-terminal domain of Stt3p at 60∼70 mg/L in E. coli. CD spectra indicate that the C-terminal Stt3p is highly helical and has a stable tertiary structure in SDS micelles. The well dispersed 2D {1H-15N}-HSQC spectrum in SDS micelles indicates that it is feasible to determine the atomic structure by NMR. The effect of the conserved D518E mutation on the conformation of the C-terminal Stt3p is particularly interesting. The comparative analysis of the fluorescence and NMR data of the mutant and the wild-type C-terminal domain of Stt3p revealed that the replacement of the key residue Asp518, which is located within the WWDYG signature motif (residues 516-520), led to a distinct tertiary structure, even though both proteins have similar overall secondary structures. This observation strongly suggests that Asp518, which was previously proposed to primarily function as a catalytic residue, also plays a critical structural role. Moreover, the activity of the protein was confirmed by Saturation Transfer Difference (STD) and NMR titration studies. PMID:20047336

  3. The third P-loop domain in cytoplasmic dynein heavy chain is essential for dynein motor function and ATP-sensitive microtubule binding.

    PubMed

    Silvanovich, Andre; Li, Min-Gang; Serr, Madeline; Mische, Sarah; Hays, Thomas S

    2003-04-01

    Sequence comparisons and structural analyses show that the dynein heavy chain motor subunit is related to the AAA family of chaperone-like ATPases. The core structure of the dynein motor unit derives from the assembly of six AAA domains into a hexameric ring. In dynein, the first four AAA domains contain consensus nucleotide triphosphate-binding motifs, or P-loops. The recent structural models of dynein heavy chain have fostered the hypothesis that the energy derived from hydrolysis at P-loop 1 acts through adjacent P-loop domains to effect changes in the attachment state of the microtubule-binding domain. However, to date, the functional significance of the P-loop domains adjacent to the ATP hydrolytic site has not been demonstrated. Our results provide a mutational analysis of P-loop function within the first and third AAA domains of the Drosophila cytoplasmic dynein heavy chain. Here we report the first evidence that P-loop-3 function is essential for dynein function. Significantly, our results further show that P-loop-3 function is required for the ATP-induced release of the dynein complex from microtubules. Mutation of P-loop-3 blocks ATP-mediated release of dynein from microtubules, but does not appear to block ATP binding and hydrolysis at P-loop 1. Combined with the recent recognition that dynein belongs to the family of AAA ATPases, the observations support current models in which the multiple AAA domains of the dynein heavy chain interact to support the translocation of the dynein motor down the microtubule lattice.

  4. Structure and Catalytic Regulatory Function of Ubiquitin Specific Protease 11 N-Terminal and Ubiquitin-like Domains

    PubMed Central

    2014-01-01

    The ubiquitin specific protease 11 (USP11) is implicated in DNA repair, viral RNA replication, and TGFβ signaling. We report the first characterization of the USP11 domain architecture and its role in regulating the enzymatic activity. USP11 consists of an N-terminal “domain present in USPs” (DUSP) and “ubiquitin-like” (UBL) domain, together referred to as DU domains, and the catalytic domain harboring a second UBL domain. Crystal structures of the DU domains show a tandem arrangement with a shortened β-hairpin at the two-domain interface and altered surface characteristics compared to the homologues USP4 and USP15. A conserved VEVY motif is a signature feature at the two-domain interface that shapes a potential protein interaction site. Small angle X-ray scattering and gel filtration experiments are consistent with the USP11DU domains and full-length USP11 being monomeric. Unexpectedly, we reveal, through kinetic assays of a series of deletion mutants, that the catalytic activity of USP11 is not regulated through intramolecular autoinhibition or activation by the N-terminal DU or UBL domains. Moreover, ubiquitin chain cleavage assays with all eight linkages reveal a preference for Lys63-, Lys6-, Lys33-, and Lys11-linked chains over Lys27-, Lys29-, and Lys48-linked and linear chains consistent with USP11’s function in DNA repair pathways that is mediated by the protease domain. Our data support a model whereby USP11 domains outside the catalytic core domain serve as protein interaction or trafficking modules rather than a direct regulatory function of the proteolytic activity. This highlights the diversity of USPs in substrate recognition and regulation of ubiquitin deconjugation. PMID:24724799

  5. Structure and catalytic regulatory function of ubiquitin specific protease 11 N-terminal and ubiquitin-like domains.

    PubMed

    Harper, Stephen; Gratton, Hayley E; Cornaciu, Irina; Oberer, Monika; Scott, David J; Emsley, Jonas; Dreveny, Ingrid

    2014-05-13

    The ubiquitin specific protease 11 (USP11) is implicated in DNA repair, viral RNA replication, and TGFβ signaling. We report the first characterization of the USP11 domain architecture and its role in regulating the enzymatic activity. USP11 consists of an N-terminal "domain present in USPs" (DUSP) and "ubiquitin-like" (UBL) domain, together referred to as DU domains, and the catalytic domain harboring a second UBL domain. Crystal structures of the DU domains show a tandem arrangement with a shortened β-hairpin at the two-domain interface and altered surface characteristics compared to the homologues USP4 and USP15. A conserved VEVY motif is a signature feature at the two-domain interface that shapes a potential protein interaction site. Small angle X-ray scattering and gel filtration experiments are consistent with the USP11DU domains and full-length USP11 being monomeric. Unexpectedly, we reveal, through kinetic assays of a series of deletion mutants, that the catalytic activity of USP11 is not regulated through intramolecular autoinhibition or activation by the N-terminal DU or UBL domains. Moreover, ubiquitin chain cleavage assays with all eight linkages reveal a preference for Lys(63)-, Lys(6)-, Lys(33)-, and Lys(11)-linked chains over Lys(27)-, Lys(29)-, and Lys(48)-linked and linear chains consistent with USP11's function in DNA repair pathways that is mediated by the protease domain. Our data support a model whereby USP11 domains outside the catalytic core domain serve as protein interaction or trafficking modules rather than a direct regulatory function of the proteolytic activity. This highlights the diversity of USPs in substrate recognition and regulation of ubiquitin deconjugation.

  6. Catalytic and substrate promiscuity: distinct multiple chemistries catalysed by the phosphatase domain of receptor protein tyrosine phosphatase.

    PubMed

    Srinivasan, Bharath; Marks, Hanna; Mitra, Sreyoshi; Smalley, David M; Skolnick, Jeffrey

    2016-07-15

    The presence of latent activities in enzymes is posited to underlie the natural evolution of new catalytic functions. However, the prevalence and extent of such substrate and catalytic ambiguity in evolved enzymes is difficult to address experimentally given the order-of-magnitude difference in the activities for native and, sometimes, promiscuous substrate/s. Further, such latent functions are of special interest when the activities concerned do not fall into the domain of substrate promiscuity. In the present study, we show a special case of such latent enzyme activity by demonstrating the presence of two mechanistically distinct reactions catalysed by the catalytic domain of receptor protein tyrosine phosphatase isoform δ (PTPRδ). The primary catalytic activity involves the hydrolysis of a phosphomonoester bond (C─O─P) with high catalytic efficiency, whereas the secondary activity is the hydrolysis of a glycosidic bond (C─O─C) with poorer catalytic efficiency. This enzyme also displays substrate promiscuity by hydrolysing diester bonds while being highly discriminative for its monoester substrates. To confirm these activities, we also demonstrated their presence on the catalytic domain of protein tyrosine phosphatase Ω (PTPRΩ), a homologue of PTPRδ. Studies on the rate, metal-ion dependence, pH dependence and inhibition of the respective activities showed that they are markedly different. This is the first study that demonstrates a novel sugar hydrolase and diesterase activity for the phosphatase domain (PD) of PTPRδ and PTPRΩ. This work has significant implications for both understanding the evolution of enzymatic activity and the possible physiological role of this new chemistry. Our findings suggest that the genome might harbour a wealth of such alternative latent enzyme activities in the same protein domain that renders our knowledge of metabolic networks incomplete. © 2016 The Author(s). published by Portland Press Limited on behalf of the

  7. Catalytic and substrate promiscuity: Distinct multiple chemistries catalyzed by the phosphatase domain of receptor protein tyrosine phosphatase

    PubMed Central

    Srinivasan, Bharath; Marks, Hanna; Mitra, Sreyoshi; Smalley, David M.; Skolnick, Jeffrey

    2016-01-01

    The presence of latent activities in enzymes is posited to underlie the natural evolution of new catalytic functions. However, the prevalence and extent of such substrate and catalytic ambiguity in evolved enzymes is difficult to address experimentally given the order-of-magnitude difference in the activities for native and, sometimes, promiscuous substrate/s. Further, such latent functions are of special interest when the activities concerned do not fall into the domain of substrate promiscuity. Here, we show a special case of such latent enzyme activity by demonstrating the presence of two mechanistically distinct reactions catalyzed by the catalytic domain of receptor protein tyrosine phosphatase isoform delta (PTPRδ). The primary catalytic activity involves the hydrolysis of a phosphomonoester bond (C-O-P) with high catalytic efficiency, while the secondary activity is the hydrolysis of a glycosidic bond (C-O-C) with poorer catalytic efficiency. This enzyme also displays substrate promiscuity by hydrolyzing diester bonds while being highly discriminative for its monoester substrates. To confirm these activities, we also demonstrated their presence on the catalytic domain of PTPRΩ, a homologue of PTPRδ. Studies on the rate, metal-ion dependence, pH dependence and inhibition of the respective activities showed that they are markedly different. This is the first study that demonstrates a novel sugar hydrolase and diesterase activity for the phosphatase domain of PTPRδ and PTPRΩ. This work has significant implications for both understanding the evolution of enzymatic activity and the possible physiological role of this new chemistry. Our findings suggest that the genome might harbor a wealth of such alternative latent enzyme activities in the same protein domain that renders our knowledge of metabolic networks incomplete. PMID:27208174

  8. Crystallization and preliminary X-ray characterization of the catalytic domain of collagenase G from Clostridium histolyticum

    SciTech Connect

    Eckhard, Ulrich Nüss, Dorota; Ducka, Paulina; Schönauer, Esther; Brandstetter, Hans

    2008-05-01

    The catalytic domain of collagenase G from C. histolyticum was expressed in E. coli BL21 (DE3) and purified using affinity and size-exclusion column-chromatographic methods. Crystals were obtained at 290 K by the sitting-drop vapour-diffusion method and diffraction data have been collected to 2.75 Å resolution. The catalytic domain of collagenase G from Clostridium histolyticum has been cloned, recombinantly expressed in Escherichia coli and purified using affinity and size-exclusion column-chromatographic methods. Crystals of the catalytic domain were obtained from 0.12 M sodium citrate and 23%(v/v) PEG 3350 at 293 K. The crystals diffracted to 2.75 Å resolution using synchrotron radiation. The crystals belong to an orthorhombic space group, with unit-cell parameters a = 57, b = 109, c = 181 Å. This unit cell is consistent with the presence of one molecule per asymmetric unit and a solvent content of approximately 53%.

  9. Domain organization and crystal structure of the catalytic domain of E.coli RluF, a pseudouridine synthase that acts on 23S rRNA

    SciTech Connect

    Sunita,S.; Zhenxing, H.; Swaathi, J.; Cygler, M.; Matte, A.; Sivaraman, J.

    2006-01-01

    Pseudouridine synthases catalyze the isomerization of uridine to pseudouridine ({psi}) in rRNA and tRNA. The pseudouridine synthase RluF from Escherichia coli (E.C. 4.2.1.70) modifies U2604 in 23S rRNA, and belongs to a large family of pseudouridine synthases present in all kingdoms of life. Here we report the domain architecture and crystal structure of the catalytic domain of E. coli RluF at 2.6 Angstroms resolution. Limited proteolysis, mass spectrometry and N-terminal sequencing indicate that RluF has a distinct domain architecture, with the catalytic domain flanked at the N and C termini by additional domains connected to it by flexible linkers. The structure of the catalytic domain of RluF is similar to those of RsuA and TruB. RluF is a member of the RsuA sequence family of {psi}-synthases, along with RluB and RluE. Structural comparison of RluF with its closest structural homologues, RsuA and TruB, suggests possible functional roles for the N-terminal and C-terminal domains of RluF.

  10. Insight into Pleiotropic Drug Resistance ATP-binding Cassette Pump Drug Transport through Mutagenesis of Cdr1p Transmembrane Domains*

    PubMed Central

    Rawal, Manpreet Kaur; Khan, Mohammad Firoz; Kapoor, Khyati; Goyal, Neha; Sen, Sobhan; Saxena, Ajay Kumar; Lynn, Andrew M.; Tyndall, Joel D. A.; Monk, Brian C.; Cannon, Richard D.; Komath, Sneha Sudha; Prasad, Rajendra

    2013-01-01

    The fungal ATP-binding cassette (ABC) transporter Cdr1 protein (Cdr1p), responsible for clinically significant drug resistance, is composed of two transmembrane domains (TMDs) and two nucleotide binding domains (NBDs). We have probed the nature of the drug binding pocket by performing systematic mutagenesis of the primary sequences of the 12 transmembrane segments (TMSs) found in the TMDs. All mutated proteins were expressed equally well and localized properly at the plasma membrane in the heterologous host Saccharomyces cerevisiae, but some variants differed significantly in efflux activity, substrate specificity, and coupled ATPase activity. Replacement of the majority of the amino acid residues with alanine or glycine yielded neutral mutations, but about 42% of the variants lost resistance to drug efflux substrates completely or selectively. A predicted three-dimensional homology model shows that all the TMSs, apart from TMS4 and TMS10, interact directly with the drug-binding cavity in both the open and closed Cdr1p conformations. However, TMS4 and TMS10 mutations can also induce total or selective drug susceptibility. Functional data and homology modeling assisted identification of critical amino acids within a drug-binding cavity that, upon mutation, abolished resistance to all drugs tested singly or in combinations. The open and closed Cdr1p models enabled the identification of amino acid residues that bordered a drug-binding cavity dominated by hydrophobic residues. The disposition of TMD residues with differential effects on drug binding and transport are consistent with a large polyspecific drug binding pocket in this yeast multidrug transporter. PMID:23824183

  11. Structure of the catalytic domain of the hepatitis C virus NS2-3 protease

    SciTech Connect

    Lorenz,I.; Marcotrigiano, J.; Dentzer, T.; Rice, C.

    2006-01-01

    Hepatitis C virus is a major global health problem affecting an estimated 170 million people worldwide. Chronic infection is common and can lead to cirrhosis and liver cancer. There is no vaccine available and current therapies have met with limited success. The viral RNA genome encodes a polyprotein that includes two proteases essential for virus replication. The NS2-3 protease mediates a single cleavage at the NS2/NS3 junction, whereas the NS3-4A protease cleaves at four downstream sites in the polyprotein. NS3-4A is characterized as a serine protease with a chymotrypsin-like fold, but the enzymatic mechanism of the NS2-3 protease remains unresolved. Here we report the crystal structure of the catalytic domain of the NS2-3 protease at 2.3 Angstroms resolution. The structure reveals a dimeric cysteine protease with two composite active sites. For each active site, the catalytic histidine and glutamate residues are contributed by one monomer, and the nucleophilic cysteine by the other. The carboxy-terminal residues remain coordinated in the two active sites, predicting an inactive post-cleavage form. Proteolysis through formation of a composite active site occurs in the context of the viral polyprotein expressed in mammalian cells. These features offer unexpected insights into polyprotein processing by hepatitis C virus and new opportunities for antiviral drug design.

  12. NMR Structure and Dynamics of the Resuscitation Promoting Factor RpfC Catalytic Domain

    PubMed Central

    Maione, Vincenzo; Ruggiero, Alessia; Russo, Luigi; De Simone, Alfonso; Pedone, Paolo Vincenzo; Malgieri, Gaetano; Berisio, Rita; Isernia, Carla

    2015-01-01

    Mycobacterium tuberculosis latent infection is maintained for years with no clinical symptoms and no adverse effects for the host. The mechanism through which dormant M. tuberculosis resuscitates and enters the cell cycle leading to tuberculosis is attracting much interest. The RPF family of proteins has been found to be responsible for bacteria resuscitation and normal proliferation. This family of proteins in M. tuberculosis is composed by five homologues (named RpfA-E) and understanding their conformational, structural and functional peculiarities is crucial to the design of therapeutic strategies.Therefore, we report the structural and dynamics characterization of the catalytic domain of RpfC from M. tubercolosis by combining Nuclear Magnetic Resonance, Circular Dichroism and Molecular Dynamics data. We also show how the formation of a disulfide bridge, highly conserved among the homologues, is likely to modulate the shape of the RpfC hydrophobic catalytic cleft. This might result in a protein function regulation via a “conformational editing” through a disulfide bond formation. PMID:26576056

  13. NMR Structure and Dynamics of the Resuscitation Promoting Factor RpfC Catalytic Domain.

    PubMed

    Maione, Vincenzo; Ruggiero, Alessia; Russo, Luigi; De Simone, Alfonso; Pedone, Paolo Vincenzo; Malgieri, Gaetano; Berisio, Rita; Isernia, Carla

    2015-01-01

    Mycobacterium tuberculosis latent infection is maintained for years with no clinical symptoms and no adverse effects for the host. The mechanism through which dormant M. tuberculosis resuscitates and enters the cell cycle leading to tuberculosis is attracting much interest. The RPF family of proteins has been found to be responsible for bacteria resuscitation and normal proliferation. This family of proteins in M. tuberculosis is composed by five homologues (named RpfA-E) and understanding their conformational, structural and functional peculiarities is crucial to the design of therapeutic strategies.Therefore, we report the structural and dynamics characterization of the catalytic domain of RpfC from M. tubercolosis by combining Nuclear Magnetic Resonance, Circular Dichroism and Molecular Dynamics data. We also show how the formation of a disulfide bridge, highly conserved among the homologues, is likely to modulate the shape of the RpfC hydrophobic catalytic cleft. This might result in a protein function regulation via a "conformational editing" through a disulfide bond formation.

  14. High resolution crystal structure of the catalytic domain of MCR-1

    PubMed Central

    Ma, Guixing; Zhu, Yifan; Yu, Zhicheng; Ahmad, Ashfaq; Zhang, Hongmin

    2016-01-01

    The newly identified mobile colistin resistant gene (mcr-1) rapidly spread among different bacterial strains and confers colistin resistance to its host, which has become a global concern. Based on sequence alignment, MCR-1 should be a phosphoethanolamine transferase, members of the YhjW/YjdB/YijP superfamily and catalyze the addition of phosphoethanolamine to lipid A, which needs to be validated experimentally. Here we report the first high-resolution crystal structure of the C-terminal catalytic domain of MCR-1 (MCR-1C) in its native state. The active pocket of native MCR-1C depicts unphosphorylated nucleophilic residue Thr285 in coordination with two Zinc ions and water molecules. A flexible adjacent active site loop (aa: Lys348-365) pose an open conformation compared to its structural homologues, suggesting of an open substrate entry channel. Taken together, this structure sets ground for further study of substrate binding and MCR-1 catalytic mechanism in development of potential therapeutic agents. PMID:28000749

  15. Structure of the catalytic domain of the Salmonella virulence factor SseI

    PubMed Central

    Bhaskaran, Shyam S.; Stebbins, C. Erec

    2012-01-01

    SseI is secreted into host cells by Salmonella and contributes to the establishment of systemic infections. The crystal structure of the C-terminal domain of SseI has been solved to 1.70 Å resolution, revealing it to be a member of the cysteine protease superfamily with a catalytic triad consisting of Cys178, His216 and Asp231 that is critical to its virulence activities. Structure-based analysis revealed that SseI is likely to possess either acyl hydrolase or acyltransferase activity, placing this virulence factor in the rapidly growing class of enzymes of this family utilized by bacterial pathogens inside eukaryotic cells. PMID:23151626

  16. Structure of the catalytic domain of human fibroblast collagenase complexed with an inhibitor.

    PubMed

    Borkakoti, N; Winkler, F K; Williams, D H; D'Arcy, A; Broadhurst, M J; Brown, P A; Johnson, W H; Murray, E J

    1994-02-01

    In rheumatoid and osteoarthritis, degradation of articular cartilage is mediated by the matrix metalloproteinases collagenase, stromelysin and gelatinase. The key event in this process is the cleavage of triple helical collagen by collagenase. We have determined the crystal structure of the catalytic domain of human recombinant fibroblast collagenase complexed with a synthetic inhibitor at 2.2 A resolution. The protein fold is similar to the amino termini of the zinc endopeptidases astacin thermolysin and elastase despite a lack of primary sequence homology. The conformation of the bound inhibitor provides a molecular basis for the design of inhibitors of collagenase and other matrix metalloproteinases. Such inhibitors should be useful in the treatment of a variety of diseases including arthritis and cancer.

  17. Engineering the catalytic domain of human protein tyrosine phosphatase beta for structure-based drug discovery.

    PubMed

    Evdokimov, Artem G; Pokross, Matthew; Walter, Richard; Mekel, Marlene; Cox, Brooke; Li, Chuiying; Bechard, Randy; Genbauffe, Frank; Andrews, Ryan; Diven, Conrad; Howard, Brian; Rastogi, Vinit; Gray, Jeffrey; Maier, Matthew; Peters, Kevin G

    2006-12-01

    Protein tyrosine phosphatases (PTPs) play roles in many biological processes and are considered to be important targets for drug discovery. As inhibitor development has proven challenging, crystal structure-based design will be very helpful to advance inhibitor potency and selectivity. Successful application of protein crystallography to drug discovery heavily relies on high-quality crystal structures of the protein of interest complexed with pharmaceutically interesting ligands. It is very important to be able to produce protein-ligand crystals rapidly and reproducibly for as many ligands as necessary. This study details our efforts to engineer the catalytic domain of human protein tyrosine phosphatase beta (HPTPbeta-CD) with properties suitable for rapid-turnaround crystallography. Structures of apo HPTPbeta-CD and its complexes with several novel small-molecule inhibitors are presented here for the first time.

  18. Human CTP:phosphoethanolamine cytidylyltransferase: enzymatic properties and unequal catalytic roles of CTP-binding motifs in two cytidylyltransferase domains.

    PubMed

    Tian, Siqi; Ohtsuka, Jun; Wang, Shipeng; Nagata, Koji; Tanokura, Masaru; Ohta, Akinori; Horiuchi, Hiroyuki; Fukuda, Ryouichi

    2014-06-20

    CTP:phosphoethanolamine cytidylyltransferase (ECT) is a key enzyme in the CDP-ethanolamine branch of the Kennedy pathway, which is the primary pathway of phosphatidylethanolamine (PE) synthesis in mammalian cells. Here, the enzymatic properties of recombinant human ECT (hECT) were characterized. The catalytic reaction of hECT obeyed Michaelis-Menten kinetics with respect to both CTP and phosphoethanolamine. hECT is composed of two tandem cytidylyltransferase (CT) domains as ECTs of other organisms. The histidines, especially the first histidine, in the CTP-binding motif HxGH in the N-terminal CT domain were critical for its catalytic activity in vitro, while those in the C-terminal CT domain were not. Overexpression of the wild-type hECT and hECT mutants containing amino acid substitutions in the HxGH motif in the C-terminal CT domain suppressed the growth defect of the Saccharomyces cerevisiae mutant of ECT1 encoding ECT in the absence of a PE supply via the decarboxylation of phosphatidylserine, but overexpression of hECT mutants of the N-terminal CT domain did not. These results suggest that the N-terminal CT domain of hECT contributes to its catalytic reaction, but C-terminal CT domain does not. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. The TriTryp phosphatome: analysis of the protein phosphatase catalytic domains.

    PubMed

    Brenchley, Rachel; Tariq, Humera; McElhinney, Helen; Szöor, Balázs; Huxley-Jones, Julie; Stevens, Robert; Matthews, Keith; Tabernero, Lydia

    2007-11-26

    The genomes of the three parasitic protozoa Trypanosoma cruzi, Trypanosoma brucei and Leishmania major are the main subject of this study. These parasites are responsible for devastating human diseases known as Chagas disease, African sleeping sickness and cutaneous Leishmaniasis, respectively, that affect millions of people in the developing world. The prevalence of these neglected diseases results from a combination of poverty, inadequate prevention and difficult treatment. Protein phosphorylation is an important mechanism of controlling the development of these kinetoplastids. With the aim to further our knowledge of the biology of these organisms we present a characterisation of the phosphatase complement (phosphatome) of the three parasites. An ontology-based scan of the three genomes was used to identify 86 phosphatase catalytic domains in T. cruzi, 78 in T. brucei, and 88 in L. major. We found interesting differences with other eukaryotic genomes, such as the low proportion of tyrosine phosphatases and the expansion of the serine/threonine phosphatase family. Additionally, a large number of atypical protein phosphatases were identified in these species, representing more than one third of the total phosphatase complement. Most of the atypical phosphatases belong to the dual-specificity phosphatase (DSP) family and show considerable divergence from classic DSPs in both the domain organisation and sequence features. The analysis of the phosphatome of the three kinetoplastids indicates that they possess orthologues to many of the phosphatases reported in other eukaryotes, including humans. However, novel domain architectures and unusual combinations of accessory domains, suggest distinct functional roles for several of the kinetoplastid phosphatases, which await further experimental exploration. These distinct traits may be exploited in the selection of suitable new targets for drug development to prevent transmission and spread of the diseases, taking

  20. Ankyrin domain of myosin 16 influences motor function and decreases protein phosphatase catalytic activity.

    PubMed

    Kengyel, András; Bécsi, Bálint; Kónya, Zoltán; Sellers, James R; Erdődi, Ferenc; Nyitrai, Miklós

    2015-05-01

    The unconventional myosin 16 (Myo16), which may have a role in regulation of cell cycle and cell proliferation, can be found in both the nucleus and the cytoplasm. It has a unique, eight ankyrin repeat containing pre-motor domain, the so-called ankyrin domain (My16Ank). Ankyrin repeats are present in several other proteins, e.g., in the regulatory subunit (MYPT1) of the myosin phosphatase holoenzyme, which binds to the protein phosphatase-1 catalytic subunit (PP1c). My16Ank shows sequence similarity to MYPT1. In this work, the interactions of recombinant and isolated My16Ank were examined in vitro. To test the effects of My16Ank on myosin motor function, we used skeletal muscle myosin or nonmuscle myosin 2B. The results showed that My16Ank bound to skeletal muscle myosin (K D ≈ 2.4 µM) and the actin-activated ATPase activity of heavy meromyosin (HMM) was increased in the presence of My16Ank, suggesting that the ankyrin domain can modulate myosin motor activity. My16Ank showed no direct interaction with either globular or filamentous actin. We found, using a surface plasmon resonance-based binding technique, that My16Ank bound to PP1cα (K D ≈ 540 nM) and also to PP1cδ (K D ≈ 600 nM) and decreased its phosphatase activity towards the phosphorylated myosin regulatory light chain. Our results suggest that one function of the ankyrin domain is probably to regulate the function of Myo16. It may influence the motor activity, and in complex with the PP1c isoforms, it can play an important role in the targeted dephosphorylation of certain, as yet unidentified, intracellular proteins.

  1. Cu-free cycloaddition for identifying catalytic active adenylation domains of nonribosomal peptide synthetases by phage display.

    PubMed

    Zou, Yekui; Yin, Jun

    2008-10-15

    To engineer the substrate specificities of nonribosomal peptide synthetases (NRPS), we developed a method to display NRPS modules on M13 phages and select catalytically active adenylation (A) domains that would load azide functionalized substrate analogs to the neighboring peptidyl carrier protein (PCP) domains. Biotin conjugated difluorinated cyclooctyne was used for copper free cycloaddition with an azide substituted substrate attached to PCP. Biotin-labeled phages were selected by binding to streptavidin.

  2. Crystallization and Preliminary X-ray Diffraction Analysis of the Glucuronoyl Esterase Catalytic Domain from Hypocrea jecorina

    USDA-ARS?s Scientific Manuscript database

    The catalytic domain of the glucuronoyl esterase from Hypocrea jecorina (anamorph Trichoderma reesei) was over-expressed, purified, and crystallized by sitting-drop vapor-diffusion method using 1.4 M sodium/potassium phosphate pH 6.9. Crystals had space group P212121 and X-ray diffraction data were...

  3. Catalytic properties of two Rhizopus oryzae 99-880 glucoamylase enzymes without starch binding domains expressed in Pichia pastoris

    USDA-ARS?s Scientific Manuscript database

    Catalytic properties of the two glucoamylases, AmyC and AmyD, without starch binding domains from Rhizopus oryzae strain 99-880 were heterologously expressed and purified to homogeneity. AmyC and AmyD demonstrate pH optima of 5.5 and 6.0, respectively, nearly 1 unit higher than most fungal glucoamy...

  4. Stromelysin-1: three-dimensional structure of the inhibited catalytic domain and of the C-truncated proenzyme.

    PubMed Central

    Becker, J. W.; Marcy, A. I.; Rokosz, L. L.; Axel, M. G.; Burbaum, J. J.; Fitzgerald, P. M.; Cameron, P. M.; Esser, C. K.; Hagmann, W. K.; Hermes, J. D.

    1995-01-01

    The proteolytic enzyme stromelysin-1 is a member of the family of matrix metalloproteinases and is believed to play a role in pathological conditions such as arthritis and tumor invasion. Stromelysin-1 is synthesized as a pro-enzyme that is activated by removal of an N-terminal prodomain. The active enzyme contains a catalytic domain and a C-terminal hemopexin domain believed to participate in macromolecular substrate recognition. We have determined the three-dimensional structures of both a C-truncated form of the proenzyme and an inhibited complex of the catalytic domain by X-ray diffraction analysis. The catalytic core is very similar in the two forms and is similar to the homologous domain in fibroblast and neutrophil collagenases, as well as to the stromelysin structure determined by NMR. The prodomain is a separate folding unit containing three alpha-helices and an extended peptide that lies in the active site of the enzyme. Surprisingly, the amino-to-carboxyl direction of this peptide chain is opposite to that adopted by the inhibitor and by previously reported inhibitors of collagenase. Comparison of the active site of stromelysin with that of thermolysin reveals that most of the residues proposed to play significant roles in the enzymatic mechanism of thermolysin have equivalents in stromelysin, but that three residues implicated in the catalytic mechanism of thermolysin are not represented in stromelysin. PMID:8535233

  5. Alternating access to the transmembrane domain of the ATP-binding cassette protein cystic fibrosis transmembrane conductance regulator (ABCC7).

    PubMed

    Wang, Wuyang; Linsdell, Paul

    2012-03-23

    The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is a member of the ATP-binding cassette (ABC) protein family, most members of which act as active transporters. Actively transporting ABC proteins are thought to alternate between "outwardly facing" and "inwardly facing" conformations of the transmembrane substrate pathway. In CFTR, it is assumed that the outwardly facing conformation corresponds to the channel open state, based on homology with other ABC proteins. We have used patch clamp recording to quantify the rate of access of cysteine-reactive probes to cysteines introduced into two different transmembrane regions of CFTR from both the intracellular and extracellular solutions. Two probes, the large [2-sulfonatoethyl]methanethiosulfonate (MTSES) molecule and permeant Au(CN)(2)(-) ions, were applied to either side of the membrane to modify cysteines substituted for Leu-102 (first transmembrane region) and Thr-338 (sixth transmembrane region). Channel opening and closing were altered by mutations in the nucleotide binding domains of the channel. We find that, for both MTSES and Au(CN)(2)(-), access to these two cysteines from the cytoplasmic side is faster in open channels, whereas access to these same sites from the extracellular side is faster in closed channels. These results are consistent with alternating access to the transmembrane regions, however with the open state facing inwardly and the closed state facing outwardly. Our findings therefore prompt revision of current CFTR structural and mechanistic models, as well as having broader implications for transport mechanisms in all ABC proteins. Our results also suggest possible locations of both functional and dysfunctional ("vestigial") gates within the CFTR permeation pathway.

  6. 1.92 Angstrom Zinc-Free APOBEC3F Catalytic Domain Crystal Structure.

    PubMed

    Shaban, Nadine M; Shi, Ke; Li, Ming; Aihara, Hideki; Harris, Reuben S

    2016-06-05

    The APOBEC3 family of DNA cytosine deaminases is capable of restricting the replication of HIV-1 and other pathogens. Here, we report a 1.92 Å resolution crystal structure of the Vif-binding and catalytic domain of APOBEC3F (A3F). This structure is distinct from the previously published APOBEC and phylogenetically related deaminase structures, as it is the first without zinc in the active site. We determined an additional structure containing zinc in the same crystal form that allows direct comparison with the zinc-free structure. In the absence of zinc, the conserved active site residues that normally participate in zinc coordination show unique conformations, including a 90 degree rotation of His249 and disulfide bond formation between Cys280 and Cys283. We found that zinc coordination is influenced by pH, and treating the protein at low pH in crystallization buffer is sufficient to remove zinc. Zinc coordination and catalytic activity are reconstituted with the addition of zinc only in a reduced environment likely due to the two active site cysteines readily forming a disulfide bond when not coordinating zinc. We show that the enzyme is active in the presence of zinc and cobalt but not with other divalent metals. These results unexpectedly demonstrate that zinc is not required for the structural integrity of A3F and suggest that metal coordination may be a strategy for regulating the activity of A3F and related deaminases. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Crystal Structure of the Catalytic Domain of Drosophila [beta]1,4-Galactosyltransferase-7

    SciTech Connect

    Ramakrishnan, Boopathy; Qasba, Pradman K.

    2010-11-03

    The {beta}1,4-galactosyltransferase-7 ({beta}4Gal-T7) enzyme, one of seven members of the {beta}4Gal-T family, transfers in the presence of manganese Gal from UDP-Gal to an acceptor sugar (xylose) that is attached to a side chain hydroxyl group of Ser/Thr residues of proteoglycan proteins. It exhibits the least protein sequence similarity with the other family members, including the well studied family member {beta}4Gal-T1, which, in the presence of manganese, transfers Gal from UDP-Gal to GlcNAc. We report here the crystal structure of the catalytic domain of {beta}4Gal-T7 from Drosophila in the presence of manganese and UDP at 1.81 {angstrom} resolution. In the crystal structure, a new manganese ion-binding motif (HXH) has been observed. Superposition of the crystal structures of {beta}4Gal-T7 and {beta}4Gal-T1 shows that the catalytic pocket and the substrate-binding sites in these proteins are similar. Compared with GlcNAc, xylose has a hydroxyl group (instead of an N-acetyl group) at C2 and lacks the CH{sub 2}OH group at C5; thus, these protein structures show significant differences in their acceptor-binding site. Modeling of xylose in the acceptor-binding site of the {beta}4Gal-T7 crystal structure shows that the aromatic side chain of Tyr{sup 177} interacts strongly with the C5 atom of xylose, causing steric hindrance to any additional group at C5. Because Drosophila Cd7 has a 73% protein sequence similarity to human Cd7, the present crystal structure offers a structure-based explanation for the mutations in human Cd7 that have been linked to Ehlers-Danlos syndrome.

  8. Crystal structure of the catalytic domain of Drosophila beta1,4-Galactosyltransferase-7.

    PubMed

    Ramakrishnan, Boopathy; Qasba, Pradman K

    2010-05-14

    The beta1,4-galactosyltransferase-7 (beta4Gal-T7) enzyme, one of seven members of the beta4Gal-T family, transfers in the presence of manganese Gal from UDP-Gal to an acceptor sugar (xylose) that is attached to a side chain hydroxyl group of Ser/Thr residues of proteoglycan proteins. It exhibits the least protein sequence similarity with the other family members, including the well studied family member beta4Gal-T1, which, in the presence of manganese, transfers Gal from UDP-Gal to GlcNAc. We report here the crystal structure of the catalytic domain of beta4Gal-T7 from Drosophila in the presence of manganese and UDP at 1.81 A resolution. In the crystal structure, a new manganese ion-binding motif (HXH) has been observed. Superposition of the crystal structures of beta4Gal-T7 and beta4Gal-T1 shows that the catalytic pocket and the substrate-binding sites in these proteins are similar. Compared with GlcNAc, xylose has a hydroxyl group (instead of an N-acetyl group) at C2 and lacks the CH(2)OH group at C5; thus, these protein structures show significant differences in their acceptor-binding site. Modeling of xylose in the acceptor-binding site of the beta4Gal-T7 crystal structure shows that the aromatic side chain of Tyr(177) interacts strongly with the C5 atom of xylose, causing steric hindrance to any additional group at C5. Because Drosophila Cd7 has a 73% protein sequence similarity to human Cd7, the present crystal structure offers a structure-based explanation for the mutations in human Cd7 that have been linked to Ehlers-Danlos syndrome.

  9. Activities of human RRP6 and structure of the human RRP6 catalytic domain

    SciTech Connect

    Januszyk, Kurt; Liu, Quansheng; Lima, Christopher D.

    2011-08-29

    The eukaryotic RNA exosome is a highly conserved multi-subunit complex that catalyzes degradation and processing of coding and noncoding RNA. A noncatalytic nine-subunit exosome core interacts with Rrp44 and Rrp6, two subunits that possess processive and distributive 3'-to-5' exoribonuclease activity, respectively. While both Rrp6 and Rrp44 are responsible for RNA processing in budding yeast, Rrp6 may play a more prominent role in processing, as it has been demonstrated to be inhibited by stable RNA secondary structure in vitro and because the null allele in budding yeast leads to the buildup of specific structured RNA substrates. Human RRP6, otherwise known as PM/SCL-100 or EXOSC10, shares sequence similarity to budding yeast Rrp6 and is proposed to catalyze 3'-to-5' exoribonuclease activity on a variety of nuclear transcripts including ribosomal RNA subunits, RNA that has been poly-adenylated by TRAMP, as well as other nuclear RNA transcripts destined for processing and/or destruction. To characterize human RRP6, we expressed the full-length enzyme as well as truncation mutants that retain catalytic activity, compared their activities to analogous constructs for Saccharomyces cerevisiae Rrp6, and determined the X-ray structure of a human construct containing the exoribonuclease and HRDC domains that retains catalytic activity. Structural data show that the human active site is more exposed when compared to the yeast structure, and biochemical data suggest that this feature may play a role in the ability of human RRP6 to productively engage and degrade structured RNA substrates more effectively than the analogous budding yeast enzyme.

  10. Structures of the human poly (ADP-ribose) glycohydrolase catalytic domain confirm catalytic mechanism and explain inhibition by ADP-HPD derivatives.

    PubMed

    Tucker, Julie A; Bennett, Neil; Brassington, Claire; Durant, Stephen T; Hassall, Giles; Holdgate, Geoff; McAlister, Mark; Nissink, J Willem M; Truman, Caroline; Watson, Martin

    2012-01-01

    Poly(ADP-ribose) glycohydrolase (PARG) is the only enzyme known to catalyse hydrolysis of the O-glycosidic linkages of ADP-ribose polymers, thereby reversing the effects of poly(ADP-ribose) polymerases. PARG deficiency leads to cell death whilst PARG depletion causes sensitisation to certain DNA damaging agents, implicating PARG as a potential therapeutic target in several disease areas. Efforts to develop small molecule inhibitors of PARG activity have until recently been hampered by a lack of structural information on PARG. We have used a combination of bio-informatic and experimental approaches to engineer a crystallisable, catalytically active fragment of human PARG (hPARG). Here, we present high-resolution structures of the catalytic domain of hPARG in unliganded form and in complex with three inhibitors: ADP-ribose (ADPR), adenosine 5'-diphosphate (hydroxymethyl)pyrrolidinediol (ADP-HPD) and 8-n-octyl-amino-ADP-HPD. Our structures confirm conservation of overall fold amongst mammalian PARG glycohydrolase domains, whilst revealing additional flexible regions in the catalytic site. These new structures rationalise a body of published mutational data and the reported structure-activity relationship for ADP-HPD based PARG inhibitors. In addition, we have developed and used biochemical, isothermal titration calorimetry and surface plasmon resonance assays to characterise the binding of inhibitors to our PARG protein, thus providing a starting point for the design of new inhibitors.

  11. Structures of the Human Poly (ADP-Ribose) Glycohydrolase Catalytic Domain Confirm Catalytic Mechanism and Explain Inhibition by ADP-HPD Derivatives

    PubMed Central

    Tucker, Julie A.; Bennett, Neil; Brassington, Claire; Durant, Stephen T.; Hassall, Giles; Holdgate, Geoff; McAlister, Mark; Nissink, J. Willem M.; Truman, Caroline; Watson, Martin

    2012-01-01

    Poly(ADP-ribose) glycohydrolase (PARG) is the only enzyme known to catalyse hydrolysis of the O-glycosidic linkages of ADP-ribose polymers, thereby reversing the effects of poly(ADP-ribose) polymerases. PARG deficiency leads to cell death whilst PARG depletion causes sensitisation to certain DNA damaging agents, implicating PARG as a potential therapeutic target in several disease areas. Efforts to develop small molecule inhibitors of PARG activity have until recently been hampered by a lack of structural information on PARG. We have used a combination of bio-informatic and experimental approaches to engineer a crystallisable, catalytically active fragment of human PARG (hPARG). Here, we present high-resolution structures of the catalytic domain of hPARG in unliganded form and in complex with three inhibitors: ADP-ribose (ADPR), adenosine 5′-diphosphate (hydroxymethyl)pyrrolidinediol (ADP-HPD) and 8-n-octyl-amino-ADP-HPD. Our structures confirm conservation of overall fold amongst mammalian PARG glycohydrolase domains, whilst revealing additional flexible regions in the catalytic site. These new structures rationalise a body of published mutational data and the reported structure-activity relationship for ADP-HPD based PARG inhibitors. In addition, we have developed and used biochemical, isothermal titration calorimetry and surface plasmon resonance assays to characterise the binding of inhibitors to our PARG protein, thus providing a starting point for the design of new inhibitors. PMID:23251397

  12. Dynamic interplay between catalytic and lectin domains of GalNAc-transferases modulates protein O-glycosylation

    NASA Astrophysics Data System (ADS)

    Lira-Navarrete, Erandi; de Las Rivas, Matilde; Compañón, Ismael; Pallarés, María Carmen; Kong, Yun; Iglesias-Fernández, Javier; Bernardes, Gonçalo J. L.; Peregrina, Jesús M.; Rovira, Carme; Bernadó, Pau; Bruscolini, Pierpaolo; Clausen, Henrik; Lostao, Anabel; Corzana, Francisco; Hurtado-Guerrero, Ramon

    2015-05-01

    Protein O-glycosylation is controlled by polypeptide GalNAc-transferases (GalNAc-Ts) that uniquely feature both a catalytic and lectin domain. The underlying molecular basis of how the lectin domains of GalNAc-Ts contribute to glycopeptide specificity and catalysis remains unclear. Here we present the first crystal structures of complexes of GalNAc-T2 with glycopeptides that together with enhanced sampling molecular dynamics simulations demonstrate a cooperative mechanism by which the lectin domain enables free acceptor sites binding of glycopeptides into the catalytic domain. Atomic force microscopy and small-angle X-ray scattering experiments further reveal a dynamic conformational landscape of GalNAc-T2 and a prominent role of compact structures that are both required for efficient catalysis. Our model indicates that the activity profile of GalNAc-T2 is dictated by conformational heterogeneity and relies on a flexible linker located between the catalytic and the lectin domains. Our results also shed light on how GalNAc-Ts generate dense decoration of proteins with O-glycans.

  13. Dynamic interplay between catalytic and lectin domains of GalNAc-transferases modulates protein O-glycosylation

    PubMed Central

    Lira-Navarrete, Erandi; de las Rivas, Matilde; Compañón, Ismael; Pallarés, María Carmen; Kong, Yun; Iglesias-Fernández, Javier; Bernardes, Gonçalo J. L.; Peregrina, Jesús M.; Rovira, Carme; Bernadó, Pau; Bruscolini, Pierpaolo; Clausen, Henrik; Lostao, Anabel; Corzana, Francisco; Hurtado-Guerrero, Ramon

    2015-01-01

    Protein O-glycosylation is controlled by polypeptide GalNAc-transferases (GalNAc-Ts) that uniquely feature both a catalytic and lectin domain. The underlying molecular basis of how the lectin domains of GalNAc-Ts contribute to glycopeptide specificity and catalysis remains unclear. Here we present the first crystal structures of complexes of GalNAc-T2 with glycopeptides that together with enhanced sampling molecular dynamics simulations demonstrate a cooperative mechanism by which the lectin domain enables free acceptor sites binding of glycopeptides into the catalytic domain. Atomic force microscopy and small-angle X-ray scattering experiments further reveal a dynamic conformational landscape of GalNAc-T2 and a prominent role of compact structures that are both required for efficient catalysis. Our model indicates that the activity profile of GalNAc-T2 is dictated by conformational heterogeneity and relies on a flexible linker located between the catalytic and the lectin domains. Our results also shed light on how GalNAc-Ts generate dense decoration of proteins with O-glycans. PMID:25939779

  14. The acidic domain of the endothelial membrane protein GPIHBP1 stabilizes lipoprotein lipase activity by preventing unfolding of its catalytic domain.

    PubMed

    Mysling, Simon; Kristensen, Kristian Kølby; Larsson, Mikael; Beigneux, Anne P; Gårdsvoll, Henrik; Fong, Loren G; Bensadouen, André; Jørgensen, Thomas Jd; Young, Stephen G; Ploug, Michael

    2016-01-03

    GPIHBP1 is a glycolipid-anchored membrane protein of capillary endothelial cells that binds lipoprotein lipase (LPL) within the interstitial space and shuttles it to the capillary lumen. The LPL•GPIHBP1 complex is responsible for margination of triglyceride-rich lipoproteins along capillaries and their lipolytic processing. The current work conceptualizes a model for the GPIHBP1•LPL interaction based on biophysical measurements with hydrogen-deuterium exchange/mass spectrometry, surface plasmon resonance, and zero-length cross-linking. According to this model, GPIHBP1 comprises two functionally distinct domains: (1) an intrinsically disordered acidic N-terminal domain; and (2) a folded C-terminal domain that tethers GPIHBP1 to the cell membrane by glycosylphosphatidylinositol. We demonstrate that these domains serve different roles in regulating the kinetics of LPL binding. Importantly, the acidic domain stabilizes LPL catalytic activity by mitigating the global unfolding of LPL's catalytic domain. This study provides a conceptual framework for understanding intravascular lipolysis and GPIHBP1 and LPL mutations causing familial chylomicronemia.

  15. Crystal structures of wild-type Trichoderma reesei Cel7A catalytic domain in open and closed states.

    PubMed

    Bodenheimer, Annette M; Meilleur, Flora

    2016-12-01

    Trichoderma reesei Cel7A efficiently hydrolyses cellulose. We report here the crystallographic structures of the wild-type TrCel7A catalytic domain (CD) in an open state and, for the first time, in a closed state. Molecular dynamics (MD) simulations indicate that the loops along the CD tunnel move in concerted motions. Together, the crystallographic and MD data suggest that the CD cycles between the tense and relaxed forms that are characteristic of work producing enzymes. Analysis of the interactions formed by R251 provides a structural rationale for the concurrent decrease in product inhibition and catalytic efficiency measured for product-binding site mutants.

  16. Structure-based engineering of histidine residues in the catalytic domain of α-amylase from Bacillus subtilis for improved protein stability and catalytic efficiency under acidic conditions.

    PubMed

    Yang, Haiquan; Liu, Long; Shin, Hyun-dong; Chen, Rachel R; Li, Jianghua; Du, Guocheng; Chen, Jian

    2013-03-10

    This work aims to improve the protein stability and catalytic efficiency of α-amylase from Bacillus subtilis under acidic conditions by site-directed mutagenesis. Based on the analysis of a three dimensional structure model, four basic histidine (His) residues His(222), His(275), His(293), and His(310) in the catalytic domain were selected as the mutation sites and were further replaced with acidic aspartic acid (Asp), respectively, yielding four mutants H222D, H275D, H293D, H310D. The mutant H222D was inactive. Double and triple mutations were further conducted and four mutants H275/293D, H275/310D, H293/310D, and H275/293/310D were obtained. The acidic stability of enzyme was significantly enhanced after mutation, and 45-92% of initial activity of mutants was retained after incubation at pH 4.5 and 25°C for 24h, while that for wild-type was only 39.5%. At pH 4.5, the specific activity of wild-type and mutants H275D, H293D, H310D, H275/293D, H275/310D, H293/310D, and H275/293/310D were 108.2, 131.8, 138.9, 196.6, 156.3, 204.6, and 216.2U/mg, respectively. The catalytic efficiency for each active mutant was much higher than that of wild-type at low pH. The kcat/Km values of the mutants H275D, H293D, H310D, H275/293D, H275/310D, H293/310D, and H275/293/310D at pH 4.5 were 3.3-, 4.3-, 6.5-, 4.5-, 11.0-, 14.5-, and 16.7-fold higher, respectively, than that of the wild-type. As revealed by the structure models of the wild-type and mutant enzymes, the hydrogen bonds and salt bridges were increased after mutation, and an obvious shift of the basic limb toward acidity was observed for mutants. These changes around the catalytic domain contributed to the significantly improved protein stability and catalytic efficiency at low pH. This work provides an effective strategy to improve the catalytic activity and stability of α-amylase under acidic conditions, and the results obtained here may be useful for the improvement of acid-resistant ability of other enzymes.

  17. Adventitious Arsenate Reductase Activity of the Catalytic Domain of the Human Cdc25B and Cdc25C Phosphatases†

    PubMed Central

    Bhattacharjee, Hiranmoy; Sheng, Ju; Ajees, A. Abdul; Mukhopadhyay, Rita; Rosen, Barry P.

    2013-01-01

    A number of eukaryotic enzymes that function as arsenate reductases are homologues of the catalytic domain of the human Cdc25 phosphatase. For example, the Leishmania major enzyme LmACR2 is both a phosphatase and an arsenate reductase, and its structure bears similarity to the structure of the catalytic domain of human Cdc25 phosphatase. These reductases contain an active site C-X5-R signature motif, where C is the catalytic cysteine, the five X residues form a phosphate binding loop, and R is a highly conserved arginine, which is also present in human Cdc25 phosphatases. We therefore investigated the possibility that the three human Cdc25 isoforms might have adventitious arsenate reductase activity. The sequences for the catalytic domains of Cdc25A, -B, and -C were cloned individually into a prokaryotic expression vector, and their gene products were purified from a bacterial host using nickel affinity chromatography. While each of the three Cdc25 catalytic domains exhibited phosphatase activity, arsenate reductase activity was observed only with Cdc25B and -C. These two enzymes reduced inorganic arsenate but not methylated pentavalent arsenicals. Alteration of either the cysteine and arginine residues of the Cys-X5-Arg motif led to the loss of both reductase and phosphatase activities. Our observations suggest that Cdc25B and -C may adventitiously reduce arsenate to the more toxic arsenite and may also provide a framework for identifying other human protein tyrosine phosphatases containing the active site Cys-X5-Arg loop that might moonlight as arsenate reductases. PMID:20025242

  18. X-ray crystallographic studies of the extracellular domain of the first plant ATP receptor, DORN1, and the orthologous protein from Camelina sativa

    SciTech Connect

    Li, Zhijie; Chakraborty, Sayan; Xu, Guozhou

    2016-10-26

    Does not respond to nucleotides 1 (DORN1) has recently been identified as the first membrane-integral plant ATP receptor, which is required for ATP-induced calcium response, mitogen-activated protein kinase activation and defense responses inArabidopsis thaliana. In order to understand DORN1-mediated ATP sensing and signal transduction, crystallization and preliminary X-ray studies were conducted on the extracellular domain of DORN1 (atDORN1-ECD) and that of an orthologous protein,Camelina sativalectin receptor kinase I.9 (csLecRK-I.9-ECD or csI.9-ECD). A variety of deglycosylation strategies were employed to optimize the glycosylated recombinant atDORN1-ECD for crystallization. In addition, the glycosylated csI.9-ECD protein was crystallized at 291 K. X-ray diffraction data were collected at 4.6 Å resolution from a single crystal. The crystal belonged to space groupC222 orC2221, with unit-cell parametersa= 94.7,b= 191.5,c= 302.8 Å. These preliminary studies have laid the foundation for structural determination of the DORN1 and I.9 receptor proteins, which will lead to a better understanding of the perception and function of extracellular ATP in plants.

  19. Structure of the Catalytic Domain of the Class I Polyhydroxybutyrate Synthase from Cupriavidus necator.

    PubMed

    Wittenborn, Elizabeth C; Jost, Marco; Wei, Yifeng; Stubbe, JoAnne; Drennan, Catherine L

    2016-11-25

    Polyhydroxybutyrate synthase (PhaC) catalyzes the polymerization of 3-(R)-hydroxybutyryl-coenzyme A as a means of carbon storage in many bacteria. The resulting polymers can be used to make biodegradable materials with properties similar to those of thermoplastics and are an environmentally friendly alternative to traditional petroleum-based plastics. A full biochemical and mechanistic understanding of this process has been hindered in part by a lack of structural information on PhaC. Here we present the first structure of the catalytic domain (residues 201-589) of the class I PhaC from Cupriavidus necator (formerly Ralstonia eutropha) to 1.80 Å resolution. We observe a symmetrical dimeric architecture in which the active site of each monomer is separated from the other by ∼33 Å across an extensive dimer interface, suggesting a mechanism in which polyhydroxybutyrate biosynthesis occurs at a single active site. The structure additionally highlights key side chain interactions within the active site that play likely roles in facilitating catalysis, leading to the proposal of a modified mechanistic scheme involving two distinct roles for the active site histidine. We also identify putative substrate entrance and product egress routes within the enzyme, which are discussed in the context of previously reported biochemical observations. Our structure lays a foundation for further biochemical and structural characterization of PhaC, which could assist in engineering efforts for the production of eco-friendly materials. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Cloning, expression, purification, and characterization of the catalytic domain of sika deer MMP-13.

    PubMed

    Zhang, Xueliang; Wang, Jiawen; Liu, Meichen; Wang, Siming; Zhang, Hui; Zhao, Yu

    2016-11-01

    Matrix metalloproteinase 13 is one of three mammalian collagenases that are capable of initiating the degradation of interstitial collagens during wound healing. Herein, we report for the first time the molecular cloning of the catalytic domain (CD) of sika deer MMP-13, followed by protein expression in Escherichia coli and purification by affinity chromatography. The final yield was approximately 90.4 mg per liter of growth culture with a purity of 91.6%. The mass recovery during the purification and renaturation were 70.2% and 81.5%, respectively. Using gelatin zymography and a degradation assay, we found that the refolded sika deer MMP-13 (CD) could digest gelatin. The optimal pH and temperature for the enzyme bioactivity was 8.0 and 37 °C, respectively. The Km value for the enzyme-catalyzed digestion of gelatin was 136+/-8 μg/mL, and the Vmax was 4.12 × 10(3) U/μg. sdMMP13 (CD) was able to completely degrade collagen II and gelatin, and partially degrade fibronectin. The sdMMP-13 (CD) activity was significantly inhibited by several chemicals including 1, 10-phenanthroline, EDTA, Fe(2+), Cu(2+), and Mn(2+). Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Expression in Escherichia coli of the catalytic domain of human proline oxidase.

    PubMed

    Tallarita, Elena; Pollegioni, Loredano; Servi, Stefano; Molla, Gianluca

    2012-04-01

    The human PRODH gene has been shown to have unique roles in regulating cell survival and apoptotic pathways and it has been related to velocardiofacial syndrome/DiGeorge syndrome and increased susceptibility to schizophrenia. It encodes for the flavoprotein proline oxidase (PO), which catalyzes the conversion of l-proline to Δ(1)-pyrroline-5-carboxylate. Despite the important physiological and medical interest in human PO, up to now only microbial homologues of PO have been expressed as recombinant protein and fully characterized. By using a bioinformatics analysis aimed at identifying the catalytic domain and the regions with a high intrinsic propensity to structural disorder, we designed deletion variants of human PO that were successfully expressed in Escherichia coli as soluble proteins in fairly high amounts (up to 10mg/L of fermentation broth). The His-tagged PO-barrelN protein was isolated as an active (the specific activity is 0.032U/mg protein), dimeric holoenzyme showing the typical spectral properties of FAD-containing flavoprotein oxidases. These results pave the way for elucidating structure-function relationships of this human flavoenzyme and clarifying the effect of the reported polymorphisms associated with disease states.

  2. Unprecedented diversity of catalytic domains in the first four modules of the putative pederin polyketide synthase.

    PubMed

    Piel, Jörn; Wen, Gaiping; Platzer, Matthias; Hui, Dequan

    2004-01-03

    Polyketides of the pederin group are highly potent antitumor compounds found in terrestrial beetles and marine sponges. Pederin is used by beetles of the genera Paederus and Paederidus as a chemical defense. We have recently identified a group of putative pederin biosynthesis genes and localized them to the genome of an as yet unculturable Pseudomonas sp. symbiont, the likely true pederin producer. However, this polyketide synthase cluster lacks several genes expected for pederin production. Here we report an additional polyketide synthase encoded on a separate region of the symbiont genome. It contains at least three novel catalytic domains that are predicted to be involved in pederin chain initiation and the formation of an unusual exomethylene bond. The region is bordered by mobility pseudogenes; this suggests that gene transposition led to the disjointed cluster organization. With this work, all putative pederin genes have been identified. Their heterologous expression in a culturable bacterium will provide important insights into how sustainable sources of invertebrate-derived drug candidates can be created.

  3. Crystal Structure of the DNA Deaminase APOBEC3B Catalytic Domain*

    PubMed Central

    Shi, Ke; Carpenter, Michael A.; Kurahashi, Kayo; Harris, Reuben S.; Aihara, Hideki

    2015-01-01

    Functional and deep sequencing studies have combined to demonstrate the involvement of APOBEC3B in cancer mutagenesis. APOBEC3B is a single-stranded DNA cytosine deaminase that functions normally as a nuclear-localized restriction factor of DNA-based pathogens. However, it is overexpressed in cancer cells and elicits an intrinsic preference for 5′-TC motifs in single-stranded DNA, which is the most frequently mutated dinucleotide in breast, head/neck, lung, bladder, cervical, and several other tumor types. In many cases, APOBEC3B mutagenesis accounts for the majority of both dispersed and clustered (kataegis) cytosine mutations. Here, we report the first structures of the APOBEC3B catalytic domain in multiple crystal forms. These structures reveal a tightly closed active site conformation and suggest that substrate accessibility is regulated by adjacent flexible loops. Residues important for catalysis are identified by mutation analyses, and the results provide insights into the mechanism of target site selection. We also report a nucleotide (dCMP)-bound crystal structure that informs a multistep model for binding single-stranded DNA. Overall, these high resolution crystal structures provide a framework for further mechanistic studies and the development of novel anti-cancer drugs to inhibit this enzyme, dampen tumor evolution, and minimize adverse outcomes such as drug resistance and metastasis. PMID:26416889

  4. Characterization of the Catalytic Domain of Clostridium novyi Alpha-Toxin

    PubMed Central

    Busch, Christian; Schömig, Kathrin; Hofmann, Fred; Aktories, Klaus

    2000-01-01

    Clostridium novyi alpha-toxin belongs to the family of large clostridial cytotoxins which act on cells through the modification of small GTP-binding proteins. We present here an analysis of the catalytic domain of alpha-toxin. A NH2-terminal 551-amino-acid fragment, α551, was found to contain the full enzyme activity of the holotoxin, whereas a slightly shortened fragment encompassing 509 amino acids showed no detectable enzyme activity. Further characterization of the enzymatically active fragment α551 revealed a substrate specificity for both UDP–N-acetylglucosamine and UDP-glucose. A Michaelis-Menten constant of 17 μM was determined for the substrate UDP–N-acetylglucosamine, while that for UDP-glucose was about 20 times higher, indicating a weaker affinity of the toxin for the latter substrate. Mutation of the aspartic acids of a conserved motif DXD within α551 reduced enzyme activity >700-fold and inhibited cytotoxicity after microinjection in cells. Inhibition of enzyme activity of the DXD mutant could be partially overcome by increased concentrations of manganese ions, suggesting the involvement of these aspartic acids in Mn2+ binding. By construction of chimeras of enzymatically active fragments of C. sordellii lethal toxin and C. novyi alpha-toxin, we located the region involved in nucleotide-sugar specificity to amino acids 133 through 517. PMID:11035748

  5. Characterization of the catalytic domain of Clostridium novyi alpha-toxin.

    PubMed

    Busch, C; Schömig, K; Hofmann, F; Aktories, K

    2000-11-01

    Clostridium novyi alpha-toxin belongs to the family of large clostridial cytotoxins which act on cells through the modification of small GTP-binding proteins. We present here an analysis of the catalytic domain of alpha-toxin. A NH(2)-terminal 551-amino-acid fragment, alpha 551, was found to contain the full enzyme activity of the holotoxin, whereas a slightly shortened fragment encompassing 509 amino acids showed no detectable enzyme activity. Further characterization of the enzymatically active fragment alpha 551 revealed a substrate specificity for both UDP-N-acetylglucosamine and UDP-glucose. A Michaelis-Menten constant of 17 microM was determined for the substrate UDP-N-acetylglucosamine, while that for UDP-glucose was about 20 times higher, indicating a weaker affinity of the toxin for the latter substrate. Mutation of the aspartic acids of a conserved motif DXD within alpha 551 reduced enzyme activity >700-fold and inhibited cytotoxicity after microinjection in cells. Inhibition of enzyme activity of the DXD mutant could be partially overcome by increased concentrations of manganese ions, suggesting the involvement of these aspartic acids in Mn(2+) binding. By construction of chimeras of enzymatically active fragments of C. sordellii lethal toxin and C. novyi alpha-toxin, we located the region involved in nucleotide-sugar specificity to amino acids 133 through 517.

  6. Crystal Structure of the APOBEC3G Catalytic Domain Reveals Potential Oligomerization Interfaces

    SciTech Connect

    Shandilya, Shivender M.D.; Nalam, Madhavi N.L.; Nalivaika, Ellen A.; Gross, Phillip J.; Valesano, Johnathan C.; Shindo, Keisuke; Li, Ming; Munson, Mary; Royer, William E.; Harjes, Elena; Kono, Takahide; Matsuo, Hiroshi; Harris, Reuben S.; Somasundaran, Mohan; Schiffer, Celia A.

    2010-02-11

    APOBEC3G is a DNA cytidine deaminase that has antiviral activity against HIV-1 and other pathogenic viruses. In this study the crystal structure of the catalytically active C-terminal domain was determined to 2.25 {angstrom}. This structure corroborates features previously observed in nuclear magnetic resonance (NMR) studies, a bulge in the second {beta} strand and a lengthening of the second {alpha} helix. Oligomerization is postulated to be critical for the function of APOBEC3G. In this structure, four extensive intermolecular interfaces are observed, suggesting potential models for APOBEC3G oligomerization. The structural and functional significance of these interfaces was probed by solution NMR and disruptive variants were designed and tested for DNA deaminase and anti-HIV activities. The variant designed to disrupt the most extensive interface lost both activities. NMR solution data provides evidence that another interface, which coordinates a novel zinc site, also exists. Thus, the observed crystallographic interfaces of APOBEC3G may be important for both oligomerization and function.

  7. A classical and ab initio study of the interaction of the myosin triphosphate binding domain with ATP.

    PubMed Central

    Minehardt, Todd J; Marzari, Nicola; Cooke, Roger; Pate, Edward; Kollman, Peter A; Car, Roberto

    2002-01-01

    We used classical molecular mechanics (MM) simulations and quantum mechanical (QM) structural relaxations to examine the active site of myosin when bound to ATP. Two conformations of myosin have been determined by x-ray crystallography. In one, there is no direct interaction between switch 2 and the nucleotide (open state). In the other (closed state), the universally conserved switch 2 glycine forms a hydrogen bond with a gamma-phosphate oxygen. MM simulations indicate that the two states are thermodynamically stable and allow us to investigate the extent to which the P-loop, switch 1, and switch 2 are involved in hydrolysis. We find that the open structure has a higher affinity for ATP than the closed structure, and that ATP is distorted toward a transition state by interactions with the protein. We also examine how the structure of the binding site changes with either MgATP or CaATP as the nucleotide in myosin in the open conformer. Our analyses suggest that higher CaATPase rates occur because the leaving phosphate (P(i)) group is more weakly bound and dissociation occurs faster. Finally, we validate the use of a particular formulation of a QM methodology (Car-Parrinello) to further refine the structures of the active site. PMID:11806909

  8. The periplasmic membrane proximal domain of MacA acts as a switch in stimulation of ATP hydrolysis by MacB transporter.

    PubMed

    Modali, Sita D; Zgurskaya, Helen I

    2011-08-01

    Escherichia coli MacAB-TolC is a tripartite macrolide efflux transporter driven by hydrolysis of ATP. In this complex, MacA is the periplasmic membrane fusion protein that stimulates the activity of MacB transporter and establishes the link with the outer membrane channel TolC. The molecular mechanism by which MacA stimulates MacB remains unknown. Here, we report that the periplasmic membrane proximal domain of MacA plays a critical role in functional MacA-MacB interactions and stimulation of MacB ATPase activity. Binding of MacA to MacB stabilizes the ATP-bound conformation of MacB, whereas interactions with both MacB and TolC affect the conformation of MacA. A single G353A substitution in the C-terminus of MacA inactivates MacAB-TolC function by changing the conformation of the membrane proximal domain of MacA and disrupting the proper assembly of the MacA-MacB complex. We propose that MacA acts in transport by promoting MacB transition into the closed ATP-bound conformation and in this respect, is similar to the periplasmic solute-binding proteins.

  9. Autoinhibition of the Nuclease ARTEMIS Is Mediated by a Physical Interaction between Its Catalytic and C-terminal Domains.

    PubMed

    Niewolik, Doris; Peter, Ingrid; Butscher, Carmen; Schwarz, Klaus

    2017-02-24

    The nuclease ARTEMIS is essential for the development of B and T lymphocytes. It is required for opening DNA hairpins generated during antigen receptor gene assembly from variable (V), diversity (D), and joining (J) subgenic elements (V(D)J recombination). As a member of the non-homologous end-joining pathway, it is also involved in repairing a subset of pathological DNA double strand breaks. Loss of ARTEMIS function therefore results in radiosensitive severe combined immunodeficiency (RS-SCID). The hairpin opening activity is dependent on the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), which can bind to and phosphorylate ARTEMIS. The ARTEMIS C terminus is dispensable for cellular V(D)J recombination and in vitro nuclease assays with C-terminally truncated ARTEMIS showing DNA-PKcs-independent hairpin opening activity. Therefore, it has been postulated that ARTEMIS is regulated via autoinhibition by its C terminus. To obtain evidence for the autoinhibition model, we performed co-immunoprecipitation experiments with combinations of ARTEMIS mutants. We show that an N-terminal fragment comprising the catalytic domain can interact both with itself and with a C-terminal fragment. Amino acid exchanges N456A+S457A+E458Q in the C terminus of full-length ARTEMIS resulted in unmasking of the N terminus and in increased ARTEMIS activity in cellular V(D)J recombination assays. Mutations in ARTEMIS-deficient patients impaired the interaction with the C terminus and also affected protein stability. The interaction between the N- and C-terminal domains was not DNA-PKcs-dependent, and phosphomimetic mutations in the C-terminal domain did not result in unmasking of the catalytic domain. Our experiments provide strong evidence that a physical interaction between the C-terminal and catalytic domains mediates ARTEMIS autoinhibition. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Crystallization and preliminary X-ray diffraction analysis of the glucuronoyl esterase catalytic domain from Hypocrea jecorina

    SciTech Connect

    Wood, S. J.; Li, X.-L.; Cotta, M. A.; Biely, P.; Duke, N. E. C.; Schiffer, M.; Pokkuluri, P. R.

    2008-04-01

    The catalytic domain of the glucuronoyl esterase from H. jecorina was overexpresssed, purified and crystallized in space group P2{sub 1}2{sub 1}2{sub 1}. X-ray diffraction data were collected to 1.9 Å resolution. The catalytic domain of the glucuronoyl esterase from Hypocrea jecorina (anamorph Trichoderma reesei) was overexpresssed, purified and crystallized by the sitting-drop vapor-diffusion method using 1.4 M sodium/potassium phosphate pH 6.9. The crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1} and X-ray diffraction data were collected to 1.9 Å resolution. This is the first enzyme with glucoronoyl esterase activity to be crystallized; its structure will be valuable in lignocellulose-degradation research.

  11. Heterologous expression and purification of catalytic domain of CESA1 from Arabidopsis thaliana

    SciTech Connect

    Vandavasi, Venu Gopal; O'Neill, Hugh Michael

    2016-10-20

    Here, heterologous expression of plant cellulose synthase (CESA) and its purification has remained a challenge for decades impeding detailed biophysical, biochemical and structural characterization of this key enzyme. An in-depth knowledge of structure and function of CESA proteins would enable us to better understand the hierarchical structure of the plant cell wall. Here, we report a detailed, and reproducible method of purification of catalytic domain of CESA1 from Arabidopsis thaliana that was recombinantly expressed in Escherichia coli. The method relies on a two stage purification procedure to obtain the catalytic domain in monomer and trimer forms. The biochemical and biophysical data including low resolution structures of the protein have been published. Currently the crystallization studies of this protein are underway.

  12. Architecture of a single membrane spanning cytochrome P450 suggests constraints that orient the catalytic domain relative to a bilayer

    PubMed Central

    Monk, Brian C.; Tomasiak, Thomas M.; Keniya, Mikhail V.; Huschmann, Franziska U.; Tyndall, Joel D. A.; O’Connell, Joseph D.; Cannon, Richard D.; McDonald, Jeffrey G.; Rodriguez, Andrew; Finer-Moore, Janet S.; Stroud, Robert M.

    2014-01-01

    Bitopic integral membrane proteins with a single transmembrane helix play diverse roles in catalysis, cell signaling, and morphogenesis. Complete monospanning protein structures are needed to show how interaction between the transmembrane helix and catalytic domain might influence association with the membrane and function. We report crystal structures of full-length Saccharomyces cerevisiae lanosterol 14α-demethylase, a membrane monospanning cytochrome P450 of the CYP51 family that catalyzes the first postcyclization step in ergosterol biosynthesis and is inhibited by triazole drugs. The structures reveal a well-ordered N-terminal amphipathic helix preceding a putative transmembrane helix that would constrain the catalytic domain orientation to lie partly in the lipid bilayer. The structures locate the substrate lanosterol, identify putative substrate and product channels, and reveal constrained interactions with triazole antifungal drugs that are important for drug design and understanding drug resistance. PMID:24613931

  13. Crystallization and preliminary X-ray characterization of the catalytic domain of collagenase G from Clostridium histolyticum

    PubMed Central

    Eckhard, Ulrich; Nüss, Dorota; Ducka, Paulina; Schönauer, Esther; Brandstetter, Hans

    2008-01-01

    The catalytic domain of collagenase G from Clostridium histolyticum has been cloned, recombinantly expressed in Escherichia coli and purified using affinity and size-exclusion column-chromatographic methods. Crystals of the catalytic domain were obtained from 0.12 M sodium citrate and 23%(v/v) PEG 3350 at 293 K. The crystals diffracted to 2.75 Å resolution using synchrotron radiation. The crystals belong to an orthorhombic space group, with unit-cell parameters a = 57, b = 109, c = 181 Å. This unit cell is consistent with the presence of one molecule per asymmetric unit and a solvent content of approximately 53%. PMID:18453715

  14. The nucleotide-binding domains of sulfonylurea receptor 2A and 2B play different functional roles in nicorandil-induced activation of ATP-sensitive K+ channels.

    PubMed

    Yamada, Mitsuhiko; Kurachi, Yoshihisa

    2004-05-01

    Nicorandil activates ATP-sensitive K(+) channels composed of Kir6.2 and either sulfonylurea receptor (SUR) 2A or 2B. Although SUR2A and SUR2B differ only in their C-terminal 42 amino acids (C42) and possess identical drug receptors and nucleotide-binding domains (NBDs), nicorandil more potently activates SUR2B/Kir6.2 than SUR2A/Kir6.2 channels. Here, we analyzed the roles of NBDs in these channels' response to nicorandil with the inside-out configuration of the patch-clamp method. Binding and hydrolysis of nucleotides by NBDs were impaired by mutations in the Walker A motif of NBD1 (K708A) and NBD2 (K1349A) and in the Walker B motif of NBD2 (D1470N). Experiments were done with internal ATP (1 mM). In SUR2A/Kir6.2 channels, the K708A mutation abolished, and the K1349A but not D1470N mutation reduced the sensitivity to nicorandil. ADP (100 microM) significantly increased the wild-type channels' sensitivity to nicorandil, which was abolished by the K1349A or D1470N mutation. Thus, the SUR2A/Kir6.2 channels' response to nicorandil critically depends on ATP-NBD1 interaction and is facilitated by interactions of ATP or ADP with NBD2. In SUR2B/Kir6.2 channels, either the K708A or K1349A mutation partially suppressed the response to nicorandil, and double mutations abolished it. The D1470N mutation also significantly impaired the response. ADP did not sensitize the channels. Thus, NBD2 hydrolyzes ATP, and NBD1 and NBD2 equally contribute to the response by interacting with ATP and ADP, accounting for the higher nicorandil sensitivity of SUR2B/Kir6.2 than SUR2A/Kir6.2 channels in the presence of ATP alone. Thus, C42 modulates the interaction of both NBDs with intracellular nucleotides.

  15. Molecular models of the structural arrangement of subunits and the mechanism of proton translocation in the membrane domain of F(1)F(0) ATP synthase.

    PubMed

    Groth, G

    2000-05-31

    Subunit c of the proton-transporting ATP synthase of Escherichia coli forms an oligomeric complex in the membrane domain that functions in transmembrane proton conduction. The arrangement of subunit c monomers in this oligomeric complex was studied by scanning mutagenesis. On the basis of these studies and structural information on subunit c, different molecular models for the potential arrangement of monomers in the c-oligomer are discussed. Intersubunit contacts in the F(0) domain that have been analysed in the past by chemical modification and mutagenesis studies are summarised. Transient contacts of the c-oligomer with subunit a might play a crucial role in the mechanism of proton translocation. Schematic models presented by several authors that interpret proton transport in the F(0) domain by a relative rotation of the c-subunit oligomer against subunit a are reviewed against the background of the molecular models of the oligomer.

  16. Modulation of thin filament activation of myosin ATP hydrolysis by N-terminal domains of cardiac myosin binding protein-C.

    PubMed

    Belknap, Betty; Harris, Samantha P; White, Howard D

    2014-10-28

    We have used enzyme kinetics to investigate the molecular mechanism by which the N-terminal domains of human and mouse cardiac MyBP-C (C0C1, C1C2, and C0C2) affect the activation of myosin ATP hydrolysis by F-actin and by native porcine thin filaments. N-Terminal domains of cMyBP-C inhibit the activation of myosin-S1 ATPase by F-actin. However, mouse and human C1C2 and C0C2 produce biphasic activating and inhibitory effects on the activation of myosin ATP hydrolysis by native cardiac thin filaments. Low ratios of MyBP-C N-terminal domains to thin filaments activate myosin-S1 ATP hydrolysis, but higher ratios inhibit ATP hydrolysis, as is observed with F-actin alone. These data suggest that low concentrations of C1C2 and C0C2 activate thin filaments by a mechanism similar to that of rigor myosin-S1, whereas higher concentrations inhibit the ATPase rate by competing with myosin-S1-ADP-Pi for binding to actin and thin filaments. In contrast to C0C2 and C1C2, the activating effects of the C0C1 domain are species-dependent: human C0C1 activates actomyosin-S1 ATPase rates, but mouse C0C1 does not produce significant activation or inhibition. Phosphorylation of serine residues in the m-linker between the C1 and C2 domains by protein kinase-A decreases the activation of thin filaments by huC0C2 at pCa > 8 but has little effect on the activation mechanism at pCa = 4. In sarcomeres, the low ratio of cMyBP-C to actin is expected to favor the activating effects of cMyBP-C while minimizing inhibition produced by competition with myosin heads.

  17. Structural and thermodynamic comparison of the catalytic domain of AMSH and AMSH-LP: nearly identical fold but different stability.

    PubMed

    Davies, Christopher W; Paul, Lake N; Kim, Myung-Il; Das, Chittaranjan

    2011-10-21

    AMSH plays a critical role in the ESCRT (endosomal sorting complexes required for transport) machinery, which facilitates the down-regulation and degradation of cell-surface receptors. It displays a high level of specificity toward cleavage of Lys63-linked polyubiquitin chains, the structural basis of which has been understood recently through the crystal structure of a highly related, but ESCRT-independent, protein AMSH-LP (AMSH-like protein). We have determined the X-ray structure of two constructs representing the catalytic domain of AMSH: AMSH244, the JAMM (JAB1/MPN/MOV34)-domain-containing polypeptide segment from residues 244 to 424, and AMSH219(E280A), an active-site mutant, Glu280 to Ala, of the segment from 219 to 424. In addition to confirming the expected zinc coordination in the protein, the structures reveal that the catalytic domains of AMSH and AMSH-LP are nearly identical; however, guanidine-hydrochloride-induced unfolding studies show that the catalytic domain of AMSH is thermodynamically less stable than that of AMSH-LP, indicating that the former is perhaps structurally more plastic. Much to our surprise, in the AMSH219(E280A) structure, the catalytic zinc was still held in place, by the compensatory effect of an aspartate from a nearby loop moving into a position where it could coordinate with the zinc, once again suggesting the plasticity of AMSH. Additionally, a model of AMSH244 bound to Lys63-linked diubiquitin reveals a type of interface for the distal ubiquitin significantly different from that seen in AMSH-LP. Altogether, we believe that our data provide important insight into the structural difference between the two proteins that may translate into the difference in their biological function. Copyright © 2011 Elsevier Ltd. All rights reserved.

  18. Structural and Thermodynamic Comparison of the Catalytic Domain of AMSH and AMSH-LP: Nearly Identical Fold but Different Stability

    SciTech Connect

    Davies, Christopher W.; Paul, Lake N.; Kim, Myung-Il; Das, Chittaranjan

    2012-02-07

    AMSH plays a critical role in the ESCRT (endosomal sorting complexes required for transport) machinery, which facilitates the down-regulation and degradation of cell-surface receptors. It displays a high level of specificity toward cleavage of Lys63-linked polyubiquitin chains, the structural basis of which has been understood recently through the crystal structure of a highly related, but ESCRT-independent, protein AMSH-LP (AMSH-like protein). We have determined the X-ray structure of two constructs representing the catalytic domain of AMSH: AMSH244, the JAMM (JAB1/MPN/MOV34)-domain-containing polypeptide segment from residues 244 to 424, and AMSH219{sup E280A}, an active-site mutant, Glu280 to Ala, of the segment from 219 to 424. In addition to confirming the expected zinc coordination in the protein, the structures reveal that the catalytic domains of AMSH and AMSH-LP are nearly identical; however, guanidine-hydrochloride-induced unfolding studies show that the catalytic domain of AMSH is thermodynamically less stable than that of AMSH-LP, indicating that the former is perhaps structurally more plastic. Much to our surprise, in the AMSH219{sup E280A} structure, the catalytic zinc was still held in place, by the compensatory effect of an aspartate from a nearby loop moving into a position where it could coordinate with the zinc, once again suggesting the plasticity of AMSH. Additionally, a model of AMSH244 bound to Lys63-linked diubiquitin reveals a type of interface for the distal ubiquitin significantly different from that seen in AMSH-LP. Altogether, we believe that our data provide important insight into the structural difference between the two proteins that may translate into the difference in their biological function.

  19. Homologous catalytic domains in a rumen fungal xylanase: evidence for gene duplication and prokaryotic origin.

    PubMed

    Gilbert, H J; Hazlewood, G P; Laurie, J I; Orpin, C G; Xue, G P

    1992-08-01

    A cDNA (xynA), encoding xylanase A (XYLA), was isolated from a cDNA library, derived from mRNA extracted from the rumen anaerobic fungus, Neocallimastix patriciarum. Recombinant XYLA, purified from Escherichia coli harbouring xynA, had a M(r) of 53,000 and hydrolysed oat-spelt xylan to xylobiose and xylose. The enzyme did not hydrolyse any cellulosic substrates. The nucleotide sequence of xynA revealed a single open reading frame of 1821 bp coding for a protein of M(r) 66,192. The predicted primary structure of XYLA comprised an N-terminal signal peptide followed by a 225-amino-acid repeated sequence, which was separated from a tandem 40-residue C-terminal repeat by a threonine/proline linker sequence. The large N-terminal reiterated regions consisted of distinct catalytic domains which displayed similar substrate specificities to the full-length enzyme. The reiterated structure of XYLA suggests that the enzyme was derived from an ancestral gene which underwent two discrete duplications. Sequence comparison analysis revealed significant homology between XYLA and bacterial xylanases belonging to cellulase/xylanase family G. One of these homologous enzymes is derived from the rumen bacterium Ruminococcus flavefaciens. The homology observed between XYLA and a rumen prokaryote xylanase could be a consequence of the horizontal transfer of genes between rumen prokaryotes and lower eukaryotes, either when the organisms were resident in the rumen, or prior to their colonization of the ruminant. It should also be noted that Neocallimastix XYLA is the first example of a xylanase which consists of reiterated sequences. It remains to be established whether this is a common phenomenon in other rumen fungal plant cell wall hydrolases.

  20. The toxic dinoflagellate Karenia brevis encodes novel type I-like polyketide synthases containing discrete catalytic domains.

    PubMed

    Monroe, Emily A; Van Dolah, Frances M

    2008-07-01

    Karenia brevis is the Florida red tide dinoflagellate responsible for detrimental effects on human and environmental health through the production of brevetoxins. Brevetoxins are thought to be synthesized by a polyketide synthase (PKS) complex, but the gene cluster for this PKS has yet to be identified. Here, eight PKS transcripts were identified in K. brevis by high throughput cDNA library screening. Full length sequences were obtained through 3' and 5' RACE, which demonstrated the presence of polyadenylation, 3'-UTRs, and an identical dinoflagellate-specific spliced leader sequence at the 5' end of PKS transcripts. Six transcripts encoded for individual ketosynthase (KS) domains, one ketoreductase (KR), and one transcript encoded both acyl carrier protein (ACP) and KS domains. Transcript lengths ranged from 1875 to 3397 nucleotides, based on sequence analysis, and were confirmed by northern blotting. Baysian phylogenetic analysis of the K. brevis KS domains placed them well within the protist type I PKS clade. Thus although most similar to type I modular PKSs, the presence of individual catalytic domains on separate transcripts suggests a protein structure more similar to type II PKSs, in which each catalytic domain resides on an individual protein. These results identify an unprecedented PKS structure in a toxic dinoflagellate.

  1. Cooperation between catalytic and DNA binding domains enhances thermostability and supports DNA synthesis at higher temperatures by thermostable DNA polymerases.

    PubMed

    Pavlov, Andrey R; Pavlova, Nadejda V; Kozyavkin, Sergei A; Slesarev, Alexei I

    2012-03-13

    We have previously introduced a general kinetic approach for comparative study of processivity, thermostability, and resistance to inhibitors of DNA polymerases [Pavlov, A. R., et al. (2002) Proc. Natl. Acad. Sci. U.S.A.99, 13510-13515]. The proposed method was successfully applied to characterize hybrid DNA polymerases created by fusing catalytic DNA polymerase domains with various sequence-nonspecific DNA binding domains. Here we use the developed kinetic analysis to assess basic parameters of DNA elongation by DNA polymerases and to further study the interdomain interactions in both previously constructed and new chimeric DNA polymerases. We show that connecting helix-hairpin-helix (HhH) domains to catalytic polymerase domains can increase thermostability, not only of DNA polymerases from extremely thermophilic species but also of the enzyme from a faculatative thermophilic bacterium Bacillus stearothermophilus. We also demonstrate that addition of Topo V HhH domains extends efficient DNA synthesis by chimerical polymerases up to 105 °C by maintaining processivity of DNA synthesis at high temperatures. We found that reversible high-temperature structural transitions in DNA polymerases decrease the rates of binding of these enzymes to the templates. Furthermore, activation energies and pre-exponential factors of the Arrhenius equation suggest that the mechanism of electrostatic enhancement of diffusion-controlled association plays a minor role in binding of templates to DNA polymerases.

  2. Nucleotide-binding domains of cystic fibrosis transmembrane conductance regulator, an ABC transporter, catalyze adenylate kinase activity but not ATP hydrolysis.

    PubMed

    Gross, Christian H; Abdul-Manan, Norzehan; Fulghum, John; Lippke, Judith; Liu, Xun; Prabhakar, Prakash; Brennan, Debra; Willis, Melissa Swope; Faerman, Carlos; Connelly, Patrick; Raybuck, Scott; Moore, Jonathan

    2006-02-17

    The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP-binding cassette (ABC) transporter family. CFTR consists of two transmembrane domains, two nucleotide-binding domains (NBD1 and NBD2), and a regulatory domain. Previous biochemical reports suggest NBD1 is a site of stable nucleotide interaction with low ATPase activity, whereas NBD2 is the site of active ATP hydrolysis. It has also been reported that NBD2 additionally possessed adenylate kinase (AK) activity. Knowledge about the intrinsic biochemical activities of the NBDs is essential to understanding the Cl(-) ion gating mechanism. We find that purified mouse NBD1, human NBD1, and human NBD2 function as adenylate kinases but not as ATPases. AK activity is strictly dependent on the addition of the adenosine monophosphate (AMP) substrate. No liberation of [(33)P]phosphate is observed from the gamma-(33)P-labeled ATP substrate in the presence or absence of AMP. AK activity is intrinsic to both human NBDs, as the Walker A box lysine mutations abolish this activity. At low protein concentration, the NBDs display an initial slower nonlinear phase in AK activity, suggesting that the activity results from homodimerization. Interestingly, the G551D gating mutation has an exaggerated nonlinear phase compared with the wild type and may indicate this mutation affects the ability of NBD1 to dimerize. hNBD1 and hNBD2 mixing experiments resulted in an 8-57-fold synergistic enhancement in AK activity suggesting heterodimer formation, which supports a common theme in ABC transporter models. A CFTR gating mechanism model based on adenylate kinase activity is proposed.

  3. Amidoligases with ATP-grasp, glutamine synthetase-like and acetyltransferase-like domains: synthesis of novel metabolites and peptide modifications of proteins

    PubMed Central

    Iyer, Lakshminarayan M.; Abhiman, Saraswathi; Burroughs, A. Maxwell; Aravind, L.

    2011-01-01

    Recent studies have shown that the ubiquitin system had its origins in ancient cofactor/amino acid biosynthesis pathways. Preliminary studies also indicated that conjugation systems for other peptide tags on proteins, such as pupylation, have evolutionary links to cofactor/amino acid biosynthesis pathways. Following up on these observations, we systematically investigated the non-ribosomal amidoligases of the ATP-grasp, glutamine synthetase-like and acetyltransferase folds by classifying the known members and identifying novel versions. We then established their contextual connections using information from domain architectures and conserved gene neighborhoods. This showed remarkable, previously uncharacterized functional links between diverse peptide ligases, several peptidases of unrelated folds and enzymes involved in synthesis of modified amino acids. Using the network of contextual connections we were able to predict numerous novel pathways for peptide synthesis and modification, amine-utilization, secondary metabolite synthesis and potential peptide-tagging systems. One potential peptide-tagging system, which is widely distributed in bacteria, involves an ATP-grasp domain and a glutamine synthetase-like ligase, both of which are circularly permuted, an NTN hydrolase fold peptidase and a novel alpha helical domain. Our analysis also elucidates key steps in the biosynthesis of antibiotics such as friulimicin, butirosin and bacilysin and cell surface structures such as capsular polymers and teichuronopeptides. We also report the discovery of several novel ribosomally synthesized bacterial peptide metabolites that are cyclized via amide and lactone linkages formed by ATP-grasp enzymes. We present an evolutionary scenario for the multiple convergent origins of peptide ligases in various folds and clarify the bacterial origin of eukaryotic peptide-tagging enzymes of the TTL family. PMID:20023723

  4. Amidoligases with ATP-grasp, glutamine synthetase-like and acetyltransferase-like domains: synthesis of novel metabolites and peptide modifications of proteins.

    PubMed

    Iyer, Lakshminarayan M; Abhiman, Saraswathi; Maxwell Burroughs, A; Aravind, L

    2009-12-01

    Recent studies have shown that the ubiquitin system had its origins in ancient cofactor/amino acid biosynthesis pathways. Preliminary studies also indicated that conjugation systems for other peptide tags on proteins, such as pupylation, have evolutionary links to cofactor/amino acid biosynthesis pathways. Following up on these observations, we systematically investigated the non-ribosomal amidoligases of the ATP-grasp, glutamine synthetase-like and acetyltransferase folds by classifying the known members and identifying novel versions. We then established their contextual connections using information from domain architectures and conserved gene neighborhoods. This showed remarkable, previously uncharacterized functional links between diverse peptide ligases, several peptidases of unrelated folds and enzymes involved in synthesis of modified amino acids. Using the network of contextual connections we were able to predict numerous novel pathways for peptide synthesis and modification, amine-utilization, secondary metabolite synthesis and potential peptide-tagging systems. One potential peptide-tagging system, which is widely distributed in bacteria, involves an ATP-grasp domain and a glutamine synthetase-like ligase, both of which are circularly permuted, an NTN-hydrolase fold peptidase and a novel alpha helical domain. Our analysis also elucidates key steps in the biosynthesis of antibiotics such as friulimicin, butirosin and bacilysin and cell surface structures such as capsular polymers and teichuronopeptides. We also report the discovery of several novel ribosomally synthesized bacterial peptide metabolites that are cyclized via amide and lactone linkages formed by ATP-grasp enzymes. We present an evolutionary scenario for the multiple convergent origins of peptide ligases in various folds and clarify the bacterial origin of eukaryotic peptide-tagging enzymes of the TTL family.

  5. ATP6V1G1 — EDRN Public Portal

    Cancer.gov

    ATP6V1G1 is a subunit of vacuolar ATPase (V-ATPase), a multisubunit enzyme. V-ATPase is an enzyme transporter that functions to acidify intracellular compartments in eukaryotic cells. This acidification process is necessary for such intracellular processes as protein sorting, zymogen activation, receptor-mediated endocytosis, and synaptic vesicle proton gradient generation. V-ATPase is ubiquitously expressed and is present in endomembrane organelles such as vacuoles, lysosomes, endosomes, the Golgi apparatus, chromaffin granules and coated vesicles, as well as in the plasma membrane. V-ATPase is composed of a cytosolic V1 domain and a transmembrane V0 domain. The V1 domain consists of three A, three B, and two G subunits, as well as a C, D, E, F, and H subunit. The V1 domain contains the ATP catalytic site.

  6. Small-angle X-ray scattering study of the ATP modulation of the structural features of the nucleotide binding domains of the CFTR in solution.

    PubMed

    Galeno, Lauretta; Galfrè, Elena; Moran, Oscar

    2011-07-01

    Nucleotide binding domains (NBD1 and NBD2) of the cystic fibrosis transmembrane conductance (CFTR), the defective protein in cystic fibrosis, are responsible for controlling the gating of the chloride channel and are the putative binding site for several candidate drugs in the disease treatment. We studied the structural properties of recombinant NBD1, NBD2, and an equimolar NBD1/NBD2 mixture in solution by small-angle X-ray scattering. We demonstrated that NBD1 or NBD2 alone have an overall structure similar to that observed for crystals. Application of 2 mM ATP induces a dimerization of NBD1 but does not modify the NBD2 monomeric conformation. An equimolar mixture of NBD1/NBD2 in solution shows a dimeric conformation, and the application of ATP to the solution causes a conformational change in the NBD1/NBD2 complex into a tight heterodimer. We hypothesize that a similar conformation change occurs in situ and that transition is part of the gating mechanism. To our knowledge, this is the first direct observation of a conformational change of the NBD1/NBD2 interaction by ATP. This information may be useful to understand the physiopathology of cystic fibrosis.

  7. The Evolutionarily Conserved Tre2/Bub2/Cdc16 (TBC), Lysin Motif (LysM), Domain Catalytic (TLDc) Domain Is Neuroprotective against Oxidative Stress.

    PubMed

    Finelli, Mattéa J; Sanchez-Pulido, Luis; Liu, Kevin X; Davies, Kay E; Oliver, Peter L

    2016-02-05

    Oxidative stress is a pathological feature of many neurological disorders; therefore, utilizing proteins that are protective against such cellular insults is a potentially valuable therapeutic approach. Oxidation resistance 1 (OXR1) has been shown previously to be critical for oxidative stress resistance in neuronal cells; deletion of this gene causes neurodegeneration in mice, yet conversely, overexpression of OXR1 is protective in cellular and mouse models of amyotrophic lateral sclerosis. However, the molecular mechanisms involved are unclear. OXR1 contains the Tre2/Bub2/Cdc16 (TBC), lysin motif (LysM), domain catalytic (TLDc) domain, a motif present in a family of proteins including TBC1 domain family member 24 (TBC1D24), a protein mutated in a range of disorders characterized by seizures, hearing loss, and neurodegeneration. The TLDc domain is highly conserved across species, although the structure-function relationship is unknown. To understand the role of this domain in the stress response, we carried out systematic analysis of all mammalian TLDc domain-containing proteins, investigating their expression and neuroprotective properties in parallel. In addition, we performed a detailed structural and functional study of this domain in which we identified key residues required for its activity. Finally, we present a new mouse insertional mutant of Oxr1, confirming that specific disruption of the TLDc domain in vivo is sufficient to cause neurodegeneration. Our data demonstrate that the integrity of the TLDc domain is essential for conferring neuroprotection, an important step in understanding the functional significance of all TLDc domain-containing proteins in the cellular stress response and disease. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Persistence of the mitochondrial permeability transition in the absence of subunit c of human ATP synthase

    PubMed Central

    He, Jiuya; Ford, Holly C.; Carroll, Joe; Ding, Shujing; Fearnley, Ian M.

    2017-01-01

    The permeability transition in human mitochondria refers to the opening of a nonspecific channel, known as the permeability transition pore (PTP), in the inner membrane. Opening can be triggered by calcium ions, leading to swelling of the organelle, disruption of the inner membrane, and ATP synthesis, followed by cell death. Recent proposals suggest that the pore is associated with the ATP synthase complex and specifically with the ring of c-subunits that constitute the membrane domain of the enzyme’s rotor. The c-subunit is produced from three nuclear genes, ATP5G1, ATP5G2, and ATP5G3, encoding identical copies of the mature protein with different mitochondrial-targeting sequences that are removed during their import into the organelle. To investigate the involvement of the c-subunit in the PTP, we generated a clonal cell, HAP1-A12, from near-haploid human cells, in which ATP5G1, ATP5G2, and ATP5G3 were disrupted. The HAP1-A12 cells are incapable of producing the c-subunit, but they preserve the characteristic properties of the PTP. Therefore, the c-subunit does not provide the PTP. The mitochondria in HAP1-A12 cells assemble a vestigial ATP synthase, with intact F1-catalytic and peripheral stalk domains and the supernumerary subunits e, f, and g, but lacking membrane subunits ATP6 and ATP8. The same vestigial complex plus associated c-subunits was characterized from human 143B ρ0 cells, which cannot make the subunits ATP6 and ATP8, but retain the PTP. Therefore, none of the membrane subunits of the ATP synthase that are involved directly in transmembrane proton translocation is involved in forming the PTP. PMID:28289229

  9. Evolution of the PPM-family protein phosphatases in Streptomyces: duplication of catalytic domain and lateral recruitment of additional sensory domains

    SciTech Connect

    Zhang, Weiwen; Shi, Liang

    2004-12-01

    Originally identified from eukaryotes, the Mg{sup 2+}- or Mn{sup 2+}-dependent protein phosphatases (PPMs) are a diverse group of enzymes whose members include eukaryotic PP2C and some prokaryotic serine/threonine phosphatases. In our previous study, an unexpectedly large number of PPMs were identified in two Streptomyces genomes. In this work, a phylogenetic analysis was performed with all the PPMs available from a wide variety of microbial sources to determine the evolutionary origin of the Streptomyces PPM proteins. Consistent with earlier hypotheses, the results suggested that the microbial PPMs were relatively recent additions from eukaryotic sources. Results also indicated that the Streptomyces PPMs were divided into two major subfamilies at an early stage of their emergence in Streptomyces genomes. The first subfamily, which contains only six Streptomyces PPMs, possesses a catalytic domain whose sequence and architecture are similar to that of eukaryotic PPMs; the second subfamily contains 89 Streptomyces PPMs that lack the 5a and 5b catalytic domain motifs, similar to the PPMs SpoIIE and RsbU of Bacillus subtilis. Significant gene duplication was observed for the PPMs in the second subfamily. In addition, more than half (54 %) of the Streptomyces PPMs from the second subfamily were found to have at least one additional sensory domain, most commonly the PAS or the GAF domain. Phylogenetic analysis showed that these domains tended to be clustered according to the putative physiological functions rather than taxonomic relationship, implying that they might have arisen as a result of domain recruitment in a late evolutionary stage. This study provides an insight into how Streptomyces spp. may have expanded their PPM-based signal transduction networks to enable them to respond to a greater range of environmental changes.

  10. ATP hydrolysis by a domain related to translation factor GTPases drives polymerization of a static bacterial morphogenetic protein.

    PubMed

    Castaing, Jean-Philippe; Nagy, Attila; Anantharaman, Vivek; Aravind, L; Ramamurthi, Kumaran S

    2013-01-08

    The assembly of static supramolecular structures is a culminating event of developmental programs. One such structure, the proteinaceous shell (called the coat) that surrounds spores of the bacterium Bacillus subtilis, is composed of about 70 different proteins and represents one of the most durable biological structures known. The coat is built atop a basement layer that contains an ATPase (SpoIVA) that forms a platform required for coat assembly. Here, we show that SpoIVA belongs to the translation factors class of P-loop GTPases and has evolutionarily lost the ability to bind GTP; instead, it uses ATP hydrolysis to drive its self-assembly into static filaments. We demonstrate that ATP hydrolysis is required by every subunit for incorporation into the growing polymer by inducing a conformational change that drives polymerization of a nucleotide-free filament. SpoIVA therefore differs from other self-organizing polymers (dynamic cytoskeletal structures and static intermediate filaments) in that it uses ATP hydrolysis to self-assemble, not disassemble, into a static polymer. We further show that polymerization requires a critical concentration that we propose is only achieved once SpoIVA is recruited to the surface of the developing spore, thereby ensuring that SpoIVA polymerization only occurs at the correct subcellular location during spore morphogenesis.

  11. An ATP-competitive inhibitor modulates the allosteric function of the HER3 pseudokinase

    PubMed Central

    Littlefield, Peter; Moasser, Mark M.; Jura, Natalia

    2014-01-01

    The human epidermal growth factor receptor 3 (HER3) is a receptor tyrosine kinase that lacks catalytic activity, but is essential for cellular homeostasis due to its ability to allosterically activate EGFR/HER2. Though catalytically inactive, HER3 binds ATP tightly, hinting at a possible role of the nucleotide-binding pocket in modulating HER3 function. We report a structure of the HER3 pseudokinase bound to the ATP-competitive inhibitor bosutinib. Previously solved structures show that bosutinib can potently interact with multiple kinase domain conformations. In complex with HER3, bosutinib binds to yet another conformation, which is nearly identical to that observed in the HER3/ATP complex. Interestingly, occupation of the ATP-binding site by bosutinib improves the ability of HER3 to act as an allosteric activator of EGFR in vitro by increasing the affinity of the HER3/EGFR heterodimer in a membrane-dependent manner. PMID:24656791

  12. Enzymatic specificities and modes of action of the two catalytic domains of the XynC xylanase from Fibrobacter succinogenes S85.

    PubMed Central

    Zhu, H; Paradis, F W; Krell, P J; Phillips, J P; Forsberg, C W

    1994-01-01

    The xylanase XynC of Fibrobacter succinogenes S85 was recently shown to contain three distinct domains, A, B, and C (F. W. Paradis, H. Zhu, P. J. Krell, J. P. Phillips, and C. W. Forsberg, J. Bacteriol. 175:7666-7672, 1993). Domains A and B each bear an active site capable of hydrolyzing xylan, while domain C has no enzymatic activity. Two truncated proteins, each containing a single catalytic domain, named XynC-A and XynC-B were purified to homogeneity. The catalytic domains A and B had similar pH and temperature parameters of 6.0 and 50 degrees C for maximum hydrolytic activity and extensively degraded birch wood xylan to xylose and xylobiose. The Km and Vmax values, respectively, were 2.0 mg ml-1 and 6.1 U mg-1 for the intact enzyme, 1.83 mg ml-1 and 689 U mg-1 for domain A, and 2.38 mg ml-1 and 91.8 U mg-1 for domain B. Although domain A had a higher specific activity than domain B, domain B exhibited a broader substrate specificity and hydrolyzed rye arabinoxylan to a greater extent than domain A. Furthermore, domain B, but not domain A, was able to release xylose at the initial stage of the hydrolysis. Both catalytic domains cleaved xylotriose, xylotetraose, and xylopentaose but had no activity on xylobiose. Bond cleavage frequencies obtained from hydrolysis of xylo-alditol substrates suggest that while both domains have a strong preference for internal linkages of the xylan backbone, domain B has fewer subsites for substrate binding than domain A and cleaves arabinoxylan more efficiently. Chemical modification with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide methiodide and N-bromosuccinimide inactivated both XynC-A and XynC-B in the absence of xylan, indicating that carboxyl groups and tryptophan residues in the catalytic site of each domain have essential roles. Images PMID:8021170

  13. A smallest 6 kda metalloprotease, mini-matrilysin, in living world: a revolutionary conserved zinc-dependent proteolytic domain- helix-loop-helix catalytic zinc binding domain (ZBD)

    PubMed Central

    2012-01-01

    Background The Aim of this study is to study the minimum zinc dependent metalloprotease catalytic folding motif, helix B Met loop-helix C, with proteolytic catalytic activities in metzincin super family. The metzincin super family share a catalytic domain consisting of a twisted five-stranded β sheet and three long α helices (A, B and C). The catalytic zinc is at the bottom of the cleft and is ligated by three His residues in the consensus sequence motif, HEXXHXXGXXH, which is located in helix B and part of the adjacent Met turn region. An interesting question is - what is the minimum portion of the enzyme that still possesses catalytic and inhibitor recognition?” Methods We have expressed a 60-residue truncated form of matrilysin which retains only the helix B-Met turn-helix C region and deletes helix A and the five-stranded β sheet which form the upper portion of the active cleft. This is only 1/4 of the full catalytic domain. The E. coli derived 6 kDa MMP-7 ZBD fragments were purified and refolded. The proteolytic activities were analyzed by Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 peptide assay and CM-transferrin zymography analysis. SC44463, BB94 and Phosphoramidon were computationally docked into the 3day structure of the human MMP7 ZBD and TAD and thermolysin using the docking program GOLD. Results This minimal 6 kDa matrilysin has been refolded and shown to have proteolytic activity in the Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 peptide assay. Triton X-100 and heparin are important factors in the refolding environment for this mini-enzyme matrilysin. This minienzyme has the proteolytic activity towards peptide substrate, but the hexamer and octamer of the mini MMP-7 complex demonstrates the CM-transferrin proteolytic activities in zymographic analysis. Peptide digestion is inhibited by SC44463, specific MMP7 inhibitors, but not phosphorimadon. Interestingly, the mini MMP-7 can be processed by autolysis and producing ~ 6 ~ 7 kDa fragments. Thus

  14. N- versus C-domain selectivity of catalytic inactivation of human angiotensin converting enzyme by lisinopril-coupled transition metal chelates.

    PubMed

    Hocharoen, Lalintip; Joyner, Jeff C; Cowan, J A

    2013-12-27

    The N- and C-terminal domains of human somatic angiotensin I converting enzyme (sACE-1) demonstrate distinct physiological functions, with resulting interest in the development of domain-selective inhibitors for specific therapeutic applications. Herein, the activity of lisinopril-coupled transition metal chelates was tested for both reversible binding and irreversible catalytic inactivation of each domain of sACE-1. C/N domain binding selectivity ratios ranged from 1 to 350, while rates of irreversible catalytic inactivation of the N- and C-domains were found to be significantly greater for the N-domain, suggesting a more optimal orientation of M-chelate-lisinopril complexes within the active site of the N-domain of sACE-1. Finally, the combined effect of binding selectivity and inactivation selectivity was assessed for each catalyst (double-filter selectivity factors), and several catalysts were found to cause domain-selective catalytic inactivation. The results of this study demonstrate the ability to optimize the target selectivity of catalytic metallopeptides through both binding and catalytic factors (double-filter effect).

  15. Effect of ATP and 2-oxoglutarate on the in vitro interaction between the NifA GAF domain and the GlnB protein of Azospirillum brasilense

    PubMed Central

    Sotomaior, P.; Araújo, L.M.; Nishikawa, C.Y.; Huergo, L.F.; Monteiro, R.A.; Pedrosa, F.O.; Chubatsu, L.S.; Souza, E.M.

    2012-01-01

    Azospirillum brasilense is a diazotroph that associates with important agricultural crops and thus has potential to be a nitrogen biofertilizer. The A. brasilense transcription regulator NifA, which seems to be constitutively expressed, activates the transcription of nitrogen fixation genes. It has been suggested that the nitrogen status-signaling protein GlnB regulates NifA activity by direct interaction with the NifA N-terminal GAF domain, preventing the inhibitory effect of this domain under conditions of nitrogen fixation. In the present study, we show that an N-terminal truncated form of NifA no longer required GlnB for activity and lost regulation by ammonium. On the other hand, in trans co-expression of the N-terminal GAF domain inhibited the N-truncated protein in response to fixed nitrogen levels. We also used pull-down assays to show in vitro interaction between the purified N-terminal GAF domain of NifA and the GlnB protein. The results showed that A. brasilense GlnB interacts directly with the NifA N-terminal domain and this interaction is dependent on the presence of ATP and 2-oxoglutarate. PMID:22983183

  16. High temperatures enhance cooperative motions between CBM and catalytic domains of a thermostable cellulase: mechanism insights from essential dynamics.

    PubMed

    Batista, Paulo Ricardo; Costa, Mauricio Garcia de Souza; Pascutti, Pedro Geraldo; Bisch, Paulo Mascarello; de Souza, Wanderley

    2011-08-14

    Cellulases from thermophiles are capable of cleaving sugar chains from cellulose efficiently at high temperatures. The thermo-resistant Cel9A-68 cellulase possesses two important domains: CBM and a catalytic domain connected by a Pro/Ser/Thr rich linker. These domains act cooperatively to allow efficient catalysis. Despite exhaustive efforts to characterize cellulase binding and mechanism of action, a detailed description of the cellulose intrinsic flexibility is still lacking. From computational simulations we studied the temperature influence on the enzyme plasticity, prior to substrate binding. Interestingly, we observed an enhancement of collective motions at high temperatures. These motions are the most representative and describe an intrinsic hinge bending transition. A detailed analysis of these motions revealed an interdomain approximation where D459 and G460, located at the linker region, are the hinge residues. Therefore, we propose a new putative site for mutagenesis targeting the modulation of such conformational transition that may be crucial for activity.

  17. Direct real-time detection of the actin-activated power stroke within the myosin catalytic domain.

    PubMed

    Muretta, Joseph M; Petersen, Karl J; Thomas, David D

    2013-04-30

    We have used transient kinetics, nanosecond time-resolved fluorescence resonance energy transfer (FRET), and kinetics simulations to resolve a structural transition in the Dictyostelium myosin II relay helix during the actin-activated power stroke. The relay helix plays a critical role in force generation in myosin, coupling biochemical changes in the ATPase site with the force-transducing rotation of the myosin light-chain domain. Previous research in the absence of actin showed that ATP binding to myosin induces a dynamic equilibrium between a bent prepower stroke state of the relay helix and a straight postpower stroke state, which dominates in the absence of ATP or when ADP is bound. We now ask whether actin binding reverses this transition and if so, how this reversal is coordinated with actin-activated phosphate release. We labeled a Cys-lite Dictyostelium myosin II motor domain with donor and acceptor probes at two engineered Cys residues designed to detect relay helix bending. We then performed transient time-resolved FRET following stopped-flow mixing of actin with labeled myosin, preincubated with ATP. We determined the kinetics of actin-activated phosphate release, using fluorescent phosphate-binding protein. The results show that actin binding to the myosin.ADP.P complex straightens the relay helix before phosphate dissociation. This actin-activated relay helix straightening is reversible, but phosphate irreversibly dissociates from the postpower stroke state, preventing reversal of the power stroke. Thus, relay helix straightening gates phosphate dissociation, whereas phosphate dissociation provides the thermodynamic driving force underlying force production.

  18. Hetero-oligomerization of C2 domains of phospholipase C-related but catalytically inactive protein and synaptotagmin-1.

    PubMed

    Wang, DaGuang; Takeuchi, Hiroshi; Gao, Jing; Zhang, Zhao; Hirata, Masato

    2015-01-01

    The C2 domain is a protein module often found in molecules that regulate exocytosis. C2 domains mediate interactions between the parental molecule and Ca(2+), phospholipids, and proteins. Although various molecules have been shown to interact with several C2 domains, no interactions between the C2 domains from different molecules have yet been reported. In the present study, we identified direct interactions between the C2 domain of PRIP (phospholipase C-related but catalytically inactive protein) and the C2 domains of other molecules. Among the C2 domains examined, those of synaptotagmin-1 (Syt1-C2A and Syt1-C2B) and phospholipase C δ-1 bound to the C2 domain of PRIP. We investigated the interactions between the C2 domain of PRIP (PRIP-C2) with Syt1-C2A and Syt1-C2B, and the mode of binding of each was Ca(2+)-dependent and -independent, respectively. We further demonstrated that the Ca(2+) dependence of the interaction between PRIP-C2 and Syt1-C2A was attributed to Ca(2+) binding with Syt1-C2A, but not PRIP-C2, using a series of mutants prepared from both C2 domains. We previously reported that the interaction between PRIP-C2 and the membrane fusion machinery suggested a critical role for PRIP in exocytosis; therefore, the results of the present study further support the importance of PRIP-C2 in the inhibitory function of PRIP in regulating exocytosis. Copyright © 2014 Elsevier Ltd. All rights reserved.

  19. Characterization of a phosphate binding domain on the alpha-subunit of chloroplast ATP synthase using the photoaffinity phosphate analogue 4-azido-2-nitrophenyl phosphate.

    PubMed

    Groth, G; Mills, D A; Christiansen, E; Richter, M L; Huchzermeyer, B

    2000-11-14

    The photoaffinity phosphate analogue 4-azido-2 nitrophenyl phosphate (ANPP) was shown previously (Pougeois, R., Lauquin, G. J.-M., and Vignais, P. V. (1983) Biochemistry 22, 1241-1245) to bind covalently and specifically to a single catalytic site on one of the three beta-subunits of the isolated chloroplast coupling factor 1 (CF(1)). Modification by ANPP strongly inhibited ATP hydrolysis activity. In this study, we examined labeling of membrane-bound CF(1) by ANPP by exposing thylakoid membranes to increasing concentrations of the reagent. ANPP exhibited saturable binding to two sites on CF(1), one on the beta-subunit and one on the alpha-subunit. Labeling by ANPP resulted in the complete inhibition of both ATP synthesis and ATP hydrolysis by the membrane-bound enzyme. Labeling of both sites by ANPP was reduced by more than 80% in the presence of P(i) (> or = 10 mM) and ATP (> or = 0.5 mM). ADP was less effective in competing with ANPP for binding, giving a maximum of approximately 35% inhibition at concentrations > or = 2 mM. ANPP-labeled tryptic peptides of the alpha-subunit were isolated and sequenced. The majority of the probe was contained in three peptides corresponding to residues Gln(173) to Arg(216), Gly(217) to Arg(253), and His(256) to Arg(272) of the alpha-subunit. In the mitochondrial F(1) (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628), all three analogous peptides are located within the nucleotide binding pocket and within close proximity to the gamma-phosphate binding site. The data indicate, however, that the azidophenyl group of bound ANPP is oriented at approximately 180 degrees in the opposite direction to the adenine binding site with reference to the phosphate binding site on the alpha-subunit. The study has confirmed that ANPP is a bona fide phosphate analogue and suggests that it specifically targets the gamma-phosphate binding site within the nucleotide binding pockets on the alpha- and beta

  20. Conserved Asp327 of Walker B motif in the N-terminal Nucleotide Binding Domain (NBD-1) of Cdr1p of Candida albicans has acquired a new role in ATP hydrolysis

    PubMed Central

    Rai, Versha; Gaur, Manisha; Shukla, Sudhanshu; Shukla, Suneet; Ambudkar, Suresh V.; Komath, Sneha Sudha; Prasad, Rajendra

    2008-01-01

    The Walker A and B motifs of nucleotide binding domains (NBDs) of Cdr1p though almost identical to all ABC transporters, has unique substitutions. We have in the past shown that Trp326 of Walker B and Cys193 of Walker A motifs of N-terminal NBD of Cdr1p have distinct roles in ATP binding and hydrolysis, respectively. In the present study, we have examined the role of a well conserved Asp327 in the Walker B motif of the N-terminal NBD which is preceded (Trp326) and followed (Asn328) by atypical amino acid substitutions and compared it with its equivalent well conserved Asp1026 of the C-terminal NBD of Cdr1p. We observed that the removal of the negative charge by D327N, D327A, D1026N, D1026A and D327N/D1026N substitutions, resulted in Cdr1p mutant variants that were severely impaired in ATPase activity and drug efflux. Importantly, all the mutant variants showed characteristics similar to those of wild type with respect to cell surface expression and photoaffinity drug analogue [125I] IAAP and [3H] azidopine labeling. While Cdr1p D327N mutant variant showed comparable binding with [α-32P] 8-azido ATP, Cdr1p D1026N and Cdr1p D327N/D1026N mutant variants were crippled in nucleotide binding. That the two conserved carboxylate residues Asp327 and Asp1026 are functionally different was further evident from the pH profile of ATPase activity. Cdr1p D327N mutant variant showed ∼40% enhancement of its residual ATPase activity at acidic pH while no such pH effect was seen with Cdr1p D1026N mutant variant. Our experimental data suggest that Asp327 of N-terminal NBD has acquired a new role to act as a catalytic base in ATP hydrolysis, a role normally conserved for Glu present adjacent to the conserved Asp in the Walker B motif of all the non-fungal transporters. PMID:17144665

  1. An antigenic domain within a catalytically active Leishmania infantum nucleoside triphosphate diphosphohydrolase (NTPDase 1) is a target of inhibitory antibodies.

    PubMed

    Maia, Ana Carolina Ribeiro Gomes; Porcino, Gabriane Nascimento; Detoni, Michelle de Lima; Emídio, Nayara Braga; Marconato, Danielle Gomes; Faria-Pinto, Priscila; Fessel, Melissa Regina; Reis, Alexandre Barbosa; Juliano, Luiz; Juliano, Maria Aparecida; Marques, Marcos José; Vasconcelos, Eveline Gomes

    2013-02-01

    We identified a shared B domain within nucleoside triphosphate diphosphohydrolases (NTPDases) of plants and parasites. Now, an NTPDase activity not affected by inhibitors of adenylate kinase and ATPases was detected in Leishmania infantum promastigotes. By non-denaturing gel electrophoresis of detergent-homogenized promastigote preparation, an active band hydrolyzing nucleosides di- and triphosphate was visualized and, following SDS-PAGE and silver staining was identified as a single polypeptide of 50kDa. By Western blots, it was recognized by immune sera raised against potato apyrase (SA), r-pot B domain (SB), a recombinant polypeptide derived from the potato apyrase, and LbB1LJ (SC) or LbB2LJ (SD), synthetic peptides derived from the Leishmania NTPDase 1, and by serum samples from dogs with visceral leishmaniasis, identifying the antigenic L. infantum NTPDase 1 and, also, its conserved B domain (r83-122). By immunoprecipitation assays and Western blots, immune sera SA and SB identified the catalytically active NTPDase 1 in promastigote preparation. In addition, the immune sera SB (44%) and SC or SD (87-99%) inhibited its activity, suggesting a direct effect on the B domain. By ELISA, 37%, 45% or 50% of 38 infected dogs were seropositive for r-pot B domain, LbB1LJ and LbB2LJ, respectively, confirming the B domain antigenicity.

  2. Solution structure and intermolecular interactions of the third metal-binding domain of ATP7A, the Menkes disease protein.

    PubMed

    Banci, Lucia; Bertini, Ivano; Cantini, Francesca; DellaMalva, Nunzia; Herrmann, Torsten; Rosato, Antonio; Wüthrich, Kurt

    2006-09-29

    The third metal-binding domain of the human Menkes protein (MNK3), a copper(I)-transporting ATPase, has been expressed in Escherichia coli and characterized in solution. The solution structure of MNK3, its copper(I)-binding properties, and its interaction with the physiological partner, HAH1, have been studied. MNK3 is the domain most dissimilar in structure from the other domains of the Menkes protein. This is reflected in a significant rearrangement of the last strand of the four-stranded beta-sheet when compared with the other known homologous proteins or protein domains. MNK3 is also peculiar with respect to its interaction with the copper(I) ion, as it was found to be a comparatively weak binder. Copper(I) transfer from metal-loaded HAH1 was observed experimentally, but the metal distribution was shifted toward binding by HAH1. This is at variance with what is observed for the other Menkes domains.

  3. Three-dimensional structure of Saccharomyces invertase: role of a non-catalytic domain in oligomerization and substrate specificity.

    PubMed

    Sainz-Polo, M Angela; Ramírez-Escudero, Mercedes; Lafraya, Alvaro; González, Beatriz; Marín-Navarro, Julia; Polaina, Julio; Sanz-Aparicio, Julia

    2013-04-05

    Invertase is an enzyme that is widely distributed among plants and microorganisms and that catalyzes the hydrolysis of the disaccharide sucrose into glucose and fructose. Despite the important physiological role of Saccharomyces invertase (SInv) and the historical relevance of this enzyme as a model in early biochemical studies, its structure had not yet been solved. We report here the crystal structure of recombinant SInv at 3.3 Å resolution showing that the enzyme folds into the catalytic β-propeller and β-sandwich domains characteristic of GH32 enzymes. However, SInv displays an unusual quaternary structure. Monomers associate in two different kinds of dimers, which are in turn assembled into an octamer, best described as a tetramer of dimers. Dimerization plays a determinant role in substrate specificity because this assembly sets steric constraints that limit the access to the active site of oligosaccharides of more than four units. Comparative analysis of GH32 enzymes showed that formation of the SInv octamer occurs through a β-sheet extension that seems unique to this enzyme. Interaction between dimers is determined by a short amino acid sequence at the beginning of the β-sandwich domain. Our results highlight the role of the non-catalytic domain in fine-tuning substrate specificity and thus supplement our knowledge of the activity of this important family of enzymes. In turn, this gives a deeper insight into the structural features that rule modularity and protein-carbohydrate recognition.

  4. Replacement and deletion mutations in the catalytic domain and belt region of Aspergillus awamori glucoamylase to enhance thermostability.

    PubMed

    Liu, H L; Doleyres, Y; Coutinho, P M; Ford, C; Reilly, P J

    2000-09-01

    Three single-residue mutations, Asp71-->Asn, Gln409-->Pro and Gly447-->Ser, two long-to-short loop replacement mutations, Gly23-Ala24-Asp25-Gly26-Ala27-Trp28- Val29-Ser30-->Asn-Pro-Pro (23-30 replacement) and Asp297-Ser298-Glu299-Ala300-Val301-->Ala-G ly-Ala (297-301 replacement) and one deletion mutation removing Glu439, Thr440 and Ser441 (Delta439-441), all based on amino acid sequence alignments, were made to improve Aspergillus awamori glucoamylase thermostability. The first and second single-residue mutations were designed to introduce a potential N:-glycosylation site and to restrict backbone bond rotation, respectively, and therefore to decrease entropy during protein unfolding. The third single-residue mutation was made to decrease flexibility and increase O:-glycosylation in the already highly O:-glycosylated belt region that extends around the globular catalytic domain. The 23-30 replacement mutation was designed to eliminate a very thermolabile extended loop on the catalytic domain surface and to bring the remainder of this region closer to the rest of the catalytic domain, therefore preventing it from unfolding. The 297-301 replacement mutant GA was made to understand the function of the random coil region between alpha-helices 9 and 10. Delta439-441 was constructed to decrease belt flexibility. All six mutations increased glucoamylase thermostability without significantly changing enzyme kinetic properties, with the 23-30 replacement mutation increasing the activation free energy for thermoinactivation by about 4 kJ/mol, which leads to a 4 degrees C increase in operating temperature at constant thermostability.

  5. Interaction of extracellular domain 2 of the human retina-specific ATP-binding cassette transporter (ABCA4) with all-trans-retinal.

    PubMed

    Biswas-Fiss, Esther E; Kurpad, Deepa S; Joshi, Kinjalben; Biswas, Subhasis B

    2010-06-18

    The retina-specific ATP-binding cassette (ABC) transporter, ABCA4, is essential for transport of all-trans-retinal from the rod outer segment discs in the retina and is associated with a broad range of inherited retinal diseases, including Stargardt disease, autosomal recessive cone rod dystrophy, and fundus flavimaculatus. A unique feature of the ABCA subfamily of ABC transporters is the presence of highly conserved, long extracellular loops or domains (ECDs) with unknown function. The high degree of sequence conservation and mapped disease-associated mutations in these domains suggests an important physiological significance. Conformational analysis using CD spectroscopy of purified, recombinant ECD2 protein demonstrated that it has an ordered and stable structure composed of 27 +/- 3% alpha-helix, 20 +/- 3% beta-pleated sheet, and 53 +/- 3% coil. Significant conformational changes were observed in disease-associated mutant proteins. Using intrinsic tryptophan fluorescence emission spectrum of ECD2 polypeptide and fluorescence anisotropy, we have demonstrated that this domain specifically interacts with all-trans-retinal. Furthermore, the retinal interaction appeared preferential for the all-trans-isomer and was directly measurable through fluorescence anisotropy analysis. Our results demonstrate that the three macular degeneration-associated mutations lead to significant changes in the secondary structure of the ECD2 domain of ABCA4, as well as in its interaction with all-trans-retinal.

  6. Conformational change induced by ATP binding in the multidrug ATP-binding cassette transporter BmrA.

    PubMed

    Orelle, Cédric; Gubellini, Francesca; Durand, Anne; Marco, Sergio; Lévy, Daniel; Gros, Philippe; Di Pietro, Attilio; Jault, Jean-Michel

    2008-02-26

    ATP-binding cassette (ABC) transporters are involved in the transport of a wide variety of substrates, and ATP-driven dimerization of their nucleotide binding domains (NBDs) has been suggested to be one of the most energetic steps of their catalytic cycle. Taking advantage of the propensity of BmrA, a bacterial multidrug resistance ABC transporter, to form stable, highly ordered ring-shaped structures [Chami et al. (2002) J. Mol. Biol. 315, 1075-1085], we show here that addition of ATP in the presence of Mg2+ prevented ring formation or destroyed the previously formed rings. To pinpoint the catalytic step responsible for such an effect, two classes of hydrolysis-deficient mutants were further studied. In contrast to hydrolytically inactive glutamate mutants that behaved essentially as the wild-type, lysine Walker A mutants formed ring-shaped structures even in the presence of ATP-Mg. Although the latter mutants still bound ATP-Mg, and even slowly hydrolyzed it for the K380R mutant, they were most likely unable to undergo a proper NBD dimerization upon ATP-Mg addition. The ATP-driven dimerization step, which was still permitted in glutamate mutants and led to a stable conformation suitable to monitor the growth of 2D crystals, appeared therefore responsible for destabilization of the BmrA ring structures. Our results provide direct visual evidence that the ATP-induced NBD dimerization triggers a conformational change large enough in BmrA to destabilize the rings, which is consistent with the assumption that this step might constitute the "power stroke" for ABC transporters.

  7. Crystallization and preliminary X-ray diffraction analysis of the glucuronoyl esterase catalytic domain from Hypocrea jecorina.

    SciTech Connect

    Wood, S. J.; Li, X. -L.; Cotta, M. A.; Biely, P.; Duke, N. E. C.; Schiffer, M.; Pokkuluri, P. R.; Biosciences Division; National Center for Agricultural Utilization Research; Slovak Academy of Sciences

    2008-01-01

    The catalytic domain of the glucuronoyl esterase from Hypocrea jecorina (anamorph Trichoderma reesei) was overexpresssed, purified and crystallized by the sitting-drop vapor-diffusion method using 1.4 M sodium/potassium phosphate pH 6.9. The crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1} and X-ray diffraction data were collected to 1.9 {angstrom} resolution. This is the first enzyme with glucoronoyl esterase activity to be crystallized; its structure will be valuable in lignocellulose-degradation research.

  8. Substructure of skeletal myosin subfragment 1. Preferential destabilization of a domain by methanol and its effect on catalytic activity.

    PubMed

    Burke, M; Sivaramakrishnan, M

    1986-09-15

    Evidence is presented that in increasing concentrations of methanol the structure of the subfragment 1 is perturbed in such a way that the Mr = 50,000 central portion of the associated heavy chain is preferentially unfolded. This unfolding is accompanied by the loss in ATPase function where the rate of inactivation can be correlated with the loss in the amount of the Mr = 50,000 fragment generated under standard tryptic digestion conditions. The residual protein appears to be a soluble aggregate of a complex of the Mr = 27,000, 21,000, and light chain with no intact Mr = 50,000 fragment. Tryptic digestions in the presence of MgATP are restricted to the usual linker regions and the Mr = 50,000 fragment is completely protected from attack. Binding of actin to subfragment 1 also results in the protection of the Mr = 50,000 segment and of the Mr = 50,000/21,000 junction from tryptic attack. The data suggest that, in terms of methanolic perturbation, the subfragment 1 appears to be comprised of two domains with differential stability. One domain appears to be the central Mr = 50,000 segment of the heavy chain which is preferentially unfolded by methanol and requires the presence of MgATP or of actin for stabilization. The other domain is more stable and appears to consist of the interacting Mr = 27,000, 21,000, and light chain. The results also suggest that the integrity of the Mr = 50,000 segment is essential for the ATPase function of the protein.

  9. A monoclonal antibody inhibits gelatinase B/MMP-9 by selective binding to part of the catalytic domain and not to the fibronectin or zinc binding domains.

    PubMed

    Martens, Erik; Leyssen, An; Van Aelst, Ilse; Fiten, Pierre; Piccard, Helene; Hu, Jialiang; Descamps, Francis J; Van den Steen, Philippe E; Proost, Paul; Van Damme, Jo; Liuzzi, Grazia Maria; Riccio, Paolo; Polverini, Eugenia; Opdenakker, Ghislain

    2007-02-01

    Gelatinase B/matrix metalloproteinase-9 (MMP-9) is a multidomain enzyme functioning in acute and chronic inflammatory and neoplastic diseases. It belongs to a family of more than 20 related zinc proteinases. Therefore, the discovery and the definition of the action mechanism of selective MMP inhibitors form the basis for future therapeutics. The monoclonal antibody REGA-3G12 is a most selective inhibitor of human gelatinase B. REGA-3G12 was found to recognize the aminoterminal part and not the carboxyterminal O-glycosylated and hemopexin protein domains. A variant of gelatinase B, lacking the two carboxyterminal domains, was expressed in insect cells and fragmented with purified proteinases. The fragments were probed by one- and two-dimensional Western blot and immunoprecipitation experiments with REGA-3G12 to map the interactions between the antibody and the enzyme. The interaction unit was identified by Edman degradation analysis as the glycosylated segment from Trp(116) to Lys(214) of gelatinase B. The sequence of this segment was analysed by hydrophobicity/hydrophilicity, accessibility and flexibility profiling. Four hydrophilic peptides were chemically synthesized and used in binding and competition assays. The peptide Gly(171)-Leu(187) in molar excess inhibited partially the binding of MMP-9 to REGA-3G12 and thus refines the structure of the conformational binding site. These results define part of the catalytic domain of gelatinase B/MMP-9, and not the zinc-binding or fibronectin domains, as target for the development of selective inhibitors.

  10. The Translocation Domain of Botulinum Neurotoxin A Moderates the Propensity of the Catalytic Domain to Interact with Membranes at Acidic pH

    PubMed Central

    Araye, Anne; Goudet, Amélie; Barbier, Julien; Pichard, Sylvain; Baron, Bruno; England, Patrick; Pérez, Javier; Zinn-Justin, Sophie; Chenal, Alexandre; Gillet, Daniel

    2016-01-01

    Botulinum neurotoxin A (BoNT/A) is composed of three domains: a catalytic domain (LC), a translocation domain (HN) and a receptor-binding domain (HC). Like most bacterial toxins BoNT/A is an amphitropic protein, produced in a soluble form that is able to interact, penetrate and/or cross a membrane to achieve its toxic function. During intoxication BoNT/A is internalized by the cell by receptor-mediated endocytosis. Then, LC crosses the membrane of the endocytic compartment and reaches the cytosol. This translocation is initiated by the low pH found in this compartment. It has been suggested that LC passes in an unfolded state through a transmembrane passage formed by HN. We report here that acidification induces no major conformational change in either secondary or tertiary structures of LC and HN of BoNT/A in solution. GdnHCl-induced denaturation experiments showed that the stability of LC and HN increases as pH drops, and that HN further stabilizes LC. Unexpectedly we found that LC has a high propensity to interact with and permeabilize anionic lipid bilayers upon acidification without the help of HN. This property is downplayed when LC is linked to HN. HN thus acts as a chaperone for LC by enhancing its stability but also as a moderator of the membrane interaction of LC. PMID:27070312

  11. The Translocation Domain of Botulinum Neurotoxin A Moderates the Propensity of the Catalytic Domain to Interact with Membranes at Acidic pH.

    PubMed

    Araye, Anne; Goudet, Amélie; Barbier, Julien; Pichard, Sylvain; Baron, Bruno; England, Patrick; Pérez, Javier; Zinn-Justin, Sophie; Chenal, Alexandre; Gillet, Daniel

    2016-01-01

    Botulinum neurotoxin A (BoNT/A) is composed of three domains: a catalytic domain (LC), a translocation domain (HN) and a receptor-binding domain (HC). Like most bacterial toxins BoNT/A is an amphitropic protein, produced in a soluble form that is able to interact, penetrate and/or cross a membrane to achieve its toxic function. During intoxication BoNT/A is internalized by the cell by receptor-mediated endocytosis. Then, LC crosses the membrane of the endocytic compartment and reaches the cytosol. This translocation is initiated by the low pH found in this compartment. It has been suggested that LC passes in an unfolded state through a transmembrane passage formed by HN. We report here that acidification induces no major conformational change in either secondary or tertiary structures of LC and HN of BoNT/A in solution. GdnHCl-induced denaturation experiments showed that the stability of LC and HN increases as pH drops, and that HN further stabilizes LC. Unexpectedly we found that LC has a high propensity to interact with and permeabilize anionic lipid bilayers upon acidification without the help of HN. This property is downplayed when LC is linked to HN. HN thus acts as a chaperone for LC by enhancing its stability but also as a moderator of the membrane interaction of LC.

  12. Mechanism for activation of the EGF receptor catalytic domain by the juxtamembrane segment

    PubMed Central

    Jura, Natalia; Endres, Nicholas F.; Engel, Kate; Deindl, Sebastian; Das, Rahul; Lamers, Meindert H.; Wemmer, David E.; Zhang, Xuewu; Kuriyan, John

    2009-01-01

    Signaling by the epidermal growth factor receptor requires an allosteric interaction between the kinase domains of two receptors, whereby one activates the other. We show that the intracellular juxtamembrane segment of the receptor, known to potentiate kinase activity, is able to dimerize the kinase domains. The C-terminal half of the juxtamembrane segment latches the activated kinase domain to the activator, and the N-terminal half of this segment further potentiates dimerization, most likely by forming an antiparallel helical dimer that engages the transmembrane helices of the activated receptor. Our data are consistent with a mechanism in which the extracellular domains block the intrinsic ability of the transmembrane and cytoplasmic domains to dimerize and activate, with ligand binding releasing this block. The formation of the activating juxtamembrane latch is prevented by the C-terminal tails in a new structure of an inactive kinase domain dimer, suggesting how alternative dimers can prevent ligand-independent activation. PMID:19563760

  13. Increasing the conformational entropy of the Ω-loop lid domain in PEPCK impairs catalysis and decreases catalytic fidelity†

    PubMed Central

    Johnson, Troy A.; Holyoak, Todd

    2010-01-01

    Many studies have shown that the dynamic motions of individual protein segments can play an important role in enzyme function. Recent structural studies on the gluconeogenic enzyme PEPCK demonstrate that PEPCK contains a 10-residue Ω-loop domain that acts as an active site lid. Based upon these structural studies we have previously proposed a model for the mechanism of PEPCK catalysis in which the conformation of this mobile lid-domain is energetically coupled to ligand binding resulting in the closed conformation of the lid, necessary for correct substrate positioning, becoming more energetically favorable as ligands associate with the enzyme. Here we test this model by the introduction of a point mutation (A467G) into the center of the Ω-loop lid that is designed to increase the entropic penalty for lid closure. Structural and kinetic characterization of this mutant enzyme demonstrates that the mutation has decreased the favorability of the enzyme adapting the closed lid conformation. As a consequence of this shift in the equilibrium defining the conformation of the active site lid, the enzyme’s ability to stabilize the reaction intermediate is reduced resulting in catalytic defect. This stabilization is initially surprising, as the lid domain makes no direct contacts with the enolate intermediate formed during the reaction. Furthermore, during the conversion of OAA to PEP, the destabilization of the lid closed conformation results in the reaction becoming decoupled as the enolate intermediate is protonated rather than phosphorylated resulting in the formation of pyruvate. Taken together, the structural and kinetic characterization of A467G-PEPCK support our model of the role of the active site lid in catalytic function and demonstrate that the shift in the lowest energy conformation between open and closed lid states is a function of the free energy available to the enzyme through ligand binding and the entropic penalty for ordering of the ten-residue

  14. Increasing the conformational entropy of the Omega-loop lid domain in phosphoenolpyruvate carboxykinase impairs catalysis and decreases catalytic fidelity .

    PubMed

    Johnson, Troy A; Holyoak, Todd

    2010-06-29

    Many studies have shown that the dynamic motions of individual protein segments can play an important role in enzyme function. Recent structural studies of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK) demonstrate that PEPCK contains a 10-residue Omega-loop domain that acts as an active site lid. On the basis of these structural studies, we have previously proposed a model for the mechanism of PEPCK catalysis in which the conformation of this mobile lid domain is energetically coupled to ligand binding, resulting in the closed conformation of the lid, necessary for correct substrate positioning, becoming more energetically favorable as ligands associate with the enzyme. Here we test this model by introducing a point mutation (A467G) into the center of the Omega-loop lid that is designed to increase the entropic penalty for lid closure. Structural and kinetic characterization of this mutant enzyme demonstrates that the mutation has decreased the favorability of the enzyme adapting the closed lid conformation. As a consequence of this shift in the equilibrium defining the conformation of the active site lid, the enzyme's ability to stabilize the reaction intermediate is weakened, resulting in catalytic defect. This stabilization is initially surprising, as the lid domain makes no direct contacts with the enolate intermediate formed during the reaction. Furthermore, during the conversion of OAA to PEP, the destabilization of the lid-closed conformation results in the reaction becoming decoupled as the enolate intermediate is protonated rather than phosphorylated, resulting in the formation of pyruvate. Taken together, the structural and kinetic characterization of A467G-PEPCK supports our model of the role of the active site lid in catalytic function and demonstrates that the shift in the lowest-energy conformation between open and closed lid states is a function of the free energy available to the enzyme through ligand binding and the entropic

  15. Constructs of human neuropathy target esterase catalytic domain containing mutations related to motor neuron disease have altered enzymatic properties.

    PubMed

    Hein, Nichole D; Stuckey, Jeanne A; Rainier, Shirley R; Fink, John K; Richardson, Rudy J

    2010-07-01

    Neuropathy target esterase (NTE) is a phospholipase/lysophospholipase associated with organophosphorus (OP) compound-induced delayed neurotoxicity (OPIDN). Distal degeneration of motor axons occurs in both OPIDN and the hereditary spastic paraplegias (HSPs). Recently, mutations within the esterase domain of NTE were identified in patients with a novel type of HSP (SPG39) designated NTE-related motor neuron disease (NTE-MND). Two of these mutations, arginine 890 to histidine (R890H) and methionine 1012 to valine (M1012V), were created in human recombinant NTE catalytic domain (NEST) to measure possible changes in catalytic properties. These mutated enzymes had decreased specific activities for hydrolysis of the artificial substrate, phenyl valerate. In addition, the M1012V mutant exhibited a reduced bimolecular rate constant of inhibition (k(i)) for all three inhibitors tested: mipafox, diisopropylphosphorofluoridate, and chlorpyrifos oxon. Finally, while both mutated enzymes inhibited by OP compounds exhibited altered time-dependent loss of their ability to be reactivated by nucleophiles (aging), more pronounced effects were seen with the M1012V mutant. Taken together, the results from specific activity, inhibition, and aging experiments suggest that the mutations found in association with NTE-MND have functional correlates in altered enzymological properties of NTE.

  16. Absence of catalytic domain in a putative protein kinase C (PkcA) suppresses tip dominance in Dictyostelium discoideum

    PubMed Central

    Mohamed, Wasima; Ray, Sibnath; Brazill, Derrick; Baskar, Ramamurthy

    2017-01-01

    A number of organisms possess several isoforms of protein kinase C but little is known about the significance of any specific isoform during embryogenesis and development. To address this we characterized a PKC ortholog (PkcA; DDB_G0288147) in Dictyostelium discoideum. pkcA expression switches from prestalk in mound to prespore in slug, indicating a dynamic expression pattern. Mutants lacking the catalytic domain of PkcA (pkcA−) did not exhibit tip dominance. A striking phenotype of pkcA− was the formation of an aggregate with a central hollow, and aggregates later fragmented to form small mounds, each becoming a fruiting body. Optical density wave patterns of cAMP in the late aggregates showed several cAMP wave generation centers. We attribute these defects in pkcA− to impaired cAMP signaling, altered cell motility and decreased expression of the cell adhesion molecules – CadA and CsaA. pkcA− slugs showed ectopic expression of ecmA in the prespore region. Further, the use of a PKC-specific inhibitor, GF109203X that inhibits the activity of catalytic domain phenocopied pkcA−. PMID:26183108

  17. Absence of catalytic domain in a putative protein kinase C (PkcA) suppresses tip dominance in Dictyostelium discoideum.

    PubMed

    Mohamed, Wasima; Ray, Sibnath; Brazill, Derrick; Baskar, Ramamurthy

    2015-09-01

    A number of organisms possess several isoforms of protein kinase C but little is known about the significance of any specific isoform during embryogenesis and development. To address this we characterized a PKC ortholog (PkcA; DDB_G0288147) in Dictyostelium discoideum. pkcA expression switches from prestalk in mound to prespore in slug, indicating a dynamic expression pattern. Mutants lacking the catalytic domain of PkcA (pkcA(-)) did not exhibit tip dominance. A striking phenotype of pkcA- was the formation of an aggregate with a central hollow, and aggregates later fragmented to form small mounds, each becoming a fruiting body. Optical density wave patterns of cAMP in the late aggregates showed several cAMP wave generation centers. We attribute these defects in pkcA(-) to impaired cAMP signaling, altered cell motility and decreased expression of the cell adhesion molecules - CadA and CsaA. pkcA(-) slugs showed ectopic expression of ecmA in the prespore region. Further, the use of a PKC-specific inhibitor, GF109203X that inhibits the activity of catalytic domain phenocopied pkcA(-).

  18. Molecular defect in factor IXBm Lake Elsinore. Substitution of Ala390 by Val in the catalytic domain.

    PubMed

    Spitzer, S G; Pendurthi, U R; Kasper, C K; Bajaj, S P

    1988-08-05

    Earlier studies with factor IXBm Lake Elsinore (IXBmLE), a nonfunctional variant of factor IX, suggested that the defect in this protein may reside in the catalytic domain of the molecule (Usharani, P., Warn-Cramer, B. J., Kasper, C. K., and Bajaj, S. P. (1985) J. Clin. Invest. 75, 76-83). In this report, genomic DNA fragments from normal IX and IXBmLE alleles were cloned into phage lambda EMBL3 and the recombinant phage identified using normal IX cDNA and synthetic oligonucleotides. Exons VI, VII, and VIII of normal IX and IXBmLE gene were also amplified using a newly developed primer-directed polymerase chain reaction method. All eight exons and flanking regions of the normal IX and IXBmLE gene were sequenced by the dideoxy chain termination method. Comparison of the normal IX and IXBmLE sequences revealed a single base substitution (C----T) in the exon VIII of the BmLE variant, which results in the replacement of Ala390 by Val in the variant molecule. Although this mutation is in the catalytic domain of the molecule, purified factor IXaBmLE is indistinguishable from normal IXa in its activity toward a small synthetic substrate, L-tosylarginine methyl ester. These data, coupled with the previous data, identify a region (around residue 390) in the normal factor IXa which appears to play a major role in the extended macromolecular substrate binding site.

  19. MMP-12 catalytic domain recognizes and cleaves at multiple sites in human skin collagen type I and type III.

    PubMed

    Taddese, Samuel; Jung, Michael C; Ihling, Christian; Heinz, Andrea; Neubert, Reinhard H H; Schmelzer, Christian E H

    2010-04-01

    Collagens of either soft connective or mineralized tissues are subject to continuous remodeling and turnover. Undesired cleavage can be the result of an imbalance between proteases and their inhibitors. Owing to their superhelical structure, collagens are resistant to many proteases and matrix metalloproteinases (MMPs) are required to initiate further degradation by other enzymes. Several MMPs are known to degrade collagens, but the action of MMP-12 has not yet been studied in detail. In this work, the potential of MMP-12 in recognizing sites in human skin collagen types I and III has been investigated. The catalytic domain of MMP-12 binds to the triple helix and cleaves the typical sites -Gly(775)-Leu(776)- in alpha-2 type I collagen and -Gly(775)-Ile(776)- in alpha-1 type I and type III collagens and at multiple other sites in both collagen types. Moreover, it was observed that the region around these typical sites contains comparatively less prolines, of which some have been proven to be only partially hydroxylated. This is of relevance since partial hydroxylation in the vicinity of a potential scissile bond may have a local effect on the conformational thermodynamics with probable consequences on the collagenolysis process. Taken together, the results of the present work confirm that the catalytic domain of MMP-12 alone binds and degrades collagens I and III. Copyright 2009 Elsevier B.V. All rights reserved.

  20. Conserved catalytic residues of the ALDH1L1 aldehyde dehydrogenase domain control binding and discharging of the coenzyme.

    PubMed

    Tsybovsky, Yaroslav; Krupenko, Sergey A

    2011-07-01

    The C-terminal domain (C(t)-FDH) of 10-formyltetrahydrofolate dehydrogenase (FDH, ALDH1L1) is an NADP(+)-dependent oxidoreductase and a structural and functional homolog of aldehyde dehydrogenases. Here we report the crystal structures of several C(t)-FDH mutants in which two essential catalytic residues adjacent to the nicotinamide ring of bound NADP(+), Cys-707 and Glu-673, were replaced separately or simultaneously. The replacement of the glutamate with an alanine causes irreversible binding of the coenzyme without any noticeable conformational changes in the vicinity of the nicotinamide ring. Additional replacement of cysteine 707 with an alanine (E673A/C707A double mutant) did not affect this irreversible binding indicating that the lack of the glutamate is solely responsible for the enhanced interaction between the enzyme and the coenzyme. The substitution of the cysteine with an alanine did not affect binding of NADP(+) but resulted in the enzyme lacking the ability to differentiate between the oxidized and reduced coenzyme: unlike the wild-type C(t)-FDH/NADPH complex, in the C707A mutant the position of NADPH is identical to the position of NADP(+) with the nicotinamide ring well ordered within the catalytic center. Thus, whereas the glutamate restricts the affinity for the coenzyme, the cysteine is the sensor of the coenzyme redox state. These conclusions were confirmed by coenzyme binding experiments. Our study further suggests that the binding of the coenzyme is additionally controlled by a long-range communication between the catalytic center and the coenzyme-binding domain and points toward an α-helix involved in the adenine moiety binding as a participant of this communication.

  1. Conserved Catalytic Residues of the ALDH1L1 Aldehyde Dehydrogenase Domain Control Binding and Discharging of the Coenzyme*

    PubMed Central

    Tsybovsky, Yaroslav; Krupenko, Sergey A.

    2011-01-01

    The C-terminal domain (Ct-FDH) of 10-formyltetrahydrofolate dehydrogenase (FDH, ALDH1L1) is an NADP+-dependent oxidoreductase and a structural and functional homolog of aldehyde dehydrogenases. Here we report the crystal structures of several Ct-FDH mutants in which two essential catalytic residues adjacent to the nicotinamide ring of bound NADP+, Cys-707 and Glu-673, were replaced separately or simultaneously. The replacement of the glutamate with an alanine causes irreversible binding of the coenzyme without any noticeable conformational changes in the vicinity of the nicotinamide ring. Additional replacement of cysteine 707 with an alanine (E673A/C707A double mutant) did not affect this irreversible binding indicating that the lack of the glutamate is solely responsible for the enhanced interaction between the enzyme and the coenzyme. The substitution of the cysteine with an alanine did not affect binding of NADP+ but resulted in the enzyme lacking the ability to differentiate between the oxidized and reduced coenzyme: unlike the wild-type Ct-FDH/NADPH complex, in the C707A mutant the position of NADPH is identical to the position of NADP+ with the nicotinamide ring well ordered within the catalytic center. Thus, whereas the glutamate restricts the affinity for the coenzyme, the cysteine is the sensor of the coenzyme redox state. These conclusions were confirmed by coenzyme binding experiments. Our study further suggests that the binding of the coenzyme is additionally controlled by a long-range communication between the catalytic center and the coenzyme-binding domain and points toward an α-helix involved in the adenine moiety binding as a participant of this communication. PMID:21540484

  2. N- vs. C-Domain Selectivity of Catalytic Inactivation of Human Angiotensin Converting Enzyme by Lisinopril-Coupled Transition Metal Chelates

    PubMed Central

    Hocharoen, Lalintip; Joyner, Jeff C.; Cowan, J. A.

    2014-01-01

    The N- and C-terminal domains of human somatic Angiotensin I Converting Enzyme (sACE-1) demonstrate distinct physiological functions, with resulting interest in the development of domain-selective inhibitors for specific therapeutic applications. Herein, the activity of lisinopril-coupled transition metal chelates were tested for both reversible binding and irreversible catalytic inactivation of sACE-1. C/N domain binding selectivity ratios ranged from 1 to 350, while rates of irreversible catalytic inactivation of the N- and C-domains were found to be significantly greater for the N-domain, suggesting a more optimal orientation of the M-chelate-lisinopril complexes within the active site of the N-domain of sACE-1. Finally, the combined effect of binding selectivity and inactivation selectivity was assessed for each catalyst (double-filter selectivity factors), and several catalysts were found to cause domain-selective catalytic inactivation. The results of this study demonstrate the ability to optimize the target selectivity of catalytic metallopeptides through both binding and orientation factors (double-filter effect). PMID:24228790

  3. Redox-coupled structural changes of the catalytic a' domain of protein disulfide isomerase.

    PubMed

    Inagaki, Koya; Satoh, Tadashi; Yagi-Utsumi, Maho; Le Gulluche, Anne-Charlotte; Anzai, Takahiro; Uekusa, Yoshinori; Kamiya, Yukiko; Kato, Koichi

    2015-09-14

    Protein disulfide isomerase functions as a folding catalyst in the endoplasmic reticulum. Its b' and a' domains provide substrate-binding sites and undergo a redox-dependent domain rearrangement coupled to an open-closed structural change. Here we determined the first solution structure of the a' domain in its oxidized form and thereby demonstrate that oxidation of the a' domain induces significant conformational changes not only in the vicinity of the active site but also in the distal b'-interfacial segment. Based on these findings, we propose that this conformational transition triggers the domain segregation coupled with the exposure of the hydrophobic surface. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  4. Molecular dissection of the domain architecture and catalytic activities of human PrimPol

    PubMed Central

    Keen, Benjamin A.; Jozwiakowski, Stanislaw K.; Bailey, Laura J.; Bianchi, Julie; Doherty, Aidan J.

    2014-01-01

    PrimPol is a primase–polymerase involved in nuclear and mitochondrial DNA replication in eukaryotic cells. Although PrimPol is predicted to possess an archaeo-eukaryotic primase and a UL52-like zinc finger domain, the role of these domains has not been established. Here, we report that the proposed zinc finger domain of human PrimPol binds zinc ions and is essential for maintaining primase activity. Although apparently dispensable for its polymerase activity, the zinc finger also regulates the processivity and fidelity of PrimPol's extension activities. When the zinc finger is disrupted, PrimPol becomes more promutagenic, has an altered translesion synthesis spectrum and is capable of faithfully bypassing cyclobutane pyrimidine dimer photolesions. PrimPol's polymerase domain binds to both single- and double-stranded DNA, whilst the zinc finger domain binds only to single-stranded DNA. We additionally report that although PrimPol's primase activity is required to restore wild-type replication fork rates in irradiated PrimPol−/− cells, polymerase activity is sufficient to maintain regular replisome progression in unperturbed cells. Together, these findings provide the first analysis of the molecular architecture of PrimPol, describing the activities associated with, and interplay between, its functional domains and defining the requirement for its primase and polymerase activities during nuclear DNA replication. PMID:24682820

  5. The properties of catalytically-inactivated Trichoderma reesei cellobiohydrolase I: Role of the cellulose binding domain

    SciTech Connect

    Woodward, J.; Donner, T.R.; Affholter, K.A.

    1993-12-31

    Cellobiohydrolase I (CBH I) was purified from a crude cellulase by preparative isoelectric focusing. Treatment of CBH I with 1-ethyl-3-3(3-dimethylaminopropyl)-carbodiimide (EDC) resulted in its catalytic inactivation but did not abolish its ability to be absorbed to microcrystalline cellulose (Avicel). CBH I thus modified possessed a pI of between 8.5 and 9.3 and decreased tryptophan fluorescence compared to native CBH I. A comparison of the effect of native and modified CBH I on the morphology of crystalline cotton cellulose fibers was made using scanning electron microscopy.

  6. Crystallization and preliminary X-ray diffraction studies on the catalytic domain of the chick retinal neurite-inhibitory factor CRYP-2

    SciTech Connect

    Girish, T. S.; Gopal, B.

    2005-04-01

    The receptor protein tyrosine phosphatase CRYP-2 has been shown to be an inhibitory factor for the growth of retinal axons in the chick. The cloning, expression, purification and crystallization of the catalytic domain of CRYP-2 are reported here. The receptor protein tyrosine phosphatase CRYP-2 has been shown to be an inhibitory factor for the growth of retinal axons in the chick. The extracellular receptor domain of CRYP-2 contains eight fibronectin repeats and studies using the extracellular domain alone demonstrated the chemorepulsive effect on retinal neurons. The precise role of the intracellular catalytic domain and the mechanism by which its activity is regulated is not known. Determination of the structure of the catalytic domain of CRYP-2 was proposed in an effort to understand the downstream signal transduction mechanism in this system. The cloning, expression, purification and crystallization of the catalytic domain of CRYP-2 are now reported. Preliminary crystallographic studies were performed on the diamond-shaped crystals, which grew under oil using the microbatch method at 298 K. Native X-ray diffraction data were collected to 2.9 Å resolution on a home source. The crystals belong to the trigonal space group P3{sub 1}21, with unit-cell parameters a = b = 68.26, c = 244.95 Å. Assuming the presence of two molecules per asymmetric unit, the V{sub M} value was 2.7 Å{sup 3} Da{sup −1} and the solvent content was 54.8%.

  7. Redirection of the immune response to the functional catalytic domain of the cystein proteinase cruzipain improves protective immunity against Trypanosoma cruzi infection.

    PubMed

    Cazorla, Silvia I; Frank, Fernanda M; Becker, Pablo D; Arnaiz, María; Mirkin, Gerardo A; Corral, Ricardo S; Guzmán, Carlos A; Malchiodi, Emilio L

    2010-07-01

    Despite the strong immune responses elicited after natural infection with Trypanosoma cruzi or vaccination against it, parasite survival suggests that these responses are insufficient or inherently inadequate. T. cruzi contains a major cystein proteinase, cruzipain, which has a catalytic N-terminal domain and a C-terminal extension. Immunizations that employed recombinant cruzipain or its N- and C-terminal domains allowed evaluation of the ability of cruzipain to circumvent responses against the catalytic domain. This phenomenon is not a property of the parasite but of cruzipain itself, because recombinant cruzipain triggers a response similar to that of cruzipain during natural or experimental infection. Cruzipain is not the only antigen with a highly immunogenic region of unknown function that somehow protects an essential domain for parasite survival. However, our studies show that this can be reverted by using the N-terminal domain as a tailored immunogen able to redirect host responses to provide enhanced protection.

  8. Crystal structure of the peptidase domain of Streptococcus ComA, a bifunctional ATP-binding cassette transporter involved in the quorum-sensing pathway.

    PubMed

    Ishii, Seiji; Yano, Takato; Ebihara, Akio; Okamoto, Akihiro; Manzoku, Miho; Hayashi, Hideyuki

    2010-04-02

    ComA of Streptococcus is a member of the bacteriocin-associated ATP-binding cassette transporter family and is postulated to be responsible for both the processing of the propeptide ComC and secretion of the mature quorum-sensing signal. The 150-amino acid peptidase domain (PEP) of ComA specifically recognizes an extended region of ComC that is 15 amino acids in length. It has been proposed that an amphipathic alpha-helix formed by the N-terminal leader region of ComC, as well as the Gly-Gly motif at the cleavage site, is critical for the PEP-ComC interaction. To elucidate the substrate recognition mechanism, we determined the three-dimensional crystal structure of Streptococcus mutans PEP and then constructed models for the PEP.ComC complexes. PEP had an overall structure similar to the papain-like cysteine proteases as has long been predicted. The active site was located at the bottom of a narrow cleft, which is suitable for binding the Gly-Gly motif. Together with the results from mutational experiments, a shallow hydrophobic concave surface of PEP was proposed as a site that accommodates the N-terminal helix of ComC. This dual mode of substrate recognition would provide the small PEP domain with an extremely high substrate specificity.

  9. Modulation of erbB kinase activity and oncogenic potential by single point mutations in the glycine loop of the catalytic domain.

    PubMed

    Shu, H K; Chang, C M; Ravi, L; Ling, L; Castellano, C M; Walter, E; Pelley, R J; Kung, H J

    1994-10-01

    Avian c-erbB is activated to a leukemia oncogene following truncation of its amino-terminal ligand-binding domain by retroviral insertion. The insertionally activated transcripts encode protein products which have constitutive tyrosine kinase activity and can induce erythroleukemia but not sarcomas. We have previously found that a valine-to-isoleucine point mutation at position 157 (V157I mutant) within the tyrosine kinase domain of this truncated erbB can dramatically activate the sarcomagenic potential of the oncogene and increase the kinase activity of this oncoprotein. This mutation lies at position 157 of the insertionally activated c-erbB product, affecting a highly conserved valine residue of the glycine loop involved in ATP binding and phosphate transfer. To investigate the functional importance of this residue in the catalytic activity of kinases, we have introduced at this position, by site-directed mutagenesis, codons representing the remaining 18 amino acid residues. Most of the mutants have diminished activity, with six of them completely devoid of kinase activity, indicating the sensitivity of this region to conformational changes. Some of these mutants displayed increased kinase activity and greater transforming potential in comparison with IA c-erbB, but none had levels as high as those of the V157I mutant. In general, the sarcomagenic potential of the various erbB mutants correlated with their autophosphorylation state and their ability to cause phosphorylation of MAP kinase. However, there are important exceptions such as the V157G mutant, which lacks enhanced autophosphorylation but is highly sarcomagenic. Studies of this and other autophosphorylation site mutants point to the existence of an autophosphorylation-independent pathway in sarcomagenesis. The requirement for leukemogenic potential is much less stringent and correlates with positivity of kinase activity. When the valine-to-isoleucine substitution was put in context of the full

  10. Structure and function of the catalytic domain of the dihydrolipoyl acetyltransferase component in Escherichia coli pyruvate dehydrogenase complex.

    PubMed

    Wang, Junjie; Nemeria, Natalia S; Chandrasekhar, Krishnamoorthy; Kumaran, Sowmini; Arjunan, Palaniappa; Reynolds, Shelley; Calero, Guillermo; Brukh, Roman; Kakalis, Lazaros; Furey, William; Jordan, Frank

    2014-05-30

    The Escherichia coli pyruvate dehydrogenase complex (PDHc) catalyzing conversion of pyruvate to acetyl-CoA comprises three components: E1p, E2p, and E3. The E2p is the five-domain core component, consisting of three tandem lipoyl domains (LDs), a peripheral subunit binding domain (PSBD), and a catalytic domain (E2pCD). Herein are reported the following. 1) The x-ray structure of E2pCD revealed both intra- and intertrimer interactions, similar to those reported for other E2pCDs. 2) Reconstitution of recombinant LD and E2pCD with E1p and E3p into PDHc could maintain at least 6.4% activity (NADH production), confirming the functional competence of the E2pCD and active center coupling among E1p, LD, E2pCD, and E3 even in the absence of PSBD and of a covalent link between domains within E2p. 3) Direct acetyl transfer between LD and coenzyme A catalyzed by E2pCD was observed with a rate constant of 199 s(-1), comparable with the rate of NADH production in the PDHc reaction. Hence, neither reductive acetylation of E2p nor acetyl transfer within E2p is rate-limiting. 4) An unprecedented finding is that although no interaction could be detected between E1p and E2pCD by itself, a domain-induced interaction was identified on E1p active centers upon assembly with E2p and C-terminally truncated E2p proteins by hydrogen/deuterium exchange mass spectrometry. The inclusion of each additional domain of E2p strengthened the interaction with E1p, and the interaction was strongest with intact E2p. E2p domain-induced changes at the E1p active site were also manifested by the appearance of a circular dichroism band characteristic of the canonical 4'-aminopyrimidine tautomer of bound thiamin diphosphate (AP).

  11. An oligodeoxyribonucleotide that supports catalytic activity in the hammerhead ribozyme domain.

    PubMed

    Chartrand, P; Harvey, S C; Ferbeyre, G; Usman, N; Cedergren, R

    1995-10-25

    A study of the activity of deoxyribonucleotide-substituted analogs of the hammerhead domain of RNA catalysis has led to the design of a 14mer oligomer composed entirely of deoxyribonucleotides that promotes the cleavage of an RNA substrate. Characterization of this reaction with sequence variants and mixed DNA/RNA oligomers shows that, although the all-deoxyribonucleotide oligomer is less efficient in catalysis, the DNA/substrate complex shares many of the properties of the all-RNA hammerhead domain such as multiple turnover kinetics and dependence on Mg2+ concentration. On the other hand, the values of kinetic parameters distinguish the DNA oligomer from the all-RNA oligomer. In addition, an analog of the oligomer having a single ribonucleotide in a strongly conserved position of the hammerhead domain is associated with more efficient catalysis than the all-RNA oligomer.

  12. An oligodeoxyribonucleotide that supports catalytic activity in the hammerhead ribozyme domain.

    PubMed Central

    Chartrand, P; Harvey, S C; Ferbeyre, G; Usman, N; Cedergren, R

    1995-01-01

    A study of the activity of deoxyribonucleotide-substituted analogs of the hammerhead domain of RNA catalysis has led to the design of a 14mer oligomer composed entirely of deoxyribonucleotides that promotes the cleavage of an RNA substrate. Characterization of this reaction with sequence variants and mixed DNA/RNA oligomers shows that, although the all-deoxyribonucleotide oligomer is less efficient in catalysis, the DNA/substrate complex shares many of the properties of the all-RNA hammerhead domain such as multiple turnover kinetics and dependence on Mg2+ concentration. On the other hand, the values of kinetic parameters distinguish the DNA oligomer from the all-RNA oligomer. In addition, an analog of the oligomer having a single ribonucleotide in a strongly conserved position of the hammerhead domain is associated with more efficient catalysis than the all-RNA oligomer. Images PMID:7479070

  13. Deciphering the catalytic domain of colicin M, a peptidoglycan lipid II-degrading enzyme.

    PubMed

    Barreteau, Hélène; Bouhss, Ahmed; Gérard, Fabien; Duché, Denis; Boussaid, Boubekeur; Blanot, Didier; Lloubès, Roland; Mengin-Lecreulx, Dominique; Touzé, Thierry

    2010-04-16

    Colicin M inhibits Escherichia coli peptidoglycan synthesis through cleavage of its lipid-linked precursors. It has a compact structure, whereas other related toxins are organized in three independent domains, each devoted to a particular function: translocation through the outer membrane, receptor binding, and toxicity, from the N to the C termini, respectively. To establish whether colicin M displays such an organization despite its structural characteristics, protein dissection experiments were performed, which allowed us to delineate an independent toxicity domain encompassing exactly the C-terminal region conserved among colicin M-like proteins and covering about half of colicin M (residues 124-271). Surprisingly, the in vitro activity of the isolated domain was 45-fold higher than that of the full-length protein, suggesting a mechanism by which the toxicity of this domain is revealed following primary protein maturation. In vivo, the isolated toxicity domain appeared as toxic as the full-length protein under conditions where the reception and translocation steps were by-passed. Contrary to the full-length colicin M, the isolated domain did not require the presence of the periplasmic FkpA protein to be toxic under these conditions, demonstrating that FkpA is involved in the maturation process. Mutational analysis further identified five residues that are essential for cytotoxicity as well as in vitro lipid II-degrading activity: Asp-229, His-235, Asp-226, Tyr-228, and Arg-236. Most of these residues are surface-exposed and located relatively close to each other, hence suggesting they belong to the colicin M active site.

  14. Refined molecular hinge between allosteric and catalytic domain determines allosteric regulation and stability of fungal chorismate mutase.

    PubMed

    Helmstaedt, Kerstin; Heinrich, Gabriele; Lipscomb, William N; Braus, Gerhard H

    2002-05-14

    The yeast chorismate mutase is regulated by tyrosine as feedback inhibitor and tryptophan as crosspathway activator. The monomer consists of a catalytic and a regulatory domain covalently linked by the loop L220s (212-226), which functions as a molecular hinge. Two monomers form the active dimeric enzyme stabilized by hydrophobic interactions in the vicinity of loop L220s. The role of loop L220s and its environment for enzyme regulation, dimerization, and stability was analyzed. Substitution of yeast loop L220s in place of the homologous loop from the corresponding and similarly regulated Aspergillus enzyme (and the reverse substitution) changed tyrosine inhibition to activation. Yeast loop L220s substituted into the Aspergillus enzyme resulted in a tryptophan-inhibitable enzyme. Monomeric yeast chorismate mutases could be generated by substituting two hydrophobic residues in and near the hinge region. The resulting Thr-212-->Asp-Phe-28-->Asp enzyme was as stable as wild type, but lost allosteric regulation and showed reduced catalytic activity. These results underline the crucial role of this molecular hinge for inhibition, activation, quaternary structure, and stability of yeast chorismate mutase.

  15. Structural and functional analyses of catalytic domain of GH10 xylanase from Thermoanaerobacterium saccharolyticum JW/SL-YS485.

    PubMed

    Han, Xu; Gao, Jian; Shang, Na; Huang, Chun-Hsiang; Ko, Tzu-Ping; Chen, Chun-Chi; Chan, Hsiu-Chien; Cheng, Ya-Shan; Zhu, Zhen; Wiegel, Juergen; Luo, Wenhua; Guo, Rey-Ting; Ma, Yanhe

    2013-07-01

    Xylanases are capable of decomposing xylans, the major components in plant cell wall, and releasing the constituent sugars for further applications. Because xylanase is widely used in various manufacturing processes, high specific activity, and thermostability are desirable. Here, the wild-type and mutant (E146A and E251A) catalytic domain of xylanase from Thermoanaerobacterium saccharolyticum JW/SL-YS485 (TsXylA) were expressed in Escherichia coli and purified subsequently. The recombinant protein showed optimal temperature and pH of 75°C and 6.5, respectively, and it remained fully active even after heat treatment at 75°C for 1 h. Furthermore, the crystal structures of apo-form wild-type TsXylA and the xylobiose-, xylotriose-, and xylotetraose-bound E146A and E251A mutants were solved by X-ray diffraction to high resolution (1.32-1.66 Å). The protein forms a classic (β/α)8 folding of typical GH10 xylanases. The ligands in substrate-binding groove as well as the interactions between sugars and active-site residues were clearly elucidated by analyzing the complex structures. According to the structural analyses, TsXylA utilizes a double displacement catalytic machinery to carry out the enzymatic reactions. In conclusion, TsXylA is effective under industrially favored conditions, and our findings provide fundamental knowledge which may contribute to further enhancement of the enzyme performance through molecular engineering.

  16. Purification, crystallization and preliminary X-ray diffraction of the N-terminal calmodulin-like domain of the human mitochondrial ATP-Mg/P{sub i} carrier SCaMC1

    SciTech Connect

    Yang, Qin Brüschweiler, Sven; Chou, James J.

    2013-12-24

    The N-terminal calmodulin-like domain of the human mitochondrial ATP-Mg/P{sub i} carrier SCaMC1 was crystallized in the presence of Ca{sup 2+}. X-ray diffraction data were collected to 2.9 Å resolution from crystals which belonged to space group P6{sub 2}22.

  17. Crystallization and preliminary diffraction analysis of the catalytic domain of major extracellular endoglucanase from Xanthomonas campestris pv. campestris

    PubMed Central

    Rosseto, Flávio R.; Puhl, Ana C.; Andrade, Maxuel O.; Polikarpov, Igor

    2013-01-01

    Cellulases, such as endoglucanases, exoglucanases and β-glucosidases, are important enzymes used in the process of enzymatic hydrolysis of plant biomass. The bacteria Xanthomonas campestris pv. campestris expresses a large number of hydrolases and the major endoglucanase (XccEG), a member of glycoside hydrolase family 5 (GH5), is the most strongly secreted extracellularly. In this work, the native XccEG was purified from the extracellular extract and crystallization assays were performed on its catalytic domain. A complete data set was collected on an in-house X-ray source. The crystal diffracted to 2.7 Å resolution and belonged to space group C2, with unit-cell parameters a = 174.66, b = 141.53, c = 108.00 Å, β = 110.49°. The Matthews coefficient suggests a solvent content of 70.1% and the presence of four protein subunits in the asymmetric unit. PMID:23385754

  18. Crystal Structure of 12-Lipoxygenase Catalytic-Domain-Inhibitor Complex Identifies a Substrate-Binding Channel for Catalysis

    SciTech Connect

    Xu, Shu; Mueser, Timothy C.; Marnett, Lawrence J.; Funk, Jr., Max O.

    2014-10-02

    Lipoxygenases are critical enzymes in the biosynthesis of families of bioactive lipids including compounds with important roles in the initiation and resolution of inflammation and in associated diseases such as diabetes, cardiovascular disease, and cancer. Crystals diffracting to high resolution (1.9 {angstrom}) were obtained for a complex between the catalytic domain of leukocyte 12-lipoxygenase and the isoform-specific inhibitor, 4-(2-oxapentadeca-4-yne)phenylpropanoic acid (OPP). In the three-dimensional structure of the complex, the inhibitor occupied a new U-shaped channel open at one end to the surface of the protein and extending past the redox-active iron site that is essential for catalysis. In models, the channel accommodated arachidonic acid, defining the binding site for the substrate of the catalyzed reaction. There was a void adjacent to the OPP binding site connecting to the surface of the enzyme and providing a plausible access channel for the other substrate, oxygen.

  19. Crystal structure of the DNA cytosine deaminase APOBEC3F: the catalytically active and HIV-1 Vif-binding domain.

    PubMed

    Bohn, Markus-Frederik; Shandilya, Shivender M D; Albin, John S; Kouno, Takahide; Anderson, Brett D; McDougle, Rebecca M; Carpenter, Michael A; Rathore, Anurag; Evans, Leah; Davis, Ahkillah N; Zhang, Jingying; Lu, Yongjian; Somasundaran, Mohan; Matsuo, Hiroshi; Harris, Reuben S; Schiffer, Celia A

    2013-06-04

    Human APOBEC3F is an antiretroviral single-strand DNA cytosine deaminase, susceptible to degradation by the HIV-1 protein Vif. In this study the crystal structure of the HIV Vif binding, catalytically active, C-terminal domain of APOBEC3F (A3F-CTD) was determined. The A3F-CTD shares structural motifs with portions of APOBEC3G-CTD, APOBEC3C, and APOBEC2. Residues identified to be critical for Vif-dependent degradation of APOBEC3F all fit within a predominantly negatively charged contiguous region on the surface of A3F-CTD. Specific sequence motifs, previously shown to play a role in Vif susceptibility and virion encapsidation, are conserved across APOBEC3s and between APOBEC3s and HIV-1 Vif. In this structure these motifs pack against each other at intermolecular interfaces, providing potential insights both into APOBEC3 oligomerization and Vif interactions.

  20. Real-time monitoring of disintegration activity of catalytic core domain of HIV-1 integrase using molecular beacon.

    PubMed

    Zhang, Da-wei; Zhao, Ming-ming; He, Hong-qiu; Guo, Shun-xing

    2013-09-15

    HIV-1 integrase, an essential enzyme for retroviral replication, is a validated target for anti-HIV therapy development. The catalytic core domain of integrase (IN-CCD) is capable of catalyzing disintegration reaction. In this work, a hairpin-shaped disintegration substrate was designed and validated by enzyme-linked immunosorbent assay; a molecular beacon-based assay was developed for disintegration reaction of IN-CCD. Results showed that the disintegration substrate could be recognized and catalyzed by IN-CCD, and the disintegration reaction can be monitored according to the increase of fluorescent signal. The assay can be applied to real-time detection of disintegration with advantages of simplicity, high sensitivity, and excellent specificity. Copyright © 2013 Elsevier Inc. All rights reserved.

  1. Crystallization and preliminary diffraction analysis of the catalytic domain of major extracellular endoglucanase from Xanthomonas campestris pv. campestris.

    PubMed

    Rosseto, Flávio R; Puhl, Ana C; Andrade, Maxuel O; Polikarpov, Igor

    2013-02-01

    Cellulases, such as endoglucanases, exoglucanases and β-glucosidases, are important enzymes used in the process of enzymatic hydrolysis of plant biomass. The bacteria Xanthomonas campestris pv. campestris expresses a large number of hydrolases and the major endoglucanase (XccEG), a member of glycoside hydrolase family 5 (GH5), is the most strongly secreted extracellularly. In this work, the native XccEG was purified from the extracellular extract and crystallization assays were performed on its catalytic domain. A complete data set was collected on an in-house X-ray source. The crystal diffracted to 2.7 Å resolution and belonged to space group C2, with unit-cell parameters a = 174.66, b = 141.53, c = 108.00 Å, β = 110.49°. The Matthews coefficient suggests a solvent content of 70.1% and the presence of four protein subunits in the asymmetric unit.

  2. Solubility of the catalytic domains of Botulinum neurotoxin serotype E subtypes.

    PubMed

    Chen, Sheng; Barbieri, Joseph T

    2016-02-01

    The Clostridium botulinum neurotoxins (BoNTs) are the most potent protein toxins known to humans. There are seven serotypes of the BoNTs (A-G), among which serotypes A, B, E and F are known to cause natural human intoxication. To date, eleven subtypes of LC/E, termed E1∼E11, have been identified. The LCs of BoNT/E were insoluble, prohibiting studies towards understanding the mechanisms of toxin action and substrate recognition. In this work, the molecular basis of insolubility of the recombinant LCs of two representative subtypes of BoNT/E, E1(Beluga) and E3 (Alaska), was determined. Hydrophobicity profile and structural modeling predicted a C-terminal candidate region responsible for the insolubility of LC/Es. Deletion of C-terminal 19 residues of LC/E(1-400) resulted in enhanced solubility, from 2 to ∼50% for LC/EAlaska and from 16 to ∼95% for LC/EBeluga. In addition, resides 230-236 were found to contribute to a different solubility level of LC/EAlaska when compared to LC/EBeluga. Substituting residues (230)TCI(232) in LC/EAlaska to the corresponding residues of (230)KYT(232) in LC/EBeluga enhanced the solubility of LC/EAlaska to a level approaching that of LC/EBeluga. Among these LC/Es and their derivatives, LC/EBeluga 1-400 was the most soluble and stable protein. Each LC/E derivative possessed similar catalytic activity, suggesting that the C-terminal region of LC/Es contributed to protein solubility, but not catalytic activity. In conclusion, this study generated a soluble and stable recombinant LC/E and provided insight into the structural components that govern the solubility and stability of the LCs of other BoNT serotypes and Tetanus toxin. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. A Structural Study of CESA1 Catalytic Domain of Arabidopsis Cellulose Synthesis Complex: Evidence for CESA Trimers1

    PubMed Central

    Zhang, Qiu; Petridis, Loukas; Nixon, B. Tracy; Haigler, Candace H.; Kalluri, Udaya; Coates, Leighton; Smith, Jeremy C.; Meiler, Jens

    2016-01-01

    A cellulose synthesis complex with a “rosette” shape is responsible for synthesis of cellulose chains and their assembly into microfibrils within the cell walls of land plants and their charophyte algal progenitors. The number of cellulose synthase proteins in this large multisubunit transmembrane protein complex and the number of cellulose chains in a microfibril have been debated for many years. This work reports a low resolution structure of the catalytic domain of CESA1 from Arabidopsis (Arabidopsis thaliana; AtCESA1CatD) determined by small-angle scattering techniques and provides the first experimental evidence for the self-assembly of CESA into a stable trimer in solution. The catalytic domain was overexpressed in Escherichia coli, and using a two-step procedure, it was possible to isolate monomeric and trimeric forms of AtCESA1CatD. The conformation of monomeric and trimeric AtCESA1CatD proteins were studied using small-angle neutron scattering and small-angle x-ray scattering. A series of AtCESA1CatD trimer computational models were compared with the small-angle x-ray scattering trimer profile to explore the possible arrangement of the monomers in the trimers. Several candidate trimers were identified with monomers oriented such that the newly synthesized cellulose chains project toward the cell membrane. In these models, the class-specific region is found at the periphery of the complex, and the plant-conserved region forms the base of the trimer. This study strongly supports the “hexamer of trimers” model for the rosette cellulose synthesis complex that synthesizes an 18-chain cellulose microfibril as its fundamental product. PMID:26556795

  4. A Structural Study of CESA1 Catalytic Domain of Arabidopsis Cellulose Synthesis Complex: Evidence for CESA Trimers

    DOE PAGES

    Vandavasi, Venu Gopal; Putnam, Daniel K.; Zhang, Qiu; ...

    2015-11-10

    In a cellulose synthesis complex a "rosette" shape is responsible for the synthesis of cellulose chains and their assembly into microfibrils within the cell walls of land plants and their charophyte algal progenitors. The number of cellulose synthase proteins in this large multisubunit transmembrane protein complex and the number of cellulose chains in a microfibril have been debated for many years. Our work reports a low resolution structure of the catalytic domain of CESA1 from Arabidopsis (Arabidopsis thaliana; AtCESA1CatD) determined by small-angle scattering techniques and provides the first experimental evidence for the self-assembly of CESA into a stable trimer inmore » solution. The catalytic domain was overexpressed in Escherichia coli, and using a two-step procedure, it was possible to isolate monomeric and trimeric forms of AtCESA1CatD. Moreover, the conformation of monomeric and trimeric AtCESA1CatD proteins were studied using small-angle neutron scattering and small-angle x-ray scattering. A series of AtCESA1CatD trimer computational models were compared with the small-angle x-ray scattering trimer profile to explore the possible arrangement of the monomers in the trimers. Several candidate trimers were identified with monomers oriented such that the newly synthesized cellulose chains project toward the cell membrane. In these models, the class-specific region is found at the periphery of the complex, and the plant-conserved region forms the base of the trimer. Finally, this study strongly supports the "hexamer of trimers" model for the rosette cellulose synthesis complex that synthesizes an 18-chain cellulose microfibril as its fundamental product.« less

  5. A Structural Study of CESA1 Catalytic Domain of Arabidopsis Cellulose Synthesis Complex: Evidence for CESA Trimers

    SciTech Connect

    Vandavasi, Venu Gopal; Putnam, Daniel K.; Zhang, Qiu; Petridis, Loukas; Heller, William T.; Nixon, B. Tracy; Haigler, Candace H.; Kalluri, Udaya; Coates, Leighton; Langan, Paul; Smith, Jeremy C.; Meiler, Jens; O’Neill, Hugh

    2015-11-10

    In a cellulose synthesis complex a "rosette" shape is responsible for the synthesis of cellulose chains and their assembly into microfibrils within the cell walls of land plants and their charophyte algal progenitors. The number of cellulose synthase proteins in this large multisubunit transmembrane protein complex and the number of cellulose chains in a microfibril have been debated for many years. Our work reports a low resolution structure of the catalytic domain of CESA1 from Arabidopsis (Arabidopsis thaliana; AtCESA1CatD) determined by small-angle scattering techniques and provides the first experimental evidence for the self-assembly of CESA into a stable trimer in solution. The catalytic domain was overexpressed in Escherichia coli, and using a two-step procedure, it was possible to isolate monomeric and trimeric forms of AtCESA1CatD. Moreover, the conformation of monomeric and trimeric AtCESA1CatD proteins were studied using small-angle neutron scattering and small-angle x-ray scattering. A series of AtCESA1CatD trimer computational models were compared with the small-angle x-ray scattering trimer profile to explore the possible arrangement of the monomers in the trimers. Several candidate trimers were identified with monomers oriented such that the newly synthesized cellulose chains project toward the cell membrane. In these models, the class-specific region is found at the periphery of the complex, and the plant-conserved region forms the base of the trimer. Finally, this study strongly supports the "hexamer of trimers" model for the rosette cellulose synthesis complex that synthesizes an 18-chain cellulose microfibril as its fundamental product.

  6. Identification of a region that assists membrane insertion and translocation of the catalytic domain of Bordetella pertussis CyaA toxin.

    PubMed

    Karst, Johanna C; Barker, Robert; Devi, Usha; Swann, Marcus J; Davi, Marilyne; Roser, Stephen J; Ladant, Daniel; Chenal, Alexandre

    2012-03-16

    The adenylate cyclase (CyaA) toxin, one of the virulence factors secreted by Bordetella pertussis, the pathogenic bacteria responsible for whooping cough, plays a critical role in the early stages of respiratory tract colonization by this bacterium. The CyaA toxin is able to invade eukaryotic cells by translocating its N-terminal catalytic domain directly across the plasma membrane of the target cells, where, activated by endogenous calmodulin, it produces supraphysiological levels of cAMP. How the catalytic domain is transferred from the hydrophilic extracellular medium into the hydrophobic environment of the membrane and then to the cell cytoplasm remains an unsolved question. In this report, we have characterized the membrane-interacting properties of the CyaA catalytic domain. We showed that a protein covering the catalytic domain (AC384, encompassing residues 1-384 of CyaA) displayed no membrane association propensity. However, a longer polypeptide (AC489), encompassing residues 1-489 of CyaA, exhibited the intrinsic property to bind to membranes and to induce lipid bilayer destabilization. We further showed that deletion of residues 375-485 within CyaA totally abrogated the toxin's ability to increase intracellular cAMP in target cells. These results indicate that, whereas the calmodulin dependent enzymatic domain is restricted to the amino-terminal residues 1-384 of CyaA, the membrane-interacting, translocation-competent domain extends up to residue 489. This thus suggests an important role of the region adjacent to the catalytic domain of CyaA in promoting its interaction with and its translocation across the plasma membrane of target cells.

  7. Identification of a Region That Assists Membrane Insertion and Translocation of the Catalytic Domain of Bordetella pertussis CyaA Toxin*

    PubMed Central

    Karst, Johanna C.; Barker, Robert; Devi, Usha; Swann, Marcus J.; Davi, Marilyne; Roser, Stephen J.; Ladant, Daniel; Chenal, Alexandre

    2012-01-01

    The adenylate cyclase (CyaA) toxin, one of the virulence factors secreted by Bordetella pertussis, the pathogenic bacteria responsible for whooping cough, plays a critical role in the early stages of respiratory tract colonization by this bacterium. The CyaA toxin is able to invade eukaryotic cells by translocating its N-terminal catalytic domain directly across the plasma membrane of the target cells, where, activated by endogenous calmodulin, it produces supraphysiological levels of cAMP. How the catalytic domain is transferred from the hydrophilic extracellular medium into the hydrophobic environment of the membrane and then to the cell cytoplasm remains an unsolved question. In this report, we have characterized the membrane-interacting properties of the CyaA catalytic domain. We showed that a protein covering the catalytic domain (AC384, encompassing residues 1–384 of CyaA) displayed no membrane association propensity. However, a longer polypeptide (AC489), encompassing residues 1–489 of CyaA, exhibited the intrinsic property to bind to membranes and to induce lipid bilayer destabilization. We further showed that deletion of residues 375–485 within CyaA totally abrogated the toxin's ability to increase intracellular cAMP in target cells. These results indicate that, whereas the calmodulin dependent enzymatic domain is restricted to the amino-terminal residues 1–384 of CyaA, the membrane-interacting, translocation-competent domain extends up to residue 489. This thus suggests an important role of the region adjacent to the catalytic domain of CyaA in promoting its interaction with and its translocation across the plasma membrane of target cells. PMID:22241477

  8. The molecular motor F-ATP synthase is targeted by the tumoricidal protein HAMLET.

    PubMed

    Ho, James; Sielaff, Hendrik; Nadeem, Aftab; Svanborg, Catharina; Grüber, Gerhard

    2015-05-22

    HAMLET (human alpha-lactalbumin made lethal to tumor cells) interacts with multiple tumor cell compartments, affecting cell morphology, metabolism, proteasome function, chromatin structure and viability. This study investigated if these diverse effects of HAMLET might be caused, in part, by a direct effect on the ATP synthase and a resulting reduction in cellular ATP levels. A dose-dependent reduction in cellular ATP levels was detected in A549 lung carcinoma cells, and by confocal microscopy, co-localization of HAMLET with the nucleotide-binding subunits α (non-catalytic) and β (catalytic) of the energy converting F1F0 ATP synthase was detected. As shown by fluorescence correlation spectroscopy, HAMLET binds to the F1 domain of the F1F0 ATP synthase with a dissociation constant (KD) of 20.5μM. Increasing concentrations of the tumoricidal protein HAMLET added to the enzymatically active α3β3γ complex of the F-ATP synthase lowered its ATPase activity, demonstrating that HAMLET binding to the F-ATP synthase effects the catalysis of this molecular motor. Single-molecule analysis was applied to study HAMLET-α3β3γ complex interaction. Whereas the α3β3γ complex of the F-ATP synthase rotated in a counterclockwise direction with a mean rotational rate of 3.8±0.7s(-1), no rotation could be observed in the presence of bound HAMLET. Our findings suggest that direct effects of HAMLET on the F-ATP synthase may inhibit ATP-dependent cellular processes. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. Incorporation of the catalytic domain of a hammerhead ribozyme into antisense RNA enhances its inhibitory effect on the replication of human immunodeficiency virus type 1.

    PubMed Central

    Homann, M; Tzortzakaki, S; Rittner, K; Sczakiel, G; Tabler, M

    1993-01-01

    The catalytic domain of a hammerhead ribozyme was incorporated into a 413 nucleotides long antisense RNA directed against the 5'-leader/gag region of the human immunodeficiency virus type 1 (HIV-1) (pos. +222 to +634). The resulting catalytic antisense RNA was shown to cleave its target RNA in vitro specifically at physiological ion strength and temperature. We compared the antiviral effectiveness of this catalytic antisense RNA with that of the corresponding unmodified antisense RNA and with a mutated catalytic antisense RNA, which did not cleave the substrate RNA in vitro. Each of these RNAs was co-transfected into human SW480 cells together with infectious complete proviral HIV-1 DNA, followed by analysis of HIV-1 replication. The presence of the catalytically active domain resulted in 4 to 7 fold stronger inhibition of HIV-1 replication as compared to the parental antisense RNA and the inactive mutant. Kinetic and structural studies performed in vitro indicated that the ability for double strand formation was not changed in catalytic antisense RNA versus parental antisense RNA. Together, these data suggest that the ability to cleave target RNA is a crucial prerequisite for the observed increase of inhibition of the replication of HIV-1. Images PMID:8332489

  10. The multifunctional peptidylglycine alpha-amidating monooxygenase gene: exon/intron organization of catalytic, processing, and routing domains.

    PubMed

    Ouafik, L H; Stoffers, D A; Campbell, T A; Johnson, R C; Bloomquist, B T; Mains, R E; Eipper, B A

    1992-10-01

    Peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) is a multifunctional protein containing two enzymes that act sequentially to catalyze the alpha-amidation of neuroendocrine peptides. Peptidylglycine alpha-hydroxylating monooxygenase (PHM) catalyzes the first step of the reaction and is dependent on copper, ascorbate, and molecular oxygen. Peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL) catalyzes the second step of the reaction. Previous studies demonstrated that alternative splicing results in the production of bifunctional PAM proteins that are integral membrane or soluble proteins as well as soluble monofunctional PHM proteins. Rat PAM is encoded by a complex single copy gene that consists of 27 exons and encompasses more than 160 kilobases (kb) of genomic DNA. The 12 exons comprising PHM are distributed over at least 76 kb genomic DNA and range in size from 49-185 base pairs; four of the introns within the PHM domain are over 10 kb in length. Alternative splicing in the PHM region can result in a truncated, inactive PHM protein (rPAM-5), or a soluble, monofunctional PHM protein (rPAM-4) instead of a bifunctional protein. The eight exons comprising PAL are distributed over at least 19 kb genomic DNA. The exons encoding PAL range in size from 54-209 base pairs and have not been found to undergo alternative splicing. The PHM and PAL domains are separated by a single alternatively spliced exon surrounded by lengthy introns; inclusion of this exon results in the production of a form of PAM (rPAM-1) in which endoproteolytic cleavage at a paired basic site can separate the two catalytic domains. The exon following the PAL domain encodes the trans-membrane domain of PAM; alternative splicing at this site produces integral membrane or soluble PAM proteins. The COOH-terminal domain of PAM is comprised of a short exon subject to alternative splicing and a long exon encoding the final 68 amino acids present in all bifunctional PAM proteins along

  11. Temperature-accelerated molecular dynamics gives insights into globular conformations sampled in the free state of the AC catalytic domain.

    PubMed

    Selwa, Edithe; Huynh, Tru; Ciccotti, Giovanni; Maragliano, Luca; Malliavin, Thérèse E

    2014-10-01

    The catalytic domain of the adenyl cyclase (AC) toxin from Bordetella pertussis is activated by interaction with calmodulin (CaM), resulting in cAMP overproduction in the infected cell. In the X-ray crystallographic structure of the complex between AC and the C terminal lobe of CaM, the toxin displays a markedly elongated shape. As for the structure of the isolated protein, experimental results support the hypothesis that more globular conformations are sampled, but information at atomic resolution is still lacking. Here, we use temperature-accelerated molecular dynamics (TAMD) simulations to generate putative all-atom models of globular conformations sampled by CaM-free AC. As collective variables, we use centers of mass coordinates of groups of residues selected from the analysis of standard molecular dynamics (MD) simulations. Results show that TAMD allows extended conformational sampling and generates AC conformations that are more globular than in the complexed state. These structures are then refined via energy minimization and further unrestrained MD simulations to optimize inter-domain packing interactions, thus resulting in the identification of a set of hydrogen bonds present in the globular conformations.

  12. Four Inserts within the Catalytic Domain Confer Extra Stability and Activity to Hyperthermostable Pyrolysin from Pyrococcus furiosus

    PubMed Central

    Gao, Xiaowei; Zeng, Jing; Yi, Huawei; Zhang, Fang

    2016-01-01

    ABSTRACT Pyrolysin from the hyperthermophilic archaeon Pyrococcus furiosus is the prototype of the pyrolysin family of the subtilisin-like serine protease superfamily (subtilases). It contains four inserts (IS147, IS29, IS27, and IS8) of unknown function in the catalytic domain. We performed domain deletions and showed that three inserts are either essential (IS147 and IS27) or important (IS8) for efficient maturation of pyrolysin at high temperatures, whereas IS29 is dispensable. The large insert IS147 contains Ca3 and Ca4, two calcium-binding Dx[DN]xDG motifs that are conserved in many pyrolysin-like proteases. Mutagenesis revealed that the Ca3 site contributes to enzyme thermostability and the Ca4 site is necessary for pyrolysin to fold into a maturation-competent conformation. Mature insert-deletion variants were characterized and showed that IS29 and IS8 contribute to enzyme activity and stability, respectively. In the presence of NaCl, pyrolysin undergoes autocleavage at two sites: one within IS29 and the other in IS27. Disrupting the ion pairs in IS27 and IS8 induces autocleavage in the absence of salts. Interestingly, autocleavage products combine noncovalently to form an active, nicked enzyme that is resistant to SDS and urea denaturation. Additionally, a single mutation in IS29 increases resistance to salt-induced autocleavage and further increases enzyme thermostability. Our results suggest that these extra structural elements play a crucial role in adapting pyrolysin to hyperthermal environments. IMPORTANCE Pyrolysin-like proteases belong to the subtilase superfamily and are characterized by large inserts and long C-terminal extensions; however, the role of the inserts in enzyme function is unclear. Our results demonstrate that four inserts in the catalytic domain of hyperthermostable pyrolysin contribute to the folding, maturation, stability, and activity of the enzyme at high temperatures. The modification of extra structural elements in pyrolysin

  13. Configuration of the catalytic GIY-YIG domain of intron endonuclease I-TevI: coincidence of computational and molecular findings.

    PubMed

    Kowalski, J C; Belfort, M; Stapleton, M A; Holpert, M; Dansereau, J T; Pietrokovski, S; Baxter, S M; Derbyshire, V

    1999-05-15

    I-TevI is a member of the GIY-YIG family of homing endonucleases. It is folded into two structural and functional domains, an N-terminal catalytic domain and a C-terminal DNA-binding domain, separated by a flexible linker. In this study we have used genetic analyses, computational sequence analysis andNMR spectroscopy to define the configuration of theN-terminal domain and its relationship to the flexible linker. The catalytic domain is an alpha/beta structure contained within the first 92 amino acids of the 245-amino acid protein followed by an unstructured linker. Remarkably, this structured domain corresponds precisely to the GIY-YIG module defined by sequence comparisons of 57 proteins including more than 30 newly reported members of the family. Although much of the unstructured linker is not essential for activity, residues 93-116 are required, raising the possibility that this region may adopt an alternate conformation upon DNA binding. Two invariant residues of the GIY-YIG module, Arg27 and Glu75, located in alpha-helices, have properties of catalytic residues. Furthermore, the GIY-YIG sequence elements for which the module is named form part of a three-stranded antiparallel beta-sheet that is important for I-TevI structure and function.

  14. Mechanism of Diphtheria Toxin Catalytic Domain Delivery to the Eukaryotic Cell Cytosol and the Cellular Factors that Directly Participate in the Process

    PubMed Central

    Murphy, John R.

    2011-01-01

    Research on diphtheria and anthrax toxins over the past three decades has culminated in a detailed understanding of their structure function relationships (e.g., catalytic (C), transmembrane (T), and receptor binding (R) domains), as well as the identification of their eukaryotic cell surface receptor, an understanding of the molecular events leading to the receptor-mediated internalization of the toxin into an endosomal compartment, and the pH triggered conformational changes required for pore formation in the vesicle membrane. Recently, a major research effort has been focused on the development of a detailed understanding of the molecular interactions between each of these toxins and eukaryotic cell factors that play an essential role in the efficient translocation of their respective catalytic domains through the trans-endosomal vesicle membrane pore and delivery into the cell cytosol. In this review, I shall focus on recent findings that have led to a more detailed understanding of the mechanism by which the diphtheria toxin catalytic domain is delivered to the eukaryotic cell cytosol. While much work remains, it is becoming increasingly clear that the entry process is facilitated by specific interactions with a number of cellular factors in an ordered sequential fashion. In addition, since diphtheria, anthrax lethal factor and anthrax edema factor all carry multiple coatomer I complex binding motifs and COPI complex has been shown to play an essential role in entry process, it is likely that the initial steps in catalytic domain entry of these divergent toxins follow a common mechanism. PMID:22069710

  15. A non-canonical lon proteinase lacking the ATPase domain employs the ser-Lys catalytic dyad to exercise broad control over the life cycle of a double-stranded RNA virus.

    PubMed Central

    Birghan, C; Mundt, E; Gorbalenya, A E

    2000-01-01

    We have identified a region related to the protease domain of bacterial and organelle ATP-dependent Lon proteases in virus protein 4 (VP4) of infectious bursal disease virus strain P2 (IBDVP2), a two-segmented double-stranded RNA virus. Unlike canonical Lons, IBDVP2 VP4 possesses a proteinase activity though it lacks an ATPase domain. Ser652 and Lys692 of IBDVP2 VP4 are conserved across the Lon/VP4 family and are essential for catalysis. Lys692 has the properties of a general base, increasing the nucleophilicity of Ser652; a similar catalytic dyad may function in the other Lons. VP4 can cleave in trans and is responsible for the interdomain proteolytic autoprocessing of the pVP2- VP4-VP3 polyprotein encoded by RNA segment A. VP2, which is later derived from pVP2, and VP3 are major capsid proteins of birnaviruses. Results of the characterization of a range of the IBDVP2 VP4 mutants in cell cultures implicate VP4 in trans-activation of the synthesis of VP1, putative RNA-dependent RNA polymerase encoded by RNA segment B, and in cleavage rate-dependent control of process(es) crucial for the generation of the infectious virus progeny. PMID:10619850

  16. Replication factor C from the hyperthermophilic archaeon Pyrococcus abyssi does not need ATP hydrolysis for clamp-loading and contains a functionally conserved RFC PCNA-binding domain.

    PubMed

    Henneke, Ghislaine; Gueguen, Yannick; Flament, Didier; Azam, Philippe; Querellou, Joël; Dietrich, Jacques; Hübscher, Ulrich; Raffin, Jean-Paul

    2002-11-08

    The molecular organization of the replication complex in archaea is similar to that in eukaryotes. Only two proteins homologous to subunits of eukaryotic replication factor C (RFC) have been detected in Pyrococcus abyssi (Pab). The genes encoding these two proteins are arranged in tandem. We cloned these two genes and co-expressed the corresponding recombinant proteins in Escherichia coli. Two inteins present in the gene encoding the small subunit (PabRFC-small) were removed during cloning. The recombinant protein complex was purified by anion-exchange and hydroxyapatite chromatography. Also, the PabRFC-small subunit could be purified, while the large subunit (PabRFC-large) alone was completely insoluble. The highly purified PabRFC complex possessed an ATPase activity, which was not enhanced by DNA. The Pab proliferating cell nuclear antigen (PCNA) activated the PabRFC complex in a DNA-dependent manner, but the PabRFC-small ATPase activity was neither DNA-dependent nor PCNA-dependent. The PabRFC complex was able to stimulate PabPCNA-dependent DNA synthesis by the Pabfamily D heterodimeric DNA polymerase. Finally, (i) the PabRFC-large fraction cross-reacted with anti-human-RFC PCNA-binding domain antibody, corroborating the conservation of the protein sequence, (ii) the human PCNA stimulated the PabRFC complex ATPase activity in a DNA-dependent way and (iii) the PabRFC complex could load human PCNA onto primed single-stranded circular DNA, suggesting that the PCNA-binding domain of RFC has been functionally conserved during evolution. In addition, ATP hydrolysis was not required either for DNA polymerase stimulation or PCNA-loading in vitro.

  17. Insights into the binding of PARP inhibitors to the catalytic domain of human tankyrase-2

    DOE PAGES

    Qiu, Wei; Lam, Robert; Voytyuk, Oleksandr; ...

    2014-07-31

    The poly(ADP-ribose) polymerase (PARP) family represents a new class of therapeutic targets with diverse potential disease indications. PARP1 and PARP2 inhibitors have been developed for breast and ovarian tumors manifesting double-stranded DNA-repair defects, whereas tankyrase 1 and 2 (TNKS1 and TNKS2, also known as PARP5a and PARP5b, respectively) inhibitors have been developed for tumors with elevated β-catenin activity. As the clinical relevance of PARP inhibitors continues to be actively explored, there is heightened interest in the design of selective inhibitors based on the detailed structural features of how small-molecule inhibitors bind to each of the PARP family members. Here, themore » high-resolution crystal structures of the human TNKS2 PARP domain in complex with 16 various PARP inhibitors are reported, including the compounds BSI-201, AZD-2281 and ABT-888, which are currently in Phase 2 or 3 clinical trials. These structures provide insight into the inhibitor-binding modes for the tankyrase PARP domain and valuable information to guide the rational design of future tankyrase-specific inhibitors.« less

  18. Insights into the binding of PARP inhibitors to the catalytic domain of human tankyrase-2.

    PubMed

    Qiu, Wei; Lam, Robert; Voytyuk, Oleksandr; Romanov, Vladimir; Gordon, Roni; Gebremeskel, Simon; Vodsedalek, Jakub; Thompson, Christine; Beletskaya, Irina; Battaile, Kevin P; Pai, Emil F; Rottapel, Robert; Chirgadze, Nickolay Y

    2014-10-01

    The poly(ADP-ribose) polymerase (PARP) family represents a new class of therapeutic targets with diverse potential disease indications. PARP1 and PARP2 inhibitors have been developed for breast and ovarian tumors manifesting double-stranded DNA-repair defects, whereas tankyrase 1 and 2 (TNKS1 and TNKS2, also known as PARP5a and PARP5b, respectively) inhibitors have been developed for tumors with elevated β-catenin activity. As the clinical relevance of PARP inhibitors continues to be actively explored, there is heightened interest in the design of selective inhibitors based on the detailed structural features of how small-molecule inhibitors bind to each of the PARP family members. Here, the high-resolution crystal structures of the human TNKS2 PARP domain in complex with 16 various PARP inhibitors are reported, including the compounds BSI-201, AZD-2281 and ABT-888, which are currently in Phase 2 or 3 clinical trials. These structures provide insight into the inhibitor-binding modes for the tankyrase PARP domain and valuable information to guide the rational design of future tankyrase-specific inhibitors.

  19. Insights into the binding of PARP inhibitors to the catalytic domain of human tankyrase-2

    PubMed Central

    Qiu, Wei; Lam, Robert; Voytyuk, Oleksandr; Romanov, Vladimir; Gordon, Roni; Gebremeskel, Simon; Vodsedalek, Jakub; Thompson, Christine; Beletskaya, Irina; Battaile, Kevin P.; Pai, Emil F.; Rottapel, Robert; Chirgadze, Nickolay Y.

    2014-01-01

    The poly(ADP-ribose) polymerase (PARP) family represents a new class of therapeutic targets with diverse potential disease indications. PARP1 and PARP2 inhibitors have been developed for breast and ovarian tumors manifesting double-stranded DNA-repair defects, whereas tankyrase 1 and 2 (TNKS1 and TNKS2, also known as PARP5a and PARP5b, respectively) inhibitors have been developed for tumors with elevated β-catenin activity. As the clinical relevance of PARP inhibitors continues to be actively explored, there is heightened interest in the design of selective inhibitors based on the detailed structural features of how small-molecule inhibitors bind to each of the PARP family members. Here, the high-resolution crystal structures of the human TNKS2 PARP domain in complex with 16 various PARP inhibitors are reported, including the compounds BSI-201, AZD-2281 and ABT-888, which are currently in Phase 2 or 3 clinical trials. These structures provide insight into the inhibitor-binding modes for the tankyrase PARP domain and valuable information to guide the rational design of future tankyrase-specific inhibitors. PMID:25286857

  20. Insights into the binding of PARP inhibitors to the catalytic domain of human tankyrase-2

    SciTech Connect

    Qiu, Wei; Lam, Robert; Voytyuk, Oleksandr; Romanov, Vladimir; Gordon, Roni; Gebremeskel, Simon; Vodsedalek, Jakub; Thompson, Christine; Beletskaya, Irina; Battaile, Kevin P.; Pai, Emil F.; Rottapel, Robert; Chirgadze, Nickolay Y.

    2014-07-31

    The poly(ADP-ribose) polymerase (PARP) family represents a new class of therapeutic targets with diverse potential disease indications. PARP1 and PARP2 inhibitors have been developed for breast and ovarian tumors manifesting double-stranded DNA-repair defects, whereas tankyrase 1 and 2 (TNKS1 and TNKS2, also known as PARP5a and PARP5b, respectively) inhibitors have been developed for tumors with elevated β-catenin activity. As the clinical relevance of PARP inhibitors continues to be actively explored, there is heightened interest in the design of selective inhibitors based on the detailed structural features of how small-molecule inhibitors bind to each of the PARP family members. Here, the high-resolution crystal structures of the human TNKS2 PARP domain in complex with 16 various PARP inhibitors are reported, including the compounds BSI-201, AZD-2281 and ABT-888, which are currently in Phase 2 or 3 clinical trials. These structures provide insight into the inhibitor-binding modes for the tankyrase PARP domain and valuable information to guide the rational design of future tankyrase-specific inhibitors.

  1. The structure of the catalytic domain of Tannerella forsythia karilysin reveals it is a bacterial xenolog of animal matrix metalloproteinases

    PubMed Central

    Cerdà-Costa, Núria; Guevara, Tibisay; Karim, Abdulkarim Y.; Ksiazek, Miroslaw; Nguyen, Ky-Anh; Arolas, Joan L.; Potempa, Jan; Gomis-Rüth, F. Xavier

    2010-01-01

    Metallopeptidases (MPs) are among virulence factors secreted by pathogenic bacteria at the site of infection. One such pathogen is Tannerella forsythia, a member of the microbial consortium that causes peridontitis, arguably the most prevalent infective chronic inflammatory disease known to mankind. The only reported MP secreted by T. forsythia is karilysin, a 52-kDa multidomain protein comprising a central 18-kDa catalytic domain (CD), termed Kly18, flanked by domains unrelated to any known protein. We analyzed the 3D structure of Kly18 in the absence and presence of Mg2+ or Ca2+, which are required for function and stability, and found that it evidences most of the structural features characteristic of the CDs of mammalian matrix metalloproteinases (MMPs). Unexpectedly, a peptide was bound to the active-site cleft of Kly18 mimicking a left-behind cleavage product, which revealed that the specificity pocket accommodates bulky hydrophobic side chains of substrates as in mammalian MMPs. In addition, Kly18 displayed a unique Mg2+ or Ca2+ binding site and two flexible segments that could play a role in substrate binding. Phylogenetic and sequence similarity studies revealed that Kly18 is evolutionarily much closer to winged-insect and mammalian MMPs than to potential bacterial counterparts found by genomic sequencing projects. Therefore, we conclude that this first structurally-characterized non-mammalian MMP is a xenolog co-opted through horizontal gene transfer during the intimate coexistence between T. forsythia and humans or other animals, in a very rare case of gene shuffling from eukaryotes to prokaryotes. Subsequently, this protein would have evolved in a bacterial environment to give rise to full-length karilysin that is furnished with unique flanking domains that do not conform to the general multidomain architecture of animal MMPs. PMID:21166898

  2. The structure of the catalytic domain of Tannerella forsythia karilysin reveals it is a bacterial xenologue of animal matrix metalloproteinases.

    PubMed

    Cerdà-Costa, Núria; Guevara, Tibisay; Karim, Abdulkarim Y; Ksiazek, Miroslaw; Nguyen, Ky-Anh; Arolas, Joan L; Potempa, Jan; Gomis-Rüth, F Xavier

    2011-01-01

    Metallopeptidases (MPs) are among virulence factors secreted by pathogenic bacteria at the site of infection. One such pathogen is Tannerella forsythia, a member of the microbial consortium that causes peridontitis, arguably the most prevalent infective chronic inflammatory disease known to mankind. The only reported MP secreted by T. forsythia is karilysin, a 52 kDa multidomain protein comprising a central 18 kDa catalytic domain (CD), termed Kly18, flanked by domains unrelated to any known protein. We analysed the 3D structure of Kly18 in the absence and presence of Mg(2+) or Ca(2+) , which are required for function and stability, and found that it evidences most of the structural features characteristic of the CDs of mammalian matrix metalloproteinases (MMPs). Unexpectedly, a peptide was bound to the active-site cleft of Kly18 mimicking a left-behind cleavage product, which revealed that the specificity pocket accommodates bulky hydrophobic side-chains of substrates as in mammalian MMPs. In addition, Kly18 displayed a unique Mg(2+) or Ca(2+) binding site and two flexible segments that could play a role in substrate binding. Phylogenetic and sequence similarity studies revealed that Kly18 is evolutionarily much closer to winged-insect and mammalian MMPs than to potential bacterial counterparts found by genomic sequencing projects. Therefore, we conclude that this first structurally characterized non-mammalian MMP is a xenologue co-opted through horizontal gene transfer during the intimate coexistence between T. forsythia and humans or other animals, in a very rare case of gene shuffling from eukaryotes to prokaryotes. Subsequently, this protein would have evolved in a bacterial environment to give rise to full-length karilysin that is furnished with unique flanking domains that do not conform to the general multidomain architecture of animal MMPs.

  3. The mechanism of ATP-dependent RNA unwinding by DEAD box proteins.

    PubMed

    Hilbert, Manuel; Karow, Anne R; Klostermeier, Dagmar

    2009-12-01

    DEAD box proteins catalyze the ATP-dependent unwinding of double-stranded RNA (dsRNA). In addition, they facilitate protein displacement and remodeling of RNA or RNA/protein complexes. Their hallmark feature is local destabilization of RNA duplexes. Here, we summarize current data on the DEAD box protein mechanism and present a model for RNA unwinding that integrates recent data on the effect of ATP analogs and mutations on DEAD box protein activity. DEAD box proteins share a conserved helicase core with two flexibly linked RecA-like domains that contain all helicase signature motifs. Variable flanking regions contribute to substrate binding and modulate activity. In the presence of ATP and RNA, the helicase core adopts a compact, closed conformation with extensive interdomain contacts and high affinity for RNA. In the closed conformation, the RecA-like domains form a catalytic site for ATP hydrolysis and a continuous RNA binding site. A kink in the backbone of the bound RNA locally destabilizes the duplex. Rearrangement of this initial complex generates a hydrolysis- and unwinding-competent state. From this complex, the first RNA strand can dissociate. After ATP hydrolysis and phosphate release, the DEAD box protein returns to a low-affinity state for RNA. Dissociation of the second RNA strand and reopening of the cleft in the helicase core allow for further catalytic cycles.

  4. Structure of the catalytic domain of the Tannerella forsythia matrix metallopeptidase karilysin in complex with a tetrapeptidic inhibitor

    PubMed Central

    Guevara, Tibisay; Ksiazek, Miroslaw; Skottrup, Peter Durand; Cerdà-Costa, Núria; Trillo-Muyo, Sergio; de Diego, Iñaki; Riise, Erik; Potempa, Jan; Gomis-Rüth, F. Xavier

    2013-01-01

    Karilysin is the only metallopeptidase identified as a virulence factor in the odontopathogen Tannerella forsythia owing to its deleterious effect on the host immune response during bacterial infection. The very close structural and sequence-based similarity of its catalytic domain (Kly18) to matrix metallo­proteinases suggests that karilysin was acquired by horizontal gene transfer from an animal host. Previous studies by phage display identified peptides with the consensus sequence XWFPXXXGGG (single-letter amino-acid codes; X represents any residue) as karilysin inhibitors with low-micromolar binding affinities. Subsequent refinement revealed that inhibition comparable to that of longer peptides could be achieved using the tetrapeptide SWFP. To analyze its binding, the high-resolution crystal structure of the complex between Kly18 and SWFP was determined and it was found that the peptide binds to the primed side of the active-site cleft in a substrate-like manner. The catalytic zinc ion is clamped by the α-amino group and the carbonyl O atom of the serine, thus distantly mimicking the general manner of binding of hydroxamate inhibitors to metallopeptidases and contributing, together with three zinc-binding histidines from the protein scaffold, to an octahedral-minus-one metal-coordination sphere. The tryptophan side chain penetrates the deep partially water-filled specificity pocket of Kly18. Together with previous serendipitous product complexes of Kly18, the present results provide the structural determinants of inhibition of karilysin and open the field for the design of novel inhibitory strategies aimed at the treatment of human periodontal disease based on a peptidic hit molecule. PMID:23695557

  5. Influence of lysophospholipid hydrolysis by the catalytic domain of neuropathy target esterase on the fluidity of bilayer lipid membranes.

    PubMed

    Greiner, Aaron J; Richardson, Rudy J; Worden, R Mark; Ofoli, Robert Y

    2010-08-01

    Neuropathy target esterase (NTE) is an integral membrane protein localized in the endoplasmic reticulum in neurons. Irreversible inhibition of NTE by certain organophosphorus compounds produces a paralysis known as organophosphorus compound-induced delayed neuropathy. In vitro, NTE has phospholipase/lysophospholipase activity that hydrolyses exogenously added single-chain lysophospholipids in preference to dual-chain phospholipids, and NTE mutations have been associated with motor neuron disease. NTE's physiological role is not well understood, although recent studies suggest that it may control the cytotoxic accumulation of lysophospholipids in membranes. We used the NTE catalytic domain (NEST) to hydrolyze palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (p-lysoPC) to palmitic acid in bilayer membranes comprising 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and the fluorophore 1-oleoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-glycero-3-phosphocholine (NBD-PC). Translational diffusion coefficients (D(L)) in supported bilayer membranes were measured by fluorescence recovery after pattern photobleaching (FRAPP). The average D(L) for DOPC/p-lysoPC membranes without NEST was 2.44 microm(2)s(-1)+/-0.09; the D(L) for DOPC/p-lysoPC membranes containing NEST and diisopropylphosphorofluoridate, an inhibitor, was nearly identical at 2.45+/-0.08. By contrast, the D(L) for membranes comprising NEST, DOPC, and p-lysoPC was 2.28+/-0.07, significantly different from the system with inhibited NEST, due to NEST hydrolysis. Likewise, a system without NEST containing the amount of palmitic acid that would have been produced by NEST hydrolysis of p-lysoPC was identical at 2.26+/-0.06. These results indicate that NTE's catalytic activity can alter membrane fluidity. Copyright 2010 Elsevier B.V. All rights reserved.

  6. Structure of the catalytic domain of the Tannerella forsythia matrix metallopeptidase karilysin in complex with a tetrapeptidic inhibitor.

    PubMed

    Guevara, Tibisay; Ksiazek, Miroslaw; Skottrup, Peter Durand; Cerdà-Costa, Núria; Trillo-Muyo, Sergio; de Diego, Iñaki; Riise, Erik; Potempa, Jan; Gomis-Rüth, F Xavier

    2013-05-01

    Karilysin is the only metallopeptidase identified as a virulence factor in the odontopathogen Tannerella forsythia owing to its deleterious effect on the host immune response during bacterial infection. The very close structural and sequence-based similarity of its catalytic domain (Kly18) to matrix metalloproteinases suggests that karilysin was acquired by horizontal gene transfer from an animal host. Previous studies by phage display identified peptides with the consensus sequence XWFPXXXGGG (single-letter amino-acid codes; X represents any residue) as karilysin inhibitors with low-micromolar binding affinities. Subsequent refinement revealed that inhibition comparable to that of longer peptides could be achieved using the tetrapeptide SWFP. To analyze its binding, the high-resolution crystal structure of the complex between Kly18 and SWFP was determined and it was found that the peptide binds to the primed side of the active-site cleft in a substrate-like manner. The catalytic zinc ion is clamped by the α-amino group and the carbonyl O atom of the serine, thus distantly mimicking the general manner of binding of hydroxamate inhibitors to metallopeptidases and contributing, together with three zinc-binding histidines from the protein scaffold, to an octahedral-minus-one metal-coordination sphere. The tryptophan side chain penetrates the deep partially water-filled specificity pocket of Kly18. Together with previous serendipitous product complexes of Kly18, the present results provide the structural determinants of inhibition of karilysin and open the field for the design of novel inhibitory strategies aimed at the treatment of human periodontal disease based on a peptidic hit molecule.

  7. The C Terminus of the Catalytic Domain of Type A Botulinum Neurotoxin May Facilitate Product Release from the Active Site*

    PubMed Central

    Mizanur, Rahman M.; Frasca, Verna; Swaminathan, Subramanyam; Bavari, Sina; Webb, Robert; Smith, Leonard A.; Ahmed, S. Ashraf

    2013-01-01

    Botulinum neurotoxins are the most toxic of all compounds. The toxicity is related to a poor zinc endopeptidase activity located in a 50-kDa domain known as light chain (Lc) of the toxin. The C-terminal tail of Lc is not visible in any of the currently available x-ray structures, and it has no known function but undergoes autocatalytic truncations during purification and storage. By synthesizing C-terminal peptides of various lengths, in this study, we have shown that these peptides competitively inhibit the normal catalytic activity of Lc of serotype A (LcA) and have defined the length of the mature LcA to consist of the first 444 residues. Two catalytically inactive mutants also inhibited LcA activity. Our results suggested that the C terminus of LcA might interact at or near its own active site. By using synthetic C-terminal peptides from LcB, LcC1, LcD, LcE, and LcF and their respective substrate peptides, we have shown that the inhibition of activity is specific only for LcA. Although a potent inhibitor with a Ki of 4.5 μm, the largest of our LcA C-terminal peptides stimulated LcA activity when added at near-stoichiometric concentration to three versions of LcA differing in their C-terminal lengths. The result suggested a product removal role of the LcA C terminus. This suggestion is supported by a weak but specific interaction determined by isothermal titration calorimetry between an LcA C-terminal peptide and N-terminal product from a peptide substrate of LcA. Our results also underscore the importance of using a mature LcA as an inhibitor screening target. PMID:23779108

  8. Elucidation of eukaryotic elongation factor-2 contact sites within the catalytic domain of Pseudomonas aeruginosa exotoxin A.

    PubMed Central

    Yates, Susan P; Merrill, Allan R

    2004-01-01

    Pseudomonas aeruginosa produces the virulence factor, ETA (exotoxin A), which catalyses an ADP-ribosyltransferase reaction of its target protein, eEF2 (eukaryotic elongation factor-2). Currently, this protein-protein interaction is poorly characterized and this study was aimed at identifying the contact sites between eEF2 and the catalytic domain of ETA (PE24H, an ETA from P. aeruginosa, a 24 kDa C-terminal fragment containing a His6 tag). Single-cysteine residues were introduced into the toxin at 21 defined surface-exposed sites and labelled with the fluorophore, IAEDANS [5-(2-iodoacetylaminoethylamino)-1-napthalenesulphonic acid]. Fluorescence quenching studies using acrylamide, and fluorescence lifetime and wavelength emission maxima analyses were conducted in the presence and absence of eEF2. Large changes in the microenvironment of the AEDANS [5-(2-aminoethylamino)-1-naphthalenesulphonic acid] probe after eEF2 binding were not observed as dictated by both fluorescence lifetime and wavelength emission maxima values. This supported the proposed minimal contact model, which suggests that only small, discrete contacts occur between these proteins. As dictated by the bimolecular quenching constant (k(q)) for acrylamide, binding of eEF2 with toxin caused the greatest change in acrylamide accessibility (>50%) when the fluorescence label was near the active site or was located within a known catalytic loop. All mutant proteins showed a decrease in accessibility to acrylamide once eEF2 bound, although the relative change varied for each labelled protein. From these data, a low-resolution model of the toxin-eEF2 complex was constructed based on the minimal contact model with the intention of enhancing our knowledge on the mode of inactivation of the ribosome translocase by the Pseudomonas toxin. PMID:14733615

  9. Reversible transport by the ATP-binding cassette multidrug export pump LmrA: ATP synthesis at the expense of downhill ethidium uptake.

    PubMed

    Balakrishnan, Lekshmy; Venter, Henrietta; Shilling, Richard A; van Veen, Hendrik W

    2004-03-19

    The ATP dependence of ATP-binding cassette (ABC) transporters has led to the widespread acceptance that these systems are unidirectional. Interestingly, in the presence of an inwardly directed ethidium concentration gradient in ATP-depleted cells of Lactococcus lactis, the ABC multidrug transporter LmrA mediated the reverse transport (or uptake) of ethidium with an apparent K(t) of 2.0 microm. This uptake reaction was competitively inhibited by the LmrA substrate vinblastine and was significantly reduced by an E314A substitution in the membrane domain of the transporter. Similar to efflux, LmrA-mediated ethidium uptake was inhibited by the E512Q replacement in the Walker B region of the nucleotide-binding domain of the protein, which strongly reduced its drug-stimulated ATPase activity, consistent with published observations for other ABC transporters. The notion that ethidium uptake is coupled to the catalytic cycle in LmrA was further corroborated by studies in LmrA-containing cells and proteoliposomes in which reverse transport of ethidium was associated with the net synthesis of ATP. Taken together, these data demonstrate that the conformational changes required for drug transport by LmrA are (i) not too far from equilibrium under ATP-depleted conditions to be reversed by appropriate changes in ligand concentrations and (ii) not necessarily coupled to ATP hydrolysis, but associated with a reversible catalytic cycle. These findings and their thermodynamic implications shed new light on the mechanism of energy coupling in ABC transporters and have implications for the development of new modulators that could enable reverse transport-associated drug delivery in cells through their ability to uncouple ATP binding/hydrolysis from multidrug efflux.

  10. Structure of the C-Terminal Half of UvrC Reveals an RNase H Endonuclease Domain with an Argonaute-like Catalytic Triad

    SciTech Connect

    Karakas,E.; Truglio, J.; Croteau, D.; Rhau, B.; Wang, L.; Van Houten, B.; Kisker, C.

    2007-01-01

    Removal and repair of DNA damage by the nucleotide excision repair pathway requires two sequential incision reactions, which are achieved by the endonuclease UvrC in eubacteria. Here, we describe the crystal structure of the C-terminal half of UvrC, which contains the catalytic domain responsible for 5' incision and a helix-hairpin-helix-domain that is implicated in DNA binding. Surprisingly, the 5' catalytic domain shares structural homology with RNase H despite the lack of sequence homology and contains an uncommon DDH triad. The structure also reveals two highly conserved patches on the surface of the protein, which are not related to the active site. Mutations of residues in one of these patches led to the inability of the enzyme to bind DNA and severely compromised both incision reactions. Based on our results, we suggest a model of how UvrC forms a productive protein-DNA complex to excise the damage from DNA.

  11. Expression, purification, and structural analysis of the trimeric form of the catalytic domain of the Escherichia coli dihydrolipoamide succinyltransferase.

    PubMed Central

    Knapp, J. E.; Carroll, D.; Lawson, J. E.; Ernst, S. R.; Reed, L. J.; Hackert, M. L.

    2000-01-01

    The dihydrolipoamide succinyltransferase (E2o) component of the alpha-ketoglutarate dehydrogenase complex catalyzes the transfer of a succinyl group from the S-succinyldihydrolipoyl moiety to coenzyme A. E2o is normally a 24-mer, but is found as a trimer when E2o is expressed with a C-terminal [His]6 tag. The crystal structure of the trimeric form of the catalytic domain (CD) of the Escherichia coli E2o has been solved to 3.0 A resolution using the Molecular Replacement method. The refined model contains an intact trimer in the asymmetric unit and has an R-factor of 0.257 (Rfree = 0.286) for 18,699 reflections between 10.0 and 3.0 A resolution. The core of tE2oCD (residues 187-396) superimposes onto that of the cubic E2oCD with an RMS difference of 0.4 A for all main-chain atoms. The C-terminal end of tE2oCD (residues 397-404) rotates by an average of 37 degrees compared to cubic E2oCD, disrupting the normal twofold interface. Despite the alteration of quaternary structure, the active site of tE2oCD shows no significant differences from that of the cubic E2oCD, although several side chains in the active site are more ordered in the trimeric form of E2oCD. Analysis of the available sequence data suggests that the majority of E2 components have active sites that resemble that of E. coli E2oCD. The remaining E2 components can be divided into three groups based on active-site sequence similarity. Analysis of the surface properties of both crystal forms of E. coli E2oCD suggests key residues that may be involved in the protein-protein contacts that occur between the catalytic and lipoyl domains of E2o. PMID:10739245

  12. Crystal structure of the anti-viral APOBEC3G catalytic domain and functional implications

    SciTech Connect

    Holden, Lauren G.; Prochnow, Courtney; Chang, Y. Paul; Bransteitter, Ronda; Chelico, Linda; Sen, Udayaditya; Stevens, Raymond C.; Goodman, Myron F.; Chen, Xiaojiang S.

    2009-04-07

    The APOBEC family members are involved in diverse biological functions. APOBEC3G restricts the replication of human immunodeficiency virus (HIV), hepatitis B virus and retroelements by cytidine deamination on single-stranded DNA or by RNA binding. Here we report the high-resolution crystal structure of the carboxy-terminal deaminase domain of APOBEC3G (APOBEC3G-CD2) purified from Escherichia coli. The APOBEC3G-CD2 structure has a five-stranded {beta}-sheet core that is common to all known deaminase structures and closely resembles the structure of another APOBEC protein, APOBEC2. A comparison of APOBEC3G-CD2 with other deaminase structures shows a structural conservation of the active-site loops that are directly involved in substrate binding. In the X-ray structure, these APOBEC3G active-site loops form a continuous 'substrate groove' around the active centre. The orientation of this putative substrate groove differs markedly (by 90 degrees) from the groove predicted by the NMR structure. We have introduced mutations around the groove, and have identified residues involved in substrate specificity, single-stranded DNA binding and deaminase activity. These results provide a basis for understanding the underlying mechanisms of substrate specificity for the APOBEC family.

  13. Intracellular catalytic domain of symbiosis receptor kinase hyperactivates spontaneous nodulation in absence of rhizobia.

    PubMed

    Saha, Sudip; Dutta, Ayan; Bhattacharya, Avisek; DasGupta, Maitrayee

    2014-12-01

    Symbiosis Receptor Kinase (SYMRK), a member of the Nod factor signaling pathway, is indispensible for both nodule organogenesis and intracellular colonization of symbionts in rhizobia-legume symbiosis. Here, we show that the intracellular kinase domain of a SYMRK (SYMRK-kd) but not its inactive or full-length version leads to hyperactivation of the nodule organogenic program in Medicago truncatula TR25 (symrk knockout mutant) in the absence of rhizobia. Spontaneous nodulation in TR25/SYMRK-kd was 6-fold higher than rhizobia-induced nodulation in TR25/SYMRK roots. The merged clusters of spontaneous nodules indicated that TR25 roots in the presence of SYMRK-kd have overcome the control over both nodule numbers and their spatial position. In the presence of rhizobia, SYMRK-kd could rescue the epidermal infection processes in TR25, but colonization of symbionts in the nodule interior was significantly compromised. In summary, ligand-independent deregulated activation of SYMRK hyperactivates nodule organogenesis in the absence of rhizobia, but its ectodomain is required for proper symbiont colonization.

  14. Crystal structure of the catalytic domain of UCHL5, a proteasome-associated human deubiquitinating enzyme, reveals an unproductive form of the enzyme

    SciTech Connect

    Maiti, Tushar K.; Permaul, Michelle; Boudreaux, David A.; Mahanic, Christina; Mauney, Sarah; Das, Chittaranjan

    2012-10-25

    Ubiquitin carboxy-terminal hydrolase L5 (UCHL5) is a proteasome-associated deubiquitinating enzyme, which, along with RPN11 and USP14, is known to carry out deubiquitination on proteasome. As a member of the ubiquitin carboxy-terminal hydrolase (UCH) family, UCHL5 is unusual because, unlike UCHL1 and UCHL3, it can process polyubiquitin chain. However, it does so only when it is bound to the proteasome; in its free form, it is capable of releasing only relatively small leaving groups from the C-terminus of ubiquitin. Such a behavior might suggest at least two catalytically distinct forms of the enzyme, an apo form incapable of chain processing activity, and a proteasome-induced activated form capable of cleaving polyubiquitin chain. Through the crystal structure analysis of two truncated constructs representing the catalytic domain (UCH domain) of this enzyme, we were able to visualize a state of this enzyme that we interpret as its inactive form, because the catalytic cysteine appears to be in an unproductive orientation. While this work was in progress, the structure of a different construct representing the UCH domain was reported; however, in that work the structure reported was that of an inactive mutant [catalytic Cys to Ala; Nishio K et al. (2009) Biochem Biophys Res Commun390, 855-860], which precluded the observation that we are reporting here. Additionally, our structures reveal conformationally dynamic parts of the enzyme that may play a role in the structural transition to the more active form.

  15. Computer modelling reveals new conformers of the ATP binding loop of Na(+)/K(+)-ATPase involved in the transphosphorylation process of the sodium pump.

    PubMed

    Tejral, Gracian; Sopko, Bruno; Necas, Alois; Schoner, Wilhelm; Amler, Evzen

    2017-01-01

    Hydrolysis of ATP by Na(+)/K(+)-ATPase, a P-Type ATPase, catalyzing active Na(+) and K(+) transport through cellular membranes leads transiently to a phosphorylation of its catalytical α-subunit. Surprisingly, three-dimensional molecular structure analysis of P-type ATPases reveals that binding of ATP to the N-domain connected by a hinge to the P-domain is much too far away from the Asp(369) to allow the transfer of ATP's terminal phosphate to its aspartyl-phosphorylation site. In order to get information for how the transfer of the γ-phosphate group of ATP to the Asp(369) is achieved, analogous molecular modeling of the M4-M5 loop of ATPase was performed using the crystal data of Na(+)/K(+)-ATPase of different species. Analogous molecular modeling of the cytoplasmic loop between Thr(338) and Ile(760) of the α2-subunit of Na(+)/K(+)-ATPase and the analysis of distances between the ATP binding site and phosphorylation site revealed the existence of two ATP binding sites in the open conformation; the first one close to Phe(475) in the N-domain, the other one close to Asp(369) in the P-domain. However, binding of Mg(2+)•ATP to any of these sites in the "open conformation" may not lead to phosphorylation of Asp(369). Additional conformations of the cytoplasmic loop were found wobbling between "open conformation" <==> "semi-open conformation <==> "closed conformation" in the absence of 2Mg(2+)•ATP. The cytoplasmic loop's conformational change to the "semi-open conformation"-characterized by a hydrogen bond between Arg(543) and Asp(611)-triggers by binding of 2Mg(2+)•ATP to a single ATP site and conversion to the "closed conformation" the phosphorylation of Asp(369) in the P-domain, and hence the start of Na(+)/K(+)-activated ATP hydrolysis.

  16. Crystal structures of wild-type Trichoderma reesei Cel7A catalytic domain in open and closed states

    SciTech Connect

    Bodenheimer, Annette M.; Meilleur, Flora

    2016-11-07

    Trichoderma reesei Cel7A efficiently hydrolyses cellulose. We report here the crystallographic structures of the wild-type TrCel7A catalytic domain (CD) in an open state and, for the first time, in a closed state. Molecular dynamics (MD) simulations indicate that the loops along the CD tunnel move in concerted motions. Together, the crystallographic and MD data suggest that the CD cycles between the tense and relaxed forms that are characteristic of work producing enzymes. Analysis of the interactions formed by R251 provides a structural rationale for the concurrent decrease in product inhibition and catalytic efficiency measured for product-binding site mutants.

  17. Purification and catalytic activities of the two domains of the allene oxide synthase-lipoxygenase fusion protein of the coral Plexaura homomalla.

    PubMed

    Boutaud, O; Brash, A R

    1999-11-19

    The conversion of fatty acid hydroperoxides to allene epoxides is catalyzed by a cytochrome P450 in plants and, in coral, by a 43-kDa catalase-related hemoprotein fused to the lipoxygenase that synthesizes the 8R-hydroperoxyeicosatetraenoic acid (8R-HPETE) substrate. We have expressed the separate lipoxygenase and allene oxide synthase (AOS) domains of the coral protein in Escherichia coli (BL21 cells) and purified the proteins; this system gives high expression (1.5 and 0.3 micromol/liter, respectively) of catalytically active enzymes. Both domains show fast reaction kinetics. Catalytic activity of the lipoxygenase domain is stimulated 5-fold by high concentrations of monovalent cations (500 mM Na(+), Li(+), or K(+)), and an additional 5-fold by 10 mM Ca(2+). The resulting rates of reaction are approximately 300 turnovers/s, 1-2 orders of magnitude faster than mammalian lipoxygenases. This makes the coral lipoxygenase well suited for partnership with the AOS domain, which shows maximum rates of approximately 1400 turnovers/s in the conversion of 8R-HPETE to the allene oxide. Some unusual catalytic activities of the two domains are described. The lipoxygenase domain converts 20.3omega6 partly to the bis-allylic hydroperoxide (10-hydroperoxyeicosa-8,11,14-trienoic acid). Metabolism of the preferred substrate of the AOS domain, 8R-HPETE, is inhibited by the enantiomer 8S-HPETE. Although the AOS domain has homology to catalase in primary structure, it is completely lacking in catalatic action on H(2)O(2); catalase itself, as expected from its preference for small hydroperoxides, is ineffective in allene oxide synthesis from 8R-HPETE.

  18. Structure of the Catalytic Domain of EZH2 Reveals Conformational Plasticity in Cofactor and Substrate Binding Sites and Explains Oncogenic Mutations

    PubMed Central

    Wu, Hong; Zeng, Hong; Dong, Aiping; Li, Fengling; He, Hao; Senisterra, Guillermo; Seitova, Alma; Duan, Shili; Brown, Peter J.; Vedadi, Masoud; Arrowsmith, Cheryl H.; Schapira, Matthieu

    2013-01-01

    Polycomb repressive complex 2 (PRC2) is an important regulator of cellular differentiation and cell type identity. Overexpression or activating mutations of EZH2, the catalytic component of the PRC2 complex, are linked to hyper-trimethylation of lysine 27 of histone H3 (H3K27me3) in many cancers. Potent EZH2 inhibitors that reduce levels of H3K27me3 kill mutant lymphoma cells and are efficacious in a mouse xenograft model of malignant rhabdoid tumors. Unlike most SET domain methyltransferases, EZH2 requires PRC2 components, SUZ12 and EED, for activity, but the mechanism by which catalysis is promoted in the PRC2 complex is unknown. We solved the 2.0 Å crystal structure of the EZH2 methyltransferase domain revealing that most of the canonical structural features of SET domain methyltransferase structures are conserved. The site of methyl transfer is in a catalytically competent state, and the structure clarifies the structural mechanism underlying oncogenic hyper-trimethylation of H3K27 in tumors harboring mutations at Y641 or A677. On the other hand, the I-SET and post-SET domains occupy atypical positions relative to the core SET domain resulting in incomplete formation of the cofactor binding site and occlusion of the substrate binding groove. A novel CXC domain N-terminal to the SET domain may contribute to the apparent inactive conformation. We propose that protein interactions within the PRC2 complex modulate the trajectory of the post-SET and I-SET domains of EZH2 in favor of a catalytically competent conformation. PMID:24367611

  19. Functional analysis of TMLH variants and definition of domains required for catalytic activity and mitochondrial targeting.

    PubMed

    Monfregola, Jlenia; Cevenini, Armando; Terracciano, Antonio; van Vlies, Naomi; Arbucci, Salvatore; Wanders, Ronald J A; D'Urso, Michele; Vaz, Frédéric M; Ursini, Matilde Valeria

    2005-09-01

    epsilon-N-Trimethyllysine hydroxylase (TMLH) (EC 1.14.11.8) is a non-heme-ferrous iron hydroxylase, Fe(++) and 2-oxoglutarate (2OG) dependent, catalyzing the first of four enzymatic reactions of the highly conserved carnitine biosynthetic pathway. Otherwise from all the other enzymes of carnitine biosynthesis, TMLH was found to be associated to the mitochondrial fraction. We here report molecular cloning of two alternative spliced forms of TMLH, which appear ubiquitously expressed in human adult and fetal tissues. The deduced proteins are designated TMLH-a and TMLH-b, and contain 421 and 399 amino acids, respectively. They share the first N-terminal 332 amino acids, including a mitochondrial targeting signal, but diverge at the C-terminal end. TMLH-a and TMLH-b exogenous expression in COS-1 cells shows that the first 15 amino acids are necessary and sufficient for mitochondrial import. Furthermore, comparative evolutionary analysis of the C-terminal portion of TMLH-a identifies a conserved domain characterized by a key triad of residues, His242-Glu244-His389 predicted to bind 2OG end. This sequence is conserved in the TMLH enzyme from all species but is partially substituted by a unique sequence in the TMLH-b variant. Indeed, TMLH-b is not functional by itself as well as a TMLH-H389L mutant produced by site directed mutagenesis. As great interest, we found that TMLH-b and TMLH-H389L, individually co-expressed with TMLH-a in COS-1 cells, negatively affect TMLH activity. Therefore, our studies on the TMLH alternative form provide relevant novel information, first that the C-terminal region of TMLH contains the main determinants for its enzymatic activity including a key H389 residue, and second that TMLH-b could act as a crucial physiological negative regulator of TMLH. Copyright 2005 Wiley-Liss, Inc.

  20. Polycomb inhibits histone acetylation by CBP by binding directly to its catalytic domain

    PubMed Central

    Tie, Feng; Banerjee, Rakhee; Fu, Chen; Stratton, Carl A.; Fang, Ming; Harte, Peter J.

    2016-01-01

    Drosophila Polycomb (PC), a subunit of Polycomb repressive complex 1 (PRC1), is well known for its role in maintaining repression of the homeotic genes and many others and for its binding to trimethylated histone H3 on Lys 27 (H3K27me3) via its chromodomain. Here, we identify a novel activity of PC: inhibition of the histone acetylation activity of CREB-binding protein (CBP). We show that PC and its mammalian CBX orthologs interact directly with the histone acetyltransferase (HAT) domain of CBP, binding to the previously identified autoregulatory loop, whose autoacetylation greatly enhances HAT activity. We identify a conserved PC motif adjacent to the chromodomain required for CBP binding and show that PC binding inhibits acetylation of histone H3. CBP autoacetylation impairs PC binding in vitro, and PC is preferentially associated with unacetylated CBP in vivo. PC knockdown elevates the acetylated H3K27 (H3K27ac) level globally and at promoter regions of some genes that are bound by both PC and CBP. Conversely, PC overexpression decreases the H3K27ac level in vivo and also suppresses CBP-dependent Polycomb phenotypes caused by overexpression of Trithorax, an antagonist of Polycomb silencing. We find that PC is physically associated with the initiating form of RNA polymerase II (Pol II) and that many promoters co-occupied by PC and CBP are associated with paused Pol II, suggesting that PC may play a role in Pol II pausing. These results suggest that PC/PRC1 inhibition of CBP HAT activity plays a role in regulating transcription of both repressed and active PC-regulated genes. PMID:26802126

  1. Rescue of HIV-1 Release by Targeting Widely Divergent NEDD4-Type Ubiquitin Ligases and Isolated Catalytic HECT Domains to Gag

    PubMed Central

    Weiss, Eric R.; Popova, Elena; Yamanaka, Hikaru; Kim, Hyung Cheol; Huibregtse, Jon M.; Göttlinger, Heinrich

    2010-01-01

    Retroviruses engage the ESCRT pathway through late assembly (L) domains in Gag to promote virus release. HIV-1 uses a PTAP motif as its primary L domain, which interacts with the ESCRT-I component Tsg101. In contrast, certain other retroviruses primarily use PPxY-type L domains, which constitute ligands for NEDD4-type ubiquitin ligases. Surprisingly, although HIV-1 Gag lacks PPxY motifs, the release of HIV-1 L domain mutants is potently enhanced by ectopic NEDD4-2s, a native isoform with a naturally truncated C2 domain that appears to account for the residual titer of L domain-defective HIV-1. The reason for the unique potency of the NEDD4-2s isoform has remained unclear. We now show that the naturally truncated C2 domain of NEDD4-2s functions as an autonomous Gag-targeting module that can be functionally replaced by the unrelated Gag-binding protein cyclophilin A (CypA). The residual C2 domain of NEDD4-2s was sufficient to transfer the ability to stimulate HIV-1 budding to other NEDD4 family members, including the yeast homologue Rsp5, and even to isolated catalytic HECT domains. The isolated catalytic domain of NEDD4-2s also efficiently promoted HIV-1 budding when targeted to Gag via CypA. We conclude that the regions typically required for substrate recognition by HECT ubiquitin ligases are all dispensable to stimulate HIV-1 release, implying that the relevant target for ubiquitination is Gag itself or can be recognized by divergent isolated HECT domains. However, the mere ability to ubiquitinate Gag was not sufficient to stimulate HIV-1 budding. Rather, our results indicate that the synthesis of K63-linked ubiquitin chains is critical for ubiquitin ligase-mediated virus release. PMID:20862313

  2. Rescue of HIV-1 release by targeting widely divergent NEDD4-type ubiquitin ligases and isolated catalytic HECT domains to Gag.

    PubMed

    Weiss, Eric R; Popova, Elena; Yamanaka, Hikaru; Kim, Hyung Cheol; Huibregtse, Jon M; Göttlinger, Heinrich

    2010-09-16

    Retroviruses engage the ESCRT pathway through late assembly (L) domains in Gag to promote virus release. HIV-1 uses a PTAP motif as its primary L domain, which interacts with the ESCRT-I component Tsg101. In contrast, certain other retroviruses primarily use PPxY-type L domains, which constitute ligands for NEDD4-type ubiquitin ligases. Surprisingly, although HIV-1 Gag lacks PPxY motifs, the release of HIV-1 L domain mutants is potently enhanced by ectopic NEDD4-2s, a native isoform with a naturally truncated C2 domain that appears to account for the residual titer of L domain-defective HIV-1. The reason for the unique potency of the NEDD4-2s isoform has remained unclear. We now show that the naturally truncated C2 domain of NEDD4-2s functions as an autonomous Gag-targeting module that can be functionally replaced by the unrelated Gag-binding protein cyclophilin A (CypA). The residual C2 domain of NEDD4-2s was sufficient to transfer the ability to stimulate HIV-1 budding to other NEDD4 family members, including the yeast homologue Rsp5, and even to isolated catalytic HECT domains. The isolated catalytic domain of NEDD4-2s also efficiently promoted HIV-1 budding when targeted to Gag via CypA. We conclude that the regions typically required for substrate recognition by HECT ubiquitin ligases are all dispensable to stimulate HIV-1 release, implying that the relevant target for ubiquitination is Gag itself or can be recognized by divergent isolated HECT domains. However, the mere ability to ubiquitinate Gag was not sufficient to stimulate HIV-1 budding. Rather, our results indicate that the synthesis of K63-linked ubiquitin chains is critical for ubiquitin ligase-mediated virus release.

  3. Structure-Guided Systems-Level Engineering of Oxidation-Prone Methionine Residues in Catalytic Domain of an Alkaline α-Amylase from Alkalimonas amylolytica for Significant Improvement of Both Oxidative Stability and Catalytic Efficiency

    PubMed Central

    Yang, Haiquan; Liu, Long; Shin, Hyun-dong; Li, Jianghua; Du, Guocheng; Chen, Jian

    2013-01-01

    High oxidative stability and catalytic efficiency are required for the alkaline α-amylases to keep the enzymatic performance under the harsh conditions in detergent industries. In this work, we attempted to significantly improve both the oxidative stability and catalytic efficiency of an alkaline α-amylase from Alkalimonas amylolytica by engineering the five oxidation-prone methionine residues around the catalytic domain via a systematic approach. Specifically, based on the tertiary structure analysis, five methionines (Met 145, Met 214, Met 229, Met 247 and Met 317) were individually substituted with oxidation-resistant threonine, isoleucine and alaline, respectively. Among the created 15 mutants, 7 mutants M145A, M145I, M214A, M229A, M229T, M247T and M317I showed significantly enhanced oxidative stability or catalytic efficiency. In previous work, we found that the replacement of M247 with leucine could significantly improve the oxidative stability. Thus, these 8 positive mutants (M145A, M145I, M214A, M229A, M229T, M247T, M247L and M317I) were used to conduct the second round of combinational mutations. Among the constructed 85 mutants (25 two-point mutants, 36 three-point mutants, 16 four-point mutants and 8 five-point mutants), the mutant M145I-214A-229T-247T-317I showed a 5.4-fold increase in oxidative stability and a 3.0-fold increase in catalytic efficiency. Interestingly, the specific activity, alkaline stability and thermal stability of this mutant were also increased. The increase of salt bridge and hydrogen bonds around the catalytic domain contributed to the significantly improved catalytic efficiency and stability, as revealed by the three-dimensional structure model of wild-type alkaline α-amylase and its mutant M145I-214A-229T-247T-317I. With the significantly improved oxidative stability and catalytic efficiency, the mutant M145I-214A-229T-247T-317I has a great potential as a detergent additive, and this structure-guided systems engineering

  4. Structure-guided systems-level engineering of oxidation-prone methionine residues in catalytic domain of an alkaline α-amylase from Alkalimonas amylolytica for significant improvement of both oxidative stability and catalytic efficiency.

    PubMed

    Yang, Haiquan; Liu, Long; Shin, Hyun-dong; Li, Jianghua; Du, Guocheng; Chen, Jian

    2013-01-01

    High oxidative stability and catalytic efficiency are required for the alkaline α-amylases to keep the enzymatic performance under the harsh conditions in detergent industries. In this work, we attempted to significantly improve both the oxidative stability and catalytic efficiency of an alkaline α-amylase from Alkalimonas amylolytica by engineering the five oxidation-prone methionine residues around the catalytic domain via a systematic approach. Specifically, based on the tertiary structure analysis, five methionines (Met 145, Met 214, Met 229, Met 247 and Met 317) were individually substituted with oxidation-resistant threonine, isoleucine and alaline, respectively. Among the created 15 mutants, 7 mutants M145A, M145I, M214A, M229A, M229T, M247T and M317I showed significantly enhanced oxidative stability or catalytic efficiency. In previous work, we found that the replacement of M247 with leucine could significantly improve the oxidative stability. Thus, these 8 positive mutants (M145A, M145I, M214A, M229A, M229T, M247T, M247L and M317I) were used to conduct the second round of combinational mutations. Among the constructed 85 mutants (25 two-point mutants, 36 three-point mutants, 16 four-point mutants and 8 five-point mutants), the mutant M145I-214A-229T-247T-317I showed a 5.4-fold increase in oxidative stability and a 3.0-fold increase in catalytic efficiency. Interestingly, the specific activity, alkaline stability and thermal stability of this mutant were also increased. The increase of salt bridge and hydrogen bonds around the catalytic domain contributed to the significantly improved catalytic efficiency and stability, as revealed by the three-dimensional structure model of wild-type alkaline α-amylase and its mutant M145I-214A-229T-247T-317I. With the significantly improved oxidative stability and catalytic efficiency, the mutant M145I-214A-229T-247T-317I has a great potential as a detergent additive, and this structure-guided systems engineering

  5. Prediction of a common beta-propeller catalytic domain for fructosyltransferases of different origin and substrate specificity.

    PubMed Central

    Pons, T.; Hernández, L.; Batista, F. R.; Chinea, G.

    2000-01-01

    The three-dimensional (3D) structure of fructan biosynthetic enzymes is still unknown. Here, we have explored folding similarities between reported microbial and plant enzymes that catalyze transfructosylation reactions. A sequence-structure compatibility search using TOPITS, SDP, 3D-PSSM, and SAM-T98 programs identified a beta-propeller fold with scores above the confidence threshold that indicate a structurally conserved catalytic domain in fructosyltransferases (FTFs) of diverse origin and substrate specificity. The predicted fold appeared related to that of neuraminidase and sialidase, of glycoside hydrolase families 33 and 34, respectively. The most reliable structural model was obtained using the crystal structure of neuraminidase (Protein Data Bank file: 5nn9) as template, and it is consistent with the location of previously identified functional residues of bacterial levansucrases (Batista et al., 1999; Song & Jacques, 1999). The sequence-sequence analysis presented here reinforces the recent inclusion of fungal and plant FTFs into glycoside hydrolase family 32, and suggests a modified sequence pattern H-x (2)-[PTV]-x (4)-[LIVMA]-[NSCAYG]-[DE]-P-[NDSC][GA]3 for this family. PMID:11305239

  6. Prediction of a common beta-propeller catalytic domain for fructosyltransferases of different origin and substrate specificity.

    PubMed

    Pons, T; Hernández, L; Batista, F R; Chinea, G

    2000-11-01

    The three-dimensional (3D) structure of fructan biosynthetic enzymes is still unknown. Here, we have explored folding similarities between reported microbial and plant enzymes that catalyze transfructosylation reactions. A sequence-structure compatibility search using TOPITS, SDP, 3D-PSSM, and SAM-T98 programs identified a beta-propeller fold with scores above the confidence threshold that indicate a structurally conserved catalytic domain in fructosyltransferases (FTFs) of diverse origin and substrate specificity. The predicted fold appeared related to that of neuraminidase and sialidase, of glycoside hydrolase families 33 and 34, respectively. The most reliable structural model was obtained using the crystal structure of neuraminidase (Protein Data Bank file: 5nn9) as template, and it is consistent with the location of previously identified functional residues of bacterial levansucrases (Batista et al., 1999; Song & Jacques, 1999). The sequence-sequence analysis presented here reinforces the recent inclusion of fungal and plant FTFs into glycoside hydrolase family 32, and suggests a modified sequence pattern H-x (2)-[PTV]-x (4)-[LIVMA]-[NSCAYG]-[DE]-P-[NDSC][GA]3 for this family.

  7. Secretory production of antimicrobial peptides in Escherichia coli using the catalytic domain of a cellulase as fusion partner.

    PubMed

    Yu, Huili; Li, Haoran; Gao, Dongfang; Gao, Cuijuan; Qi, Qingsheng

    2015-11-20

    Antimicrobial peptides (AMPs) are small molecules which serve as essential components of the innate immune system in various organisms. AMPs possess a broad spectrum of antimicrobial activities. However, the scaled production of such peptides in Escherichia coli faces many difficulties because of their small size and toxicity to the host. Here, we described a new fusion strategy to extracellularly produce significant amounts of these antimicrobial peptides in recombinant E. coli at significant amount. Employing the catalytic domain of a cellulase (Cel-CD) from Bacillus subtilis KSM-64 as the fusion partner, five recombinant antimicrobial peptides were confirmed to accumulate in the culture medium at concentrations ranging from 184 mg/L to 297 mg/L. The radical diffusion experiment demonstrated that the released model antimicrobial peptide, bombinin, had antibacterial activities against both E. coli and Staphylococcus aureus. This strategy will be suitable for the production of antimicrobial peptides and other toxicity proteins. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Multiple isoforms of Arabidopsis casein kinase I combine conserved catalytic domains with variable carboxyl-terminal extensions.

    PubMed Central

    Klimczak, L J; Farini, D; Lin, C; Ponti, D; Cashmore, A R; Giuliano, G

    1995-01-01

    Three cDNA clones encoding isoforms of casein kinase I (CKI) were isolated from Arabidopsis thaliana. One full-length clone, designated CKI1, contained an open reading frame of 1371 bp encoding a protein of 51,949 D with an isoelectric point of 9.7. In addition to the highly conserved catalytic domain (of about 300 amino acids), the Arabidopsis CKI isoforms contain 150 to 180 amino acid carboxyl-terminal extensions, which show among themselves a lower level of sequence conservation. These extensions do not show any sequence similarity to nonplant CKI isoforms, such as rat testis CKI delta, which is their closest isolated homolog, or to yeast CKI isoforms. Three additional isoforms of Arabidopsis CKI were found in the data bases of expressed sequence tags and/or were isolated serendipitously in nonspecific screening procedures by others. One of them also shows a carboxyl-terminal extension, but of only 80 amino acids. Casein kinase activity was detected in the soluble fraction of Escherichia coli strains expressing the CKI1 protein. This activity showed the crucial properties of CKI, including the ability to phosphorylate the D4 peptide, a specific substrate of CKI, and inhibition by N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide, a specific CKI inhibitor. Like several recombinant CKI isoforms from yeast, CKI1 was able to phosphorylate tyrosine-containing acidic polymers. PMID:7480353

  9. Factor IX Amagasaki: A new mutation in the catalytic domain resulting in the loss of both coagulant and esterase activities

    SciTech Connect

    Miyata, Toshiyuki; Iwanaga, Sadaaki ); Sakai, Toshiyuki; Sugimoto, Mitsuhiko; Naka, Hiroyuki; Yamamoto, Kazukuni; Yoshioka, Akira; Fukui, Hiromu ); Mitsui, Kotoko; Kamiya, Kensyu; Umeyama, Hideaki )

    1991-11-26

    Factor IX Amagasaki (AMG) is a naturally occurring mutant of factor IX having essentially no coagulant activity, even though normal levels of antigen are detected in plasma. Factor IX AMG was purified from the patient's plasma by immunoaffinity chromatography with an anti-factor IX monoclonal antibody column. Factor IX AMG was cleaved normally by factor VIIa-tissue factor complex, yielding a two-chain factor IXa. Amino acid composition and sequence analysis of one of the tryptic peptides isolated from factor IX AMG revealed that Gly-311 had been replaced by Glu. The authors identified a one-base substitution of guanine to adenine in exon VIII by amplifying exon VIII using the polymerase chain reaction method and sequencing the product. This base mutation also supported the replacement of Gly-311 by Glu. In the purified system, factor IXa AMG did not activate for factor X in the presence of factor VIII, phospholipids, and Ca{sup 2+}, and no esterase activity toward Z-Arg-p-nitrobenzyl ester was observed. The model building of the serine protease domain of factor IXa suggests that the Gly-311 {yields} Glu exchange would disrupt the specific conformational state in the active site environment, resulting in the substrate binding site not forming properly. This is the first report to show the experimental evidence for importance of a highly conserved Gly-142 (chymotrypsinogen numbering) located in the catalytic site of mammalian serine proteases so far known.

  10. Non-catalytic motor domains enable processive movement and functional diversification of the kinesin-14 Kar3

    PubMed Central

    Mieck, Christine; Molodtsov, Maxim I; Drzewicka, Katarzyna; van der Vaart, Babet; Litos, Gabriele; Schmauss, Gerald; Vaziri, Alipasha; Westermann, Stefan

    2015-01-01

    Motor proteins of the conserved kinesin-14 family have important roles in mitotic spindle organization and chromosome segregation. Previous studies have indicated that kinesin-14 motors are non-processive enzymes, working in the context of multi-motor ensembles that collectively organize microtubule networks. In this study, we show that the yeast kinesin-14 Kar3 generates processive movement as a heterodimer with the non-motor proteins Cik1 or Vik1. By analyzing the single-molecule properties of engineered motors, we demonstrate that the non-catalytic domain has a key role in the motility mechanism by acting as a ‘foothold’ that allows Kar3 to bias translocation towards the minus end. This mechanism rivals the speed and run length of conventional motors, can support transport of the Ndc80 complex in vitro and is critical for Kar3 function in vivo. Our findings provide an example for a non-conventional translocation mechanism and can explain how Kar3 substitutes for key functions of Dynein in the yeast nucleus. DOI: http://dx.doi.org/10.7554/eLife.04489.001 PMID:25626168

  11. ATP synthase.

    PubMed

    Junge, Wolfgang; Nelson, Nathan

    2015-01-01

    Oxygenic photosynthesis is the principal converter of sunlight into chemical energy. Cyanobacteria and plants provide aerobic life with oxygen, food, fuel, fibers, and platform chemicals. Four multisubunit membrane proteins are involved: photosystem I (PSI), photosystem II (PSII), cytochrome b6f (cyt b6f), and ATP synthase (FOF1). ATP synthase is likewise a key enzyme of cell respiration. Over three billion years, the basic machinery of oxygenic photosynthesis and respiration has been perfected to minimize wasteful reactions. The proton-driven ATP synthase is embedded in a proton tight-coupling membrane. It is composed of two rotary motors/generators, FO and F1, which do not slip against each other. The proton-driven FO and the ATP-synthesizing F1 are coupled via elastic torque transmission. Elastic transmission decouples the two motors in kinetic detail but keeps them perfectly coupled in thermodynamic equilibrium and (time-averaged) under steady turnover. Elastic transmission enables operation with different gear ratios in different organisms.

  12. Purification, crystallization and preliminary X-ray diffraction of the N-terminal calmodulin-like domain of the human mitochondrial ATP-Mg/Pi carrier SCaMC1.

    PubMed

    Yang, Qin; Brüschweiler, Sven; Chou, James J

    2014-01-01

    SCaMC is an ATP-Mg/Pi carrier protein located at the mitochondrial inner membrane. SCaMC has an unusual N-terminal Ca(2+)-binding domain (NTD) in addition to its characteristic six-helix transmembrane bundle. The NTD of human SCaMC1 (residues 1-193) was expressed and purified in order to study its role in Ca(2+)-regulated ATP-Mg/Pi transport mediated by its transmembrane domain. While Ca(2+)-bound NTD could be crystallized, the apo state resisted extensive crystallization trials. Selenomethionine-labeled Ca(2+)-bound NTD crystals, which belonged to space group P6(2)22 with one molecule per asymmetric unit, diffracted X-rays to 2.9 Å resolution.

  13. Crystal structure of the catalytic domain of RluD, the only rRNA pseudouridine synthase required for normal growth of Escherichia coli

    PubMed Central

    DEL CAMPO, MARK; OFENGAND, JAMES; MALHOTRA, ARUN

    2004-01-01

    Escherichia coli pseudouridine synthase RluD makes pseudouridines 1911, 1915, and 1917 in the loop of helix 69 in 23S RNA. These are the most highly conserved ribosomal pseudouridines known. Of 11 pseudouridine synthases in E. coli, only cells lacking RluD have severe growth defects and abnormal ribosomes. We have determined the 2.0 Å structure of the catalytic domain of RluD (residues 77–326), the first structure of an RluA family member. The catalytic domain folds into a mainly antiparallel β-sheet flanked by several loops and helices. A positively charged cleft that presumably binds RNA leads to the conserved Asp 139. The RluD N-terminal S4 domain, connected by a flexible linker, is disordered in our structure. RluD is very similar in both catalytic domain structure and active site arrangement to the pseudouridine synthases RsuA, TruB, and TruA. We identify five sequence motifs, two of which are novel, in the RluA, RsuA, TruB, and TruA families, uniting them as one superfamily. These results strongly suggest that four of the five families of pseudouridine synthases arose by divergent evolution. The RluD structure also provides insight into its multisite specificity. PMID:14730022

  14. Stability of the domain interface contributes towards the catalytic function at the H-site of class alpha glutathione transferase A1-1.

    PubMed

    Balchin, David; Fanucchi, Sylvia; Achilonu, Ikechukwu; Adamson, Roslin J; Burke, Jonathan; Fernandes, Manuel; Gildenhuys, Samantha; Dirr, Heini W

    2010-12-01

    Cytosolic glutathione transferases (GSTs) are major detoxification enzymes in aerobes. Each subunit has two distinct domains and an active site consisting of a G-site for binding GSH and an H-site for an electrophilic substrate. While the active site is located at the domain interface, the role of the stability of this interface in the catalytic function of GSTs is poorly understood. Domain 1 of class alpha GSTs has a conserved tryptophan (Trp21) in helix 1 that forms a major interdomain contact with helices 6 and 8 in domain 2. Replacing Trp21 with an alanine is structurally non-disruptive but creates a cavity between helices 1, 6 and 8 thus reducing the packing density and van der Waals contacts at the domain interface. This results in destabilization of the protein and a marked reduction in catalytic activity. While functionality at the G-site is not adversely affected by the W21A mutation, the H-site becomes more accessible to solvent and less favorable for the electrophilic substrate 1-chloro-2,4-dinitrobenzene (CDNB). Not only does the mutation result in a reduction in the energy for stabilizing the transition state formed in the S(N)Ar reaction between the substrates GSH and CDNB, it also compromises the ability of the enzyme to form and stabilize a transition state analogue (Meisenheimer complex) formed between GSH and 1,3,5-trinitrobenzene (TNB). The study demonstrates that the stability of the domain-domain interface plays a role in mediating the catalytic functionality of the active site, particularly the H-site, of class alpha GSTs. Copyright © 2010 Elsevier B.V. All rights reserved.

  15. Cholinesterase-like domains in enzymes and structural proteins: functional and evolutionary relationships and identification of a catalytically essential aspartic acid.

    PubMed Central

    Krejci, E; Duval, N; Chatonnet, A; Vincens, P; Massoulié, J

    1991-01-01

    Primary sequences of cholinesterases and related proteins have been systematically compared. The cholinesterase-like domain of these proteins, about 500 amino acids, may fulfill a catalytic and a structural function. We identified an aspartic acid residue that is conserved among esterases and lipases (Asp-397 in Torpedo acetylcholinesterase) but that had not been considered to be involved in the catalytic mechanism. Site-directed mutagenesis demonstrated that this residue is necessary for activity. Analysis of evolutionary relationships shows that the noncatalytic members of the family do not constitute a separate subgroup, suggesting that loss of catalytic activity occurred independently on several occasions, probably from bifunctional molecules. Cholinesterases may thus be involved in cell-cell interactions in addition to the hydrolysis of acetylcholine. This would explain their specific expression in well-defined territories during embryogenesis before the formation of cholinergic synapses and their presence in noncholinergic tissues. Images PMID:1862088

  16. Solution NMR structure of the NlpC/P60 domain of lipoprotein Spr from Escherichia coli: structural evidence for a novel cysteine peptidase catalytic triad.

    PubMed

    Aramini, James M; Rossi, Paolo; Huang, Yuanpeng J; Zhao, Li; Jiang, Mei; Maglaqui, Melissa; Xiao, Rong; Locke, Jessica; Nair, Rajesh; Rost, Burkhard; Acton, Thomas B; Inouye, Masayori; Montelione, Gaetano T

    2008-09-16

    Escherichia coli Spr is a membrane-anchored cell wall hydrolase. The solution NMR structure of the C-terminal NlpC/P60 domain of E. coli Spr described here reveals that the protein adopts a papain-like alpha+beta fold and identifies a substrate-binding cleft featuring several highly conserved residues. The active site features a novel Cys-His-His catalytic triad that appears to be a unique structural signature of this cysteine peptidase family. Moreover, the relative orientation of these catalytic residues is similar to that observed in the analogous Ser-His-His triad, a variant of the classic Ser-His-Asp charge relay system, suggesting the convergent evolution of a catalytic mechanism in quite distinct peptidase families.

  17. Role of the P-Type ATPases, ATP7A and ATP7B in brain copper homeostasis.

    PubMed

    Telianidis, Jonathon; Hung, Ya Hui; Materia, Stephanie; Fontaine, Sharon La

    2013-01-01

    Over the past two decades there have been significant advances in our understanding of copper homeostasis and the pathological consequences of copper dysregulation. Cumulative evidence is revealing a complex regulatory network of proteins and pathways that maintain copper homeostasis. The recognition of copper dysregulation as a key pathological feature in prominent neurodegenerative disorders such as Alzheimer's, Parkinson's, and prion diseases has led to increased research focus on the mechanisms controlling copper homeostasis in the brain. The copper-transporting P-type ATPases (copper-ATPases), ATP7A and ATP7B, are critical components of the copper regulatory network. Our understanding of the biochemistry and cell biology of these complex proteins has grown significantly since their discovery in 1993. They are large polytopic transmembrane proteins with six copper-binding motifs within the cytoplasmic N-terminal domain, eight transmembrane domains, and highly conserved catalytic domains. These proteins catalyze ATP-dependent copper transport across cell membranes for the metallation of many essential cuproenzymes, as well as for the removal of excess cellular copper to prevent copper toxicity. A key functional aspect of these copper transporters is their copper-responsive trafficking between the trans-Golgi network and the cell periphery. ATP7A- and ATP7B-deficiency, due to genetic mutation, underlie the inherited copper transport disorders, Menkes and Wilson diseases, respectively. Their importance in maintaining brain copper homeostasis is underscored by the severe neuropathological deficits in these disorders. Herein we will review and update our current knowledge of these copper transporters in the brain and the central nervous system, their distribution and regulation, their role in normal brain copper homeostasis, and how their absence or dysfunction contributes to disturbances in copper homeostasis and neurodegeneration.

  18. Role of the P-Type ATPases, ATP7A and ATP7B in brain copper homeostasis

    PubMed Central

    Telianidis, Jonathon; Hung, Ya Hui; Materia, Stephanie; Fontaine, Sharon La

    2013-01-01

    Over the past two decades there have been significant advances in our understanding of copper homeostasis and the pathological consequences of copper dysregulation. Cumulative evidence is revealing a complex regulatory network of proteins and pathways that maintain copper homeostasis. The recognition of copper dysregulation as a key pathological feature in prominent neurodegenerative disorders such as Alzheimer’s, Parkinson’s, and prion diseases has led to increased research focus on the mechanisms controlling copper homeostasis in the brain. The copper-transporting P-type ATPases (copper-ATPases), ATP7A and ATP7B, are critical components of the copper regulatory network. Our understanding of the biochemistry and cell biology of these complex proteins has grown significantly since their discovery in 1993. They are large polytopic transmembrane proteins with six copper-binding motifs within the cytoplasmic N-terminal domain, eight transmembrane domains, and highly conserved catalytic domains. These proteins catalyze ATP-dependent copper transport across cell membranes for the metallation of many essential cuproenzymes, as well as for the removal of excess cellular copper to prevent copper toxicity. A key functional aspect of these copper transporters is their copper-responsive trafficking between the trans-Golgi network and the cell periphery. ATP7A- and ATP7B-deficiency, due to genetic mutation, underlie the inherited copper transport disorders, Menkes and Wilson diseases, respectively. Their importance in maintaining brain copper homeostasis is underscored by the severe neuropathological deficits in these disorders. Herein we will review and update our current knowledge of these copper transporters in the brain and the central nervous system, their distribution and regulation, their role in normal brain copper homeostasis, and how their absence or dysfunction contributes to disturbances in copper homeostasis and neurodegeneration. PMID:23986700

  19. Mutagenic definition of a papain-like catalytic triad, sufficiency of the N-terminal domain for single-site core catalytic enzyme acylation, and C-terminal domain for augmentative metal activation of a eukaryotic phytochelatin synthase.

    PubMed

    Romanyuk, Nataliya D; Rigden, Daniel J; Vatamaniuk, Olena K; Lang, Albert; Cahoon, Rebecca E; Jez, Joseph M; Rea, Philip A

    2006-07-01

    in the case of AtPCS1, for formation of the biosynthetically competent gamma-Glu-Cys enzyme acyl intermediate, the primary data from experiments directed at determining whether the other two residues, His-162 and Asp-180 of the putative papain-like catalytic triad of AtPCS1, are essential for catalysis have yet to be presented. This shortfall in our basic understanding of AtPCS1 is addressed here by the results of systematic site-directed mutagenesis studies that demonstrate that not only Cys-56 but also His-162 and Asp-180 are indeed required for net PC synthesis. It is therefore established experimentally that AtPCS1 and, by implication, other eukaryotic PC synthases are papain Cys protease superfamily members but ones, unlike their prokaryotic counterparts, which, in addition to having a papain-like N-terminal catalytic domain that undergoes primary gamma-Glu-Cys acylation, contain an auxiliary metal-sensing C-terminal domain that undergoes secondary gamma-Glu-Cys acylation.

  20. Specificity and versatility of substrate binding sites in four catalytic domains of human N-terminal acetyltransferases.

    PubMed

    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 [Formula: see text] that tightens under substrate binding. Finally, by comparing the four structures we propose maps of the peptide-enzyme interactions

  1. Catalytic roles of lysines (K9, K27, K31) in the N-terminal domain in human adenylate kinase by random site-directed mutagenesis.

    PubMed

    Ayabe, T; Park, S K; Takenaka, H; Sumida, M; Uesugi, S; Takenaka, O; Hamada, M

    1996-11-01

    To elucidate lysine residues in the N-terminal domain of human cytosolic adenylate kinase (hAK1, EC 2.7.4.3), random site-directed mutagenesis of K9, K27, and K31 residues was performed, and six mutants were analyzed by steady-state kinetics. K9 residue may play an important role in catalysis by interacting with AMP2-. K27 and K31 residues appear to play a functional role in catalysis by interacting with MgATP2-. In human AK, the epsilon-amino group in the side chain of these lysine residues would be essential for phosphoryl transfer between MgATP2- and AMP2- during transition state.

  2. Plasmodium falciparum CTP:phosphocholine cytidylyltransferase possesses two functional catalytic domains and is inhibited by a CDP-choline analog selected from a virtual screening.

    PubMed

    Contet, Alicia; Pihan, Emilie; Lavigne, Marina; Wengelnik, Kai; Maheshwari, Sweta; Vial, Henri; Douguet, Dominique; Cerdan, Rachel

    2015-04-13

    Phosphatidylcholine is the major lipid component of the malaria parasite membranes and is required for parasite multiplication in human erythrocytes. Plasmodium falciparum CTP:phosphocholine cytidylyltransferase (PfCCT) is the rate-limiting enzyme of the phosphatidylcholine biosynthesis pathway and thus considered as a potential antimalarial target. In contrast to its mammalian orthologs, PfCCT contains a duplicated catalytic domain. Here, we show that both domains are catalytically active with similar kinetic parameters. A virtual screening strategy allowed the identification of a drug-size molecule competitively inhibiting the enzyme. This compound also prevented phosphatidylcholine biosynthesis in parasites and exerted an antimalarial effect. This study constitutes the first step towards a rationalized design of future new antimalarial agents targeting PfCCT.

  3. PRIP (Phospholipase C-related but Catalytically Inactive Protein) Inhibits Exocytosis by Direct Interactions with Syntaxin 1 and SNAP-25 through Its C2 Domain*

    PubMed Central

    Zhang, Zhao; Takeuchi, Hiroshi; Gao, Jing; Wang, DaGuang; James, Declan J.; Martin, Thomas F. J.; Hirata, Masato

    2013-01-01

    Membrane fusion for exocytosis is mediated by SNAREs, forming trans-ternary complexes to bridge vesicle and target membranes. There is an array of accessory proteins that directly interact with and regulate SNARE proteins. PRIP (phospholipase C-related but catalytically inactive protein) is likely one of these proteins; PRIP, consisting of multiple functional modules including pleckstrin homology and C2 domains, inhibited exocytosis, probably via the binding to membrane phosphoinositides through the pleckstrin homology domain. However, the roles of the C2 domain have not yet been investigated. In this study, we found that the C2 domain of PRIP directly interacts with syntaxin 1 and SNAP-25 but not with VAMP2. The C2 domain promoted PRIP to co-localize with syntaxin 1 and SNAP-25 in PC12 cells. The binding profile of the C2 domain to SNAP-25 was comparable with that of synaptotagmin I, and PRIP inhibited synaptotagmin I in binding to SNAP-25 and syntaxin 1. It was also shown that the C2 domain was required for PRIP to suppress SDS-resistant ternary SNARE complex formation and inhibit high K+-induced noradrenalin release from PC12 cells. These results suggest that PRIP inhibits regulated exocytosis through the interaction of its C2 domain with syntaxin 1 and SNAP-25, potentially competing with other SNARE-binding, C2 domain-containing accessory proteins such as synaptotagmin I and by directly inhibiting trans-SNARE complex formation. PMID:23341457

  4. The catalytic subunit of Dictyostelium cAMP-dependent protein kinase -- role of the N-terminal domain and of the C-terminal residues in catalytic activity and stability.

    PubMed

    Etchebehere, L C; Van Bemmelen, M X; Anjard, C; Traincard, F; Assemat, K; Reymond, C; Véron, M

    1997-09-15

    The C subunit of Dictyostelium cAMP-dependent protein kinase (PKA) is unusually large (73 kDa) due to the presence of 330 amino acids N-terminal to the conserved catalytic core. The sequence following the core, including a C-terminal -Phe-Xaa-Xaa-Phe-COOH motif, is highly conserved. We have characterized the catalytic activity and stability of C subunits mutated in sequences outside the catalytic core and we have analyzed their ability to interact with the R subunit and with the heat-stable protein-kinase inhibitor PKI. Mutants carrying deletions in the N-terminal domain displayed little difference in their kinetic properties and retained their capacity to be inhibited by R subunit and by PKI. In contrast, the mutation of one or both of the phenylalanine residues in the C-terminal motif resulted in a decrease of catalytic activity and stability of the proteins. Inhibition by the R subunit or by PKI were however unaffected. Sequence-comparison analysis of other protein kinases revealed that a -Phe-Xaa-Xaa-Phe- motif is present in many Ser/Thr protein kinases, although its location at the very end of the polypeptide is a particular feature of the PKA family. We propose that the presence of this motif may serve to identify isoforms of protein kinases.

  5. Predicted unfolding order of the 13 alpha-helices in the catalytic domain of glucoamylase from Aspergillus awamori var. X100 by molecular dynamics simulations.

    PubMed

    Liu, Hsuan-Liang; Wang, Wen-Chi

    2003-01-01

    The unfolding mechanism of the 13 alpha-helices in the catalytic domain of Aspergillus awamori var. X100 glucoamylase was investigated by 200 ps molecular dynamics simulations in explicit water with temperature jump technique. Rather than a simultaneous event, the unfolding of these 13 alpha-helices followed a random ordered mechanism as alpha8-->alpha1-->alpha11-->alpha7-->alpha10-->alpha3-->alpha12-->alpha13-->alpha4-->alpha5-->alpha9-->alpha6-->alpha2. No significant relationships were found between the unfolding order and the length and the hydrophobicity of the helix. alpha-Helix 8 located in the inner region of the catalytic domain was predicted to be the first helix to unfold, indicating that the destruction of the secondary structure motif was initiated from the inner region of the catalytic domain. The dynamic behavior of these alpha-helices induced by increased kinetic energy during the unfolding process is considered to be similar to the expansion and compression of a series of springs under the influence of mechanical stress.

  6. Improved catalytic efficiency, thermophilicity, anti-salt and detergent tolerance of keratinase KerSMD by partially truncation of PPC domain

    PubMed Central

    Fang, Zhen; Zhang, Juan; Du, Guocheng; Chen, Jian

    2016-01-01

    The keratinase from Stenotrophomonas maltophilia (KerSMD) is known for its high activity and pH stability in keratin degradation. However, catalytic efficiency and detergent tolerability need to be improved in order to be used for industrial application. In this work, we obtained several keratinase variants with enhanced catalytic efficiency, thermophilicity, and anti-salt and detergent tolerability by partially truncating the PPC domain of KerSMD. The variants all showed improved catalytic efficiency to synthetic substrate AAPF, with the V355 variant having the highest kcat /Km value of 143.6 s−1 mM−1. The truncation of keratinase had little effect on alkaline stability but obviously decreased collagenase activity, developing its potential application in leather treatment. The variants V380, V370, and V355 were thermophilic, with a 1.7-fold enhancement of keratinlytic activity at 60 °C when compared to the wild type. The entire truncation of PPC domain obtained the variant V355 with improved tolerance to alkalinity, salt, chaotropic agents, and detergents. The V355 variant showed more than a 40% improvement in activity under 15% (w/v) NaCl or 4% (w/v) SDS solution, showing excellent stability under harsh washing and unhairing conditions. Our work investigated how protein engineering affects the function of PPC domain of KerSMD. PMID:27298079

  7. Quantum Mechanics and Molecular Mechanics Study of the Catalytic Mechanism of Human AMSH-LP Domain Deubiquitinating Enzymes.

    PubMed

    Zhu, Wenyou; Liu, Yongjun; Ling, Baoping

    2015-08-25

    Deubiquitinating enzymes (DUBs) catalyze the cleavage of the isopeptide bond in polyubiquitin chains to control and regulate the deubiquitination process in all known eukaryotic cells. The human AMSH-LP DUB domain specifically cleaves the isopeptide bonds in the Lys63-linked polyubiquitin chains. In this article, the catalytic mechanism of AMSH-LP has been studied using a combined quantum mechanics and molecular mechanics method. Two possible hydrolysis processes (Path 1 and Path 2) have been considered. Our calculation results reveal that the activation of Zn(2+)-coordinated water molecule is the essential step for the hydrolysis of isopeptide bond. In Path 1, the generated hydroxyl first attacks the carbonyl group of Gly76, and then the amino group of Lys63 is protonated, which is calculated to be the rate limiting step with an energy barrier of 13.1 kcal/mol. The energy barrier of the rate limiting step and the structures of intermediate and product are in agreement with the experimental results. In Path 2, the protonation of amino group of Lys63 is prior to the nucleophilic attack of activated hydroxyl. The two proton transfer processes in Path 2 correspond to comparable overall barriers (33.4 and 36.1 kcal/mol), which are very high for an enzymatic reaction. Thus, Path 2 can be ruled out. During the reaction, Glu292 acts as a proton transfer mediator, and Ser357 mainly plays a role in stabilizing the negative charge of Gly76. Besides acting as a Lewis acid, Zn(2+) also influences the reaction by coordinating to the reaction substrates (W1 and Gly76).

  8. Three Cdk1 sites in the kinesin-5 Cin8 catalytic domain coordinate motor localization and activity during anaphase.

    PubMed

    Goldstein, Alina; Siegler, Nurit; Goldman, Darya; Judah, Haim; Valk, Ervin; Kõivomägi, Mardo; Loog, Mart; Gheber, Larisa

    2017-09-01

    The bipolar kinesin-5 motors perform essential functions in mitotic spindle dynamics. We previously demonstrated that phosphorylation of at least one of the Cdk1 sites in the catalytic domain of the Saccharomyces cerevisiae kinesin-5 Cin8 (S277, T285, S493) regulates its localization to the anaphase spindle. The contribution of these three sites to phospho-regulation of Cin8, as well as the timing of such contributions, remains unknown. Here, we examined the function and spindle localization of phospho-deficient (serine/threonine to alanine) and phospho-mimic (serine/threonine to aspartic acid) Cin8 mutants. In vitro, the three Cdk1 sites undergo phosphorylation by Clb2-Cdk1. In cells, phosphorylation of Cin8 affects two aspects of its localization to the anaphase spindle, translocation from the spindle-pole bodies (SPBs) region to spindle microtubules (MTs) and the midzone, and detachment from the mitotic spindle. We found that phosphorylation of S277 is essential for the translocation of Cin8 from SPBs to spindle MTs and the subsequent detachment from the spindle. Phosphorylation of T285 mainly affects the detachment of Cin8 from spindle MTs during anaphase, while phosphorylation at S493 affects both the translocation of Cin8 from SPBs to the spindle and detachment from the spindle. Only S493 phosphorylation affected the anaphase spindle elongation rate. We conclude that each phosphorylation site plays a unique role in regulating Cin8 functions and postulate a model in which the timing and extent of phosphorylation of the three sites orchestrates the anaphase function of Cin8.

  9. Cooperation between Catalytic and DNA-binding Domains Enhances Thermostability and Supports DNA Synthesis at Higher Temperatures by Thermostable DNA Polymerases

    PubMed Central

    Pavlov, Andrey R.; Pavlova, Nadejda V.; Kozyavkin, Sergei A.; Slesarev, Alexei I.

    2012-01-01

    We have previously introduced a general kinetic approach for comparative study of processivity, thermostability, and resistance to inhibitors of DNA polymerases (Pavlov et. al., (2002) Proc. Natl. Acad. Sci. USA 99, 13510–13515). The proposed method was successfully applied to characterize hybrid DNA polymerases created by fusing catalytic DNA polymerase domains with various non-specific DNA binding domains. Here we use the developed kinetic analysis to assess basic parameters of DNA elongation by DNA polymerases and to further study the interdomain interactions in both previously constructed and new chimeric DNA polymerases. We show that connecting Helix-hairpin-Helix (HhH) domains to catalytic polymerase domains can increase thermostability, not only of DNA polymerases from extremely thermophilic species, but also of the enzyme from a faculatative thermophilic bacterium Bacillus stearothermophilus. We also demonstrate that addition of TopoV HhH domains extends efficient DNA synthesis by chimerical polymerases up to 105°C by maintaining processivity of DNA synthesis at high temperatures. We also found that reversible high-temperature structural transitions in DNA polymerases decrease the rates of binding of these enzymes to the templates. Furthermore, activation energies and pre-exponential factors of the Arrhenius equation suggest that the mechanism of electrostatic enhancement of diffusion-controlled association plays a minor role in binding templates to DNA polymerases. PMID:22320201

  10. Homologous xylanases from Clostridium thermocellum: evidence for bi-functional activity, synergism between xylanase catalytic modules and the presence of xylan-binding domains in enzyme complexes.

    PubMed Central

    Fernandes, A C; Fontes, C M; Gilbert, H J; Hazlewood, G P; Fernandes, T H; Ferreira, L M

    1999-01-01

    Clostridium thermocellum produces a consortium of plant-cell-wall hydrolases that form a cell-bound multi-enzyme complex called the cellulosome. In the present study two similar xylanase genes, xynU and xynV, were cloned from C. thermocellum strain YS and sequenced. The deduced primary structures of both xylanases, xylanase U (XylU) and xylanase V (XylV), were homologous with the previously characterized xylanases from C. thermocellum strain F1. Truncated derivatives of XylV were produced and their biochemical properties were characterized. The xylanases were shown to be remarkably thermostable and resistant to proteolytic inactivation. The catalytic domains hydrolysed xylan by a typical endo-mode of action. The type VI cellulose-binding domain (CBD) homologue of XylV bound xylan and, to a smaller extent, Avicel and acid-swollen cellulose. Deletion of the CBD from XylV abolished the capacity of the enzymes to bind polysaccharides. The polysaccharide-binding domain was shown to have a key role in the hydrolysis of insoluble substrates by XylV. The C-terminal domain of XylV, which is absent from XylU, removed acetyl groups from acetylated xylan and acted in synergy with the glycosyl hydrolase catalytic domain of the enzyme to elicit the hydrolysis of acetylated xylan. PMID:10432306

  11. Crystal Structures of YkuI and Its Complex with Second Messenger Cyclic Di-GMP Suggest Catalytic Mechanism of Phosphodiester Bond Cleavage by EAL Domains*

    PubMed Central

    Minasov, George; Padavattan, Sivaraman; Shuvalova, Ludmilla; Brunzelle, Joseph S.; Miller, Darcie J.; Baslé, Arnaud; Massa, Claudia; Collart, Frank R.; Schirmer, Tilman; Anderson, Wayne F.

    2009-01-01

    Cyclic di-GMP (c-di-GMP) is a ubiquitous bacterial second messenger that is involved in the regulation of cell surface-associated traits and the persistence of infections. Omnipresent GGDEF and EAL domains, which occur in various combinations with regulatory domains, catalyze c-di-GMP synthesis and degradation, respectively. The crystal structure of full-length YkuI from Bacillus subtilis, composed of an EAL domain and a C-terminal PAS-like domain, has been determined in its native form and in complex with c-di-GMP and Ca2+. The EAL domain exhibits a triose-phosphate isomerase-barrel fold with one antiparallel β-strand. The complex with c-di-GMP-Ca2+ defines the active site of the putative phosphodiesterase located at the C-terminal end of the β-barrel. The EAL motif is part of the active site with Glu-33 of the motif being involved in cation coordination. The structure of the complex allows the proposal of a phosphodiesterase mechanism, in which the divalent cation and the general base Glu-209 activate a catalytic water molecule for nucleophilic in-line attack on the phosphorus. The C-terminal domain closely resembles the PAS-fold. Its pocket-like structure could accommodate a yet unknown ligand. YkuI forms a tight dimer via EAL-EAL and trans EAL-PAS-like domain association. The possible regulatory significance of the EAL-EAL interface and a mechanism for signal transduction between sensory and catalytic domains of c-di-GMP-specific phosphodiesterases are discussed. PMID:19244251

  12. Structural and Enzymatic Insights into the ATP Binding and Autophosphorylation Mechanism of a Sensor Histidine Kinase*

    PubMed Central

    Trajtenberg, Felipe; Graña, Martin; Ruétalo, Natalia; Botti, Horacio; Buschiazzo, Alejandro

    2010-01-01

    DesK is a sensor histidine kinase (HK) that allows Bacillus subtilis to respond to cold shock, triggering the adaptation of membrane fluidity via transcriptional control of a fatty acid desaturase. It belongs to the HK family HPK7, which includes the nitrogen metabolism regulators NarX/Q and the antibiotic sensor LiaS among other important sensor kinases. Structural information on different HK families is still scarce and several questions remain, particularly concerning the molecular features that determine HK specificity during its catalytic autophosphorylation and subsequent response-regulator phosphotransfer reactions. To analyze the ATP-binding features of HPK7 HKs and dissect their mechanism of autophosphorylation at the molecular level, we have studied DesK in complex with ATP using high resolution structural approaches in combination with biochemical studies. We report the first crystal structure of an HK in complex with its natural nucleotidic substrate. The general fold of the ATP-binding domain of DesK is conserved, compared with well studied members of other families. Yet, DesK displays a far more compact structure at the ATP-binding pocket: the ATP lid loop is much shorter with no secondary structural organization and becomes ordered upon ATP loading. Sequence conservation mapping onto the molecular surface, semi-flexible protein-protein docking simulations, and structure-based point mutagenesis allow us to propose a specific domain-domain geometry during autophosphorylation catalysis. Supporting our hypotheses, we have been able to trap an autophosphorylating intermediate state, by protein engineering at the predicted domain-domain interaction surface. PMID:20507988

  13. RipD (Rv1566c) from Mycobacterium tuberculosis: adaptation of an NlpC/p60 domain to a non-catalytic peptidoglycan-binding function.

    PubMed

    Böth, Dominic; Steiner, Eva Maria; Izumi, Atsushi; Schneider, Gunter; Schnell, Robert

    2014-01-01

    Enzymes carrying NlpC/p60 domains, for instance RipA and RipB from Mycobacterium tuberculosis, are bacterial peptidoglycan hydrolases that cleave the peptide stems and contribute to cell wall remodelling during cell division. A member of this protein family, RipD (Rv1566c) from M. tuberculosis described in the present study, displays sequence alterations in the NlpC/p60 catalytic triad and carries a pentapeptide repeat at its C-terminus. Bioinformatics analysis revealed RipD-like proteins in eleven mycobacterial genomes, whereas similar pentapeptide repeats occur in cell-wall-localized bacterial proteins and in a mycobacteriophage. In contrast with previously known members of the NlpC/p60 family, RipD does not show peptidoglycan hydrolase activity, which is consistent with the sequence alterations at the catalytic site. A strong interaction of the catalytically inactive core domain with peptidoglycan is however retained, presenting the first example of the NlpC/p60 domains that evolved to a non-catalytic peptidoglycan-binding function. Full-length RipD carrying the C-terminal repeat shows, however, a decrease in binding affinity to peptidoglycan, suggesting that the C-terminal tail modulates the interaction with bacterial cell wall components. The pentapeptide repeat at the C-terminus does not adopt a defined secondary structure in solution which is in accordance with results from the 1.17 Å (1 Å=0.1 nm) crystal structure of the protein carrying two repeat units.

  14. Two ENU-induced alleles of Atp2b2 cause deafness in mice.

    PubMed

    Carpinelli, Marina R; Manning, Michael G; Kile, Benjamin T; Burt, Rachel A; Rachel, A Burt

    2013-01-01

    Over 120 loci are known to cause inherited hearing loss in humans. The deafness gene has been identified for only half of these loci. With the aim of identifying some of the remaining deafness genes, we performed an ethylnitrosourea mutagenesis screen for deaf mice. We isolated two mutants with semi-dominant hearing loss, Deaf11 and Deaf13. Both contained causative mutations in Atp2b2, which encodes the plasma membrane calcium ATPase 2. The Atp2b2 (Deaf11) mutation leads to a p. I1023S substitution in the tenth transmembrane domain. The Atp2b2 (Deaf13) mutation leads to a p. R561S substitution in the catalytic core. Mice homozygous for these mutations display profound hearing loss. Heterozygotes display mild to moderate, progressive hearing loss.

  15. Two ENU-Induced Alleles of Atp2b2 Cause Deafness in Mice

    PubMed Central

    Carpinelli, Marina R.; Manning, Michael G.; Kile, Benjamin T.; Rachel, A. Burt

    2013-01-01

    Over 120 loci are known to cause inherited hearing loss in humans. The deafness gene has been identified for only half of these loci. With the aim of identifying some of the remaining deafness genes, we performed an ethylnitrosourea mutagenesis screen for deaf mice. We isolated two mutants with semi-dominant hearing loss, Deaf11 and Deaf13. Both contained causative mutations in Atp2b2, which encodes the plasma membrane calcium ATPase 2. The Atp2b2Deaf11 mutation leads to a p. I1023S substitution in the tenth transmembrane domain. The Atp2b2Deaf13 mutation leads to a p. R561S substitution in the catalytic core. Mice homozygous for these mutations display profound hearing loss. Heterozygotes display mild to moderate, progressive hearing loss. PMID:23826306

  16. The prolyl isomerase domain of PpiD from Escherichia coli shows a parvulin fold but is devoid of catalytic activity

    PubMed Central

    Weininger, Ulrich; Jakob, Roman P; Kovermann, Michael; Balbach, Jochen; Schmid, Franz X

    2010-01-01

    PpiD is a periplasmic folding helper protein of Escherichia coli. It consists of an N-terminal helix that anchors PpiD in the inner membrane near the SecYEG translocon, followed by three periplasmic domains. The second domain (residues 264–357) shows homology to parvulin-like prolyl isomerases. This domain is a well folded, stable protein and follows a simple two-state folding mechanism. In its solution structure, as determined by NMR spectroscopy, it resembles most closely the first parvulin domain of the SurA protein, which resides in the periplasm of E. coli as well. A previously reported prolyl isomerase activity of PpiD could not be reproduced when using improved protease-free peptide assays or assays with refolding proteins as substrates. The parvulin domain of PpiD interacts, however, with a proline-containing tetrapeptide, and the binding site, as identified by NMR resonance shift analysis, colocalized with the catalytic sites of other parvulins. In its structure, the parvulin domain of PpiD resembles most closely the inactive first parvulin domain of SurA, which is part of the chaperone unit of this protein and presumably involved in substrate recognition. PMID:19866485

  17. The N-terminal shuttle domain of Erv1 determines the affinity for Mia40 and mediates electron transfer to the catalytic Erv1 core in yeast mitochondria.

    PubMed

    Lionaki, Eirini; Aivaliotis, Michalis; Pozidis, Charalambos; Tokatlidis, Kostas

    2010-11-01

    Erv1 and Mia40 constitute the two important components of the disulfide relay system that mediates oxidative protein folding in the mitochondrial intermembrane space. Mia40 is the import receptor that recognizes the substrates introducing disulfide bonds while it is reduced. A key function of Erv1 is to recycle Mia40 to its active oxidative state. Our aims here were to dissect the domain of Erv1 that mediates the protein-protein interaction with Mia40 and to investigate the interactions between the shuttle domain of Erv1 and its catalytic core and their relevance for the interaction with Mia40. We purified these domains separately as well as cysteine mutants in the shuttle and the active core domains. The noncovalent interaction of Mia40 with Erv1 was measured by isothermal titration calorimetry, whereas their covalent mixed disulfide intermediate was analyzed in reconstitution experiments in vitro and in organello. We established that the N-terminal shuttle domain of Erv1 is necessary and sufficient for interaction to occur. Furthermore, we provide direct evidence for the intramolecular electron transfer from the shuttle cysteine pair of Erv1 to the core domain. Finally, we reconstituted the system by adding in trans the N- and C- terminal domains of Erv1 together with its substrate Mia40.

  18. Crystal structure of full-length human collagenase 3 (MMP-13) with peptides in the active site defines exosites in the catalytic domain

    PubMed Central

    Stura, Enrico A.; Visse, Robert; Cuniasse, Philippe; Dive, Vincent; Nagase, Hideaki

    2013-01-01

    Matrix metalloproteinase (MMP)-13 is one of the mammalian collagenases that play key roles in tissue remodelling and repair and in progression of diseases such as cancer, arthritis, atherosclerosis, and aneurysm. For collagenase to cleave triple helical collagens, the triple helical structure has to be locally unwound before hydrolysis, but this process is not well understood. We report crystal structures of catalytically inactive full-length human MMP-13(E223A) in complex with peptides of 14–26 aa derived from the cleaved prodomain during activation. Peptides are bound to the active site of the enzyme by forming an extended β-strand with Glu40 or Tyr46 inserted into the S1′ specificity pocket. The structure of the N-terminal part of the peptides is variable and interacts with different parts of the catalytic domain. Those areas are designated substrate-dependent exosites, in that they accommodate different peptide structures, whereas the precise positioning of the substrate backbone is maintained in the active site. These modes of peptide-MMP-13 interactions have led us to propose how triple helical collagen strands fit into the active site cleft of the collagenase.—Stura, E. A., Visse, R., Cuniasse, P., Dive, V., Nagase, H. Crystal structure of full-length human collagenase 3 (MMP-13) with peptides in the active site defines exosites in the catalytic domain. PMID:23913860

  19. Catalytic domain of plasmid pAD1 relaxase TraX defines a group of relaxases related to restriction endonucleases.

    PubMed

    Francia, María Victoria; Clewell, Don B; de la Cruz, Fernando; Moncalián, Gabriel

    2013-08-13

    Plasmid pAD1 is a 60-kb conjugative element commonly found in clinical isolates of Enterococcus faecalis. The relaxase TraX and the primary origin of transfer oriT2 are located close to each other and have been shown to be essential for conjugation. The oriT2 site contains a large inverted repeat (where the nic site is located) adjacent to a series of short direct repeats. TraX does not show any of the typical relaxase sequence motifs but is the prototype of a unique family of relaxases (MOBC). The present study focuses on the genetic, biochemical, and structural analysis of TraX, whose 3D structure could be predicted by protein threading. The structure consists of two domains: (i) an N-terminal domain sharing the topology of the DNA binding domain of the MarR family of transcriptional regulators and (ii) a C-terminal catalytic domain related to the PD-(D/E)XK family of restriction endonucleases. Alignment of MOBC relaxase amino acid sequences pointed to several conserved polar amino acid residues (E28, D152, E170, E172, K176, R180, Y181, and Y203) that were mutated to alanine. Functional analysis of these mutants (in vivo DNA transfer and cleavage assays) revealed the importance of these residues for relaxase activity and suggests Y181 as a potential catalytic residue similarly to His-hydrophobe-His relaxases. We also show that TraX binds specifically to dsDNA containing the oriT2 direct repeat sequences, confirming their role in transfer specificity. The results provide insights into the catalytic mechanism of MOBC relaxases, which differs radically from that of His-hydrophobe-His relaxases.

  20. Role of the two catalytic domains of DSR-E dextransucrase and their involvement in the formation of highly alpha-1,2 branched dextran.

    PubMed

    Fabre, Emeline; Bozonnet, Sophie; Arcache, Audrey; Willemot, René-Marc; Vignon, Michel; Monsan, Pierre; Remaud-Simeon, Magali

    2005-01-01

    The dsrE gene from Leuconostoc mesenteroides NRRL B-1299 was shown to encode a very large protein with two potentially active catalytic domains (CD1 and CD2) separated by a glucan binding domain (GBD). From sequence analysis, DSR-E was classified in glucoside hydrolase family 70, where it is the only enzyme to have two catalytic domains. The recombinant protein DSR-E synthesizes both alpha-1,6 and alpha-1,2 glucosidic linkages in transglucosylation reactions using sucrose as the donor and maltose as the acceptor. To investigate the specific roles of CD1 and CD2 in the catalytic mechanism, truncated forms of dsrE were cloned and expressed in Escherichia coli. Gene products were then small-scale purified to isolate the various corresponding enzymes. Dextran and oligosaccharide syntheses were performed. Structural characterization by (13)C nuclear magnetic resonance and/or high-performance liquid chromatography showed that enzymes devoid of CD2 synthesized products containing only alpha-1,6 linkages. On the other hand, enzymes devoid of CD1 modified alpha-1,6 linear oligosaccharides and dextran acceptors through the formation of alpha-1,2 linkages. Therefore, each domain is highly regiospecific, CD1 being specific for the synthesis of alpha-1,6 glucosidic bonds and CD2 only catalyzing the formation of alpha-1,2 linkages. This finding permitted us to elucidate the mechanism of alpha-1,2 branching formation and to engineer a novel transglucosidase specific for the formation of alpha-1,2 linkages. This enzyme will be very useful to control the rate of alpha-1,2 linkage synthesis in dextran or oligosaccharide production.

  1. Catalytic domain of plasmid pAD1 relaxase TraX defines a group of relaxases related to restriction endonucleases

    PubMed Central

    Francia, María Victoria; Clewell, Don B.; de la Cruz, Fernando; Moncalián, Gabriel

    2013-01-01

    Plasmid pAD1 is a 60-kb conjugative element commonly found in clinical isolates of Enterococcus faecalis. The relaxase TraX and the primary origin of transfer oriT2 are located close to each other and have been shown to be essential for conjugation. The oriT2 site contains a large inverted repeat (where the nic site is located) adjacent to a series of short direct repeats. TraX does not show any of the typical relaxase sequence motifs but is the prototype of a unique family of relaxases (MOBC). The present study focuses on the genetic, biochemical, and structural analysis of TraX, whose 3D structure could be predicted by protein threading. The structure consists of two domains: (i) an N-terminal domain sharing the topology of the DNA binding domain of the MarR family of transcriptional regulators and (ii) a C-terminal catalytic domain related to the PD-(D/E)XK family of restriction endonucleases. Alignment of MOBC relaxase amino acid sequences pointed to several conserved polar amino acid residues (E28, D152, E170, E172, K176, R180, Y181, and Y203) that were mutated to alanine. Functional analysis of these mutants (in vivo DNA transfer and cleavage assays) revealed the importance of these residues for relaxase activity and suggests Y181 as a potential catalytic residue similarly to His-hydrophobe-His relaxases. We also show that TraX binds specifically to dsDNA containing the oriT2 direct repeat sequences, confirming their role in transfer specificity. The results provide insights into the catalytic mechanism of MOBC relaxases, which differs radically from that of His-hydrophobe-His relaxases. PMID:23904483

  2. ATP-driven Rad50 conformations regulate DNA tethering, end resection, and ATM checkpoint signaling.

    PubMed

    Deshpande, Rajashree A; Williams, Gareth J; Limbo, Oliver; Williams, R Scott; Kuhnlein, Jeff; Lee, Ji-Hoon; Classen, Scott; Guenther, Grant; Russell, Paul; Tainer, John A; Paull, Tanya T

    2014-03-03

    The Mre11-Rad50 complex is highly conserved, yet the mechanisms by which Rad50 ATP-driven states regulate the sensing, processing and signaling of DNA double-strand breaks are largely unknown. Here we design structure-based mutations in Pyrococcus furiosus Rad50 to alter protein core plasticity and residues undergoing ATP-driven movements within the catalytic domains. With this strategy we identify Rad50 separation-of-function mutants that either promote or destabilize the ATP-bound state. Crystal structures, X-ray scattering, biochemical assays, and functional analyses of mutant PfRad50 complexes show that the ATP-induced 'closed' conformation promotes DNA end binding and end tethering, while hydrolysis-induced opening is essential for DNA resection. Reducing the stability of the ATP-bound state impairs DNA repair and Tel1 (ATM) checkpoint signaling in Schizosaccharomyces pombe, double-strand break resection in Saccharomyces cerevisiae, and ATM activation by human Mre11-Rad50-Nbs1 in vitro, supporting the generality of the P. furiosus Rad50 structure-based mutational analyses. These collective results suggest that ATP-dependent Rad50 conformations switch the Mre11-Rad50 complex between DNA tethering, ATM signaling, and 5' strand resection, revealing molecular mechanisms regulating responses to DNA double-strand breaks.

  3. ATP-dependent interplay between local and global conformational changes in the myosin motor.

    PubMed

    Kiani, Farooq Ahmad; Fischer, Stefan

    2016-11-01

    The ATPase active site of myosin is located at the core of the motor head. During the Lymn-Taylor actomyosin contractile cycle, small conformational changes in the active site upon ATP binding, ATP hydrolysis and ADP/Pi release are accompanied by large conformational transitions of the motor domains, such as opening and closing of the actin binding cleft and the movement of lever arm. Here, our previous computational studies of myosin are summarized in a comprehensive model at the level of atomic detail. Molecular movies show how the successive domain motions during the ATP induced actin dissociation and the recovery stroke are coupled with the precise positioning of the key catalytic groups in the active site. This leads to a precise timing of the activation of the ATPase function: it allows ATP hydrolysis only after unbinding from actin and the priming of the lever arm, both pre-requisites for an efficient functioning of the motor during the subsequent power stroke. These coupling mechanisms constitute essential principles of every myosin motor, of which the ATP-site can be seen as the central allosteric control unit. © 2016 Wiley Periodicals, Inc.

  4. Structure of the mitochondrial ATP synthase from Pichia angusta determined by electron cryo-microscopy

    PubMed Central

    Vinothkumar, Kutti R.; Montgomery, Martin G.; Liu, Sidong

    2016-01-01

    The structure of the intact monomeric ATP synthase from the fungus, Pichia angusta, has been solved by electron cryo-microscopy. The structure provides insights into the mechanical coupling of the transmembrane proton motive force across mitochondrial membranes in the synthesis of ATP. This mechanism requires a strong and integral stator, consisting of the catalytic α3β3-domain, peripheral stalk, and, in the membrane domain, subunit a and associated supernumerary subunits, kept in contact with the rotor turning at speeds up to 350 Hz. The stator’s integrity is ensured by robust attachment of both the oligomycin sensitivity conferral protein (OSCP) to the catalytic domain and the membrane domain of subunit b to subunit a. The ATP8 subunit provides an additional brace between the peripheral stalk and subunit a. At the junction between the OSCP and the apparently stiff, elongated α-helical b-subunit and associated d- and h-subunits, an elbow or joint allows the stator to bend to accommodate lateral movements during the activity of the catalytic domain. The stator may also apply lateral force to help keep the static a-subunit and rotating c10-ring together. The interface between the c10-ring and the a-subunit contains the transmembrane pathway for protons, and their passage across the membrane generates the turning of the rotor. The pathway has two half-channels containing conserved polar residues provided by a bundle of four α-helices inclined at ∼30° to the plane of the membrane, similar to those described in other species. The structure provides more insights into the workings of this amazing machine. PMID:27791192

  5. A 2.2 Å resolution structure of the USP7 catalytic domain in a new space group elaborates upon structural rearrangements resulting from ubiquitin binding.

    PubMed

    Molland, Katrina; Zhou, Qing; Mesecar, Andrew D

    2014-03-01

    A sparse-matrix screen for new crystallization conditions for the USP7 catalytic domain (USP7CD) led to the identification of a condition in which crystals grow reproducibly in 24-48 h. Variation of the halide metal, growth temperature and seed-stock concentration resulted in a shift in space group from P21 with two molecules in the asymmetric unit to C2 with one molecule in the asymmetric unit. Representative structures from each space group were determined to 2.2 Å resolution and these structures support previous findings that the catalytic triad and switching loop are likely to be in unproductive conformations in the absence of ubiquitin (Ub). Importantly, the new structures reveal previously unobserved electron density for blocking loop 1 (BL1) residues 410-419. The new structures indicate a distinct rearrangement of the USP7 BL1 compared with its position in the presence of bound Ub.

  6. Identification of cytoplasmic subdomains that control pH-sensing of the Na+/H+ exchanger (NHE1): pH-maintenance, ATP-sensitive, and flexible loop domains.

    PubMed

    Ikeda, T; Schmitt, B; Pouysségur, J; Wakabayashi, S; Shigekawa, M

    1997-02-01

    To precisely identify the cytoplasmic subdomains that are responsible for the intracellular pH (pHi)-sensitivity, ATP depletion-induced inhibition and Ca2+ activation of the Na+/H+ exchanger (NHE1), we generated a set of deletion mutants of carboxyl-terminated cytoplasmic domain and expressed them in the exchanger-deficient cell line PS120. We evaluated pHi-sensitivity of these mutants by measuring the resting pHi in cells placed in an acidic medium (pH 6.0) and pHi-dependence of 5-(N-ethyl-N-isopropyl)amiloride-sensitive 22Na+ uptake. Detailed analysis revealed that the cytoplasmic domain of NHE1 is consists of at least four subdomains in terms of pHi-sensitivity of the unstimulated NHE1: I, aa 516-590/595; II, aa 596-635; III aa 636-659; and IV, aa 660-815. Subdomains II and IV were silent for pHi-sensitivity. Subdomain I had a pHi-maintenance function, preserving pHi-sensitivity in a physiological range, whereas subdomain III, overlapping with the high affinity calmodulin (CaM)-binding site, exhibited an autoinhibitory function. Deletion of subdomain I abolished the decrease of pHi-sensitivity induced by cell ATP depletion, indicating that domain I plays a crucial role in this phenomenon. Deletion of subdomain III rendered the inhibition by ATP depletion less efficient, suggesting the possible interaction between subdomains I and III. On the other hand, tandem elongation of subdomain II by insertion did not affect either the inhibitory function of domain III or the removal of this inhibition by ionomycin or thrombin. However, deletion of subdomain II partially abolished the inhibitory effect of subdomain III. Subdomain II thus seems to function as a mobile "flexible loop," permitting the CaM-binding subdomain III to exert its normal function. These findings, together with our previous data, support a concept that cell ATP, Ca2+, and growth factors regulate NHE1 via a mechanism involving direct or indirect interactions of specific cytoplasmic subdomains with the

  7. Crystallization and atomic resolution X-ray diffraction of the catalytic domain of the large sialidase, nanI, from Clostridium perfringens.

    PubMed

    Newstead, Simon; Chien, Chin Hsiang; Taylor, Margaret; Taylor, Garry

    2004-11-01

    Sialidases catalyse the removal of terminal sialic acids from a range of glycoproteins, glycolipids and oligosaccharides. They have been found in bacteria, viruses and parasites, where they play important roles in pathogenesis and/or microbial nutrition, and in mammalian cells, where they modulate cell-surface glycosylation associated with a range of cellular activities. Clostridium perfringens, a causative agent of gas gangrene and peritonitis in humans, possesses three sialidases: nanH, nanI and nanJ, with molecular weights of 42, 77 and 129 kDa, respectively. The two larger enzymes are secreted by the bacterium and are involved in the pathogenesis and nutrition of Clostridium. As part of a study to examine the structures of all three enzymes, crystallization of the 77 kDa nanI isoenzyme was attempted. The expressed full-length protein was found to degrade easily; a stable 50 kDa catalytic domain was therefore subcloned. This domain was overexpressed in Escherichia coli and produced crystals belonging to space group P2(1)2(1)2(1), with unit-cell parameters a = 96.98, b = 69.41, c = 72.69 A and one monomer per asymmetric unit. The crystals diffract to at least 0.92 A. A molecular-replacement solution was obtained using the catalytic domain of the sialidase from the leech Macrobdella decora.

  8. Oxidative Unfolding of the Rubredoxin Domain and the Natively Disordered N-terminal Region Regulate the Catalytic Activity of Mycobacterium tuberculosis Protein Kinase G.

    PubMed

    Wittwer, Matthias; Luo, Qi; Kaila, Ville R I; Dames, Sonja A

    2016-12-30

    Mycobacterium tuberculosis escapes killing in human macrophages by secreting protein kinase G (PknG). PknG intercepts host signaling to prevent fusion of the phagosome engulfing the mycobacteria with the lysosome and, thus, their degradation. The N-terminal NORS (no regulatory secondary structure) region of PknG (approximately residues 1-75) has been shown to play a role in PknG regulation by (auto)phosphorylation, whereas the following rubredoxin-like metal-binding motif (RD, residues ∼74-147) has been shown to interact tightly with the subsequent catalytic domain (approximately residues 148-420) to mediate its redox regulation. Deletions or mutations in NORS or the redox-sensitive RD significantly decrease PknG survival function. Based on combined NMR spectroscopy, in vitro kinase assay, and molecular dynamics simulation data, we provide novel insights into the regulatory roles of the N-terminal regions. The NORS region is indeed natively disordered and rather dynamic. Consistent with most earlier data, autophosphorylation occurs in our assays only when the NORS region is present and, thus, in the NORS region. Phosphorylation of it results only in local conformational changes and does not induce interactions with the subsequent RD. Although the reduced, metal-bound RD makes tight interactions with the following catalytic domain in the published crystal structures, it can also fold in its absence. Our data further suggest that oxidation-induced unfolding of the RD regulates substrate access to the catalytic domain and, thereby, PknG function under different redox conditions, e.g. when exposed to increased levels of reactive oxidative species in host macrophages.

  9. GTP Binding and Oncogenic Mutations May Attenuate Hypervariable Region (HVR)-Catalytic Domain Interactions in Small GTPase K-Ras4B, Exposing the Effector Binding Site*

    PubMed Central

    Lu, Shaoyong; Banerjee, Avik; Jang, Hyunbum; Zhang, Jian; Gaponenko, Vadim; Nussinov, Ruth

    2015-01-01

    K-Ras4B, a frequently mutated oncogene in cancer, plays an essential role in cell growth, differentiation, and survival. Its C-terminal membrane-associated hypervariable region (HVR) is required for full biological activity. In the active GTP-bound state, the HVR interacts with acidic plasma membrane (PM) headgroups, whereas the farnesyl anchors in the membrane; in the inactive GDP-bound state, the HVR may interact with both the PM and the catalytic domain at the effector binding region, obstructing signaling and nucleotide exchange. Here, using molecular dynamics simulations and NMR, we aim to figure out the effects of nucleotides (GTP and GDP) and frequent (G12C, G12D, G12V, G13D, and Q61H) and infrequent (E37K and R164Q) oncogenic mutations on full-length K-Ras4B. The mutations are away from or directly at the HVR switch I/effector binding site. Our results suggest that full-length wild-type GDP-bound K-Ras4B (K-Ras4BWT-GDP) is in an intrinsically autoinhibited state via tight HVR-catalytic domain interactions. The looser association in K-Ras4BWT-GTP may release the HVR. Some of the oncogenic mutations weaken the HVR-catalytic domain association in the K-Ras4B-GDP/-GTP bound states, which may facilitate the HVR disassociation in a nucleotide-independent manner, thereby up-regulating oncogenic Ras signaling. Thus, our results suggest that mutations can exert their effects in more than one way, abolishing GTP hydrolysis and facilitating effector binding. PMID:26453300

  10. (1)H, (13)C and (15)N backbone resonance assignment for the 40.5 kDa catalytic domain of Ubiquitin Specific Protease 7 (USP7).

    PubMed

    Di Lello, Paola; Rougé, Lionel; Pan, Borlan; Maurer, Till

    2016-10-01

    The deubiquitinase Ubiquitin Specific Protease 7 (USP7) is part of the regulatory cascade of proteins that modulates the activity of the tumor suppressor protein p53. Deubiquitination of its target Murine Double Minute 2 (MDM2) leads to increased proteosomal degradation of p53. Consequently, USP7 has emerged as an attractive oncology target because its inhibition stabilizes p53, thereby promoting p53-dependent apoptosis in cancer cells. Here we report the backbone resonance assignment for the 40.5 kDa catalytic domain of USP7.

  11. Distortion of the catalytic domain of tissue-type plasminogen activator by plasminogen activator inhibitor-1 coincides with the formation of stable serpin-proteinase complexes.

    PubMed

    Perron, Michel J; Blouse, Grant E; Shore, Joseph D

    2003-11-28

    Plasminogen activator inhibitor-1 (PAI-1) is a typical member of the serpin family that kinetically traps its target proteinase as a covalent complex by distortion of the proteinase domain. Incorporation of the fluorescently silent 4-fluorotryptophan analog into PAI-1 permitted us to observe changes in the intrinsic tryptophan fluorescence of two-chain tissue-type plasminogen activator (tPA) and the proteinase domain of tPA during the inhibition reaction. We demonstrated three distinct conformational changes of the proteinase that occur during complex formation and distortion. A conformational change occurred during the initial formation of the non-covalent Michaelis complex followed by a large conformational change associated with the distortion of the proteinase catalytic domain that occurs concurrently with the formation of stable proteinase-inhibitor complexes. Following distortion, a very slow structural change occurs that may be involved in the stabilization or regulation of the trapped complex. Furthermore, by comparing the inhibition rates of two-chain tPA and the proteinase domain of tPA by PAI-1, we demonstrate that the accessory domains of tPA play a prominent role in the initial formation of the non-covalent Michaelis complex.

  12. Profiling Protein Kinases and Other ATP Binding Proteins in Arabidopsis Using Acyl-ATP Probes*

    PubMed Central

    Villamor, Joji Grace; Kaschani, Farnusch; Colby, Tom; Oeljeklaus, Julian; Zhao, David; Kaiser, Markus; Patricelli, Matthew P.; van der Hoorn, Renier A. L.

    2013-01-01

    Many protein activities are driven by ATP binding and hydrolysis. Here, we explore the ATP binding proteome of the model plant Arabidopsis thaliana using acyl-ATP (AcATP)1 probes. These probes target ATP binding sites and covalently label lysine residues in the ATP binding pocket. Gel-based profiling using biotinylated AcATP showed that labeling is dependent on pH and divalent ions and can be competed by nucleotides. The vast majority of these AcATP-labeled proteins are known ATP binding proteins. Our search for labeled peptides upon in-gel digest led to the discovery that the biotin moiety of the labeled peptides is oxidized. The in-gel analysis displayed kinase domains of two receptor-like kinases (RLKs) at a lower than expected molecular weight, indicating that these RLKs lost the extracellular domain, possibly as a result of receptor shedding. Analysis of modified peptides using a gel-free platform identified 242 different labeling sites for AcATP in the Arabidopsis proteome. Examination of each individual labeling site revealed a preference of labeling in ATP binding pockets for a broad diversity of ATP binding proteins. Of these, 24 labeled peptides were from a diverse range of protein kinases, including RLKs, mitogen-activated protein kinases, and calcium-dependent kinases. A significant portion of the labeling sites could not be assigned to known nucleotide binding sites. However, the fact that labeling could be competed with ATP indicates that these labeling sites might represent previously uncharacterized nucleotide binding sites. A plot of spectral counts against expression levels illustrates the high specificity of AcATP probes for protein kinases and known ATP binding proteins. This work introduces profiling of ATP binding activities of a large diversity of proteins in plant proteomes. The data have been deposited in ProteomeXchange with the identifier PXD000188. PMID:23722185

  13. Insights into the Mechanistic Basis of Plasmid-Mediated Colistin Resistance from Crystal Structures of the Catalytic Domain of MCR-1

    PubMed Central

    Hinchliffe, Philip; Yang, Qiu E.; Portal, Edward; Young, Tom; Li, Hui; Tooke, Catherine L.; Carvalho, Maria J.; Paterson, Neil G.; Brem, Jürgen; Niumsup, Pannika R.; Tansawai, Uttapoln; Lei, Lei; Li, Mei; Shen, Zhangqi; Wang, Yang; Schofield, Christopher J.; Mulholland, Adrian J; Shen, Jianzhong; Fey, Natalie; Walsh, Timothy R.; Spencer, James

    2017-01-01

    The polymixin colistin is a “last line” antibiotic against extensively-resistant Gram-negative bacteria. Recently, the mcr-1 gene was identified as a plasmid-mediated resistance mechanism in human and animal Enterobacteriaceae, with a wide geographical distribution and many producer strains resistant to multiple other antibiotics. mcr-1 encodes a membrane-bound enzyme catalysing phosphoethanolamine transfer onto bacterial lipid A. Here we present crystal structures revealing the MCR-1 periplasmic, catalytic domain to be a zinc metalloprotein with an alkaline phosphatase/sulphatase fold containing three disulphide bonds. One structure captures a phosphorylated form representing the first intermediate in the transfer reaction. Mutation of residues implicated in zinc or phosphoethanolamine binding, or catalytic activity, restores colistin susceptibility of recombinant E. coli. Zinc deprivation reduces colistin MICs in MCR-1-producing laboratory, environmental, animal and human E. coli. Conversely, over-expression of the disulphide isomerase DsbA increases the colistin MIC of laboratory E. coli. Preliminary density functional theory calculations on cluster models suggest a single zinc ion may be sufficient to support phosphoethanolamine transfer. These data demonstrate the importance of zinc and disulphide bonds to MCR-1 activity, suggest that assays under zinc-limiting conditions represent a route to phenotypic identification of MCR-1 producing E. coli, and identify key features of the likely catalytic mechanism. PMID:28059088

  14. Crystal structure of the catalytic domain of Clostridium perfringens neuraminidase in complex with a non-carbohydrate-based inhibitor, 2-(cyclohexylamino)ethanesulfonic acid.

    PubMed

    Lee, Youngjin; Youn, Hyung-Seop; Lee, Jung-Gyu; An, Jun Yop; Park, Kyoung Ryoung; Kang, Jung Youn; Ryu, Young Bae; Jin, Mi Sun; Park, Ki Hun; Eom, Soo Hyun

    2017-03-16

    Anti-bacterial and anti-viral neuraminidase agents inhibit neuraminidase activity catalyzing the hydrolysis of terminal N-acetylneuraminic acid (Neu5Ac) from glycoconjugates and help to prevent the host pathogenesis that lead to fatal infectious diseases including influenza, bacteremia, sepsis, and cholera. Emerging antibiotic and drug resistances to commonly used anti-neuraminidase agents such as oseltamivir (Tamiflu) and zanamivir (Relenza) have highlighted the need to develop new anti-neuraminidase drugs. We obtained a serendipitous complex crystal of the catalytic domain of Clostridium perfringens neuraminidase (CpNanICD) with 2-(cyclohexylamino)ethanesulfonic acid (CHES) as a buffer. Here, we report the crystal structure of CpNanICD in complex with CHES at 1.24 Å resolution. Amphipathic CHES binds to the catalytic site of CpNanICD similar to the substrate (Neu5Ac) binding site. The 2-aminoethanesulfonic acid moiety and cyclohexyl groups of CHES interact with the cluster of three arginine residues and with the hydrophobic pocket of the CpNanICD catalytic site. In addition, a structural comparison with other bacterial and human neuraminidases suggests that CHES could serve as a scaffold for the development of new anti-neuraminidase agents targeting CpNanI.

  15. An Insight into the Interaction Mode Between CheB and Chemoreceptor from Two Crystal Structures of CheB Methylesterase Catalytic Domain

    SciTech Connect

    K Cho; B Crane; S Park

    2011-12-31

    We have determined 2.2 {angstrom} resolution crystal structure of Thermotoga maritima CheB methylesterase domain to provide insight into the interaction mode between CheB and chemoreceptors. T. maritima CheB methylesterase domain has identical topology of a modified doubly-wound {alpha}/{beta} fold that was observed from the previously reported Salmonella typhimurium counterpart, but the analysis of the electrostatic potential surface near the catalytic triad indicated considerable charge distribution difference. As the CheB demethylation consensus sites of the chemoreceptors, the CheB substrate, are not uniquely conserved between T. maritima and S. typhimurium, such surfaces with differing electrostatic properties may reflect CheB regions that mediate protein-protein interaction. Via the computational docking of the two T. maritima and S. typhimurium CheB structures to the respective T. maritima and Escherichia coli chemoreceptors, we propose a CheB:chemoreceptor interaction mode.

  16. Examination of the catalytic fitness of the hammerhead ribozyme by in vitro selection.

    PubMed Central

    Tang, J; Breaker, R R

    1997-01-01

    We have designed a self-cleaving ribozyme construct that is rendered inactive during preparative in vitro transcription by allosteric interactions with ATP. This allosteric ribozyme was constructed by joining a hammerhead domain to an ATP-binding RNA aptamer, thereby creating a ribozyme whose catalytic rate can be controlled by ATP. Upon purification by PAGE, the engineered ribozyme undergoes rapid self-cleavage when incubated in the absence of ATP. This strategy of "allosteric delay" was used to prepare intact hammerhead ribozymes that would otherwise self-destruct during transcription. Using a similar strategy, we have prepared a combinatorial pool of RNA in order to assess the catalytic fitness of ribozymes that carry the natural consensus sequence for the hammerhead. Using in vitro selection, this comprehensive RNA pool was screened for sequence variants of the hammerhead ribozyme that also display catalytic activity. We find that sequences that comprise the core of naturally occurring hammerhead dominate the population of selected RNAs, indicating that the natural consensus sequence of this ribozyme is optimal for catalytic function. PMID:9257650

  17. Homology modeling, molecular docking and molecular dynamics studies of the catalytic domain of chitin deacetylase from Cryptococcus laurentii strain RY1.

    PubMed

    Sarkar, Soumyadev; Gupta, Suchetana; Chakraborty, Writachit; Senapati, Sanjib; Gachhui, Ratan

    2017-03-15

    This study provides structural insights into chitin deacetylase, over-expressing under nitrogen limiting condition in Cryptococcus laurentii strain RY1. The enzyme converts chitin, the second most abundant natural biopolymer, to chitosan, which offers tremendous applications in diverse fields. To elucidate the structure-function relationship of this biologically and industrially important enzyme, a homology model of the catalytic domain was constructed. The stability of the structure was assessed by molecular dynamics simulation studies. Tryptophan 151 of the domain was identified to form hydrogen bond and stacking interaction with chitin upon docking. In Silico substitution of Tryptophan (W) to Alanine (A), Phenylalanine (F) and Aspartate (D) corroborated the importance of the Tryptophan residue in interaction with the substrate. This is the first report of unravelling the structural characteristics of chitin deacetylase from Cryptococcus and understanding the approach of the enzyme towards its substrate. Our results would be helpful to perform experimental validations and apply quantum mechanics/molecular mechanics techniques to determine the detailed catalytic mechanism and enhance the industrial potency of the enzyme.

  18. Solution structure of the parvulin-type PPIase domain of Staphylococcus aureus PrsA – Implications for the catalytic mechanism of parvulins

    PubMed Central

    Heikkinen, Outi; Seppala, Raili; Tossavainen, Helena; Heikkinen, Sami; Koskela, Harri; Permi, Perttu; Kilpeläinen, Ilkka

    2009-01-01

    Background Staphylococcus aureus is a Gram-positive pathogenic bacterium causing many kinds of infections from mild respiratory tract infections to life-threatening states as sepsis. Recent emergence of S. aureus strains resistant to numerous antibiotics has created a need for new antimicrobial agents and novel drug targets. S. aureus PrsA is a membrane associated extra-cytoplasmic lipoprotein which contains a parvulin-type peptidyl-prolyl cis-trans isomerase domain. PrsA is known to act as an essential folding factor for secreted proteins in Gram-positive bacteria and thus it is a potential target for antimicrobial drugs against S. aureus. Results We have solved a high-resolution solution structure of the parvulin-type peptidyl-prolyl cis-trans isomerase domain of S. aureus PrsA (PrsA-PPIase). The results of substrate peptide titrations pinpoint the active site and demonstrate the substrate preference of the enzyme. With detailed NMR spectroscopic investigation of the orientation and tautomeric state of the active site histidines we are able to give further insight into the structure of the catalytic site. NMR relaxation analysis gives information on the dynamic behaviour of PrsA-PPIase. Conclusion Detailed structural description of the S. aureus PrsA-PPIase lays the foundation for structure-based design of enzyme inhibitors. The structure resembles hPin1-type parvulins both structurally and regarding substrate preference. Even though a wealth of structural data is available on parvulins, the catalytic mechanism has yet to be resolved. The structure of S. aureus PrsA-PPIase and our findings on the role of the conserved active site histidines help in designing further experiments to solve the detailed catalytic mechanism. PMID:19309529

  19. Probing the ATP hydrolysis cycle of the ABC multidrug transporter LmrA by pulsed EPR spectroscopy.

    PubMed

    Hellmich, Ute A; Lyubenova, Sevdalina; Kaltenborn, Eva; Doshi, Rupak; van Veen, Hendrik W; Prisner, Thomas F; Glaubitz, Clemens

    2012-04-04

    Members of the ATP binding cassette (ABC) transporter superfamily translocate various types of molecules across the membrane at the expense of ATP. This requires cycling through a number of catalytic states. Here, we report conformational changes throughout the catalytic cycle of LmrA, a homodimeric multidrug ABC transporter from L. lactis. Using site-directed spin labeling and pulsed electron-electron double resonance (PELDOR/DEER) spectroscopy, we have probed the reorientation of the nucleotide binding domains and transmembrane helix 6 which is of particular relevance to drug binding and part of the dimerization interface. Our data show that LmrA samples a very large conformational space in its apo state, which is significantly reduced upon nucleotide binding. ATP binding but not hydrolysis is required to trigger this conformational change, which results in a relatively fixed orientation of both the nucleotide binding domains and transmembrane helices 6. This orientation is maintained throughout the ATP hydrolysis cycle until the protein cycles back to its apo state. Our data present strong evidence that switching between two dynamically and structurally distinct states is required for substrate translocation.

  20. Torque Generation and Utilization in Motor Enzyme F0F1-ATP Synthase

    PubMed Central

    Usukura, Eiji; Suzuki, Toshiharu; Furuike, Shou; Soga, Naoki; Saita, Ei-ichiro; Hisabori, Toru; Kinosita, Kazuhiko; Yoshida, Masasuke

    2012-01-01

    ATP synthase (F0F1) is made of two motors, a proton-driven motor (F0) and an ATP-driven motor (F1), connected by a common rotary shaft, and catalyzes proton flow-driven ATP synthesis and ATP-driven proton pumping. In F1, the central γ subunit rotates inside the α3β3 ring. Here we report structural features of F1 responsible for torque generation and the catalytic ability of the low-torque F0F1. (i) Deletion of one or two turns in the α-helix in the C-terminal domain of catalytic β subunit at the rotor/stator contact region generates mutant F1s, termed F1(1/2)s, that rotate with about half of the normal torque. This helix would support the helix-loop-helix structure acting as a solid “pushrod” to push the rotor γ subunit, but the short helix in F1(1/2)s would fail to accomplish this task. (ii) Three different half-torque F0F1(1/2)s were purified and reconstituted into proteoliposomes. They carry out ATP-driven proton pumping and build up the same small transmembrane ΔpH, indicating that the final ΔpH is directly related to the amount of torque. (iii) The half-torque F0F1(1/2)s can catalyze ATP synthesis, although slowly. The rate of synthesis varies widely among the three F0F1(1/2)s, which suggests that the rate reflects subtle conformational variations of individual mutants. PMID:22128167

  1. Novel stand-alone RAM domain protein-mediated catalytic control of anthranilate phosphoribosyltransferase in tryptophan biosynthesis in Thermus thermophilus.

    PubMed

    Kubota, Tetsuo; Matsushita, Hajime; Tomita, Takeo; Kosono, Saori; Yoshida, Minoru; Kuzuyama, Tomohisa; Nishiyama, Makoto

    2017-01-01

    Regulation of amino acid metabolism (RAM) domains are widely distributed among prokaryotes. In most cases, a RAM domain fuses with a DNA-binding domain to act as a transcriptional regulator. The extremely thermophilic bacterium, Thermus thermophilus, only carries a single gene encoding a RAM domain-containing protein on its genome. This protein is a stand-alone RAM domain protein (SraA) lacking a DNA-binding domain. Therefore, we hypothesized that SraA, which senses amino acids through its RAM domain, may interact with other proteins to modify its functions. In the present study, we identified anthranilate phosphoribosyltransferase (AnPRT), the second enzyme in the tryptophan biosynthetic pathway, as a partner protein that interacted with SraA in T. thermophilus. In the presence of tryptophan, SraA was assembled to a decamer and exhibited the ability to form a stable hetero-complex with AnPRT. An enzyme assay revealed that AnPRT was only inhibited by tryptophan in the presence of SraA. This result suggests a novel feedback control mechanism for tryptophan biosynthesis through an inter-RAM domain interaction in bacteria.

  2. About a switch: how P-glycoprotein (ABCB1) harnesses the energy of ATP binding and hydrolysis to do mechanical work.

    PubMed

    Sauna, Zuben E; Ambudkar, Suresh V

    2007-01-01

    The efflux of drugs by the multidrug transporter P-glycoprotein (Pgp; ABCB1) is one of the principal means by which cancer cells evade chemotherapy and exhibit multidrug resistance. Mechanistic studies of Pgp-mediated transport, however, transcend the importance of this protein per se as they help us understand the transport pathway of the ATP-binding cassette proteins in general. The ATP-binding cassette proteins comprise one of the largest protein families, are central to cellular physiology, and constitute important drug targets. The functional unit of Pgp consists of two nucleotide-binding domains (NBD) and two transmembrane domains that are involved in the transport of drug substrates. Early studies postulated that conformational changes as a result of ATP hydrolysis were transmitted to the transmembrane domains bringing about drug transport. More recent structural and biochemical studies on the other hand suggested that ATP binds at the interface of the two NBDs and induces the formation of a closed dimer, and it has been hypothesized that this dimerization and subsequent ATP hydrolysis powers transport. Based on the mutational and biochemical work on Pgp and structural studies with isolated NBDs, we review proposed schemes for the catalytic cycle of ATP hydrolysis and the transport pathway.

  3. alpha3beta3gamma complex of F1-ATPase from thermophilic Bacillus PS3 can maintain steady-state ATP hydrolysis activity depending on the number of non-catalytic sites.

    PubMed Central

    Amano, T; Matsui, T; Muneyuki, E; Noji, H; Hara, K; Yoshida, M; Hisabori, T

    1999-01-01

    Homogeneous preparations of alpha(3)beta(3)gamma complexes with one, two or three non-competent non-catalytic site(s) were performed as described [Amano, Hisabori, Muneyuki, and Yoshida (1996) J. Biol. Chem. 271, 18128-18133] and their properties were compared with those of the wild-type complex. The ATPase activity of the complex with three non-competent non-catalytic sites decayed rapidly to an inactivated state, as reported previously [Matsui, Muneyuki, Honda, Allison, Dou, and Yoshida (1997) J. Biol. Chem. 272, 8215-8221]. In contrast, the complex with one or two non-competent non-catalytic sites displayed a substantial steady-state phase activity depending on the number of non-competent non-catalytic sites in the complex. This result indicates that one competent non-catalytic site can maintain the continuous catalytic turnover of the enzyme and can potentially relieve all three catalytic sites from inhibition by MgADP(-). Furthermore, the results suggest that the interaction between three non-catalytic sites might not be as strong as that between catalytic sites, which are all strictly required for a continuous catalytic turnover. PMID:10493921

  4. Histidine 114 Is Critical for ATP Hydrolysis by the Universally Conserved ATPase YchF*

    PubMed Central

    Rosler, Kirsten S.; Mercier, Evan; Andrews, Ian C.; Wieden, Hans-Joachim

    2015-01-01

    GTPases perform a wide range of functions, ranging from protein synthesis to cell signaling. Of all known GTPases, only eight are conserved across all three domains of life. YchF is one of these eight universally conserved GTPases; however, its cellular function and enzymatic properties are poorly understood. YchF differs from the classical GTPases in that it has a higher affinity for ATP than for GTP and is a functional ATPase. As a hydrophobic amino acid-substituted ATPase, YchF does not possess the canonical catalytic Gln required for nucleotide hydrolysis. To elucidate the catalytic mechanism of ATP hydrolysis by YchF, we have taken a two-pronged approach combining classical biochemical and in silico techniques. The use of molecular dynamics simulations allowed us to complement our biochemical findings with information about the structural dynamics of YchF. We have thereby identified the highly conserved His-114 as critical for the ATPase activity of YchF from Escherichia coli. His-114 is located in a flexible loop of the G-domain, which undergoes nucleotide-dependent conformational changes. The use of a catalytic His is also observed in the hydrophobic amino acid-substituted GTPase RbgA and is an identifier of the translational GTPase family. PMID:26018081

  5. Histidine 114 Is Critical for ATP Hydrolysis by the Universally Conserved ATPase YchF.

    PubMed

    Rosler, Kirsten S; Mercier, Evan; Andrews, Ian C; Wieden, Hans-Joachim

    2015-07-24

    GTPases perform a wide range of functions, ranging from protein synthesis to cell signaling. Of all known GTPases, only eight are conserved across all three domains of life. YchF is one of these eight universally conserved GTPases; however, its cellular function and enzymatic properties are poorly understood. YchF differs from the classical GTPases in that it has a higher affinity for ATP than for GTP and is a functional ATPase. As a hydrophobic amino acid-substituted ATPase, YchF does not possess the canonical catalytic Gln required for nucleotide hydrolysis. To elucidate the catalytic mechanism of ATP hydrolysis by YchF, we have taken a two-pronged approach combining classical biochemical and in silico techniques. The use of molecular dynamics simulations allowed us to complement our biochemical findings with information about the structural dynamics of YchF. We have thereby identified the highly conserved His-114 as critical for the ATPase activity of YchF from Escherichia coli. His-114 is located in a flexible loop of the G-domain, which undergoes nucleotide-dependent conformational changes. The use of a catalytic His is also observed in the hydrophobic amino acid-substituted GTPase RbgA and is an identifier of the translational GTPase family. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. WD40 domain of Apc1 is critical for the coactivator-induced allosteric transition that stimulates APC/C catalytic activity

    PubMed Central

    Li, Qiuhong; Chang, Leifu; Aibara, Shintaro; Yang, Jing; Zhang, Ziguo; Barford, David

    2016-01-01

    The anaphase-promoting complex/cyclosome (APC/C) is a large multimeric cullin–RING E3 ubiquitin ligase that orchestrates cell-cycle progression by targeting cell-cycle regulatory proteins for destruction via the ubiquitin proteasome system. The APC/C assembly comprises two scaffolding subcomplexes: the platform and the TPR lobe that together coordinate the juxtaposition of the catalytic and substrate-recognition modules. The platform comprises APC/C subunits Apc1, Apc4, Apc5, and Apc15. Although the role of Apc1 as an APC/C scaffolding subunit has been characterized, its specific functions in contributing toward APC/C catalytic activity are not fully understood. Here, we report the crystal structure of the N-terminal domain of human Apc1 (Apc1N) determined at 2.2-Å resolution and provide an atomic-resolution description of the architecture of its WD40 (WD40 repeat) domain (Apc1WD40). To understand how Apc1WD40 contributes to APC/C activity, a mutant form of the APC/C with Apc1WD40 deleted was generated and evaluated biochemically and structurally. We found that the deletion of Apc1WD40 abolished the UbcH10-dependent ubiquitination of APC/C substrates without impairing the Ube2S-dependent ubiquitin chain elongation activity. A cryo-EM structure of an APC/C–Cdh1 complex with Apc1WD40 deleted showed that the mutant APC/C is locked into an inactive conformation in which the UbcH10-binding site of the catalytic module is inaccessible. Additionally, an EM density for Apc15 is not visible. Our data show that Apc1WD40 is required to mediate the coactivator-induced conformational change of the APC/C that is responsible for stimulating APC/C catalytic activity by promoting UbcH10 binding. In contrast, Ube2S activity toward APC/C substrates is not dependent on the initiation-competent conformation of the APC/C. PMID:27601667

  7. Inhibition of Multidrug Resistance-Linked P-Glycoprotein (ABCB1) Function by 5′-Fluorosulfonylbenzoyl 5′-Adenosine: Evidence for an ATP Analog That Interacts With Both Drug-Substrate- and Nucleotide-Binding Sites†

    PubMed Central

    Ohnuma, Shinobu; Chufan, Eduardo; Nandigama, Krishnamachary; Miller Jenkins, Lisa M.; Durell, Stewart R.; Appella, Ettore; Sauna, Zuben E.; Ambudkar, Suresh V.

    2011-01-01

    5′-fluorosulfonylbenzonyl 5′-adenosine (FSBA) is an ATP analog that covalently modifies several residues in the nucleotide-binding domains (NBDs) of several ATPases, kinases and other proteins. P-glycoprotein (P-gp, ABCB1) is a member of the ATP-binding cassette (ABC) transporter superfamily that utilizes energy from ATP hydrolysis for the efflux of amphipathic anticancer agents from cancer cells. We investigated the interactions of FSBA with P-gp to study the catalytic cycle of ATP hydrolysis. Incubation of P-gp with FSBA inhibited ATP hydrolysis (IC50= 0.21 mM) and the binding of 8-azido[α–32P]ATP (IC50= 0.68 mM). In addition, 14C-FSBA crosslinks to P-gp, suggesting that FSBA-mediated inhibition of ATP hydrolysis is irreversible due to covalent modification of P-gp. However, when the NBDs were occupied with a saturating concentration of ATP prior to treatment, FSBA stimulated ATP hydrolysis by P-gp. Furthermore, FSBA inhibited the photocrosslinking of P-gp with [125I]-Iodoaryl-azidoprazosin (IAAP; IC50 = 0.17 mM). As IAAP is a transport substrate for P-gp, this suggests that FSBA affects not only the NBDs, but also the transport-substrate site in the transmembrane domains. Consistent with these results, FSBA blocked efflux of rhodamine 123 from P-gp-expressing cells. Additionally, mass spectrometric analysis identified FSBA crosslinks to residues within or nearby the NBDs but not in the transmembrane domains and docking of FSBA in a homology model of human P-gp NBDs supports the biochemical studies. Thus, FSBA is an ATP analog that interacts with both the drug-binding and ATP-binding sites of P-gp, but fluorosulfonyl-mediated crosslinking is observed only at the NBDs. PMID:21452853

  8. Kinetic equivalence of transmembrane pH and electrical potential differences in ATP synthesis.

    PubMed

    Soga, Naoki; Kinosita, Kazuhiko; Yoshida, Masasuke; Suzuki, Toshiharu

    2012-03-16

    ATP synthase is the key player of Mitchell's chemiosmotic theory, converting the energy of transmembrane proton flow into the high energy bond between ADP and phosphate. The proton motive force that drives this reaction consists of two components, the pH difference (ΔpH) across the membrane and transmembrane electrical potential (Δψ). The two are considered thermodynamically equivalent, but kinetic equivalence in the actual ATP synthesis is not warranted, and previous experimental results vary. Here, we show that with the thermophilic Bacillus PS3 ATP synthase that lacks an inhibitory domain of the ε subunit, ΔpH imposed by acid-base transition and Δψ produced by valinomycin-mediated K(+) diffusion potential contribute equally to the rate of ATP synthesis within the experimental range examined (ΔpH -0.3 to 2.2, Δψ -30 to 140 mV, pH around the catalytic domain 8.0). Either ΔpH or Δψ alone can drive synthesis, even when the other slightly opposes. Δψ was estimated from the Nernst equation, which appeared valid down to 1 mm K(+) inside the proteoliposomes, due to careful removal of K(+) from the lipid.

  9. Crystal Structure of the Human Pol α B Subunit in Complex with the C-terminal Domain of the Catalytic Subunit.

    PubMed

    Suwa, Yoshiaki; Gu, Jianyou; Baranovskiy, Andrey G; Babayeva, Nigar D; Pavlov, Youri I; Tahirov, Tahir H

    2015-06-05

    In eukaryotic DNA replication, short RNA-DNA hybrid primers synthesized by primase-DNA polymerase α (Prim-Pol α) are needed to start DNA replication by the replicative DNA polymerases, Pol δ and Pol ϵ. The C terminus of the Pol α catalytic subunit (p180C) in complex with the B subunit (p70) regulates the RNA priming and DNA polymerizing activities of Prim-Pol α. It tethers Pol α and primase, facilitating RNA primer handover from primase to Pol α. To understand these regulatory mechanisms and to reveal the details of human Pol α organization, we determined the crystal structure of p70 in complex with p180C. The structured portion of p70 includes a phosphodiesterase (PDE) domain and an oligonucleotide/oligosaccharide binding (OB) domain. The N-terminal domain and the linker connecting it to the PDE domain are disordered in the reported crystal structure. The p180C adopts an elongated asymmetric saddle shape, with a three-helix bundle in the middle and zinc-binding modules (Zn1 and Zn2) on each side. The extensive p180C-p70 interactions involve 20 hydrogen bonds and a number of hydrophobic interactions resulting in an extended buried surface of 4080 Å(2). Importantly, in the structure of the p180C-p70 complex with full-length p70, the residues from the N-terminal to the OB domain contribute to interactions with p180C. The comparative structural analysis revealed both the conserved features and the differences between the human and yeast Pol α complexes. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. The Gly-Arg-rich C-terminal domain of pea nucleolin is a DNA helicase that catalytically translocates in the 5'- to 3'-direction.

    PubMed

    Nasirudin, Khondaker M; Ehtesham, Nasreen Z; Tuteja, Renu; Sopory, Sudhir K; Tuteja, Narendra

    2005-02-15

    Nucleolin is a major nucleolar phosphoprotein of exponentially growing eukaryotic cells. Here we report the cloning, purification, and characterization of the C-terminal glycine/arginine-rich (GAR) domain of pea nucleolin. The purified recombinant protein (17 kDa) shows ATP-/Mg(2+)-dependent DNA helicase and ssDNA-/Mg(2+)-dependent ATPase activities. The enzyme unwinds DNA in the 5'- to 3'-direction, which is the first report in plant for this directional activity. It unwinds forked/non-forked DNA with equal efficiency. The anti-nucleolin antibodies immunodepleted the activities of the enzyme. The DNA interacting ligands nogalamycin, daunorubicin, actinomycin C1, and ethidium bromide were inhibitory to DNA unwinding (with K(i) values of 0.40, 2.21, 8.0, and 9.0 microM, respectively) and ATPase (with K(i) values of 0.43, 1.65, 4.6, and 7.0 microM, respectively) activities of the enzyme. This study confirms that the unwinding and ATPase activities of pea nucleolin resided in the GAR domain. This study should make important contribution to our better understanding of DNA transaction in plants, mechanism of DNA unwinding, and the mechanism by which these ligands can disturb genome integrity.

  11. Loss of autoinhibition of the plasma membrane Ca(2+) pump by substitution of aspartic 170 by asparagin. A ctivation of plasma membrane calcium ATPase 4 without disruption of the interaction between the catalytic core and the C-terminal regulatory domain.

    PubMed

    Bredeston, Luis M; Adamo, Hugo P

    2004-10-01

    The plasma membrane calcium ATPase (PMCA) actively transports Ca(2+) from the cytosol to the extra cellular space. The C-terminal segment of the PMCA functions as an inhibitory domain by interacting with the catalytic core. Ca(2+)-calmodulin binds to the C-terminal segment and stops inhibition. Here we showed that residue Asp(170), in the putative "A" domain of human PMCA isoform 4xb, plays a critical role in autoinhibition. In the absence of calmodulin a PMCA containing a site-specific mutation of D170N had 80% of the maximum activity of the calmodulin-activated PMCA and a similar high affinity for Ca(2+). The mutation did not change the activation of the PMCA by ATP. Deletion of the C-terminal segment further downstream of the calmodulin-binding site led to an additional increase in the maximal activity of the mutant, which suggests that the mutation did not affect the inhibition because of this portion of the C-terminal segment. The calmodulin-activated PMCA was more sensitive to vanadate inhibition than the autoinhibited enzyme. In contrast, inhibition of the D170N mutant required higher concentrations of vanadate and was not affected by calmodulin. Despite its higher basal activity, the mutant had an apparent affinity for calmodulin similar to that of the wild type enzyme, and its rate of proteolysis at the C-terminal segment was still calmodulin-dependent. Altogether these results suggest that activation by mutation D170N does not involve the displacement of the calmodulin-binding autoinhibitory domain from the catalytic core and may arise directly from changes in the accessibility to the calcium-binding residues of the pump.

  12. Improving the reversibility of thermal denaturation and catalytic efficiency of Bacillus licheniformis α-amylase through stabilizing a long loop in domain B

    PubMed Central

    Li, Zhu; Duan, Xuguo; Chen, Sheng; Wu, Jing

    2017-01-01

    The reversibility of thermal denaturation and catalytic efficiency of Bacillus licheniformis α-amylase were improved through site-directed mutagenesis. By using multiple sequence alignment and PoPMuSiC algorithm, Ser187 and Asn188, which located within a long loop in Domain B of Bacillus licheniformis α-amylase, were selected for mutation. In addition, Ala269, which is adjacent to Ser187 and Asn188, was also investigated. Seven mutants carrying the mutations S187D, N188T, N188S, A269K, A269K/S187D, S187D/N188T, and A269K/S187D/N188T were generated and characterized. The most thermostable mutant, A269K/S187D/N188T, exhibited a 9-fold improvement in half-life at 95°C and pH 5.5, compared with that of the wild-type enzyme. Mutant A269K/S187D/N188T also exhibited improved catalytic efficiency. The catalytic efficiency of mutant A269K/S187D/N188T reached 5.87×103±0.17 g·L-1·s-1 at pH 5.5, which is 1.84-fold larger than the corresponding value determined for the wild-type enzyme. Furthermore, the structure analysis showed that immobilization of the loop containing Ser187 and Asn188 plays a significant role in developing the properties of Bacillus licheniformis α-amylase. PMID:28253342

  13. The catalytic domain CysPc of the DEK1 calpain is functionally conserved in land plants.

    PubMed

    Liang, Zhe; Demko, Viktor; Wilson, Robert C; Johnson, Kenneth A; Ahmad, Rafi; Perroud, Pierre-François; Quatrano, Ralph; Zhao, Sen; Shalchian-Tabrizi, Kamran; Otegui, Marisa S; Olsen, Odd-Arne; Johansen, Wenche

    2013-09-01

    DEK1, the single calpain of land plants, is a member of the ancient membrane bound TML-CysPc-C2L calpain family that dates back 1.5 billion years. Here we show that the CysPc-C2L domains of land plant calpains form a separate sub-clade in the DEK1 clade of the phylogenetic tree of plants. The charophycean alga Mesostigma viride DEK1-like gene is clearly divergent from those in land plants, suggesting that a major evolutionary shift in DEK1 occurred during the transition to land plants. Based on genetic complementation of the Arabidopsis thaliana dek1-3 mutant using CysPc-C2L domains of various origins, we show that these two domains have been functionally conserved within land plants for at least 450 million years. This conclusion is based on the observation that the CysPc-C2L domains of DEK1 from the moss Physcomitrella patens complements the A. thaliana dek1-3 mutant phenotype. In contrast, neither the CysPc-C2L domains from M. viride nor chimeric animal-plant calpains complement this mutant. Co-evolution analysis identified differences in the interactions between the CysPc-C2L residues of DEK1 and classical calpains, supporting the view that the two enzymes are regulated by fundamentally different mechanisms. Using the A. thaliana dek1-3 complementation assay, we show that four conserved amino acid residues of two Ca²⁺-binding sites in the CysPc domain of classical calpains are conserved in land plants and functionally essential in A. thaliana DEK1.

  14. Structural elucidation of transmembrane domain zero (TMD0) of EcdL: A multidrug resistance-associated protein (MRP) family of ATP-binding cassette transporter protein revealed by atomistic simulation.

    PubMed

    Bera, Krishnendu; Rani, Priyanka; Kishor, Gaurav; Agarwal, Shikha; Kumar, Antresh; Singh, Durg Vijay

    2017-09-20

    ATP-Binding cassette (ABC) transporters play an extensive role in the translocation of diverse sets of biologically important molecules across membrane. EchnocandinB (antifungal) and EcdL protein of Aspergillus rugulosus are encoded by the same cluster of genes. Co-expression of EcdL and echinocandinB reflects tightly linked biological functions. EcdL belongs to Multidrug Resistance associated Protein (MRP) subfamily of ABC transporters with an extra transmembrane domain zero (TMD0). Complete structure of MRP subfamily comprising of TMD0 domain, at atomic resolution is not known. We hypothesized that the transportation of echonocandinB is mediated via EcdL protein. Henceforth, it is pertinent to know the topological arrangement of TMD0, with other domains of protein and its possible role in transportation of echinocandinB. Absence of effective template for TMD0 domain lead us to model by I-TASSER, further structure has been refined by multiple template modelling using homologous templates of remaining domains (TMD1, NBD1, TMD2, NBD2). The modelled structure has been validated for packing, folding and stereochemical properties. MD simulation for 0.1 μs has been carried out in the biphasic environment for refinement of modelled protein. Non-redundant structures have been excavated by clustering of MD trajectory. The structural alignment of modelled structure has shown Z-score -37.9; 31.6, 31.5 with RMSD; 2.4, 4.2, 4.8 with ABC transporters; PDB ID 4F4C, 4M1 M, 4M2T, respectively, reflecting the correctness of structure. EchinocandinB has been docked to the modelled as well as to the clustered structures, which reveals interaction of echinocandinB with TMD0 and other TM helices in the translocation path build of TMDs.

  15. Unraveling the molecular structure of the catalytic domain of matrix metalloproteinase-2 in complex with a triple-helical peptide by means of molecular dynamics simulations.

    PubMed

    Díaz, Natalia; Suárez, Dimas; Valdés, Haydeé

    2013-11-26

    Herein, we present the results of a computational study that employed various simulation methodologies to build and validate a series of molecular models of a synthetic triple-helical peptide (fTHP-5) both in its native state and in a prereactive complex with the catalytic domain of the MMP-2 enzyme. First, the structure and dynamical properties of the fTHP-5 substrate are investigated by means of molecular dynamics (MD) simulations. Then, the propensity of each of the three peptide chains in fTHP-5 to be distorted around the scissile peptide bond is assessed by carrying out potential of mean force calculations. Subsequently, the distorted geometries of fTHP-5 are docked within the MMP-2 active site following a semirigid protocol, and the most stable docked structures are fully relaxed and characterized by extensive MD simulations in explicit solvent. Following a similar approach, we also investigate a hypothetical ternary complex formed between two MMP-2 catalytic units and a single fTHP-5 molecule. Overall, our models for the MMP-2/fTHP-5 complexes unveil the extent to which the triple helix is distorted to allow the accommodation of an individual peptide chain within the MMP active site.

  16. Critical Role of a Loop at C-Terminal Domain on the Conformational Stability and Catalytic Efficiency of Chondroitinase ABC I.

    PubMed

    Akram Shirdel, S; Khalifeh, Khosrow; Golestani, Abolfazl; Ranjbar, Bijan; Khajeh, Khosro

    2015-08-01

    We used a combination of protein engineering and spectroscopic methods to investigate the effect of a long length loop on the conformational stability and activity of chondroitinase ABC I. This study involves manipulation of interactions around Asp(689) as a key residue in the central region of the loop containing residues 681-695 located at C-terminal domain of the enzyme. According to the equilibrium unfolding experiments and considering thermodynamic m value and ΔG(H2O), we found that the folded state of H700N, L701T, and H700N/L701T are more compact relative to the folded state of wild-type protein and they become stabilized upon mutation. However, the compactness and stability of other variants are less than those of wild-type protein. According to enzyme activity measurements, we found that the catalytic efficiency of structurally stabilized variants is decreased, while that of destabilized mutants is improved.

  17. Conformational movements and cooperativity upon amino acid, ATP and tRNA binding in threonyl-tRNA synthetase.

    PubMed

    Torres-Larios, Alfredo; Sankaranarayanan, Rajan; Rees, Bernard; Dock-Bregeon, Anne Catherine; Moras, Dino

    2003-08-01

    The crystal structures of threonyl-tRNA synthetase (ThrRS) from Staphylococcus aureus, with ATP and an analogue of threonyl adenylate, are described. Together with the previously determined structures of Escherichia coli ThrRS with different substrates, they allow a comprehensive analysis of the effect of binding of all the substrates: threonine, ATP and tRNA. The tRNA, by inserting its acceptor arm between the N-terminal domain and the catalytic domain, causes a large rotation of the former. Within the catalytic domain, four regions surrounding the active site display significant conformational changes upon binding of the different substrates. The binding of threonine induces the movement of as much as 50 consecutive amino acid residues. The binding of ATP triggers a displacement, as large as 8A at some C(alpha) positions, of a strand-loop-strand region of the core beta-sheet. Two other regions move in a cooperative way upon binding of threonine or ATP: the motif 2 loop, which plays an essential role in the first step of the aminoacylation reaction, and the ordering loop, which closes on the active site cavity when the substrates are in place. The tRNA interacts with all four mobile regions, several residues initially bound to threonine or ATP switching to a position in which they can contact the tRNA. Three such conformational switches could be identified, each of them in a different mobile region. The structural analysis suggests that, while the small substrates can bind in any order, they must be in place before productive tRNA binding can occur.

  18. The Rotary Mechanism of the ATP Synthase

    PubMed Central

    Nakamoto, Robert K.; Scanlon, Joanne A. Baylis; Al-Shawi, Marwan K.

    2008-01-01

    The FOF1 ATP synthase is a large complex of at least 22 subunits, more than half of which are in the membranous FO sector. This nearly ubiquitous transporter is responsible for the majority of ATP synthesis in oxidative and photo-phosphorylation, and its overall structure and mechanism have remained conserved throughout evolution. Most examples utilize the proton motive force to drive ATP synthesis except for a few bacteria, which use a sodium motive force. A remarkable feature of the complex is the rotary movement of an assembly of subunits that plays essential roles in both transport and catalytic mechanisms. This review addresses the role of rotation in catalysis of ATP synthesis/hydrolysis and the transport of protons or sodium. PMID:18515057

  19. Human cap methyltransferase (RNMT) N-terminal non-catalytic domain mediates recruitment to transcription initiation sites

    PubMed Central

    Aregger, Michael; Cowling, Victoria H.

    2013-01-01

    Gene expression in eukaryotes is dependent on the mRNA methyl cap which mediates mRNA processing and translation initiation. Synthesis of the methyl cap initiates with the addition of 7-methylguanosine to the initiating nucleotide of RNA pol II (polymerase II) transcripts, which occurs predominantly during transcription and in mammals is catalysed by RNGTT (RNA guanylyltransferase and 5′ phosphatase) and RNMT (RNA guanine-7 methyltransferase). RNMT has a methyltransferase domain and an N-terminal domain whose function is unclear; it is conserved in mammals, but not required for cap methyltransferase activity. In the present study we report that the N-terminal domain is necessary and sufficient for RNMT recruitment to transcription initiation sites and that recruitment occurs in a DRB (5,6-dichloro-1-β-D-ribofuranosylbenzimidazole)-dependent manner. The RNMT-activating subunit, RAM (RNMT-activating miniprotein), is also recruited to transcription initiation sites via an interaction with RNMT. The RNMT N-terminal domain is required for transcript expression, translation and cell proliferation. PMID:23863084

  20. Evidence for Two Catalytic Sites in the Functional Unit of H+-ATPase from Higher Plants.

    PubMed Central

    Roberts, G.; Berberian, G.; Beauge, L.

    1995-01-01

    We investigated the nature of the complex ATP activation kinetics of plant H+-ATPases. To this aim we analyzed that activation in three isolated isoforms (AHA1, AHA2, and AHA3) of H+-ATPase from Arabidopsis thaliana. The isoforms were obtained by heterologous expression in endoplasmic reticulum of yeast. ATP stimulation was always with low affinity (K0.5 between 500 and 1800 [mu]M). In addition, the curves were not Michaelian and displayed positive cooperativity. Detailed studies with AHA2 showed that (a) enzyme solubilized with lysophosphatidylcholine exhibited Michaelian behavior even in the presence of soybean lecithin liposomes free of enzyme, (b) solubilized enzyme incorporated into the same liposomes displayed two-site kinetics with negative cooperativity, and (c) enzyme partially digested with trypsin lost the C-terminal portion of the molecule. Under this condition the ATP activation kinetics was Michaelian or had a slight negative cooperativity and the K0.5ATP was reduced 3-fold. These data suggest that the functional unit of the H+-ATPase has two catalytic ATP sites with variable cooperativity and kinetics competence of the E(ATP) and E(ATP)2 complexes. Such variability is likely modulated by the association of the enzyme with membrane structures and by a regulatory domain in the C terminus of the enzyme molecule. PMID:12228512

  1. Crystal structure of the catalytic domain of PigE: a transaminase involved in the biosynthesis of 2-methyl-3-n-amyl-pyrrole (MAP) from Serratia sp. FS14.

    PubMed

    Lou, Xiangdi; Ran, Tingting; Han, Ning; Gao, Yanyan; He, Jianhua; Tang, Lin; Xu, Dongqing; Wang, Weiwu

    2014-04-25

    Prodigiosin, a tripyrrole red pigment synthesized by Serratia and some other microbes through a bifurcated biosynthesis pathway, MBC (4-methoxy-2,2'-bipyrrole-5-carbaldehyde) and MAP (2-methyl-3-n-amyl-pyrrole) are synthesized separately and then condensed by PigC to form prodigiosin. MAP is synthesized sequentially by PigD, PigE and PigB. PigE catalyzes the transamination of an amino group to the aldehyde group of 3-acetyloctanal, resulting in an aminoketone, which spontaneously cyclizes to form H2MAP. Here we report the crystal structure of the catalytic domain of PigE which involved in the biosynthesis of prodigiosin precursor MAP for the first time to a resolution of 2.3Å with a homodimer in the asymmetric unit. The monomer of PigE catalytic domain is composed of three domains with PLP as cofactor: a small N-terminal domain connecting the catalytic domain with the front part of PigE, a large PLP-binding domain and a C-terminal domain. The residues from both monomers build the PLP binding site at the interface of the dimer which resembles the other PLP-dependent enzymes. Structural comparison of PigE with Thermus thermophilus AcOAT showed a higher hydrophobic and smaller active site of PigE, these differences may be the reason for substrate specificity.

  2. ATP6AP1 — EDRN Public Portal

    Cancer.gov

    ATP6AP1 is an integral membrane protein composed of at least 10 subunits. It is responsible for acidifying various eukaryotic intracellular organelles. ATP6AP1, also known as vacuolar ATPase or V-ATPase, has a cytosolic V1 domain and a transmembrane V0 domain. The acidification performed by ATP6AP1 is necessary for intracellular processes such as protein sorting, zymogen activation, and receptor-mediated endocytosis.

  3. Purification and crystallization of the catalytic PRONE domain of RopGEF8 and its complex with Rop4 from Arabidopsis thaliana

    SciTech Connect

    Thomas, Christoph; Weyand, Michael; Wittinghofer, Alfred; Berken, Antje

    2006-06-01

    Crystals of the catalytic PRONE domain of the guanine nucleotide exchange factor RopGEF8 and its complex with the Rho-family protein Rop4 from A. thaliana were obtained that diffract to 2.2 and 3.1 Å resolution, respectively. The PRONE domain of the guanine nucleotide exchange factor RopGEF8 (PRONE8) was purified and crystallized free and in complex with the Rho-family protein Rop4 using the hanging-drop vapour-diffusion method. PRONE8 crystals were obtained using NaCl as precipitating agent and belong to the hexagonal space group P6{sub 5}22. Native and anomalous data sets were collected using synchrotron radiation at 100 K to 2.2 and 2.8 Å resolution, respectively. Crystals of the Rop4–PRONE8 complex belonging to space group P6{sub 3} were obtained using Tacsimate and PEG 3350 as precipitating agents and diffracted to 3.1 Å resolution.

  4. Lactobacillus plantarum WCFS1 β-Fructosidase: Evidence for an Open Funnel-Like Channel Through the Catalytic Domain with Importance for the Substrate Selectivity.

    PubMed

    Mendoza-Llerenas, Edgar Omar; Pérez, David Javier; Gómez-Sandoval, Zeferino; Escalante-Minakata, Pilar; Ibarra-Junquera, Vrani; Razo-Hernández, Rodrigo Said; Capozzi, Vittorio; Russo, Pasquale; Spano, Giuseppe; Fiocco, Daniela; Osuna-Castro, Juan Alberto; Moreno, Abel

    2016-11-01

    β-Fructosidase, a glycoside hydrolase of a biotechnologically important strain, was studied for its biochemical, physicochemical, and three-dimensional structure characteristics. This enzyme was heterologously expressed in Escherichia coli as a C-terminal His-tagged protein (SacB). β-Fructosidase catalyzes the cleavage of glycoside bonds toward certain carbohydrates with β-fructofuranosyl linkages; however, SacB exhibited selectivity toward sucrose and an optimum activity at pH 6.0-6.5 and 37 °C. In such optimum enzymatic activity conditions, the SacB was commonly observed as a monodisperse protein by dynamic light scattering (DLS). As β-fructosidase belongs to glycoside hydrolase family 32 (GH32), a β-sandwich and a five-bladed β-propeller domain are typical predicted folds in its structure. Docking and molecular dynamic simulations revealed for the first time a funnel-like channel perfectly exposed in the β-propeller domain of the Lactobacillus plantarum β-fructosidase (this allows the interaction between its entire catalytic triad and substrates that are larger than sucrose). In contrast, SacB showed a closed central tunnel collaterally induced by its His-tag.

  5. Dynamic regulation of extracellular ATP in Escherichia coli.

    PubMed

    Alvarez, Cora Lilia; Corradi, Gerardo; Lauri, Natalia; Marginedas-Freixa, Irene; Leal Denis, María Florencia; Enrique, Nicolás; Mate, Sabina María; Milesi, Verónica; Ostuni, Mariano Anibal; Herlax, Vanesa; Schwarzbaum, Pablo Julio

    2017-04-04

    We studied the kinetics of extracellular ATP (ATPe) in Escherichia coli and their outer membrane vesicles (OMVs) stimulated with amphipatic peptides melittin (MEL) and mastoparan 7 (MST7). Real-time luminometry was used to measure ATPe kinetics, ATP release, and ATPase activity. The latter was also determined by following [(32)P]Pi released from [γ-(32)P]ATP. E. coli was studied alone, co-incubated with Caco-2 cells, or in rat jejunum segments. In E. coli, the addition of [γ-(32)P]ATP led to the uptake and subsequent hydrolysis of ATPe. Exposure to peptides caused an acute 3-fold (MST7) and 7-fold (MEL) increase in [ATPe]. In OMVs, ATPase activity increased linearly with [ATPe] (0.1-1 µM). Exposure to MST7 and MEL enhanced ATP release by 3-7 fold, with similar kinetics to that of bacteria. In Caco-2 cells, the addition of ATP to the apical domain led to a steep [ATPe] increase to a maximum, with subsequent ATPase activity. The addition of bacterial suspensions led to a 6-7 fold increase in [ATPe], followed by an acute decrease. In perfused jejunum segments, exposure to E. coli increased luminal ATP 2 fold. ATPe regulation of E. coli depends on the balance between ATPase activity and ATP release. This balance can be altered by OMVs, which display their own capacity to regulate ATPe. E. coli can activate ATP release from Caco-2 cells and intestinal segments, a response which in vivo might lead to intestinal release of ATP from the gut lumen.

  6. 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

  7. Structural model of the cytosolic domain of the plant ethylene receptor 1 (ETR1).

    PubMed

    Mayerhofer, Hubert; Panneerselvam, Saravanan; Kaljunen, Heidi; Tuukkanen, Anne; Mertens, Haydyn D T; Mueller-Dieckmann, Jochen

    2015-01-30

    Ethylene initiates important aspects of plant growth and development through disulfide-linked receptor dimers located in the endoplasmic reticulum. The receptors feature a small transmembrane, ethylene binding domain followed by a large cytosolic domain, which serves as a scaffold for the assembly of large molecular weight complexes of different ethylene receptors and other cellular participants of the ethylene signaling pathway. Here we report the crystallographic structures of the ethylene receptor 1 (ETR1) catalytic ATP-binding and the ethylene response sensor 1 dimerization histidine phosphotransfer (DHp) domains and the solution structure of the entire cytosolic domain of ETR1, all from Arabidopsis thaliana. The isolated dimeric ethylene response sensor 1 DHp domain is asymmetric, the result of different helical bending angles close to the conserved His residue. The structures of the catalytic ATP-binding, DHp, and receiver domains of ethylene receptors and of a homologous, but dissimilar, GAF domain were refined against experimental small angle x-ray scattering data, leading to a structural model of the entire cytosolic domain of the ethylene receptor 1. The model illustrates that the cytosolic domain is shaped like a dumbbell and that the receiver domain is flexible and assumes a position different from those observed in prokaryotic histidine kinases. Furthermore the cytosolic domain of ETR1 plays a key role, interacting with all other receptors and several participants of the ethylene signaling pathway. Our model, therefore, provides the first step toward a detailed understanding of the molecular mechanics of this important signal transduction process in plants.

  8. Magnetic field affects enzymatic ATP synthesis.

    PubMed

    Buchachenko, Anatoly L; Kuznetsov, Dmitry A

    2008-10-01

    The rate of ATP synthesis by creatine kinase extracted from V. xanthia venom was shown to depend on the magnetic field. The yield of ATP produced by enzymes with 24Mg2+ and 26Mg2+ ions in catalytic sites increases by 7-8% at 55 mT and then decreases at 80 mT. For enzyme with 25Mg2+ ion in a catalytic site, the ATP yield increases by 50% and 70% in the fields 55 and 80 mT, respectively. In the Earth field the rate of ATP synthesis by enzyme, in which Mg2+ ion has magnetic nucleus 25Mg, is 2.5 times higher than that by enzymes, in which Mg2+ ion has nonmagnetic, spinless nuclei 24Mg or 26Mg. Both magnetic field effect and magnetic isotope effect demonstrate that the ATP synthesis is an ion-radical process, affected by Zeeman interaction and hyperfine coupling in the intermediate ion-radical pair.

  9. Systematic Domain Swaps of Iterative, Nonreducing Polyketide Synthases Provide a Mechanistic Understanding and Rationale For Catalytic Reprogramming

    PubMed Central

    2015-01-01

    Iterative, nonreducing polyketide synthases (NR-PKSs) are multidomain enzymes responsible for the construction of the core architecture of aromatic polyketide natural products in fungi. Engineering these enzymes for the production of non-native metabolites has been a long-standing goal. We conducted a systematic survey of in vitro “domain swapped” NR-PKSs using an enzyme deconstruction approach. The NR-PKSs were dissected into mono- to multidomain fragments and recombined as noncognate pairs in vitro, reconstituting enzymatic activity. The enzymes used in this study produce aromatic polyketides that are representative of the four main chemical features set by the individual NR-PKS: starter unit selection, chain-length control, cyclization register control, and product release mechanism. We found that boundary conditions limit successful chemistry, which are dependent on a set of underlying enzymatic mechanisms. Crucial for successful redirection of catalysis, the rate of productive chemistry must outpace the rate of spontaneous derailment and thioesterase-mediated editing. Additionally, all of the domains in a noncognate system must interact efficiently if chemical redirection is to proceed. These observations refine and further substantiate current understanding of the mechanisms governing NR-PKS catalysis. PMID:24815013

  10. Structural Insight into the Mechanism of Substrate Specificity and Catalytic Activity of an HD-Domain Phosphohydrolase: The 5;#8242;-Deoxyribonucleotidase YfbR from Escherichia coli

    SciTech Connect

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

    2011-08-16

    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-{angstrom}-resolution crystal structures (one in the free state and two complexed with natural substrates) of an HD-domain phosphohydrolase, the Escherichia 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 Co{sup 2+} and either thymidine-5'-monophosphate or 2'-deoxyriboadenosine-5'-monophosphate disclose the novel binding mode of deoxyribonucleotides in the active site. Residue R18 stabilizes the phosphate on the Co{sup 2+}, 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 deoxyriboadenosine-5'-monophosphate and thymidine-5'-monophosphate 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 an HD-domain phosphohydrolase based directly on substrate-bound crystal structures.

  11. The catalytic domains of Clostridium sordellii lethal toxin and related large clostridial glucosylating toxins specifically recognize the negatively charged phospholipids phosphatidylserine and phosphatidic acid.

    PubMed

    Varela Chavez, Carolina; Hoos, Sylviane; Haustant, Georges Michel; Chenal, Alexandre; England, Patrick; Blondel, Arnaud; Pauillac, Serge; Lacy, D Borden; Popoff, Michel Robert

    2015-10-01

    Clostridium sordellii lethal toxin (TcsL) is a potent virulence factor belonging to the large clostridial glucosylating toxin family. TcsL enters target cells via receptor-mediated endocytosis and delivers the N-terminal catalytic domain (TcsL-cat) into the cytosol upon an autoproteolytic process. TcsL-cat inactivates small GTPases including Rac and Ras by glucosylation with uridine-diphosphate (UDP)-glucose as cofactor leading to drastic changes in cytoskeleton and cell viability. TcsL-cat was found to preferentially bind to phosphatidylserine (PS)-containing membranes and to increase the glucosylation of Rac anchored to lipid membrane. We here report binding affinity measurements of TcsL-cat for brain PS-containing membranes by surface plasmon resonance and enzyme-linked immunosorbent assay (ELISA). In addition, TcsL-cat bound to phosphatidic acid (PA) and, to a lesser extent, to other anionic lipids, but not to neutral lipids, sphingolipids or sterol. We further show that the lipid unsaturation status influenced TcsL-cat binding to phospholipids, PS with unsaturated acyl chains and PA with saturated acyl chains being the preferred bindingsubstrates. Phospholipid binding site is localized at the N-terminal four helical bundle structure (1-93 domain). However, TcsL-1-93 bound to a broad range of substrates, whereas TcsL-cat, which is the active domain physiologically delivered into the cytosol, selectively bound to PS and PA. Similar findings were observed with the other large clostridial glucosylating toxins from C. difficile, C. novyi and C. perfringens. © 2015 John Wiley & Sons Ltd.

  12. Communication between catalytic and regulatory subunits in Ni(II)- and Co(II)-aspartate transcarbamoylase. Ligand-promoted structural alterations at the intersubunit bonding domains.

    PubMed

    Johnson, R S; Schachman, H K

    1983-03-25

    The cooperativity with respect to substrates exhibited by allosteric enzymes as well as their inhibition and activation by effectors is mediated through the propagation of conformational changes from the site of ligand binding on one polypeptide chain to other, unliganded chains. This "communication" between subunits in the enzyme is achieved in part by changes at the intersubunit bonding domains as the oligomeric enzymes are converted from the low affinity state to a conformation of high affinity. Structural alterations at the bonding domains between the catalytic and regulatory subunits of aspartate transcarbamoylase from Escherichia coli were detected in derivatives containing either nickel or cobalt in place of the endogenous zinc ion bound to each of the six regulatory chains. These metal-substituted derivatives exhibited physical-chemical and allosteric properties identical to those of the native enzyme. Spectral analyses indicated that both nickel and cobalt are bound to the enzyme in a distorted tetrahedral environment. With the nickel ions serving as spectral probes for structural alterations at the intersubunit bonding domains, it was observed that the substrate, carbamoyl phosphate, and the bisubstrate ligand, N-(phosphonacetyl)-L-aspartate caused different changes. The substrate analog, succinate, alone had no effect. However, the combination of both carbamoyl phosphate and succinate produced the same spectral change as that caused by the bisubstrate analog. Circular dichroism measurements on the nickel derivative showed that the perturbation at 360 nm was directly proportional to the extent of occupancy of the six active sites by the bisubstrate ligand. In contrast, the changes at 406 and 460 nm coincided with the gross conformational change measured by the decrease in the sedimentation coefficient of the enzyme. These latter changes in the circular dichroism and the alterations in the absorption spectrum were complete even though about one-third of

  13. Interactions between beta D372 and gamma subunit N-terminus residues gamma K9 and gamma S12 are important to catalytic activity catalyzed by Escherichia coli F1F0-ATP synthase.

    PubMed

    Lowry, David S; Frasch, Wayne D

    2005-05-17

    Substitution of Escherichia coli F(1)F(0) ATP synthase residues betaD372 or gammaS12 with groups that are unable to form a hydrogen bond at this location decreased ATP synthase-dependent cell growth by 2 orders of magnitude, eliminated the ability of F(1)F(0) to catalyze ATPase-dependent proton pumping in inverted E. coli membranes, caused a 15-20% decrease in the coupling efficiency of the membranes as measured by the extent of succinate-dependent acridine orange fluorescence quenching, but increased soluble F(1)-ATPase activity by about 10%. Substitution of gammaK9 to eliminate the ability to form a salt bridge with betaD372 decreased soluble F(1)-ATPase activity and ATPase-driven proton pumping by 2-fold but had no effect on the proton gradient induced by addition of succinate. Mutations to eliminate the potential to form intersubunit hydrogen bonds and salt bridges between other less highly conserved residues on the gamma subunit N-terminus and the beta subunits had little effect on ATPase or ATP synthase activities. These results suggest that the betaD372-gammaK9 salt bridge contributes significantly to the rate-limiting step in ATP hydrolysis of soluble F(1) while the betaD372-gammaS12 hydrogen bond may serve as a component of an escapement mechanism for ATP synthesis in which alphabetagamma intersubunit interactions provide a means to make substrate binding a prerequisite of proton gradient-driven gamma subunit rotation.

  14. ATP dependent charge movement in ATP7B Cu+-ATPase is demonstrated by pre-steady state electrical measurements.

    PubMed

    Tadini-Buoninsegni, Francesco; Bartolommei, Gianluca; Moncelli, Maria Rosa; Pilankatta, Rajendra; Lewis, David; Inesi, Giuseppe

    2010-11-19

    ATP7B is a copper dependent P-type ATPase, required for copper homeostasis. Taking advantage of high yield heterologous expression of recombinant protein, we investigated charge transfer in ATP7B. We detected charge displacement within a single catalytic cycle upon ATP addition and formation of phosphoenzyme intermediate. We attribute this charge displacement to movement of bound copper within ATP7B. Based on specific mutations, we demonstrate that enzyme activation by copper requires occupancy of a site in the N-terminus extension which is not present in other transport ATPases, as well as of a transmembrane site corresponding to the cation binding site of other ATPases.

  15. Parasite-specific inserts in the bifunctional S-adenosylmethionine decarboxylase/ornithine decarboxylase of Plasmodium falciparum modulate catalytic activities and domain interactions.

    PubMed Central

    Birkholtz, Lyn-Marie; Wrenger, Carsten; Joubert, Fourie; Wells, Gordon A; Walter, Rolf D; Louw, Abraham I

    2004-01-01

    Polyamine biosynthesis of the malaria parasite, Plasmodium falciparum, is regulated by a single, hinge-linked bifunctional PfAdoMetDC/ODC [ P. falciparum AdoMetDC (S-adenosylmethionine decarboxylase)/ODC (ornithine decarboxylase)] with a molecular mass of 330 kDa. The bifunctional nature of AdoMetDC/ODC is unique to Plasmodia and is shared by at least three species. The PfAdoMetDC/ODC contains four parasite-specific regions ranging in size from 39 to 274 residues. The significance of the parasite-specific inserts for activity and protein-protein interactions of the bifunctional protein was investigated by a single- and multiple-deletion strategy. Deletion of these inserts in the bifunctional protein diminished the corresponding enzyme activity and in some instances also decreased the activity of the neighbouring, non-mutated domain. Intermolecular interactions between AdoMetDC and ODC appear to be vital for optimal ODC activity. Similar results have been reported for the bifunctional P. falciparum dihydrofolate reductase-thymidylate synthase [Yuvaniyama, Chitnumsub, Kamchonwongpaisan, Vanichtanankul, Sirawaraporn, Taylor, Walkinshaw and Yuthavong (2003) Nat. Struct. Biol. 10, 357-365]. Co-incubation of the monofunctional, heterotetrameric approximately 150 kDa AdoMetDC domain with the monofunctional, homodimeric ODC domain (approximately 180 kDa) produced an active hybrid complex of 330 kDa. The hinge region is required for bifunctional complex formation and only indirectly for enzyme activities. Deletion of the smallest, most structured and conserved insert in the ODC domain had the biggest impact on the activities of both decarboxylases, homodimeric ODC arrangement and hybrid complex formation. The remaining large inserts are predicted to be non-globular regions located on the surface of these proteins. The large insert in AdoMetDC in contrast is not implicated in hybrid complex formation even though distinct interactions between this insert and the two domains

  16. Thiol oxidation of mitochondrial F0-c subunits: a way to switch off antimicrobial drug targets of the mitochondrial ATP synthase.

    PubMed

    Nesci, S; Ventrella, V; Trombetti, F; Pirini, M; Pagliarani, A

    2014-08-01

    A primary goal in antimicrobial drug design is to find molecules which inhibit key proteins in bacteria without affecting mammalian homologues. To this aim, structural differences between eukaryotic and prokaryotic enzyme proteins involved in life processes are widely exploited. The membrane-bound enzyme complex ATP synthase synthesizes the energy currency molecule of the cell. Due to its bioenergetic role, it represents "the enzyme of life" of all living beings. The enzyme complex has the unique bi-functional property of exploiting either the electrochemical transmembrane gradient to make ATP or, conversely, the free energy of ATP hydrolysis to build an electrochemical gradient across the membrane. The catalytic mechanism of ATP synthesis/hydrolysis, based on the coupling between the two rotary sectors FO and F1 is shared by eukaryotes and prokaryotes. However slight structural differences distinguish prokaryotic ATP synthases, embedded in cell membrane, from eukaryotic ones localized in the mitochondrial inner membrane. In spite of its fundamental task in living organisms, up to now the ATP synthase has been poorly exploited as target in antibacterial therapy, mainly due to harmful effects on patients. Recent advances shoulder the use of drugs targeting the ATP synthase to fight mycobacteria and treat human tuberculosis. Macrolide antibiotics and other antimicrobial drugs specifically bind to the c-ring of the membrane-embedded FO domain, thus blocking ion translocation through FO which is essential for both ATP synthesis and ATP hydrolysis. Our findings show that, once bound to the ATP synthase, probably through different binding sites on a common binding region on FO, the macrolide antibiotics oligomycin, venturicidin and bafilomycin behave as enzyme inhibitors. Interestingly, the c subunits of mitochondrial ATP synthase contain conserved cysteine residues which are absent in bacteria. We pointed out that when these crucial cysteine thiols are oxidized, the

  17. In silico rational design and systems engineering of disulfide bridges in the catalytic domain of an alkaline α-amylase from Alkalimonas amylolytica to improve thermostability.

    PubMed

    Liu, Long; Deng, Zhuangmei; Yang, Haiquan; Li, Jianghua; Shin, Hyun-dong; Chen, Rachel R; Du, Guocheng; Chen, Jian

    2014-02-01

    High thermostability is required for alkaline α-amylases to maintain high catalytic activity under the harsh conditions used in textile production. In this study, we attempted to improve the thermostability of an alkaline α-amylase from Alkalimonas amylolytica through in silico rational design and systems engineering of disulfide bridges in the catalytic domain. Specifically, 7 residue pairs (P35-G426, Q107-G167, G116-Q120, A147-W160, G233-V265, A332-G370, and R436-M480) were chosen as engineering targets for disulfide bridge formation, and the respective residues were replaced with cysteines. Three single disulfide bridge mutants-P35C-G426C, G116C-Q120C, and R436C-M480C-of the 7 showed significantly enhanced thermostability. Combinational mutations were subsequently assessed, and the triple mutant P35C-G426C/G116C-Q120C/R436C-M480C showed a 6-fold increase in half-life at 60°C and a 5.2°C increase in melting temperature compared with the wild-type enzyme. Interestingly, other biochemical properties of this mutant also improved: the optimum temperature increased from 50°C to 55°C, the optimum pH shifted from 9.5 to 10.0, the stable pH range extended from 7.0 to 11.0 to 6.0 to 12.0, and the catalytic efficiency (kcat/Km) increased from 1.8 × 10(4) to 2.4 × 10(4) liters/g · min. The possible mechanism responsible for these improvements was explored through comparative analysis of the model structures of wild-type and mutant enzymes. The disulfide bridge engineering strategy used in this work may be applied to improve the thermostability of other industrial enzymes.

  18. In Silico Rational Design and Systems Engineering of Disulfide Bridges in the Catalytic Domain of an Alkaline α-Amylase from Alkalimonas amylolytica To Improve Thermostability

    PubMed Central

    Liu, Long; Deng, Zhuangmei; Yang, Haiquan; Li, Jianghua; Shin, Hyun-dong; Chen, Rachel R.

    2014-01-01

    High thermostability is required for alkaline α-amylases to maintain high catalytic activity under the harsh conditions used in textile production. In this study, we attempted to improve the thermostability of an alkaline α-amylase from Alkalimonas amylolytica through in silico rational design and systems engineering of disulfide bridges in the catalytic domain. Specifically, 7 residue pairs (P35-G426, Q107-G167, G116-Q120, A147-W160, G233-V265, A332-G370, and R436-M480) were chosen as engineering targets for disulfide bridge formation, and the respective residues were replaced with cysteines. Three single disulfide bridge mutants—P35C-G426C, G116C-Q120C, and R436C-M480C—of the 7 showed significantly enhanced thermostability. Combinational mutations were subsequently assessed, and the triple mutant P35C-G426C/G116C-Q120C/R436C-M480C showed a 6-fold increase in half-life at 60°C and a 5.2°C increase in melting temperature compared with the wild-type enzyme. Interestingly, other biochemical properties of this mutant also improved: the optimum temperature increased from 50°C to 55°C, the optimum pH shifted from 9.5 to 10.0, the stable pH range extended from 7.0 to 11.0 to 6.0 to 12.0, and the catalytic efficiency (kcat/Km) increased from 1.8 × 104 to 2.4 × 104 liters/g · min. The possible mechanism responsible for these improvements was explored through comparative analysis of the model structures of wild-type and mutant enzymes. The disulfide bridge engineering strategy used in this work may be applied to improve the thermostability of other industrial enzymes. PMID:24212581

  19. Conserved mechanisms of microtubule-stimulated ADP release, ATP binding, and force generation in transport kinesins

    PubMed Central

    Atherton, Joseph; Farabella, Irene; Yu, I-Mei; Rosenfeld, Steven S; Houdusse, Anne; Topf, Maya; Moores, Carolyn A

    2014-01-01

    Kinesins are a superfamily of microtubule-based ATP-powered motors, important for multiple, essential cellular functions. How microtubule binding stimulates their ATPase and controls force generation is not understood. To address this fundamental question, we visualized microtubule-bound kinesin-1 and kinesin-3 motor domains at multiple steps in their ATPase cycles—including their nucleotide-free states—at ∼7 Å resolution using cryo-electron microscopy. In both motors, microtubule binding promotes ordered conformations of conserved loops that stimulate ADP release, enhance microtubule affinity and prime the catalytic site for ATP binding. ATP binding causes only small shifts of these nucleotide-coordinating loops but induces large conformational changes elsewhere that allow force generation and neck linker docking towards the microtubule plus end. Family-specific differences across the kinesin–microtubule interface account for the distinctive properties of each motor. Our data thus provide evidence for a conserved ATP-driven mechanism for kinesins and reveal the critical mechanistic contribution of the microtubule interface. DOI: http://dx.doi.org/10.7554/eLife.03680.001 PMID:25209998

  20. The c-Ring of the F1FO-ATP Synthase: Facts and Perspectives.

    PubMed

    Nesci, Salvatore; Trombetti, Fabiana; Ventrella, Vittoria; Pagliarani, Alessandra

    2016-04-01

    The F1FO-ATP synthase is the only enzyme in nature endowed with bi-functional catalytic mechanism of synthesis and hydrolysis of ATP. The enzyme functions, not only confined to energy transduction, are tied to three intrinsic features of the annular arrangement of c subunits which constitutes the so-called c-ring, the core of the membrane-embedded FO domain: (i) the c-ring constitution is linked to the number of ions (H(+) or Na(+)) channeled across the membrane during the dissipation of the transmembrane electrochemical gradient, which in turn determines the species-specific bioenergetic cost of ATP, the "molecular currency unit" of energy transfer in all living beings; (ii) the c-ring is increasingly involved in the mitochondrial permeability transition, an event linked to cell death and to most mitochondrial dysfunctions; (iii) the c subunit species-specific amino acid sequence and susceptibility to post-translational modifications can address antibacterial drug design according to the model of enzyme inhibitors which target the c subunits. Therefore, the simple c-ring structure not only allows the F1FO-ATP synthase to perform the two opposite tasks of molecular machine of cell life and death, but it also amplifies the enzyme's potential role as a drug target.

  1. The activity of the murine DNA methyltransferase Dnmt1 is controlled by interaction of the catalytic domain with the N-terminal part of the enzyme leading to an allosteric activation of the enzyme after binding to methylated DNA.

    PubMed

    Fatemi, M; Hermann, A; Pradhan, S; Jeltsch, A

    2001-06-22

    The mammalian DNA methyltransferase Dnmt1 is responsible for the maintenance of the pattern of DNA methylation in vivo. It is a large multidomain enzyme comprising 1620 amino acid residues. We have purified and characterized individual domains of Dnmt1 (NLS-containing domain, NlsD, amino acid residues: 1-343; replication foci-directing domain, 350-609; Zn-binding domain (ZnD), 613-748; polybromo domain, 746-1110; and the catalytic domain (CatD), 1124-1620). CatD, ZnD and NlsD bind to DNA, demonstrating the existence of three independent DNA-binding sites in Dnmt1. CatD shows a preference for binding to hemimethylated CpG-sites; ZnD prefers methylated CpGs; and NlsD specifically binds to CpG-sites, but does not discriminate between unmethylated and methylated DNA. These results are not compatible with the suggestion that the target recognition domain of Dnmt1 resides in the N terminus of the enzyme. We show by protein-protein interaction assays that ZnD and CatD interact with each other. The isolated catalytic domain does not methylate DNA, neither alone nor in combination with other domains. Full-length Dnmt1 was purified from baculovirus-infected insect cells. Under the experimental conditions, Dnmt1 has a strong (50-fold) preference for hemimethylated DNA. Dnmt1 is stimulated to methylate unmodified CpG sites by the addition of fully methylated DNA. This effect is dependent on Zn, suggesting that binding of methylated DNA to ZnD triggers the allosteric activation of the catalytic center of Dnmt1. The allosteric activation model can explain kinetic data obtained by others. It suggests that Dnmt1 might be responsible for spreading of methylation, a process that is observed during aging and carcenogenesis but may be important for de novo methylation of DNA. Copyright 2001 Academic Press.

  2. Entropy increases from different sources support the high-affinity binding of the N-terminal inhibitory domains of tissue inhibitors of metalloproteinases to the catalytic domains of matrix metalloproteinases-1 and -3.

    PubMed

    Wu, Ying; Wei, Shuo; Van Doren, Steven R; Brew, Keith

    2011-05-13

    The avid binding of tissue inhibitors of metalloproteinases (TIMPs) to matrix metalloproteinases (MMPs) is crucial for the regulation of pericellular and extracellular proteolysis. The interactions of the catalytic domain (cd) of MMP-1 with the inhibitory domains of TIMP-1 and TIMP-2 (N-TIMPs) and MMP-3cd with N-TIMP-2 have been characterized by isothermal titration calorimetry and compared with published data for the N-TIMP-1/MMP-3cd interaction. All interactions are largely driven by increases in entropy but there are significant differences in the profiles for the interactions of both N-TIMPs with MMP-1cd as compared with MMP-3cd; the enthalpy change ranges from small for MMP-1cd to highly unfavorable for MMP-3cd (-0.1 ± 0.7 versus 6.0 ± 0.5 kcal mol(-1)). The heat capacity change (ΔC(p)) of binding to MMP-1cd (temperature dependence of ΔH) is large and negative (-210 ± 20 cal K(-1) mol(-1)), indicating a large hydrophobic contribution, whereas the ΔC(p) values for the binding to MMP-3cd are much smaller (-53 ± 3 cal K(-1) mol(-1)), and some of the entropy increase may arise from increased conformational entropy. Apart from differences in ionization effects, it appears that the properties of the MMP may have a predominant influence in the thermodynamic profiles for these N-TIMP/MMP interactions.

  3. Computer modelling reveals new conformers of the ATP binding loop of Na+/K+-ATPase involved in the transphosphorylation process of the sodium pump

    PubMed Central

    Tejral, Gracian; Sopko, Bruno; Necas, Alois; Schoner, Wilhelm

    2017-01-01

    Hydrolysis of ATP by Na+/K+-ATPase, a P-Type ATPase, catalyzing active Na+ and K+ transport through cellular membranes leads transiently to a phosphorylation of its catalytical α-subunit. Surprisingly, three-dimensional molecular structure analysis of P-type ATPases reveals that binding of ATP to the N-domain connected by a hinge to the P-domain is much too far away from the Asp369 to allow the transfer of ATP’s terminal phosphate to its aspartyl-phosphorylation site. In order to get information for how the transfer of the γ-phosphate group of ATP to the Asp369 is achieved, analogous molecular modeling of the M4–M5 loop of ATPase was performed using the crystal data of Na+/K+-ATPase of different species. Analogous molecular modeling of the cytoplasmic loop between Thr338 and Ile760 of the α2-subunit of Na+/K+-ATPase and the analysis of distances between the ATP binding site and phosphorylation site revealed the existence of two ATP binding sites in the open conformation; the first one close to Phe475 in the N-domain, the other one close to Asp369 in the P-domain. However, binding of Mg2+•ATP to any of these sites in the “open conformation” may not lead to phosphorylation of Asp369. Additional conformations of the cytoplasmic loop were found wobbling between “open conformation” <==> “semi-open conformation <==> “closed conformation” in the absence of 2Mg2+•ATP. The cytoplasmic loop’s conformational change to the “semi-open conformation”—characterized by a hydrogen bond between Arg543 and Asp611—triggers by binding of 2Mg2+•ATP to a single ATP site and conversion to the “closed conformation” the phosphorylation of Asp369 in the P-domain, and hence the start of Na+/K+-activated ATP hydrolysis. PMID:28316890

  4. Characterization of the molecular basis of group II intron RNA recognition by CRS1-CRM domains.

    PubMed

    Keren, Ido; Klipcan, Liron; Bezawork-Geleta, Ayenachew; Kolton, Max; Shaya, Felix; Ostersetzer-Biran, Oren

    2008-08-22

    CRM (chloroplast RNA splicing and ribosome maturation) is a recently recognized RNA-binding domain of ancient origin that has been retained in eukaryotic genomes only within the plant lineage. Whereas in bacteria CRM domains exist as single domain proteins involved in ribosome maturation, in plants they are found in a family of proteins that contain between one and four repeats. Several members of this family with multiple CRM domains have been shown to be required for the splicing of specific plastidic group II introns. Detailed biochemical analysis of one of these factors in maize, CRS1, demonstrated its high affinity and specific binding to the single group II intron whose splicing it facilitates, the plastid-encoded atpF intron RNA. Through its association with two intronic regions, CRS1 guides the folding of atpF intron RNA into its predicted "catalytically active" form. To understand how multiple CRM domains cooperate to achieve high affinity sequence-specific binding to RNA, we analyzed the RNA binding affinity and specificity associated with each individual CRM domain in CRS1; whereas CRM3 bound tightly to the RNA, CRM1 associated specifically with a unique region found within atpF intron domain I. CRM2, which demonstrated only low binding affinity, also seems to form specific interactions with regions localized to domains I, III, and IV. We further show that CRM domains share structural similarities and RNA binding characteristics with the well known RNA recognition motif domain.

  5. Polyphosphate-dependent synthesis of ATP and ADP by the family-2 polyphosphate kinases in bacteria.

    PubMed

    Nocek, Boguslaw; Kochinyan, Samvel; Proudfoot, Michael; Brown, Greg; Evdokimova, Elena; Osipiuk, Jerzy; Edwards, Aled M; Savchenko, Alexei; Joachimiak, Andrzej; Yakunin, Alexander F

    2008-11-18

    Inorganic polyphosphate (polyP) is a linear polymer of tens or hundreds of phosphate residues linked by high-energy bonds. It is found in all organisms and has been proposed to serve as an energy source in a pre-ATP world. This ubiquitous and abundant biopolymer plays numerous and vital roles in metabolism and regulation in prokaryotes and eukaryotes, but the underlying molecular mechanisms for most activities of polyP remain unknown. In prokaryotes, the synthesis and utilization of polyP are catalyzed by 2 families of polyP kinases, PPK1 and PPK2, and polyphosphatases. Here, we present structural and functional characterization of the PPK2 family. Proteins with a single PPK2 domain catalyze polyP-dependent phosphorylation of ADP to ATP, whereas proteins containing 2 fused PPK2 domains phosphorylate AMP to ADP. Crystal structures of 2 representative proteins, SMc02148 from Sinorhizobium meliloti and PA3455 from Pseudomonas aeruginosa, revealed a 3-layer alpha/beta/alpha sandwich fold with an alpha-helical lid similar to the structures of microbial thymidylate kinases, suggesting that these proteins share a common evolutionary origin and catalytic mechanism. Alanine replacement mutagenesis identified 9 conserved residues, which are required for activity and include the residues from both Walker A and B motifs and the lid. Thus, the PPK2s represent a molecular mechanism, which potentially allow bacteria to use polyP as an intracellular energy reserve for the generation of ATP and survival.

  6. Unique Biological Properties of Catalytic Domain Directed Human Anti-CAIX Antibodies Discovered through Phage-Display Technology

    PubMed Central

    Xu, Chen; Lo, Agnes; Yammanuru, Anuradha; Tallarico, Aimee St. Clair; Brady, Kristen; Murakami, Akikazu; Barteneva, Natasha; Zhu, Quan; Marasco, Wayne A.

    2010-01-01

    Carbonic anhydrase IX (CAIX, gene G250/MN-encoded transmembrane protein) is highly expressed in various human epithelial tumors such as renal clear cell carcinoma (RCC), but absent from the corresponding normal tissues. Besides the CA signal transduction activity, CAIX may serve as a biomarker in early stages of oncogenesis and also as a reliable marker of hypoxia, which is associated with tumor resistance to chemotherapy and radiotherapy. Although results from preclinical and clinical studies have shown CAIX as a promising target for detection and therapy for RCC, only a limited number of murine monoclonal antibodies (mAbs) and one humanized mAb are available for clinical testing and development. In this study, paramagnetic proteoliposomes of CAIX (CAIX-PMPLs) were constructed and used for anti-CAIX antibody selection from our 27 billion human single-chain antibody (scFv) phage display libraries. A panel of thirteen human scFvs that specifically recognize CAIX expressed on cell surface was identified, epitope mapped primarily to the CA domain, and affinity-binding constants (KD) determined. These human anti-CAIX mAbs are diverse in their functions including induction of surface CAIX internalization into endosomes and inhibition of the carbonic anhydrase activity, the latter being a unique feature that has not been previously reported for anti-CAIX antibodies. These human anti-CAIX antibodies are important reagents for development of new immunotherapies and diagnostic tools for RCC treatment as well as extending our knowledge on the basic structure-function relationships of the CAIX molecule. PMID:20224781

  7. An efficient approach to identify ilvA mutations reveals an amino-terminal catalytic domain in biosynthetic threonine deaminase from Escherichia coli.

    PubMed Central

    Fisher, K E; Eisenstein, E

    1993-01-01

    High-level expression of the regulatory enzyme threonine deaminase in Escherichia coli strains grown on minimal medium that are deficient in the activities of enzymes needed for branched-chain amino acid biosynthesis result in growth inhibition, possibly because of the accumulation of toxic levels of alpha-ketobutyrate, the product of the committed step in isoleucine biosynthesis. This condition affords a means for selecting genetic variants of threonine deaminase that are deficient in catalysis by suppression of growth inhibition. Strains harboring mutations in ilvA that decreased the catalytic activity of threonine deaminase were found to grow more rapidly than isogenic strains containing wild-type ilvA. Modification of the ilvA gene to introduce additional unique, evenly spaced restriction enzyme sites facilitated the identification of suppressor mutations by enabling small DNA fragments to be subcloned for sequencing. The 10 mutations identified in ilvA code for enzymes with significantly reduced activity relative to that of wild-type threonine deaminase. Values for their specific activities range from 40% of that displayed by wild-type enzyme to complete inactivation as evidenced by failure to complement an ilvA deletion strain to isoleucine prototrophy. Moreover, some mutant enzymes showed altered allosteric properties with respect to valine activation and isoleucine inhibition. The location of the 10 mutations in the 5' two-thirds of the ilvA gene is consistent with suggestions that threonine deaminase is organized functionally with an amino-terminal domain that is involved in catalysis and a carboxy-terminal domain that is important for regulation. Images PMID:8407838

  8. Conformational transitions of the catalytic domain of heme-regulated eukaryotic initiation factor 2α kinase, a key translational regulatory molecule.

    PubMed

    Sreejith, R K; Suresh, C G; Bhosale, Siddharth H; Bhavnani, Varsha; Kumar, Avinash; Gaikwad, Sushama M; Pal, Jayanta K

    2012-01-01

    In mammalian cells, the heme-regulated inhibitor (HRI) plays a critical role in the regulation of protein synthesis at the initiation step through phosphorylation of α-subunit of the eukaryotic initiation factor 2 (eIF2). In this study we have cloned and performed biophysical characterization of the kinase catalytic domain (KD) of rabbit HRI. The KD described here comprises kinase 1, the kinase insertion domain (KI) and kinase 2. We report here the existence of an active and stable monomer of HRI (KD). The HRI (KD) containing three tryptophan residues was examined for its conformational transitions occurring under various denaturing conditions using steady-state and time-resolved tryptophan fluorescence, circular dichroism (CD) and hydrophobic dye binding. The parameter A and phase diagram analysis revealed multi-state unfolding and existence of three stable intermediates during guanidine hydrochloride (Gdn-HCl) induced unfolding of HRI (KD). The protein treated with 6 M Gdn-HCl showed collisional and static mechanism of acrylamide quenching and the constants (K(sv) = 3.08 M(-1) and K(s)= 5.62 M(-1)) were resolved using time resolved fluorescence titration. Based on pH, guanidine hydrochloride and temperature mediated transitions, HRI (KD) appears to exemplify a rigid molten globule-like intermediate with compact secondary structure, altered tertiary structure and exposed hydrophobic patches at pH 3.0. The results indicate the inherent structural stability of HRI (KD), a member of the class of stress response proteins.

  9. Aerobic Growth of Escherichia coli Is Reduced, and ATP Synthesis Is Selectively Inhibited when Five C-terminal Residues Are Deleted from the ϵ Subunit of ATP Synthase.

    PubMed

    Shah, Naman B; Duncan, Thomas M

    2015-08-21

    F-type ATP synthases are rotary nanomotor enzymes involved in cellular energy metabolism in eukaryotes and eubacteria. The ATP synthase from Gram-positive and -negative model bacteria can be autoinhibited by the C-terminal domain of its ϵ subunit (ϵCTD), but the importance of ϵ inhibition in vivo is unclear. Functional rotation is thought to be blocked by insertion of the latter half of the ϵCTD into the central cavity of the catalytic complex (F1). In the inhibited state of the Escherichia coli enzyme, the final segment of ϵCTD is deeply buried but has few specific interactions with other subunits. This region of the ϵCTD is variable or absent in other bacteria that exhibit strong ϵ-inhibition in vitro. Here, genetically deleting the last five residues of the ϵCTD (ϵΔ5) caused a greater defect in respiratory growth than did the complete absence of the ϵCTD. Isolated membranes with ϵΔ5 generated proton-motive force by respiration as effectively as with wild-type ϵ but showed a nearly 3-fold decrease in ATP synthesis rate. In contrast, the ϵΔ5 truncation did not change the intrinsic rate of ATP hydrolysis with membranes. Further, the ϵΔ5 subunit retained high affinity for isolated F1 but reduced the maximal inhibition of F1-ATPase by ϵ from >90% to ∼20%. The results suggest that the ϵCTD has distinct regulatory interactions with F1 when rotary catalysis operates in opposite directions for the hydrolysis or synthesis of ATP.

  10. CDKL5 gene status in female patients with epilepsy and Rett-like features: two new mutations in the catalytic domain

    PubMed Central

    2012-01-01

    Background Mutations in the cyclin-dependent kinase-like 5 gene (CDKL5) located in the Xp22 region have been shown to cause a subset of atypical Rett syndrome with infantile spasms or early seizures starting in the first postnatal months. Methods We performed mutation screening of CDKL5 in 60 female patients who had been identified as negative for the methyl CpG-binding protein 2 gene (MECP2) mutations, but who had current or past epilepsy, regardless of the age of onset, type, and severity. All the exons in the CDKL5 gene and their neighbouring sequences were examined, and CDKL5 rearrangements were studied by multiplex ligation-dependent probe amplification (MLPA). Results Six previously unidentified DNA changes were detected, two of which were disease-causing mutations in the catalytic domain: a frameshift mutation (c.509_510insGT; p.Glu170GlyfsX36) and a complete deletion of exon 10. Both were found in patients with seizures that started in the first month of life. Conclusions This study demonstrated the importance of CDKL5 mutations as etiological factors in neurodevelopmental disorders, and indicated that a thorough analysis of the CDKL5 gene sequence and its rearrangements should be considered in females with Rett syndrome-like phenotypes, severe encephalopathy and epilepsy with onset before 5 months of age. This study also confirmed the usefulness of MLPA as a diagnostic screening method for use in clinical practice. PMID:22867051

  11. Nuclear dynamics of topoisomerase IIβ reflects its catalytic activity that is regulated by binding of RNA to the C-terminal domain

    PubMed Central

    Onoda, Akihisa; Hosoya, Osamu; Sano, Kuniaki; Kiyama, Kazuko; Kimura, Hiroshi; Kawano, Shinji; Furuta, Ryohei; Miyaji, Mary; Tsutsui, Ken; Tsutsui, Kimiko M.

    2014-01-01

    DNA topoisomerase II (topo II) changes DNA topology by cleavage/re-ligation cycle(s) and thus contributes to various nuclear DNA transactions. It is largely unknown how the enzyme is controlled in a nuclear context. Several studies have suggested that its C-terminal domain (CTD), which is dispensable for basal relaxation activity, has some regulatory influence. In this work, we examined the impact of nuclear localization on regulation of activity in nuclei. Specifically, human cells were transfected with wild-type and mutant topo IIβ tagged with EGFP. Activity attenuation experiments and nuclear localization data reveal that the endogenous activity of topo IIβ is correlated with its subnuclear distribution. The enzyme shuttles between an active form in the nucleoplasm and a quiescent form in the nucleolus in a dynamic equilibrium. Mechanistically, the process involves a tethering event with RNA. Isolated RNA inhibits the catalytic activity of topo IIβ in vitro through the interaction with a specific 50-residue region of the CTD (termed the CRD). Taken together, these results suggest that both the subnuclear distribution and activity regulation of topo IIβ are mediated by the interplay between cellular RNA and the CRD. PMID:25034690

  12. Distinct functional and conformational states of the human lymphoid tyrosine phosphatase catalytic domain can be targeted by choice of the inhibitor chemotype

    NASA Astrophysics Data System (ADS)

    Vidović, Dušica; Xie, Yuli; Rinderspacher, Alison; Deng, Shi-Xian; Landry, Donald W.; Chung, Caty; Smith, Deborah H.; Tautz, Lutz; Schürer, Stephan C.

    2011-09-01

    The lymphoid tyrosine phosphatase (LYP), encoded by the PTPN22 gene, has recently been identified as a promising drug target for human autoimmunity diseases. Like the majority of protein-tyrosine phosphatases LYP can adopt two functionally distinct forms determined by the conformation of the WPD-loop. The WPD-loop plays an important role in the catalytic dephosphorylation by protein-tyrosine phosphatases. Here we investigate the binding modes of two chemotypes of small molecule LYP inhibitors with respect to both protein conformations using computational modeling. To evaluate binding in the active form, we built a LYP protein structure model of high quality. Our results suggest that the two different compound classes investigated, bind to different conformations of the LYP phosphatase domain. Binding to the closed form is facilitated by an interaction with Asp195 in the WPD-loop, presumably stabilizing the active conformation. The analysis presented here is relevant for the design of inhibitors that specifically target either the closed or the open conformation of LYP in order to achieve better selectivity over phosphatases with similar binding sites.

  13. Imaging Adenosine Triphosphate (ATP)

    PubMed Central

    Rajendran, Megha; Dane, Eric; Conley, Jason; Tantama, Mathew

    2016-01-01

    Adenosine triphosphate (ATP) is a universal mediator of metabolism and signaling across unicellular and multicellular species. There is a fundamental interdependence between the dynamics of ATP and the physiology that occurs inside and outside the cell. Characterizing and understanding ATP dynamics provides valuable mechanistic insight into processes that range from neurotransmission to the chemotaxis of immune cells. Therefore, we require the methodology to interrogate both temporal and spatial components of ATP dynamics from the subcellular to organismal levels in live specimens. Over the last several decades, a number of molecular probes that are specific for ATP have been developed. These probes have been combined with imaging approaches, particularly optical microscopy, to enable qualitative and quantitative detection of this critical molecule. In this review, we survey current examples of technologies that are available to visualize ATP in living cells and identify areas where new tools and approaches are needed to expand our capabilities. PMID:27638696

  14. Functional domains of plant chimeric calcium/calmodulin-dependent protein kinase: regulation by autoinhibitory and visinin-like domains

    NASA Technical Reports Server (NTRS)

    Ramachandiran, S.; Takezawa, D.; Wang, W.; Poovaiah, B. W.

    1997-01-01

    A novel calcium-binding calcium/calmodulin-dependent protein kinase (CCaMK) with a catalytic domain, calmodulin-binding domain, and a neural visinin-like domain was cloned and characterized from plants [Patil et al., (1995) Proc. Natl. Acad. Sci. USA 92, 4797-4801; Takezawa et al. (1996) J. Biol. Chem. 271, 8126-8132]. The mechanisms of CCaMK activation by calcium and calcium/calmodulin were investigated using various deletion mutants. The use of deletion mutants of CCaMK lacking either one, two, or all three calcium-binding EF hands indicated that all three calcium-binding sites in the visinin-like domain were crucial for the full calcium/calmodulin-dependent kinase activity. As each calcium-binding EF hand was deleted, there was a gradual reduction in calcium/calmodulin-dependent kinase activity from 100 to 4%. Another mutant (amino acids 1-322) which lacks both the visinin-like domain containing three EF hands and the calmodulin-binding domain was constitutively active, indicating the presence of an autoinhibitory domain around the calmodulin-binding domain. By using various synthetic peptides and the constitutively active mutant, we have shown that CCaMK contains an autoinhibitory domain within the residues 322-340 which overlaps its calmodulin-binding domain. Kinetic studies with both ATP and the GS peptide substrate suggest that the autoinhibitory domain of CCaMK interacts only with the peptide substrate binding motif of the catalytic domain, but not with the ATP-binding motif.

  15. Functional domains of plant chimeric calcium/calmodulin-dependent protein kinase: regulation by autoinhibitory and visinin-like domains

    NASA Technical Reports Server (NTRS)

    Ramachandiran, S.; Takezawa, D.; Wang, W.; Poovaiah, B. W.

    1997-01-01

    A novel calcium-binding calcium/calmodulin-dependent protein kinase (CCaMK) with a catalytic domain, calmodulin-binding domain, and a neural visinin-like domain was cloned and characterized from plants [Patil et al., (1995) Proc. Natl. Acad. Sci. USA 92, 4797-4801; Takezawa et al. (1996) J. Biol. Chem. 271, 8126-8132]. The mechanisms of CCaMK activation by calcium and calcium/calmodulin were investigated using various deletion mutants. The use of deletion mutants of CCaMK lacking either one, two, or all three calcium-binding EF hands indicated that all three calcium-binding sites in the visinin-like domain were crucial for the full calcium/calmodulin-dependent kinase activity. As each calcium-binding EF hand was deleted, there was a gradual reduction in calcium/calmodulin-dependent kinase activity from 100 to 4%. Another mutant (amino acids 1-322) which lacks both the visinin-like domain containing three EF hands and the calmodulin-binding domain was constitutively active, indicating the presence of an autoinhibitory domain around the calmodulin-binding domain. By using various synthetic peptides and the constitutively active mutant, we have shown that CCaMK contains an autoinhibitory domain within the residues 322-340 which overlaps its calmodulin-binding domain. Kinetic studies with both ATP and the GS peptide substrate suggest that the autoinhibitory domain of CCaMK interacts only with the peptide substrate binding motif of the catalytic domain, but not with the ATP-binding motif.

  16. Mechanically driven ATP synthesis by F1-ATPase

    NASA Astrophysics Data System (ADS)

    Itoh, Hiroyasu; Takahashi, Akira; Adachi, Kengo; Noji, Hiroyuki; Yasuda, Ryohei; Yoshida, Masasuke; Kinosita, Kazuhiko

    2004-01-01

    ATP, the main biological energy currency, is synthesized from ADP and inorganic phosphate by ATP synthase in an energy-requiring reaction. The F1 portion of ATP synthase, also known as F1-ATPase, functions as a rotary molecular motor: in vitro its γ-subunit rotates against the surrounding α3β3 subunits, hydrolysing ATP in three separate catalytic sites on the β-subunits. It is widely believed that reverse rotation of the γ-subunit, driven by proton flow through the associated Fo portion of ATP synthase, leads to ATP synthesis in biological systems. Here we present direct evidence for the chemical synthesis of ATP driven by mechanical energy. We attached a magnetic bead to the γ-subunit of isolated F1 on a glass surface, and rotated the bead using electrical magnets. Rotation in the appropriate direction resulted in the appearance of ATP in the medium as detected by the luciferase-luciferin reaction. This shows that a vectorial force (torque) working at one particular point on a protein machine can influence a chemical reaction occurring in physically remote catalytic sites, driving the reaction far from equilibrium.

  17. Catalytic Function and Substrate Specificity of the Papain-Like Protease Domain of nsp3 from the Middle East Respiratory Syndrome Coronavirus

    PubMed Central

    Báez-Santos, Yahira M.; Mielech, Anna M.; Deng, Xufang; Baker, Susan

    2014-01-01

    ABSTRACT The papain-like protease (PLpro) domain from the deadly Middle East respiratory syndrome coronavirus (MERS-CoV) was overexpressed and purified. MERS-CoV PLpro constructs with and without the putative ubiquitin-like (UBL) domain at the N terminus were found to possess protease, deubiquitinating, deISGylating, and interferon antagonism activities in transfected HEK293T cells. The quaternary structure and substrate preferences of MERS-CoV PLpro were determined and compared to those of severe acute respiratory syndrome coronavirus (SARS-CoV) PLpro, revealing prominent differences between these closely related enzymes. Steady-state kinetic analyses of purified MERS-CoV and SARS-CoV PLpros uncovered significant differences in their rates of hydrolysis of 5-aminomethyl coumarin (AMC) from C-terminally labeled peptide, ubiquitin, and ISG15 substrates, as well as in their rates of isopeptide bond cleavage of K48- and K63-linked polyubiquitin chains. MERS-CoV PLpro was found to have 8-fold and 3,500-fold higher catalytic efficiencies for hydrolysis of ISG15-AMC than for hydrolysis of the Ub-AMC and Z-RLRGG-AMC substrates, respectively. A similar trend was observed for SARS-CoV PLpro, although it was much more efficient than MERS-CoV PLpro toward ISG15-AMC and peptide-AMC substrates. MERS-CoV PLpro was found to process K48- and K63-linked polyubiquitin chains at similar rates and with similar debranching patterns, producing monoubiquitin species. However, SARS-CoV PLpro much preferred K48-linked polyubiquitin chains to K63-linked chains, and it rapidly produced di-ubiquitin molecules from K48-linked chains. Finally, potent inhibitors of SARS-CoV PLpro were found to have no effect on MERS-CoV PLpro. A homology model of the MERS-CoV PLpro structure was generated and compared to the X-ray structure of SARS-CoV PLpro to provide plausible explanations for differences in substrate and inhibitor recognition. IMPORTANCE Unlocking the secrets of how coronavirus (CoV) papain

  18. The β Subunit Loop That Couples Catalysis and Rotation in ATP Synthase Has a Critical Length*

    PubMed Central

    Mnatsakanyan, Nelli; Kemboi, Silas K.; Salas, Jasmin; Weber, Joachim

    2011-01-01

    ATP synthase uses a unique rotational mechanism to convert chemical energy into mechanical energy and back into chemical energy. The helix-turn-helix structure in the C-terminal domain of the β subunit containing the conserved DELSEED motif, termed “DELSEED-loop,” was suggested to be involved in coupling between catalysis and rotation. If this is indeed the role of the loop, it must have a critical length, the minimum length required to sustain its function. Here, the critical length of the DELSEED-loop was determined by functional analysis of mutants of Bacillus PS3 ATP synthase that had 7–14 amino acids within the loop deleted. A 10 residue deletion lost the ability to catalyze ATP synthesis, but was still an active ATPase. Deletion of 14 residues abolished any enzymatic activity. Modeling indicated that in both deletion mutants the DELSEED-loop was shortened by ∼10 Å; fluorescence resonance energy transfer experiments confirmed the modeling results. This appears to define the minimum length for DELSEED-loop required for coupling of catalysis and rotation. In addition, we could demonstrate that the loss of high-affinity binding to the catalytic site(s) that had been observed previously in two deletion mutants with 3–4 residues removed was not due to the loss of negative charged residues of the DELSEED motif in these mutants. An AALSAAA mutant in which all negative charges of the DELSEED motif were removed showed a normal pattern for MgATP binding to the catalytic sites, with a clearly present high-affinity site. PMID:21705326

  19. Electron transfer precedes ATP hydrolysis during nitrogenase catalysis

    PubMed Central

    Duval, Simon; Danyal, Karamatullah; Shaw, Sudipta; Lytle, Anna K.; Dean, Dennis R.; Hoffman, Brian M.; Antony, Edwin; Seefeldt, Lance C.

    2013-01-01

    The biological reduction of N2 to NH3 catalyzed by Mo-dependent nitrogenase requires at least eight rounds of a complex cycle of events associated with ATP-driven electron transfer (ET) from the Fe protein to the catalytic MoFe protein, with each ET coupled to the hydrolysis of two ATP molecules. Although steps within this cycle have been studied for decades, the nature of the coupling between ATP hydrolysis and ET, in particular the order of ET and ATP hydrolysis, has been elusive. Here, we have measured first-order rate constants for each key step in the reaction sequence, including direct measurement of the ATP hydrolysis rate constant: kATP = 70 s−1, 25 °C. Comparison of the rate constants establishes that the reaction sequence involves four sequential steps: (i) conformationally gated ET (kET = 140 s−1, 25 °C), (ii) ATP hydrolysis (kATP = 70 s−1, 25 °C), (iii) Phosphate release (kPi = 16 s−1, 25 °C), and (iv) Fe protein dissociation from the MoFe protein (kdiss = 6 s−1, 25 °C). These findings allow completion of the thermodynamic cycle undergone by the Fe protein, showing that the energy of ATP binding and protein–protein association drive ET, with subsequent ATP hydrolysis and Pi release causing dissociation of the complex between the Feox(ADP)2 protein and the reduced MoFe protein. PMID:24062462

  20. Application of the Principle of Linked Functions to ATP-Driven Ion Pumps: Kinetics of Activation by ATP

    NASA Astrophysics Data System (ADS)

    Reynolds, Jacqueline A.; Johnson, Edward A.; Tanford, Charles

    1985-06-01

    If a ligand binds with unequal affinity to two distinct states of a protein, then the equilibrium between the two states becomes a function of the concentration of the ligand. A necessary consequence is that the ligand must also affect the forward and/or reverse rate constants for transition between the two states. For an enzyme or transport protein with such a transition as a slow step in the catalytic cycle, the overall rate also becomes a function of ligand concentration. These conclusions are independent of whether or not the ligand is a direct participant in the reaction. If it is a direct partitipant, then the kinetic effect arising from the principle of linked functions is distinct from the direct catalytic effect. These principles suffice to account for the biphasic response of the hydrolytic activity of ATP-driven ion pumps to the concentration of ATP, without the need to invoke more than one ATP binding site per catalytic center.

  1. Domain-confined catalytic soot combustion over Co3O4 anchored on a TiO2 nanotube array catalyst prepared by mercaptoacetic acid induced surface-grafting.

    PubMed

    Ren, Jiale; Yu, Yifu; Dai, Fangfang; Meng, Ming; Zhang, Jing; Zheng, Lirong; Hu, Tiandou

    2013-12-21

    Herein, we introduce a specially designed domain-confined macroporous catalyst, namely, the Co3O4 nanocrystals anchored on a TiO2 nanotube array catalyst, which was synthesized by using the mercaptoacetic acid induced surface-grafting method. This catalyst exhibits much better performance for catalytic soot combustion than the conventional TiO2 powder supported one in gravitational contact mode (GMC).

  2. Characterization of zinc-binding sites in human stromelysin-1: stoichiometry of the catalytic domain and identification of a cysteine ligand in the proenzyme.

    PubMed

    Salowe, S P; Marcy, A I; Cuca, G C; Smith, C K; Kopka, I E; Hagmann, W K; Hermes, J D

    1992-05-19

    A determination of the zinc stoichiometry of the catalytic domain of the human matrix metalloproteinase stromelysin-1 has been carried out using enzyme purified from recombinant Escherichia coli that express C-terminally truncated protein. Atomic absorption spectrometry revealed that both the proenzyme (prostrom255) and the mature active form (strom255) contained nearly 2 mol of Zn/mol of protein. Full-length prostromelysin purified from a mammalian cell culture line also contained zinc in excess of 1 equiv. While zinc in prostrom255 could not be removed by dialysis against o-phenanthroline, similar treatment of mature strom255 resulted in the loss of one-half of the original zinc content. The peptidase activity of the zinc-depleted protein was reduced by greater than 85% but could be restored upon addition of Zn2+ or Co2+. Addition of a thiol-containing inhibitor to a CoZn hybrid enzyme resulted in marked spectral changes in both the visible and ultraviolet regions characteristic of sulfur ligation to Co2+. This direct evidence for an integral role in catalysis and inhibitor binding confirms the location of the exchangeable metal at the active site. To examine the environment of zinc in the proenzyme, a fully cobalt-substituted proenzyme was prepared by in vivo metal replacement. The absorbance features of dicobalt prostrom255 were consistent with metal coordination by the single cysteine present in the propeptide, although the data do not allow assignment to a particular zinc site.(ABSTRACT TRUNCATED AT 250 WORDS)

  3. Photo-activated psoralen binds the ErbB2 catalytic kinase domain, blocking ErbB2 signaling and triggering tumor cell apoptosis.

    PubMed

    Xia, Wenle; Gooden, David; Liu, Leihua; Zhao, Sumin; Soderblom, Erik J; Toone, Eric J; Beyer, Wayne F; Walder, Harold; Spector, Neil L

    2014-01-01

    Photo-activation of psoralen with UVA irradiation, referred to as PUVA, is used in the treatment of proliferative skin disorders. The anti-proliferative effects of PUVA have been largely attributed to psoralen intercalation of DNA, which upon UV treatment, triggers the formation of interstrand DNA crosslinks (ICL) that inhibit transcription and DNA replication. Here, we show that PUVA exerts antitumor effects in models of human breast cancer that overexpress the ErbB2 receptor tyrosine kinase oncogene, through a new mechanism. Independent of ICL formation, the antitumor effects of PUVA in ErbB2+ breast cancer models can instead be mediated through inhibition of ErbB2 activation and signaling. Using a mass spectroscopy-based approach, we show for the first time that photo-activated 8MOP (8-methoxypsoralen) interacts with the ErbB2 catalytic autokinase domain. Furthermore, PUVA can reverse therapeutic resistance to lapatinib and other ErbB2 targeted therapies, including resistance mediated via expression of a phosphorylated, truncated form of ErbB2 (p85(ErbB2)) that is preferentially expressed in tumor cell nuclei. Current ErbB2 targeted therapies, small molecule kinase inhibitors or antibodies, do not block the phosphorylated, activated state of p85(ErbB2). Here we show that PUVA reduced p85(ErbB2) phosphorylation leading to tumor cell apoptosis. Thus, in addition to its effects on DNA and the formation of ICL, PUVA represents a novel ErbB2 targeted therapy for the treatment of ErbB2+ breast cancers, including those that have developed resistance to other ErbB2 targeted therapies.

  4. Transmembrane/cytoplasmic, rather than catalytic, domains of Mmp14 signal to MAPK activation and mammary branching morphogenesis via binding to integrin β1

    PubMed Central

    Mori, Hidetoshi; Lo, Alvin T.; Inman, Jamie L.; Alcaraz, Jordi; Ghajar, Cyrus M.; Mott, Joni D.; Nelson, Celeste M.; Chen, Connie S.; Zhang, Hui; Bascom, Jamie L.; Seiki, Motoharu; Bissell, Mina J.

    2013-01-01

    Epithelial cell invasion through the extracellular matrix (ECM) is a crucial step in branching morphogenesis. The mechanisms by which the mammary epithelium integrates cues from the ECM with intracellular signaling in order to coordinate invasion through the stroma to make the mammary tree are poorly understood. Because the cell membrane-bound matrix metalloproteinase Mmp14 is known to play a key role in cancer cell invasion, we hypothesized that it could also be centrally involved in integrating signals for mammary epithelial cells (MECs) to navigate the collagen 1 (CL-1)-rich stroma of the mammary gland. Expression studies in nulliparous mice that carry a NLS-lacZ transgene downstream of the Mmp14 promoter revealed that Mmp14 is expressed in MECs at the tips of the branches. Using both mammary organoids and 3D organotypic cultures, we show that MMP activity is necessary for invasion through dense CL-1 (3 mg/ml) gels, but dispensable for MEC branching in sparse CL-1 (1 mg/ml) gels. Surprisingly, however, Mmp14 without its catalytic activity was still necessary for branching. Silencing Mmp14 prevented cell invasion through CL-1 and disrupted branching altogether; it also reduced integrin β1 (Itgb1) levels and attenuated MAPK signaling, disrupting Itgb1-dependent invasion/branching within CL-1 gels. FRET imaging revealed that Mmp14 associates directly with Itgb1. We identified a domain of Mmp14 that is required for modulating the levels of Itgb1, MEC signaling and the rate of invasion within CL-1. These results shed light on hitherto undescribed non-proteolytic activities of Mmp14 that are necessary for the Itgb1-dependent biochemical and mechanical signals that regulate branching in the mammary epithelium. PMID:23250208

  5. ATP-dependent motor activity of the transcription termination factor Rho from Mycobacterium tuberculosis

    PubMed Central

    D'Heygère, François; Schwartz, Annie; Coste, Franck; Castaing, Bertrand; Boudvillain, Marc

    2015-01-01

    The bacterial transcription termination factor Rho—a ring-shaped molecular motor displaying directional, ATP-dependent RNA helicase/translocase activity—is an interesting therapeutic target. Recently, Rho from Mycobacterium tuberculosis (MtbRho) has been proposed to operate by a mechanism uncoupled from molecular motor action, suggesting that the manner used by Rho to dissociate transcriptional complexes is not conserved throughout the bacterial kingdom. Here, however, we demonstrate that MtbRho is a bona fide molecular motor and directional helicase which requires a catalytic site competent for ATP hydrolysis to disrupt RNA duplexes or transcription elongation complexes. Moreover, we show that idiosyncratic features of the MtbRho enzyme are conferred by a large, hydrophilic insertion in its N-terminal ‘RNA binding’ domain and by a non-canonical R-loop residue in its C-terminal ‘motor’ domain. We also show that the ‘motor’ domain of MtbRho has a low apparent affinity for the Rho inhibitor bicyclomycin, thereby contributing to explain why M. tuberculosis is resistant to this drug. Overall, our findings support that, in spite of adjustments of the Rho motor to specific traits of its hosting bacterium, the basic principles of Rho action are conserved across species and could thus constitute pertinent screening criteria in high-throughput searches of new Rho inhibitors. PMID:25999346

  6. ATP-dependent motor activity of the transcription termination factor Rho from Mycobacterium tuberculosis.

    PubMed

    D'Heygère, François; Schwartz, Annie; Coste, Franck; Castaing, Bertrand; Boudvillain, Marc

    2015-07-13

    The bacterial transcription termination factor Rho-a ring-shaped molecular motor displaying directional, ATP-dependent RNA helicase/translocase activity-is an interesting therapeutic target. Recently, Rho from Mycobacterium tuberculosis (MtbRho) has been proposed to operate by a mechanism uncoupled from molecular motor action, suggesting that the manner used by Rho to dissociate transcriptional complexes is not conserved throughout the bacterial kingdom. Here, however, we demonstrate that MtbRho is a bona fide molecular motor and directional helicase which requires a catalytic site competent for ATP hydrolysis to disrupt RNA duplexes or transcription elongation complexes. Moreover, we show that idiosyncratic features of the MtbRho enzyme are conferred by a large, hydrophilic insertion in its N-terminal 'RNA binding' domain and by a non-canonical R-loop residue in its C-terminal 'motor' domain. We also show that the 'motor' domain of MtbRho has a low apparent affinity for the Rho inhibitor bicyclomycin, thereby contributing to explain why M. tuberculosis is resistant to this drug. Overall, our findings support that, in spite of adjustments of the Rho motor to specific traits of its hosting bacterium, the basic principles of Rho action are conserved across species and could thus constitute pertinent screening criteria in high-throughput searches of new Rho inhibitors.

  7. Structural and Functional Characterization of the JH2 Pseudokinase Domain of JAK Family Tyrosine Kinase 2 (TYK2).

    PubMed

    Min, Xiaoshan; Ungureanu, Daniela; Maxwell, Sarah; Hammarén, Henrik; Thibault, Steve; Hillert, Ellin-Kristina; Ayres, Merrill; Greenfield, Brad; Eksterowicz, John; Gabel, Chris; Walker, Nigel; Silvennoinen, Olli; Wang, Zhulun

    2015-11-06

    JAK (Janus family of cytoplasmic tyrosine kinases) family tyrosine kinase 2 (TYK2) participates in signaling through cytokine receptors involved in immune responses and inflammation. JAKs are characterized by dual kinase domain: a tyrosine kinase domain (JH1) that is preceded by a pseudokinase domain (JH2). The majority of disease-associated mutations in JAKs map to JH2, demonstrating its central regulatory function. JH2s were considered catalytically inactive, but JAK2 JH2 was found to have low autoregulatory catalytic activity. Whether the other JAK JH2s share ATP binding and enzymatic activity has been unclear. Here we report the crystal structure of TYK2 JH2 in complex with adenosine 5'-O-(thiotriphosphate) (ATP-γS) and characterize its nucleotide binding by biochemical and biophysical methods. TYK2 JH2 did not show phosphotransfer activity, but it binds ATP and the nucleotide binding stabilizes the protein without inducing major conformational changes. Mutation of the JH2 ATP-binding pocket increased basal TYK2 phosphorylation and downstream signaling. The overall structural characteristics of TYK2 JH2 resemble JAK2 JH2, but distinct stabilizing molecular interactions around helix αAL in the activation loop provide a structural basis for differences in substrate access and catalytic activities among JAK family JH2s. The structural and biochemical data suggest that ATP binding is functionally important for both TYK2 and JAK2 JH2s, whereas the regulatory phosphorylation appears to be a unique property of JAK2. Finally, the co-crystal structure of TYK2 JH2 complexed with a small molecule inhibitor demonstrates that JH2 is accessible to ATP-competitive compounds, which offers novel approaches for targeting cytokine signaling as well as potential therapeutic applications. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Understanding structure, function, and mutations in the mitochondrial ATP synthase

    PubMed Central

    Xu, Ting; Pagadala, Vijayakanth; Mueller, David M.

    2015-01-01

    The mitochondrial ATP synthase is a multimeric enzyme complex with an overall molecular weight of about 600,000 Da. The ATP synthase is a molecular motor composed of two separable parts: F1 and Fo. The F1 portion contains the catalytic sites for ATP synthesis and protrudes into the mitochondrial matrix. Fo forms a proton turbine that is embedded in the inner membrane and connected to the rotor of F1. The flux of protons flowing down a potential gradient powers the rotation of the rotor driving the synthesis of ATP. Thus, the flow of protons though Fo is coupled to the synthesis of ATP. This review will discuss the structure/function relationship in the ATP synthase as determined by biochemical, crystallographic, and genetic studies. An emphasis will be placed on linking the structure/function relationship with understanding how disease causing mutations or putative single nucleotide polymorphisms (SNPs) in genes encoding the subunits of the ATP synthase, will affect the function of the enzyme and the health of the individual. The review will start by summarizing the current understanding of the subunit composition of the enzyme and the role of the subunits followed by a discussion on known mutations and their effect on the activity of the ATP synthase. The review will conclude with a summary of mutations in genes encoding subunits of the ATP synthase that are known to be responsible for human disease, and a brief discussion on SNPs. PMID:25938092

  9. Dynamically-driven enhancement of the catalytic machinery of the SARS 3C-like protease by the S284-T285-I286/A mutations on the extra domain.

    PubMed

    Lim, Liangzhong; Shi, Jiahai; Mu, Yuguang; Song, Jianxing

    2014-01-01

    Previously we revealed that the extra domain of SARS 3CLpro mediated the catalysis via different mechanisms. While the R298A mutation completely abolished the dimerization, thus resulting in the inactive catalytic machinery, N214A inactivated the enzyme by altering its dynamics without significantly perturbing its structure. Here we studied another mutant with S284-T285-I286 replaced by Ala (STI/A) with a 3.6-fold activity increase and slightly enhanced dimerization. We determined its crystal structure, which still adopts the dimeric structure almost identical to that of the wild-type (WT), except for slightly tighter packing between two extra-domains. We then conducted 100-ns molecular dynamics (MD) simulations for both STI/A and WT, the longest reported so far for 3CLpro. In the simulations, two STI/A extra domains become further tightly packed, leading to a significant volume reduction of the nano-channel formed by residues from both catalytic and extra domains. The enhanced packing appears to slightly increase the dynamic stability of the N-finger and the first helix residues, which subsequently triggers the redistribution of dynamics over residues directly contacting them. This ultimately enhances the dynamical stability of the residues constituting the catalytic dyad and substrate-binding pockets. Further correlation analysis reveals that a global network of the correlated motions exists in the protease, whose components include all residues identified so far to be critical for the dimerization and catalysis. Most strikingly, the N214A mutation globally decouples this network while the STI/A mutation alters the correlation pattern. Together with previous results, the present study establishes that besides the classic structural allostery, the dynamic allostery also operates in the SARS 3CLpro, which is surprisingly able to relay the perturbations on the extra domain onto the catalytic machinery to manifest opposite catalytic effects. Our results thus imply a

  10. Structural Basis of GLUT1 Inhibition by Cytoplasmic ATP

    PubMed Central

    Blodgett, David M.; De Zutter, Julie K.; Levine, Kara B.; Karim, Pusha; Carruthers, Anthony

    2007-01-01

    Cytoplasmic ATP inhibits human erythrocyte glucose transport protein (GLUT1)–mediated glucose transport in human red blood cells by reducing net glucose transport but not exchange glucose transport (Cloherty, E.K., D.L. Diamond, K.S. Heard, and A. Carruthers. 1996. Biochemistry. 35:13231–13239). We investigated the mechanism of ATP regulation of GLUT1 by identifying GLUT1 domains that undergo significant conformational change upon GLUT1–ATP interaction. ATP (but not GTP) protects GLUT1 against tryptic digestion. Immunoblot analysis indicates that ATP protection extends across multiple GLUT1 domains. Peptide-directed antibody binding to full-length GLUT1 is reduced by ATP at two specific locations: exofacial loop 7–8 and the cytoplasmic C terminus. C-terminal antibody binding to wild-type GLUT1 expressed in HEK cells is inhibited by ATP but binding of the same antibody to a GLUT1–GLUT4 chimera in which loop 6–7 of GLUT1 is substituted with loop 6–7 of GLUT4 is unaffected. ATP reduces GLUT1 lysine covalent modification by sulfo-NHS-LC-biotin by 40%. AMP is without effect on lysine accessibility but antagonizes ATP inhibition of lysine modification. Tandem electrospray ionization mass spectrometry analysis indicates that ATP reduces covalent modification of lysine residues 245, 255, 256, and 477, whereas labeling at lysine residues 225, 229, and 230 is unchanged. Exogenous, intracellular GLUT1 C-terminal peptide mimics ATP modulation of transport whereas C-terminal peptide-directed IgGs inhibit ATP modulation of glucose transport. These findings suggest that transport regulation involves ATP-dependent conformational changes in (or interactions between) the GLUT1 C terminus and the C-terminal half of GLUT1 cytoplasmic loop 6–7. PMID:17635959

  11. Customized ATP towpreg

    NASA Astrophysics Data System (ADS)

    Sandusky, Donald A.; Marchello, Joseph M.; Baucom, Robert M.; Johnston, Norman J.

    Automated tow placement (ATP) utilizes robotic technology to lay down adjacent polymer-matrix-impregnated carbon fiber tows on a tool surface. Consolidation and cure during ATP requires that void elimination and polymer matrix adhesion be accomplished in the short period of heating and pressure rolling that follows towpreg ribbon placeme