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Sample records for atp catalytic domain

  1. The conserved lysine of the catalytic domain of protein kinases is actively involved in the phosphotransfer reaction and not required for anchoring ATP.

    PubMed Central

    Carrera, A C; Alexandrov, K; Roberts, T M

    1993-01-01

    The study of the various protein kinases reveals that, despite their considerably diversity, they have evolved from a common origin. Eleven conserved subdomains have been described that encompass the catalytic core of these enzymes. One of these conserved regions, subdomain II, contains an invariant lysine residue present in all known protein kinase catalytic domains. Two facts have suggested that this conserved lysine of subdomain II is essential for binding ATP: (i) several investigators have demonstrated that this residue is physically proximal to the ATP molecule, and (ii) conservative substitutions at this site render the kinase inactive. However, these results are also consistent with a functional role of the conserved lysine of subdomain II in orienting or facilitating the transfer of phosphate. To study in more detail the role of subdomain II, we have generated mutants of the protein-tyrosine kinase pp56lck that have single amino acid substitutions within the area surrounding the conserved residue Lys-273 in subdomain II. When compared with wild-type pp56lck, these mutants displayed profound reductions in their phosphotransfer efficiencies and small differences in their affinities for ATP. Further, the substitution of arginine for Lys-273 resulted in a mutant protein unable to transfer the gamma-phosphate of ATP but able to bind 8-azido-ATP with an efficiency similar to that of wild-type pp56lck. These results suggest that the region including Lys-273 of subdomain II is involved in the enzymatic process of phosphate transfer, rather than in anchoring ATP. Images PMID:8421674

  2. 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. PMID:26666844

  3. 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. PMID:27296627

  4. 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.

  5. Functional domains of an ATP-dependent DNA ligase.

    PubMed

    Doherty, A J; Wigley, D B

    1999-01-01

    The crystal structure of an ATP-dependent DNA ligase from bacteriophage T7 revealed that the protein comprised two structural domains. In order to investigate the biochemical activities of these domains, we have overexpressed them separately and purified them to homogeneity. The larger N-terminal domain retains adenylation and ligase activities, though both at a reduced level. The adenylation activity of the large domain is stimulated by the presence of the smaller domain, suggesting that a conformational change is required for adenylation in the full length protein. The DNA binding properties of the two fragments have also been studied. The larger domain is able to band shift both single and double-stranded DNA, while the smaller fragment is only able to bind to double-stranded DNA. These data suggest that the specificity of DNA ligases for nick sites in DNA is produced by a combination of these different DNA binding activities in the intact enzyme. PMID:9878388

  6. 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

  7. Inactivation of Multiple Bacterial Histidine Kinases by Targeting the ATP-Binding Domain

    PubMed Central

    2015-01-01

    Antibacterial agents that exploit new targets will be required to combat the perpetual rise of bacterial resistance to current antibiotics. We are exploring the inhibition of histidine kinases, constituents of two-component systems. Two-component systems are the primary signaling pathways that bacteria utilize to respond to their environment. They are ubiquitous in bacteria and trigger various pathogenic mechanisms. To attenuate these signaling pathways, we sought to broadly target the histidine kinase family by focusing on their highly conserved ATP-binding domain. Development of a fluorescence polarization displacement assay facilitated high-throughput screening of ∼53 000 diverse small molecules for binding to the ATP-binding pocket. Of these compounds, nine inhibited the catalytic activity of two or more histidine kinases. These scaffolds could provide valuable starting points for the design of broadly effective HK inhibitors, global reduction of bacterial signaling, and ultimately, a class of antibiotics that function by a new mechanism of action. PMID:25531939

  8. Catalytic signal amplification for the discrimination of ATP and ADP using functionalised gold nanoparticles.

    PubMed

    Pezzato, Cristian; Chen, Jack L-Y; Galzerano, Patrizia; Salvi, Michela; Prins, Leonard J

    2016-07-12

    Diagnostic assays that incorporate a signal amplification mechanism permit the detection of analytes with enhanced selectivity. Herein, we report a gold nanoparticle-based chemical system able to differentiate ATP from ADP by means of catalytic signal amplification. The discrimination between ATP and ADP is of relevance for the development of universal assays for the detection of enzymes which consume ATP. For example, protein kinases are a class of enzymes critical for the regulation of cellular functions, and act to modulate the activity of other proteins by transphosphorylation, transferring a phosphate group from ATP to give ADP as a byproduct. The system described here exploits the ability of cooperative catalytic head groups on gold nanoparticles to very efficiently catalyze chromogenic reactions such as the transphosphorylation of 2-hydroxypropyl-4-nitrophenyl phosphate (HPNPP). A series of chromogenic substrates have been synthesized and evaluated by means of Michaelis-Menten kinetics (compounds 2, 4-6). 2-Hydroxypropyl-(3-trifluoromethyl-4-nitro)phenyl phosphate (5) was found to display higher reactivity (kcat) and higher binding affinity (KM) when compared to HPNPP. This higher binding affinity allows phosphate 5 to compete with ATP and ADP to different extents for binding on the monolayer surface, thus enabling a catalytically amplified signal only when ATP is absent. Overall, this represents a viable new approach for monitoring the conversion of ATP into ADP with high sensitivity. PMID:27336846

  9. Copper directs ATP7B to the apical domain of hepatic cells via basolateral endosomes.

    PubMed

    Nyasae, Lydia K; Schell, Michael J; Hubbard, Ann L

    2014-12-01

    Physiologic Cu levels regulate the intracellular location of the Cu ATPase ATP7B. Here, we determined the routes of Cu-directed trafficking of endogenous ATP7B in the polarized hepatic cell line WIF-B and in the liver in vivo. Copper (10 µm) caused ATP7B to exit the trans-Golgi network (TGN) in vesicles, which trafficked via large basolateral endosomes to the apical domain within 1 h. Although perturbants of luminal acidification had little effect on the TGN localization of ATP7B in low Cu, they blocked delivery to the apical membrane in elevated Cu. If the vesicular proton-pump inhibitor bafilomycin-A1 (Baf) was present with Cu, ATP7B still exited the TGN, but accumulated in large endosomes located near the coverslip, in the basolateral region. Baf washout restored ATP7B trafficking to the apical domain. If ATP7B was staged apically in high Cu, Baf addition promoted the accumulation of ATP7B in subapical endosomes, indicating a blockade of apical recycling, with concomitant loss of ATP7B at the apical membrane. The retrograde pathway to the TGN, induced by Cu removal, was far less affected by Baf than the anterograde (Cu-stimulated) case. Overall, loss of acidification-impaired Cu-regulated trafficking of ATP7B at two main sites: (i) sorting and exit from large basolateral endosomes and (ii) recycling via endosomes near the apical membrane. PMID:25243755

  10. 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.

  11. Rotary movements within the ATP synthase do not constitute an obligatory element of the catalytic mechanism.

    PubMed

    Berden, Jan A

    2003-08-01

    After a brief history of the proposals for the mechanism of the ATP synthase, the main experimental arguments for a rotational mechanism of catalysis are analyzed and on the basis of this analysis it is concluded that no evidence has been provided for rotation as an obligatory element of the catalytic mechanism. On the other hand, the experimental evidence in favor of a two-sites catalytic mechanism, derived from various approaches and not compatible with a three-sites rotary mechanism, appear to be very solid. Finally a brief characterization of the various nucleotide binding sites is provided and a suggestion is made how the enzyme has evolutionarily developed from a rotating machine into an asymmetrical device for energy conservation. PMID:14609203

  12. Gating of the CFTR Cl- channel by ATP-driven nucleotide-binding domain dimerisation.

    PubMed

    Hwang, Tzyh-Chang; Sheppard, David N

    2009-05-15

    The cystic fibrosis transmembrane conductance regulator (CFTR) plays a fundamental role in fluid and electrolyte transport across epithelial tissues. Based on its structure, function and regulation, CFTR is an ATP-binding cassette (ABC) transporter. These transporters are assembled from two membrane-spanning domains (MSDs) and two nucleotide-binding domains (NBDs). In the vast majority of ABC transporters, the NBDs form a common engine that utilises the energy of ATP hydrolysis to pump a wide spectrum of substrates through diverse transmembrane pathways formed by the MSDs. By contrast, in CFTR the MSDs form a pathway for passive anion flow that is gated by cycles of ATP binding and hydrolysis by the NBDs. Here, we consider how the interaction of ATP with two ATP-binding sites, formed by the NBDs, powers conformational changes in CFTR structure to gate the channel pore. We explore how conserved sequences from both NBDs form ATP-binding sites at the interface of an NBD dimer and highlight the distinct roles that each binding site plays during the gating cycle. Knowledge of how ATP gates the CFTR Cl- channel is critical for understanding CFTR's physiological role, its malfunction in disease and the mechanism of action of small molecules that modulate CFTR channel gating. PMID:19332488

  13. Gating of the CFTR Cl− channel by ATP-driven nucleotide-binding domain dimerisation

    PubMed Central

    Hwang, Tzyh-Chang; Sheppard, David N

    2009-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) plays a fundamental role in fluid and electrolyte transport across epithelial tissues. Based on its structure, function and regulation, CFTR is an ATP-binding cassette (ABC) transporter. These transporters are assembled from two membrane-spanning domains (MSDs) and two nucleotide-binding domains (NBDs). In the vast majority of ABC transporters, the NBDs form a common engine that utilises the energy of ATP hydrolysis to pump a wide spectrum of substrates through diverse transmembrane pathways formed by the MSDs. By contrast, in CFTR the MSDs form a pathway for passive anion flow that is gated by cycles of ATP binding and hydrolysis by the NBDs. Here, we consider how the interaction of ATP with two ATP-binding sites, formed by the NBDs, powers conformational changes in CFTR structure to gate the channel pore. We explore how conserved sequences from both NBDs form ATP-binding sites at the interface of an NBD dimer and highlight the distinct roles that each binding site plays during the gating cycle. Knowledge of how ATP gates the CFTR Cl− channel is critical for understanding CFTR's physiological role, its malfunction in disease and the mechanism of action of small molecules that modulate CFTR channel gating. PMID:19332488

  14. 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.

  15. Recombinant preparation and functional studies of EspI ATP binding domain from Mycobacterium tuberculosis.

    PubMed

    Chen, Hanyu; Wang, Huilin; Sun, Tao; Tian, Shuangliang; Lin, Donghai; Guo, Chenyun

    2016-07-01

    The ESX-1 secretion system of Mycobacterium tuberculosis is required for the virulence of tubercle bacillus. EspI, the ESX-1 secretion-associated protein in Mycobacterium tuberculosis (MtEspI), is involved in repressing the activity of ESX-1-mediated secretion when the cellular ATP level is low. The ATP binding domain of MtEspI plays a crucial role in this regulatory process. However, further structural and functional studies of MtEspI are hindered due to the bottleneck of obtaining stable and pure recombinant protein. In this study, we systematically analyzed the structure and function of MtEspI using bioinformatics tools and tried various expression constructs to recombinantly express full-length and truncated MtEspI ATP binding domain. Finally, we prepared pure and stable MtEspI ATP binding domain, MtEspI415-493, in Escherichia coli by fusion expression and purification with dual tag, Glutathione S-transferase (GST) tag and (His)6 tag. (31)P NMR titration assay indicated that MtEspI415-493 possessed a moderate affinity (∼μM) for ATP and the residue K425 was located at the binding site. The protocol described here may provide a train of thought for recombinant preparation of other ESX-1 secretion-associated proteins. PMID:27017992

  16. ATP binding to the pseudokinase domain of JAK2 is critical for pathogenic activation.

    PubMed

    Hammarén, Henrik M; Ungureanu, Daniela; Grisouard, Jean; Skoda, Radek C; Hubbard, Stevan R; Silvennoinen, Olli

    2015-04-14

    Pseudokinases lack conserved motifs typically required for kinase activity. Nearly half of pseudokinases bind ATP, but only few retain phosphotransfer activity, leaving the functional role of nucleotide binding in most cases unknown. Janus kinases (JAKs) are nonreceptor tyrosine kinases with a tandem pseudokinase-kinase domain configuration, where the pseudokinase domain (JAK homology 2, JH2) has important regulatory functions and harbors mutations underlying hematological and immunological diseases. JH2 of JAK1, JAK2, and TYK2 all bind ATP, but the significance of this is unclear. We characterize the role of nucleotide binding in normal and pathogenic JAK signaling using comprehensive structure-based mutagenesis. Disruption of JH2 ATP binding in wild-type JAK2 has only minor effects, and in the presence of type I cytokine receptors, the mutations do not affect JAK2 activation. However, JH2 mutants devoid of ATP binding ameliorate the hyperactivation of JAK2 V617F. Disrupting ATP binding in JH2 also inhibits the hyperactivity of other pathogenic JAK2 mutants, as well as of JAK1 V658F, and prevents induction of erythrocytosis in a JAK2 V617F myeloproliferative neoplasm mouse model. Molecular dynamic simulations and thermal-shift analysis indicate that ATP binding stabilizes JH2, with a pronounced effect on the C helix region, which plays a critical role in pathogenic activation of JAK2. Taken together, our results suggest that ATP binding to JH2 serves a structural role in JAKs, which is required for aberrant activity of pathogenic JAK mutants. The inhibitory effect of abrogating JH2 ATP binding in pathogenic JAK mutants may warrant novel therapeutic approaches. PMID:25825724

  17. A stable ATP binding to the nucleotide binding domain is important for reliable gating cycle in an ABC transporter CFTR.

    PubMed

    Shimizu, Hiroyasu; Yu, Ying-Chun; Kono, Koichi; Kubota, Takahiro; Yasui, Masato; Li, Min; Hwang, Tzyh-Chang; Sohma, Yoshiro

    2010-09-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) anion channel, a member of ABC transporter superfamily, gates following ATP-dependent conformational changes of the nucleotide binding domains (NBD). Reflecting the hundreds of milliseconds duration of the channel open state corresponding to the dimerization of two NBDs, macroscopic WT-CFTR currents usually showed a fast, single exponential relaxation upon removal of cytoplasmic ATP. Mutations of tyrosine1219, a residue critical for ATP binding in second NBD (NBD2), induced a significant slow phase in the current relaxation, suggesting that weakening ATP binding affinity at NBD2 increases the probability of the stable open state. The slow phase was effectively diminished by a higher affinity ATP analogue. These data suggest that a stable binding of ATP to NBD2 is required for normal CFTR gating cycle, andthat the instability of ATP binding frequently halts the gating cycle in the open state presumably through a failure of ATP hydrolysis at NBD2. PMID:20628841

  18. A stable ATP binding to the nucleotide binding domain is important for reliable gating cycle in an ABC transporter CFTR

    PubMed Central

    Shimizu, Hiroyasu; Yu, Ying-Chun; Kono, Koichi; Kubota, Takahiro; Yasui, Masato; Li, Min

    2016-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) anion channel, a member of ABC transporter superfamily, gates following ATP-dependent conformational changes of the nucleotide binding domains (NBD). Reflecting the hundreds of milliseconds duration of the channel open state corresponding to the dimerization of two NBDs, macroscopic WT-CFTR currents usually showed a fast, single exponential relaxation upon removal of cytoplasmic ATP. Mutations of tyrosine1219, a residue critical for ATP binding in second NBD (NBD2), induced a significant slow phase in the current relaxation, suggesting that weakening ATP binding affinity at NBD2 increases the probability of the stable open state. The slow phase was effectively diminished by a higher affinity ATP analogue. These data suggest that a stable binding of ATP to NBD2 is required for normal CFTR gating cycle, andthat the instability of ATP binding frequently halts the gating cycle in the open state presumably through a failure of ATP hydrolysis at NBD2. PMID:20628841

  19. Timing of inorganic phosphate release modulates the catalytic activity of ATP-driven rotary motor protein

    NASA Astrophysics Data System (ADS)

    Watanabe, Rikiya; Noji, Hiroyuki

    2014-04-01

    F1-ATPase is a rotary motor protein driven by ATP hydrolysis. The rotary motion of F1-ATPase is tightly coupled to catalysis, in which the catalytic sites strictly obey the reaction sequences at the resolution of elementary reaction steps. This fine coordination of the reaction scheme is thought to be important to achieve extremely high chemomechanical coupling efficiency and reversibility, which is the prominent feature of F1-ATPase among molecular motor proteins. In this study, we intentionally change the reaction scheme by using single-molecule manipulation, and we examine the resulting effect on the rotary motion of F1-ATPase. When the sequence of the products released, that is, ADP and inorganic phosphate, is switched, we find that F1 frequently stops rotating for a long time, which corresponds to inactivation of catalysis. This inactive state presents MgADP inhibition, and thus, we find that an improper reaction sequence of F1-ATPase catalysis induces MgADP inhibition.

  20. Evolving Catalytic Properties of the MLL Family SET Domain

    PubMed Central

    Zhang, Ying; Mittal, Anshumali; Reid, James; Reich, Stephanie; Gamblin, Steven J.; Wilson, Jon R.

    2015-01-01

    Summary Methylation of histone H3 lysine-4 is a hallmark of chromatin associated with active gene expression. The activity of H3K4-specific modification enzymes, in higher eukaryotes the MLL (or KMT2) family, is tightly regulated. The MLL family has six members, each with a specialized function. All contain a catalytic SET domain that associates with a core multiprotein complex for activation. These SET domains segregate into three classes that correlate with the arrangement of targeting domains that populate the rest of the protein. Here we show that, unlike MLL1, the MLL4 SET domain retains significant activity without the core complex. We also present the crystal structure of an inactive MLL4-tagged SET domain construct and describe conformational changes that account for MLL4 intrinsic activity. Finally, our structure explains how the MLL SET domains are able to add multiple methyl groups to the target lysine, despite having the sequence characteristics of a classical monomethylase. PMID:26320581

  1. Mg2+ -dependent ATP occlusion at the first nucleotide-binding domain (NBD1) of CFTR does not require the second (NBD2).

    PubMed

    Aleksandrov, Luba; Aleksandrov, Andrei; Riordan, John R

    2008-11-15

    ATP binding to the first and second NBDs (nucleotide-binding domains) of CFTR (cystic fibrosis transmembrane conductance regulator) are bivalent-cation-independent and -dependent steps respectively [Aleksandrov, Aleksandrov, Chang and Riordan (2002) J. Biol. Chem. 277, 15419-15425]. Subsequent to the initial binding, Mg(2+) drives rapid hydrolysis at the second site, while promoting non-exchangeable trapping of the nucleotide at the first site. This occlusion at the first site of functional wild-type CFTR is somewhat similar to that which occurs when the catalytic glutamate residues in both of the hydrolytic sites of P-glycoprotein are mutated, which has been proposed to be the result of dimerization of the two NBDs and represents a transient intermediate formed during ATP hydrolysis [Tombline and Senior (2005) J. Bioenerg. Biomembr. 37, 497-500]. To test the possible relevance of this interpretation to CFTR, we have now characterized the process by which NBD1 occludes [(32)P]N(3)ATP (8-azido-ATP) and [(32)P]N(3)ADP (8-azido-ADP). Only N(3)ATP, but not N(3)ADP, can be bound initially at NBD1 in the absence of Mg(2+). Despite the lack of a requirement for Mg(2+) for ATP binding, retention of the NTP at 37 degrees C was dependent on the cation. However, at reduced temperature (4 degrees C), N(3)ATP remains locked in the binding pocket with virtually no reduction over a 1 h period, even in the absence of Mg(2+). Occlusion occurred identically in a DeltaNBD2 construct, but not in purified recombinant NBD1, indicating that the process is dependent on the influence of regions of CFTR in addition to NBD1, but not NBD2. PMID:18605986

  2. Robust and tunable circadian rhythms from differentially sensitive catalytic domains

    PubMed Central

    Phong, Connie; Markson, Joseph S.; Wilhoite, Crystal M.; Rust, Michael J.

    2013-01-01

    Circadian clocks are ubiquitous biological oscillators that coordinate an organism’s behavior with the daily cycling of the external environment. To ensure synchronization with the environment, the period of the clock must be maintained near 24 h even as amplitude and phase are altered by input signaling. We show that, in a reconstituted circadian system from cyanobacteria, these conflicting requirements are satisfied by distinct functions for two domains of the central clock protein KaiC: the C-terminal autokinase domain integrates input signals through the ATP/ADP ratio, and the slow N-terminal ATPase acts as an input-independent timer. We find that phosphorylation in the C-terminal domain followed by an ATPase cycle in the N-terminal domain is required to form the inhibitory KaiB•KaiC complexes that drive the dynamics of the clock. We present a mathematical model in which this ATPase-mediated delay in negative feedback gives rise to a compensatory mechanism that allows a tunable phase and amplitude while ensuring a robust circadian period. PMID:23277568

  3. Robust and tunable circadian rhythms from differentially sensitive catalytic domains.

    PubMed

    Phong, Connie; Markson, Joseph S; Wilhoite, Crystal M; Rust, Michael J

    2013-01-15

    Circadian clocks are ubiquitous biological oscillators that coordinate an organism's behavior with the daily cycling of the external environment. To ensure synchronization with the environment, the period of the clock must be maintained near 24 h even as amplitude and phase are altered by input signaling. We show that, in a reconstituted circadian system from cyanobacteria, these conflicting requirements are satisfied by distinct functions for two domains of the central clock protein KaiC: the C-terminal autokinase domain integrates input signals through the ATP/ADP ratio, and the slow N-terminal ATPase acts as an input-independent timer. We find that phosphorylation in the C-terminal domain followed by an ATPase cycle in the N-terminal domain is required to form the inhibitory KaiB•KaiC complexes that drive the dynamics of the clock. We present a mathematical model in which this ATPase-mediated delay in negative feedback gives rise to a compensatory mechanism that allows a tunable phase and amplitude while ensuring a robust circadian period. PMID:23277568

  4. Normal gating of CFTR requires ATP binding to both nucleotide-binding domains and hydrolysis at the second nucleotide-binding domain.

    PubMed

    Berger, Allan L; Ikuma, Mutsuhiro; Welsh, Michael J

    2005-01-11

    ATP interacts with the two nucleotide-binding domains (NBDs) of CFTR to control gating. However, it is unclear whether gating involves ATP binding alone, or also involves hydrolysis at each NBD. We introduced phenylalanine residues into nonconserved positions of each NBD Walker A motif to sterically prevent ATP binding. These mutations blocked [alpha-(32)P]8-N(3)-ATP labeling of the mutated NBD and reduced channel opening rate without changing burst duration. Introducing cysteine residues at these positions and modifying with N-ethylmaleimide produced the same gating behavior. These results indicate that normal gating requires ATP binding to both NBDs, but ATP interaction with one NBD is sufficient to support some activity. We also studied mutations of the conserved Walker A lysine residues (K464A and K1250A) that prevent hydrolysis. By combining substitutions that block ATP binding with Walker A lysine mutations, we could differentiate the role of ATP binding vs. hydrolysis at each NBD. The K1250A mutation prolonged burst duration; however, blocking ATP binding prevented the long bursts. These data indicate that ATP binding to NBD2 allowed channel opening and that closing was delayed in the absence of hydrolysis. The corresponding NBD1 mutations showed relatively little effect of preventing ATP hydrolysis but a large inhibition of blocking ATP binding. These data suggest that ATP binding to NBD1 is required for normal activity but that hydrolysis has little effect. Our results suggest that both NBDs contribute to channel gating, NBD1 binds ATP but supports little hydrolysis, and ATP binding and hydrolysis at NBD2 are key for normal gating. PMID:15623556

  5. Normal gating of CFTR requires ATP binding to both nucleotide-binding domains and hydrolysis at the second nucleotide-binding domain

    PubMed Central

    Berger, Allan L.; Ikuma, Mutsuhiro; Welsh, Michael J.

    2005-01-01

    ATP interacts with the two nucleotide-binding domains (NBDs) of CFTR to control gating. However, it is unclear whether gating involves ATP binding alone, or also involves hydrolysis at each NBD. We introduced phenylalanine residues into nonconserved positions of each NBD Walker A motif to sterically prevent ATP binding. These mutations blocked [α-32P]8-N3-ATP labeling of the mutated NBD and reduced channel opening rate without changing burst duration. Introducing cysteine residues at these positions and modifying with N-ethylmaleimide produced the same gating behavior. These results indicate that normal gating requires ATP binding to both NBDs, but ATP interaction with one NBD is sufficient to support some activity. We also studied mutations of the conserved Walker A lysine residues (K464A and K1250A) that prevent hydrolysis. By combining substitutions that block ATP binding with Walker A lysine mutations, we could differentiate the role of ATP binding vs. hydrolysis at each NBD. The K1250A mutation prolonged burst duration; however, blocking ATP binding prevented the long bursts. These data indicate that ATP binding to NBD2 allowed channel opening and that closing was delayed in the absence of hydrolysis. The corresponding NBD1 mutations showed relatively little effect of preventing ATP hydrolysis but a large inhibition of blocking ATP binding. These data suggest that ATP binding to NBD1 is required for normal activity but that hydrolysis has little effect. Our results suggest that both NBDs contribute to channel gating, NBD1 binds ATP but supports little hydrolysis, and ATP binding and hydrolysis at NBD2 are key for normal gating. PMID:15623556

  6. Conformational changes of the bacterial type I ATP-binding cassette importer HisQMP2 at distinct steps of the catalytic cycle.

    PubMed

    Heuveling, Johanna; Frochaux, Violette; Ziomkowska, Joanna; Wawrzinek, Robert; Wessig, Pablo; Herrmann, Andreas; Schneider, Erwin

    2014-01-01

    Prokaryotic solute binding protein-dependent ATP-binding cassette import systems are divided into type I and type II and mechanistic differences in the transport process going along with this classification are under intensive investigation. Little is known about the conformational dynamics during the catalytic cycle especially concerning the transmembrane domains. The type I transporter for positively charged amino acids from Salmonella enterica serovar Typhimurium (LAO-HisQMP2) was studied by limited proteolysis in detergent solution in the absence and presence of co-factors including ATP, ADP, LAO/arginine, and Mg(2+) ions. Stable peptide fragments could be obtained and differentially susceptible cleavage sites were determined by mass spectrometry as Lys-258 in the nucleotide-binding subunit, HisP, and Arg-217/Arg-218 in the transmembrane subunit, HisQ. In contrast, transmembrane subunit HisM was gradually degraded but no stable fragment could be detected. HisP and HisQ were equally resistant under pre- and post-hydrolysis conditions in the presence of arginine-loaded solute-binding protein LAO and ATP/ADP. Some protection was also observed with LAO/arginine alone, thus reflecting binding to the transporter in the apo-state and transmembrane signaling. Comparable digestion patterns were obtained with the transporter reconstituted into proteoliposomes and nanodiscs. Fluorescence lifetime spectroscopy confirmed the change of HisQ(R218) to a more apolar microenvironment upon ATP binding and hydrolysis. Limited proteolysis was subsequently used as a tool to study the consequences of mutations on the transport cycle. Together, our data suggest similar conformational changes during the transport cycle as described for the maltose ABC transporter of Escherichia coli, despite distinct structural differences between both systems. PMID:24021237

  7. 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.

  8. The Intrinsic Reactivity of ATP and the Catalytic Proficiencies of Kinases Acting on Glucose, N-Acetylgalactosamine, and Homoserine

    PubMed Central

    Stockbridge, Randy B.; Wolfenden, Richard

    2009-01-01

    To evaluate the rate enhancements produced by representative kinases and their thermodynamic basis, rate constants were determined as a function of changing temperature for 1) the spontaneous methanolysis of ATP and 2) reactions catalyzed by kinases to which different mechanisms of action have been ascribed. For each of these enzymes, the minor effects of changing viscosity indicate that kcat/Km is governed by the central chemical events in the enzyme-substrate complex rather than by enzyme-substrate encounter. Individual Arrhenius plots, obtained at intervals between pH 4.8 and 11.0, yielded ΔH‡ and TΔS‡ for the nonenzymatic methanolysis of ATP2−, ATP3−, and ATP4− in the absence of Mg2+. The addition of Mg2+ led to partly compensating changes in ΔH‡ and TΔS‡, accelerating the nonenzymatic methanolysis of ATP 11-fold at pH 7 and 25 °C. The rate enhancements produced by yeast hexokinase, homoserine kinase, and N-acetylgalactosamine kinase (obtained by comparison of their kcat/Km values in the presence of saturating phosphoryl acceptor with the second order rate constant for methanolysis of MgATP) ranged between 1012- and 1014-fold. Their nominal affinities for the altered substrates in the transition state were 2.1 × 10−16 m for N-acetylgalactosamine kinase, 7.4 × 10−17 m for homoserine kinase, and 6.4 × 10−18 m for hexokinase. Compared with nonenzymatic phosphoryl transfer, all three kinases were found to produce major reductions in the entropy of activation, in accord with the likelihood that substrate juxtaposition and desolvation play prominent roles in their catalytic action. PMID:19531469

  9. 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.

  10. 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.

  11. GroEL and CCT are catalytic unfoldases mediating out-of-cage polypeptide refolding without ATP.

    PubMed

    Priya, Smriti; Sharma, Sandeep Kumar; Sood, Vishal; Mattoo, Rayees U H; Finka, Andrija; Azem, Abdussalam; De Los Rios, Paolo; Goloubinoff, Pierre

    2013-04-30

    Chaperonins are cage-like complexes in which nonnative polypeptides prone to aggregation are thought to reach their native state optimally. However, they also may use ATP to unfold stably bound misfolded polypeptides and mediate the out-of-cage native refolding of large proteins. Here, we show that even without ATP and GroES, both GroEL and the eukaryotic chaperonin containing t-complex polypeptide 1 (CCT/TRiC) can unfold stable misfolded polypeptide conformers and readily release them from the access ways to the cage. Reconciling earlier disparate experimental observations to ours, we present a comprehensive model whereby following unfolding on the upper cavity, in-cage confinement is not needed for the released intermediates to slowly reach their native state in solution. As over-sticky intermediates occasionally stall the catalytic unfoldase sites, GroES mobile loops and ATP are necessary to dissociate the inhibitory species and regenerate the unfolding activity. Thus, chaperonin rings are not obligate confining antiaggregation cages. They are polypeptide unfoldases that can iteratively convert stable off-pathway conformers into functional proteins. PMID:23584019

  12. GroEL and CCT are catalytic unfoldases mediating out-of-cage polypeptide refolding without ATP

    PubMed Central

    Priya, Smriti; Sharma, Sandeep Kumar; Sood, Vishal; Mattoo, Rayees U. H.; Finka, Andrija; Azem, Abdussalam; De Los Rios, Paolo; Goloubinoff, Pierre

    2013-01-01

    Chaperonins are cage-like complexes in which nonnative polypeptides prone to aggregation are thought to reach their native state optimally. However, they also may use ATP to unfold stably bound misfolded polypeptides and mediate the out-of-cage native refolding of large proteins. Here, we show that even without ATP and GroES, both GroEL and the eukaryotic chaperonin containing t-complex polypeptide 1 (CCT/TRiC) can unfold stable misfolded polypeptide conformers and readily release them from the access ways to the cage. Reconciling earlier disparate experimental observations to ours, we present a comprehensive model whereby following unfolding on the upper cavity, in-cage confinement is not needed for the released intermediates to slowly reach their native state in solution. As over-sticky intermediates occasionally stall the catalytic unfoldase sites, GroES mobile loops and ATP are necessary to dissociate the inhibitory species and regenerate the unfolding activity. Thus, chaperonin rings are not obligate confining antiaggregation cages. They are polypeptide unfoldases that can iteratively convert stable off-pathway conformers into functional proteins. PMID:23584019

  13. The C2 Domain and Altered ATP-Binding Loop Phosphorylation at Ser359 Mediate the Redox-Dependent Increase in Protein Kinase C-δ Activity

    PubMed Central

    Gong, Jianli; Yao, Yongneng; Zhang, Pingbo; Udayasuryan, Barath; Komissarova, Elena V.; Chen, Ju; Sivaramakrishnan, Sivaraj; Van Eyk, Jennifer E.

    2015-01-01

    The diverse roles of protein kinase C-δ (PKCδ) in cellular growth, survival, and injury have been attributed to stimulus-specific differences in PKCδ signaling responses. PKCδ exerts membrane-delimited actions in cells activated by agonists that stimulate phosphoinositide hydrolysis. PKCδ is released from membranes as a Tyr313-phosphorylated enzyme that displays a high level of lipid-independent activity and altered substrate specificity during oxidative stress. This study identifies an interaction between PKCδ's Tyr313-phosphorylated hinge region and its phosphotyrosine-binding C2 domain that controls PKCδ's enzymology indirectly by decreasing phosphorylation in the kinase domain ATP-positioning loop at Ser359. We show that wild-type (WT) PKCδ displays a strong preference for substrates with serine as the phosphoacceptor residue at the active site when it harbors phosphomimetic or bulky substitutions at Ser359. In contrast, PKCδ-S359A displays lipid-independent activity toward substrates with either a serine or threonine as the phosphoacceptor residue. Additional studies in cardiomyocytes show that oxidative stress decreases Ser359 phosphorylation on native PKCδ and that PKCδ-S359A overexpression increases basal levels of phosphorylation on substrates with both phosphoacceptor site serine and threonine residues. Collectively, these studies identify a C2 domain-pTyr313 docking interaction that controls ATP-positioning loop phosphorylation as a novel, dynamically regulated, and physiologically relevant structural determinant of PKCδ catalytic activity. PMID:25755284

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

    PubMed

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

    2012-07-01

    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(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. PMID:22751669

  16. Crystal Structure of Yeast DNA Polymerase ε Catalytic Domain

    PubMed Central

    Jain, Rinku; Rajashankar, Kanagalaghatta R.; Buku, Angeliki; Johnson, Robert E.; Prakash, Louise; Prakash, Satya; Aggarwal, Aneel K.

    2014-01-01

    DNA polymerase ε (Polε) is a multi-subunit polymerase that contributes to genomic stability via its roles in leading strand replication and the repair of damaged DNA. Here we report the ternary structure of the Polε catalytic subunit (Pol2) bound to a nascent G:C base pair (Pol2G:C). Pol2G:C has a typical B-family polymerase fold and embraces the template-primer duplex with the palm, fingers, thumb and exonuclease domains. The overall arrangement of domains is similar to the structure of Pol2T:A reported recently, but there are notable differences in their polymerase and exonuclease active sites. In particular, we observe Ca2+ ions at both positions A and B in the polymerase active site and also observe a Ca2+ at position B of the exonuclease site. We find that the contacts to the nascent G:C base pair in the Pol2G:C structure are maintained in the Pol2T:A structure and reflect the comparable fidelity of Pol2 for nascent purine-pyrimidine and pyrimidine-purine base pairs. We note that unlike that of Pol3, the shape of the nascent base pair binding pocket in Pol2 is modulated from the major grove side by the presence of Tyr431. Together with Pol2T:A, our results provide a framework for understanding the structural basis of high fidelity DNA synthesis by Pol2. PMID:24733111

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

    PubMed

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

    2015-10-01

    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

  18. 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

  19. PROPERTIES OF CATALYTIC, LINKER AND CHITIN-BINDING DOMAINS OF INSECT CHITINASE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Manduca sexta (tobacco hornworm) chitinase is a glycoprotein that consists of an N-terminal catalytic domain, a Ser/Thr-rich linker region, and a C-terminal chitin-binding domain. To delineate the properties of these domains, we have generated truncated forms of chitinase, which were expressed in i...

  20. Cytochemical localization of ATP diphosphohydrolase from Leishmania (Viannia) braziliensis promastigotes and identification of an antigenic and catalytically active isoform.

    PubMed

    Rezende-Soares, F A; Carvalho-Campos, C; Marques, M J; Porcino, G N; Giarola, N L L; Costa, B L S; Taunay-Rodrigues, A; Faria-Pinto, P; Souza, M A; Diniz, V A; Corte-Real, S; Juliano, M A; Juliano, L; Vasconcelos, E G

    2010-04-01

    An ATP diphosphohydrolase (EC 3.6.1.5) activity was identified in a Leishmania (Viannia) braziliensis promastigotes preparation (Lb). Ultrastructural cytochemical microscopy showed this protein on the parasite surface and also stained a possible similar protein at the mitochondrial membrane. Isolation of an active ATP diphosphohydrolase isoform from Lb was obtained by cross-immunoreactivity with polyclonal anti-potato apyrase antibodies. These antibodies, immobilized on Protein A-Sepharose, immunoprecipitated a polypeptide of approximately 48 kDa and, in lower amount, a polypeptide of approximately 43 kDa, and depleted 83% ATPase and 87% of the ADPase activities from detergent-homogenized Lb. Potato apyrase was recognized in Western blots by IgG antibody from American cutaneous leishmaniasis (ACL) patients, suggesting that the parasite and vegetable proteins share antigenic conserved epitopes. Significant IgG seropositivity in serum samples diluted 1:50 from ACL patients (n=20) for Lb (65%) and potato apyrase (90%) was observed by ELISA technique. Significant IgG antibody reactivity was also observed against synthetic peptides belonging to a conserved domain from L. braziliensis NDPase (80% seropositivity) and its potato apyrase counterpart (50% seropositivity), in accordance with the existence of shared antigenic epitopes and demonstrating that in leishmaniasis infection the domain r82-103 from L. braziliensis NDPase is a target for the human immune response. PMID:19961654

  1. 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.

    PubMed

    Milgrom, Elena M; Milgrom, Yakov M

    2012-06-29

    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(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. PMID:22659742

  2. NMR characterization of the interaction of the endonuclease domain of MutL with divalent metal ions and ATP.

    PubMed

    Mizushima, Ryota; Kim, Ju Yaen; Suetake, Isao; Tanaka, Hiroaki; Takai, Tomoyo; Kamiya, Narutoshi; Takano, Yu; Mishima, Yuichi; Tajima, Shoji; Goto, Yuji; Fukui, Kenji; Lee, Young-Ho

    2014-01-01

    MutL is a multi-domain protein comprising an N-terminal ATPase domain (NTD) and C-terminal dimerization domain (CTD), connected with flexible linker regions, that plays a key role in DNA mismatch repair. To expand understanding of the regulation mechanism underlying MutL endonuclease activity, our NMR-based study investigated interactions between the CTD of MutL, derived from the hyperthermophilic bacterium Aquifex aeolicus (aqMutL-CTD), and putative binding molecules. Chemical shift perturbation analysis with the model structure of aqMutL-CTD and circular dichroism results revealed that tight Zn(2+) binding increased thermal stability without changing secondary structures to function at high temperatures. Peak intensity analysis exploiting the paramagnetic relaxation enhancement effect indicated the binding site for Mn(2+), which shared binding sites for Zn(2+). The coexistence of these two metal ions appears to be important for the function of MutL. Chemical shift perturbation analysis revealed a novel ATP binding site in aqMutL-CTD. A docking simulation incorporating the chemical shift perturbation data provided a putative scheme for the intermolecular interactions between aqMutL-CTD and ATP. We proposed a simple and understandable mechanical model for the regulation of MutL endonuclease activity in MMR based on the relative concentrations of ATP and CTD through ATP binding-regulated interdomain interactions between CTD and NTD. PMID:24901533

  3. NMR Characterization of the Interaction of the Endonuclease Domain of MutL with Divalent Metal Ions and ATP

    PubMed Central

    Mizushima, Ryota; Kim, Ju Yaen; Suetake, Isao; Tanaka, Hiroaki; Takai, Tomoyo; Kamiya, Narutoshi; Takano, Yu; Mishima, Yuichi; Tajima, Shoji; Goto, Yuji; Fukui, Kenji; Lee, Young-Ho

    2014-01-01

    MutL is a multi-domain protein comprising an N-terminal ATPase domain (NTD) and C-terminal dimerization domain (CTD), connected with flexible linker regions, that plays a key role in DNA mismatch repair. To expand understanding of the regulation mechanism underlying MutL endonuclease activity, our NMR-based study investigated interactions between the CTD of MutL, derived from the hyperthermophilic bacterium Aquifex aeolicus (aqMutL-CTD), and putative binding molecules. Chemical shift perturbation analysis with the model structure of aqMutL-CTD and circular dichroism results revealed that tight Zn2+ binding increased thermal stability without changing secondary structures to function at high temperatures. Peak intensity analysis exploiting the paramagnetic relaxation enhancement effect indicated the binding site for Mn2+, which shared binding sites for Zn2+. The coexistence of these two metal ions appears to be important for the function of MutL. Chemical shift perturbation analysis revealed a novel ATP binding site in aqMutL-CTD. A docking simulation incorporating the chemical shift perturbation data provided a putative scheme for the intermolecular interactions between aqMutL-CTD and ATP. We proposed a simple and understandable mechanical model for the regulation of MutL endonuclease activity in MMR based on the relative concentrations of ATP and CTD through ATP binding-regulated interdomain interactions between CTD and NTD. PMID:24901533

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

    PubMed

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

    2015-09-25

    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 Mg(2+) 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

  5. The DUSP–Ubl domain of USP4 enhances its catalytic efficiency by promoting ubiquitin exchange

    PubMed Central

    Clerici, Marcello; Luna-Vargas, Mark P. A.; Faesen, Alex C.; Sixma, Titia K.

    2014-01-01

    Ubiquitin-specific protease USP4 is emerging as an important regulator of cellular pathways, including the TGF-β response, NF-κB signalling and splicing, with possible roles in cancer. Here we show that USP4 has its catalytic triad arranged in a productive conformation. Nevertheless, it requires its N-terminal DUSP–Ubl domain to achieve full catalytic turnover. Pre-steady-state kinetics measurements reveal that USP4 catalytic domain activity is strongly inhibited by slow dissociation of ubiquitin after substrate hydrolysis. The DUSP–Ubl domain is able to enhance ubiquitin dissociation, hence promoting efficient turnover. In a mechanism that requires all USP4 domains, binding of the DUSP–Ubl domain promotes a change of a switching loop near the active site. This ‘allosteric regulation of product discharge’ provides a novel way of regulating deubiquitinating enzymes that may have relevance for other enzyme classes. PMID:25404403

  6. Interactions between Metal-binding Domains Modulate Intracellular Targeting of Cu(I)-ATPase ATP7B, as Revealed by Nanobody Binding*

    PubMed Central

    Huang, Yiping; Nokhrin, Sergiy; Hassanzadeh-Ghassabeh, Gholamreza; Yu, Corey H.; Yang, Haojun; Barry, Amanda N.; Tonelli, Marco; Markley, John L.; Muyldermans, Serge; Dmitriev, Oleg Y.; Lutsenko, Svetlana

    2014-01-01

    The biologically and clinically important membrane transporters are challenging proteins to study because of their low level of expression, multidomain structure, and complex molecular dynamics that underlies their activity. ATP7B is a copper transporter that traffics between the intracellular compartments in response to copper elevation. The N-terminal domain of ATP7B (N-ATP7B) is involved in binding copper, but the role of this domain in trafficking is controversial. To clarify the role of N-ATP7B, we generated nanobodies that interact with ATP7B in vitro and in cells. In solution NMR studies, nanobodies revealed the spatial organization of N-ATP7B by detecting transient functionally relevant interactions between metal-binding domains 1–3. Modulation of these interactions by nanobodies in cells enhanced relocalization of the endogenous ATP7B toward the plasma membrane linking molecular and cellular dynamics of the transporter. Stimulation of ATP7B trafficking by nanobodies in the absence of elevated copper provides direct evidence for the important role of N-ATP7B structural dynamics in regulation of ATP7B localization in a cell. PMID:25253690

  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. 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.

  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. PMID:25012190

  10. 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.

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

    DOE PAGESBeta

    Olek, Anna T.; Rayon, Catherine; Makowski, Lee; Kim, Hyung Rae; Ciesielski, Peter; Badger, John; Paul, Lake N.; Ghosh, Subhangi; Kihara, Daisuke; Crowley, Michael; et al

    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

  12. Structure-function relationships in the catalytic and starch binding domains of glucoamylase.

    PubMed

    Coutinho, P M; Reilly, P J

    1994-03-01

    Sixteen primary sequences from five sub-families of fungal, yeast and bacterial glucoamylases were related to structural information from the model of the catalytic domain of Aspergillus awamori var. X100 glucoamylase obtained by protein crystallography. This domain is composed of thirteen alpha-helices, with five conserved regions defining the active site. Interactions between methyl alpha-maltoside and active site residues were modelled, and the importance of these residues on the catalytic action of different glucoamylases was shown by their presence in each primary sequence. The overall structure of the starch binding domain of some fungal glucoamylases was determined based on homology to the C-terminal domains of Bacillus cyclodextrin glucosyl-transferases. Crystallography indicated that this domain contains 6-8 beta-strands and homology allowed the attribution of a disulfide bridge in the glucoamylase starch binding domain. Glucoamylase residues Thr525, Asn530 and Trp560, homologous to Bacillus stearothermophilus cyclodextrin glucosyltransferase residues binding to maltose in the C-terminal domain, could be involved in raw-starch binding. The structure and length of the linker region between the catalytic and starch binding domains in fungal glucoamylases can vary substantially, a further indication of the functional independence of the two domains. PMID:8177888

  13. 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. PMID:7656014

  14. Time domain computational modeling of viscothermal acoustic propagation in catalytic converter substrates with porous walls

    NASA Astrophysics Data System (ADS)

    Dickey, N. S.; Selamet, A.; Miazgowicz, K. D.; Tallio, K. V.; Parks, S. J.

    2005-08-01

    Models for viscothermal effects in catalytic converter substrates are developed for time domain computational methods. The models are suitable for use in one-dimensional approaches for the prediction of exhaust system performance (engine tuning characteristics) and radiated sound levels. Starting with the ``low reduced frequency'' equations for viscothermal acoustic propagation in capillary tubes, time domain submodels are developed for the frequency-dependent wall friction, frequency-dependent wall heat transfer, and porous wall effects exhibited by catalytic converter substrates. Results from a time domain computational approach employing these submodels are compared to available analytical solutions for the low reduced frequency equations. The computational results are shown to agree well with the analytical solutions for capillary geometries representative of automotive catalytic converter substrates.

  15. Domain function dissection and catalytic properties of Listeria monocytogenes p60 protein with bacteriolytic activity.

    PubMed

    Yu, Minfeng; Zuo, Jinrong; Gu, Hao; Guo, Minliang; Yin, Yuelan

    2015-12-01

    The major extracellular protein p60 of Listeria monocytogenes (Lm-p60) is an autolysin that can hydrolyze the peptidoglycan of bacterial cell wall and has been shown to be required for L. monocytogenes virulence. The predicted three-dimensional structure of Lm-p60 showed that Lm-p60 could be split into two independent structural domains at the amino acid residue 270. Conserved motif analysis showed that V30, D207, S395, and H444 are the key amino acid residues of the corresponding motifs. However, not only the actual functions of these two domains but also the catalytic properties of Lm-p60 are unclear. We try to express recombinant Lm-p60 and identify the functions of two domains by residue substitution (V30A, D207A, S395A, and H444A) and peptide truncation. The C-terminal domain was identified as catalytic element and N-terminal domain as substrate recognition and binding element. Either N-terminal domain truncation or C-terminal domain truncation presents corresponding biological activity. The catalytic activity of Lm-p60 with a malfunctioned substrate-binding domain was decreased, while the substrate binding was not affected by a mulfunctioned catalytic domain. With turbidimetric method, we determined the optimal conditions for the bacteriolytic activity of Lm-p60 against Micrococcus lysodeikficus. The assay for the effect of Lm-p60 on the bacteriolytic activity of lysozyme revealed that the combined use of Lm-p60 protein with lysozyme showed a strong synergistic effect on the bacteriolytic activity. PMID:26363556

  16. Dynamics of endoglucanase catalytic domains: implications towards thermostability.

    PubMed

    Yennamalli, Ragothaman M; Wolt, Jeffrey D; Sen, Taner Z

    2011-12-01

    Thermostable endoglucanases play a crucial role in the production of biofuels to breakdown plant cellulose. Analyzing their structure-dynamics relationship can inform about the origins of their thermostability. Although tertiary structures of many endoglucanase proteins are available, the relationship between thermostability, structure, and dynamics is not explored fully. We have generated elastic network models for thermostable and mesostable endoglucanases with the (αβ)₈ fold in substrate bound and unbound states. The comparative analyses shed light on the relation between protein dynamics, thermostability, and substrate binding. We observed specific differences in the dynamic behavior of catalytic residues in slow modes: while both the nucleophile and the acid/base donor residues show positively correlated motions in the thermophile, their dynamics is uncoupled in the mesophile. Our proof-of-concept comparison study suggests that global dynamics can be harnessed to further our understanding of thermostability. PMID:22066537

  17. 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.

  18. 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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  20. 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

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

    2016-03-01

    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

  1. 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

    Maharana, Jitendra; Sahoo, Bikash Ranjan; Bej, Aritra; Jena, Itishree; Parida, Arunima; 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

  2. 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

  3. The Non-Catalytic Domains of Drosophila Katanin Regulate Its Abundance and Microtubule-Disassembly Activity

    PubMed Central

    Grode, Kyle D.; Rogers, Stephen L.

    2015-01-01

    Microtubule severing is a biochemical reaction that generates an internal break in a microtubule and regulation of microtubule severing is critical for cellular processes such as ciliogenesis, morphogenesis, and meiosis and mitosis. Katanin is a conserved heterodimeric ATPase that severs and disassembles microtubules, but the molecular determinants for regulation of microtubule severing by katanin remain poorly defined. Here we show that the non-catalytic domains of Drosophila katanin regulate its abundance and activity in living cells. Our data indicate that the microtubule-interacting and trafficking (MIT) domain and adjacent linker region of the Drosophila katanin catalytic subunit Kat60 cooperate to regulate microtubule severing in two distinct ways. First, the MIT domain and linker region of Kat60 decrease its abundance by enhancing its proteasome-dependent degradation. The Drosophila katanin regulatory subunit Kat80, which is required to stabilize Kat60 in cells, conversely reduces the proteasome-dependent degradation of Kat60. Second, the MIT domain and linker region of Kat60 augment its microtubule-disassembly activity by enhancing its association with microtubules. On the basis of our data, we propose that the non-catalytic domains of Drosophila katanin serve as the principal sites of integration of regulatory inputs, thereby controlling its ability to sever and disassemble microtubules. PMID:25886649

  4. Structure of the catalytic domain of Plasmodium falciparum ARF GTPase-activating protein (ARFGAP)

    SciTech Connect

    Cook, William J.; Senkovich, Olga; Chattopadhyay, Debasish

    2012-03-26

    The crystal structure of the catalytic domain of the ADP ribosylation factor GTPase-activating protein (ARFGAP) from Plasmodium falciparum has been determined and refined to 2.4 {angstrom} resolution. Multiwavelength anomalous diffraction (MAD) data were collected utilizing the Zn{sup 2+} ion bound at the zinc-finger domain and were used to solve the structure. The overall structure of the domain is similar to those of mammalian ARFGAPs. However, several amino-acid residues in the area where GAP interacts with ARF1 differ in P. falciparum ARFGAP. Moreover, a number of residues that form the dimer interface in the crystal structure are unique in P. falciparum ARFGAP.

  5. Expression, purification, and characterization of human osteoclastic protein-tyrosine phosphatase catalytic domain in Escherichia coli.

    PubMed

    Jiang, Huan; Sui, Yuan; Cui, Yue; Lin, Peng; Li, Wannan; Xing, Shu; Wang, Deli; Hu, Min; Fu, Xueqi

    2015-03-01

    Osteoclastic protein tyrosine phosphatase (PTP-oc) is a structurally unique transmembrane protein tyrosine phosphatase (PTP) that contains only a relatively small intracellular PTP catalytic domain, does not have an extracellular domain, and lacks a signal peptide proximal to the NH2 terminus. The present study reports the expression, purification, and characterization of the intracellular catalytic domain of PTP-oc (ΔPTP-oc). ΔPTP-oc was expressed in Escherichia coli cells as a fusion with a six-histidine tag and was purified via nickel affinity chromatography. When with para-nitrophenylphosphate (p-NPP) as a substrate, ΔPTP-oc exhibited classical Michaelis-Menten kinetics. Its responses to temperature and ionic strength were similar to those of other PTPs. The optimal pH value of ΔPTP-oc is approximately 7.0, unlike other PTPs, whose optimal pH values are approximately 5.0. PMID:25462809

  6. 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.

  7. Biochemical properties and catalytic domain structure of the CcmH protein from Escherichia coli.

    PubMed

    Zheng, Xue-Ming; Hong, Jing; Li, Hai-Yin; Lin, Dong-Hai; Hu, Hong-Yu

    2012-12-01

    In the Gram-negative bacterium of Escherichia coli, eight genes organized as a ccm operon (ccmABCDEFGH) are involved in the maturation of c-type cytochromes. The proteins encoded by the last three genes ccmFGH are believed to form a lyase complex functioning in the reduction of apocytochrome c and haem attachment. Among them, CcmH is a membrane-associated protein; its N-terminus is a catalytic domain with the active CXXC motif and the C-terminus is predicted as a TPR-like domain with unknown function. By using SCAM (scanning cysteine accessibility mutagenesis) and Gaussia luciferase fusion assays, we provide experimental evidence for the entire topological structure of E. coli CcmH. The mature CcmH is a periplasm-resident oxidoreductase anchored to the inner membrane by two transmembrane segments. Both N- and C-terminal domains are located and function in the periplasmic compartment. Moreover, the N-terminal domain forms a monomer in solution, while the C-terminal domain is a compact fold with helical structures. The NMR solution structure of the catalytic domain in reduced form exhibits mainly a three-helix bundle, providing further information for the redox mechanism. The redox potential suggests that CcmH exhibits a strong reductase that may function in the last step of reduction of apocytochrome c for haem attachment. PMID:22789558

  8. ATP-dependent partitioning of the DNA template into supercoiled domains by Escherichia coli UvrAB

    SciTech Connect

    Koo, Hyeon-Sook; Liu, L.F.; Claassen, L.; Grossman, L. )

    1991-02-15

    The helicase action of the Escherichia coli UvrAB complex on a covalently closed circular DNA template was monitored using bacterial DNA topoisomerase I, which specifically removes negative supercoils. In the presence of E. coli DNA topoisomerase I and ATP, the UvrAB complex gradually introduced positive supercoils into the input relaxed plasmid DNA template. Positive supercoils were not produced when E. coli DNA topoisomerase I was replaced by eukaryotic DNA topoisomerase I or when both E. coli and eukaryotic DNA topoisomerases I were added simultaneously. These results suggest that like other DNA helix-tracking processes, the ATP-dependent action of the UvrAM complex on duplex DNA simultaneously generates both positive and negative supercoils, which are not constrained by protein binding but are torsionally strained. The supercoiling activity of UvrAB on UV-damaged DNA was also studied using UV-damaged plasmid DNA and a mutant UvrA protein that lacks the 40 C-terminal amino acids and is defective in preferential binding to UV-damaged DNA. UvrAB was found to preferentially supercoil the UV-damaged DNA template, whereas the mutant protein supercoiled UV-damaged and undamaged DNA with equal efficiency. The authors results therefore suggest that the DNA helix-tracking activity of UvrAB may be involved in searching and/or prepriming the damaged DNA for UvrC incision. A possible role of supercoiled domains in the incision process is discussed.

  9. Biochemical characterization and NMR studies of the nucleotide-binding domain 1 of multidrug-resistance-associated protein 1: evidence for interaction between ATP and Trp653.

    PubMed Central

    Ramaen, Odile; Masscheleyn, Sandrine; Duffieux, Francis; Pamlard, Olivier; Oberkampf, Marine; Lallemand, Jean-Yves; Stoven, Véronique; Jacquet, Eric

    2003-01-01

    Multidrug-resistance-associated protein 1 (MRP1/ABCC1) is a human ATP-binding cassette transporter that confers cell resistance to antitumour drugs. Its NBDs (nucleotide-binding domains) bind/hydrolyse ATP, a key step in the activation of MRP1 function. To relate its intrinsic functional features to the mechanism of action of the full-size transporter, we expressed the N-terminal NBD1 domain (Asn(642) to Ser(871)) in Escherichia coli. NBD1 was highly purified under native conditions and was characterized as a soluble monomer. (15)N-labelling allowed recording of the first two-dimensional NMR spectra of this domain. The NMR study showed that NBD1 was folded, and that Trp(653) was a key residue in the NBD1-ATP interaction. Thus, interaction of NBD1 with ATP/ADP was studied by intrinsic tryptophan fluorescence. The affinity for ATP and ADP were in the same range (K (d(ATP))=118 microM and K (d(ADP))=139 microM). Binding of nucleotides did not influence the monomeric state of NBD1. The ATPase activity of NBD1 was magnesium-dependent and very low [V (max) and K (m) values of 5x10(-5) pmol of ATP x (pmol NBD1)(-1) x s(-1) and 833 microM ATP respectively]. The present study suggests that NBD1 has a low contribution to the ATPase activity of full-length MRP1 and/or that this activity requires NBD1-NBD2 heterodimer formation. PMID:12954082

  10. Biochemical characterization and NMR studies of the nucleotide-binding domain 1 of multidrug-resistance-associated protein 1: evidence for interaction between ATP and Trp653.

    PubMed

    Ramaen, Odile; Masscheleyn, Sandrine; Duffieux, Francis; Pamlard, Olivier; Oberkampf, Marine; Lallemand, Jean-Yves; Stoven, Véronique; Jacquet, Eric

    2003-12-15

    Multidrug-resistance-associated protein 1 (MRP1/ABCC1) is a human ATP-binding cassette transporter that confers cell resistance to antitumour drugs. Its NBDs (nucleotide-binding domains) bind/hydrolyse ATP, a key step in the activation of MRP1 function. To relate its intrinsic functional features to the mechanism of action of the full-size transporter, we expressed the N-terminal NBD1 domain (Asn(642) to Ser(871)) in Escherichia coli. NBD1 was highly purified under native conditions and was characterized as a soluble monomer. (15)N-labelling allowed recording of the first two-dimensional NMR spectra of this domain. The NMR study showed that NBD1 was folded, and that Trp(653) was a key residue in the NBD1-ATP interaction. Thus, interaction of NBD1 with ATP/ADP was studied by intrinsic tryptophan fluorescence. The affinity for ATP and ADP were in the same range (K (d(ATP))=118 microM and K (d(ADP))=139 microM). Binding of nucleotides did not influence the monomeric state of NBD1. The ATPase activity of NBD1 was magnesium-dependent and very low [V (max) and K (m) values of 5x10(-5) pmol of ATP x (pmol NBD1)(-1) x s(-1) and 833 microM ATP respectively]. The present study suggests that NBD1 has a low contribution to the ATPase activity of full-length MRP1 and/or that this activity requires NBD1-NBD2 heterodimer formation. PMID:12954082

  11. Expression and purification of correctly processed, active human TACE catalytic domain in Saccharomyces cerevisiae.

    PubMed

    Clarke, H R; Wolfson, M F; Rauch, C T; Castner, B J; Huang, C P; Gerhart, M J; Johnson, R S; Cerretti, D P; Paxton, R J; Price, V L; Black, R A

    1998-06-01

    Human tumor necrosis factor-alpha (TNF alpha) converting enzyme (TACE) releases soluble TNF alpha from cells. It is a member of the adamalysin family of metalloproteases. A truncated form of TACE cDNA was expressed in Saccharomyces cerevisiae and purified to homogeneity in order to study TACE structure and function. Recombinant TACE was expressed as a preproprotein including the pro- and catalytic (PROCAT) domains fused to the yeast alpha-factor leader. A C-terminal immunoreactive FLAG peptide was added for Western blot detection and anti-FLAG antibody column purification. We constructed two glycosylation mutant PROCAT TACE isoforms to facilitate purification. A PROCAT isoform, mutated to eliminate two N-linked glycosylation sites, was buffer exchanged and purified to homogeneity by ion exchange chromatography and an anti-FLAG antibody affinity step. N-terminal sequence analysis showed that the mutant preproprotein was processed in yeast at the furin protease cleavage site and yielded an active catalytic domain which has TNF alpha peptide-specific protease activity. Mass spectrometry of the purified catalytic domain showed that removal of both N-linked sites results in a homogeneous sized polypeptide lacking further posttranslational modifications. PMID:9631522

  12. Spectrum of mutations in the ATP binding domain of ATP7B gene of Wilson Disease in a regional Indian cohort.

    PubMed

    Guggilla, Sreenivasa Rao; Senagari, Jalandhar Reddy; Rao, P N; Madireddi, Sujatha

    2015-09-10

    Wilson disease is an autosomal recessive disorder of abnormal copper accumulation in the liver, brain, kidney and cornea, resulting in hepatic and neurological abnormalities, which results from impaired ATP7B protein function due to mutations in candidate ATP7B gene, till date more than 500 disease causing mutations were found. In India most disease causing mutations were identified in ATP-BD. DNA samples of the 101 WD cases and 100 control population were analyzed for mutations. 11 mutations were identified in 57 chromosomes. Three novel mutations, c.3310T>A (p.Cys1104Ser), c.3337C>A (p.Leu1113Met) on exon 15 and c.3877G>A (p.Glu1293Lys) on exon 18 were identified for the first time in the ATP7B gene. Two mutations, c.3121C>T (p.Arg1041Trp) and c.3128T>C (p.Leu1043Pro) on exon 14 were discovered for the first time in Indian Wilson disease patients. Four previously reported mutations c.3008C>T, c.3029A>G on exon 13, c.3182G>A on exon 14 and c.3809A>G on exon 18 from South India were also found in this study. Our research has enriched the spectrum of mutations of the ATP7B gene in the south Indian population. The detection of new mutations in the ATP7B gene can aid in genetic counseling and clinical or/prenatal diagnosis. PMID:25982861

  13. The auto-inhibitory domain and ATP-independent microtubule-binding region of Kinesin heavy chain are major functional domains for transport in the Drosophila germline

    PubMed Central

    Williams, Lucy S.; Ganguly, Sujoy; Loiseau, Philippe; Ng, Bing Fu; Palacios, Isabel M.

    2014-01-01

    The major motor Kinesin-1 provides a key pathway for cell polarization through intracellular transport. Little is known about how Kinesin works in complex cellular surroundings. Several cargos associate with Kinesin via Kinesin light chain (KLC). However, KLC is not required for all Kinesin transport. A putative cargo-binding domain was identified in the C-terminal tail of fungal Kinesin heavy chain (KHC). The tail is conserved in animal KHCs and might therefore represent an alternative KLC-independent cargo-interacting region. By comprehensive functional analysis of the tail during Drosophila oogenesis we have gained an understanding of how KHC achieves specificity in its transport and how it is regulated. This is, to our knowledge, the first in vivo structural/functional analysis of the tail in animal Kinesins. We show that the tail is essential for all functions of KHC except Dynein transport, which is KLC dependent. These tail-dependent KHC activities can be functionally separated from one another by further characterizing domains within the tail. In particular, our data show the following. First, KHC is temporally regulated during oogenesis. Second, the IAK domain has an essential role distinct from its auto-inhibitory function. Third, lack of auto-inhibition in itself is not necessarily detrimental to KHC function. Finally, the ATP-independent microtubule-binding motif is required for cargo localization. These results stress that two unexpected highly conserved domains, namely the auto-inhibitory IAK and the auxiliary microtubule-binding motifs, are crucial for transport by Kinesin-1 and that, although not all cargos are conserved, their transport involves the most conserved domains of animal KHCs. PMID:24257625

  14. Studies on the catalytic domains of multiple JmjC oxygenases using peptide substrates

    PubMed Central

    Williams, Sophie T; Walport, Louise J; Hopkinson, Richard J; Madden, Sarah K; Chowdhury, Rasheduzzaman; Schofield, Christopher J; Kawamura, Akane

    2014-01-01

    The JmjC-domain-containing 2-oxoglutarate-dependent oxygenases catalyze protein hydroxylation and Nε-methyllysine demethylation via hydroxylation. A subgroup of this family, the JmjC lysine demethylases (JmjC KDMs) are involved in histone modifications at multiple sites. There are conflicting reports as to the substrate selectivity of some JmjC oxygenases with respect to KDM activities. In this study, a panel of modified histone H3 peptides was tested for demethylation against 15 human JmjC-domain-containing proteins. The results largely confirmed known Nε-methyllysine substrates. However, the purified KDM4 catalytic domains showed greater substrate promiscuity than previously reported (i.e., KDM4A was observed to catalyze demethylation at H3K27 as well as H3K9/K36). Crystallographic analyses revealed that the Nε-methyllysine of an H3K27me3 peptide binds similarly to Nε-methyllysines of H3K9me3/H3K36me3 with KDM4A. A subgroup of JmjC proteins known to catalyze hydroxylation did not display demethylation activity. Overall, the results reveal that the catalytic domains of the KDM4 enzymes may be less selective than previously identified. They also draw a distinction between the Nε-methyllysine demethylation and hydroxylation activities within the JmjC subfamily. These results will be of use to those working on functional studies of the JmjC enzymes. PMID:25625844

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  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. Structural basis for the ATP-induced isomerization of kinesin.

    PubMed

    Chang, Qing; Nitta, Ryo; Inoue, Shigeyuki; Hirokawa, Nobutaka

    2013-06-12

    Kinesin superfamily proteins (KIFs) are microtubule-based molecular motors driven by the energy derived from the hydrolysis of ATP. Previous studies have revealed that the ATP binding step is crucial both for the power stroke to produce motility and for the inter-domain regulation of ATPase activity to guarantee the processive movement of dimeric KIFs. Here, we report the first crystal structure of KIF4 complexed with the non-hydrolyzable ATP analog, AMPPNP (adenylyl imidodiphosphate), at 1.7Å resolution. By combining our structure with previously solved KIF1A structures complexed with two ATP analogs, molecular snapshots during ATP binding reveal that the closure of the nucleotide-binding pocket during ATP binding is achieved by closure of the backdoor. Closure of the backdoor stabilizes two mobile regions, switch I and switch II, to generate the phosphate tube from which hydrolyzed phosphate is released. Through the stabilization of switch II, the local conformational change at the catalytic center is further relayed to the neck-linker element that fully docks to the catalytic core to produce the power stroke. Because the neck linker is a sole element that connects the partner heads in dimeric KIFs, this tight structural coordination between the catalytic center and neck linker enables inter-domain communication between the partner heads. This study also revealed the putative microtubule-binding site of KIF4, thus providing structural insights that describe the specific binding of KIF4 to the microtubule. PMID:23500491

  18. [Molecular engineering of cellulase catalytic domain based on glycoside hydrolase family].

    PubMed

    Zhang, Xiaomei; Li, Dandan; Wang, Lushan; Zhao, Yue; Chen, Guanjun

    2013-04-01

    Molecular engineering of cellulases can improve enzymatic activity and efficiency. Recently, the Carbohydrate-Active enZYmes Database (CAZy), including glycoside hydrolase (GH) families, has been established with the development of Omics and structural measurement technologies. Molecular engineering based on GH families can obviously decrease the probing space of target sequences and structures, and increase the odds of experimental success. Besides, the study of cellulase active-site architecture paves the way toward the explanation of catalytic mechanism. This review focuses on the main GH families and the latest progresses in molecular engineering of catalytic domain. Based on the combination of analysis of a large amount of data in the same GH family and their conservative active-site architecture information, rational design will be an important direction for molecular engineering and promote the rapid development of the conversion of biomass. PMID:23894816

  19. 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

  20. 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.

  1. 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. PMID:27183903

  2. Correlated Mutation Analysis on the Catalytic Domains of Serine/Threonine Protein Kinases

    PubMed Central

    Xu, Feng; Du, Pan; Shen, Hongbo; Hu, Hairong; Wu, Qi; Xie, Jun; Yu, Long

    2009-01-01

    Background Protein kinases (PKs) have emerged as the largest family of signaling proteins in eukaryotic cells and are involved in every aspect of cellular regulation. Great progresses have been made in understanding the mechanisms of PKs phosphorylating their substrates, but the detailed mechanisms, by which PKs ensure their substrate specificity with their structurally conserved catalytic domains, still have not been adequately understood. Correlated mutation analysis based on large sets of diverse sequence data may provide new insights into this question. Methodology/Principal Findings Statistical coupling, residue correlation and mutual information analyses along with clustering were applied to analyze the structure-based multiple sequence alignment of the catalytic domains of the Ser/Thr PK family. Two clusters of highly coupled sites were identified. Mapping these positions onto the 3D structure of PK catalytic domain showed that these two groups of positions form two physically close networks. We named these two networks as θ-shaped and γ-shaped networks, respectively. Conclusions/Significance The θ-shaped network links the active site cleft and the substrate binding regions, and might participate in PKs recognizing and interacting with their substrates. The γ-shaped network is mainly situated in one side of substrate binding regions, linking the activation loop and the substrate binding regions. It might play a role in supporting the activation loop and substrate binding regions before catalysis, and participate in product releasing after phosphoryl transfer. Our results exhibit significant correlations with experimental observations, and can be used as a guide to further experimental and theoretical studies on the mechanisms of PKs interacting with their substrates. PMID:19526051

  3. 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

  4. 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.

  5. Quaternary structure of K[ssubscript ATP] channel SUR2A nucleotide binding domains resolved by synchrotron radiation X-ray scattering

    SciTech Connect

    Park, Sungjo; Terzic, Andre

    2010-05-25

    Heterodimeric nucleotide binding domains NBD1/NBD2 distinguish the ATP-binding cassette protein SUR2A, a recognized regulatory subunit of cardiac ATP-sensitive K{sup +} (K{sub ATP}) channels. The tandem function of these core domains ensures metabolism-dependent gating of the Kir6.2 channel pore, yet their structural arrangement has not been resolved. Here, purified monodisperse and interference-free recombinant particles were subjected to synchrotron radiation small-angle X-ray scattering (SAXS) in solution. Intensity function analysis of SAXS profiles resolved NBD1 and NBD2 as octamers. Implemented by ab initio simulated annealing, shape determination prioritized an oblong envelope wrapping NBD1 and NBD2 with respective dimensions of 168 x 80 x 37 {angstrom}{sup 3} and 175 x 81 x 37 {angstrom}{sup 3} based on symmetry constraints, validated by atomic force microscopy. Docking crystal structure homology models against SAXS data reconstructed the NBD ensemble surrounding an inner cleft suitable for Kir6.2 insertion. Human heart disease-associated mutations introduced in silico verified the criticality of the mapped protein-protein interface. The resolved quaternary structure delineates thereby a macromolecular arrangement of K{sub ATP} channel SUR2A regulatory domains.

  6. 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

  7. Phosphorylation of the TOR ATP binding domain by AGC kinase constitutes a novel mode of TOR inhibition.

    PubMed

    Hálová, Lenka; Du, Wei; Kirkham, Sara; Smith, Duncan L; Petersen, Janni

    2013-11-25

    TOR (target of rapamycin) signaling coordinates cell growth, metabolism, and cell division through tight control of signaling via two complexes, TORC1 and TORC2. Here, we show that fission yeast TOR kinases and mTOR are phosphorylated on an evolutionarily conserved residue of their ATP-binding domain. The Gad8 kinase (AKT homologue) phosphorylates fission yeast Tor1 at this threonine (T1972) to reduce activity. A T1972A mutation that blocked phosphorylation increased Tor1 activity and stress resistance. Nitrogen starvation of fission yeast inhibited TOR signaling to arrest cell cycle progression in G1 phase and promoted sexual differentiation. Starvation and a Gad8/T1972-dependent decrease in Tor1 (TORC2) activity was essential for efficient cell cycle arrest and differentiation. Experiments in human cell lines recapitulated these yeast observations, as mTOR was phosphorylated on T2173 in an AKT-dependent manner. In addition, a T2173A mutation increased mTOR activity. Thus, TOR kinase activity can be reduced through AGC kinase-controlled phosphorylation to generate physiologically significant changes in TOR signaling. PMID:24247430

  8. Structure of the HHARI Catalytic Domain Shows Glimpses of a HECT E3 Ligase

    PubMed Central

    Spratt, Donald E.; Mercier, Pascal; Shaw, Gary S.

    2013-01-01

    The ubiquitin-signaling pathway utilizes E1 activating, E2 conjugating, and E3 ligase enzymes to sequentially transfer the small modifier protein ubiquitin to a substrate protein. During the last step of this cascade different types of E3 ligases either act as scaffolds to recruit an E2 enzyme and substrate (RING), or form an ubiquitin-thioester intermediate prior to transferring ubiquitin to a substrate (HECT). The RING-inBetweenRING-RING (RBR) proteins constitute a unique group of E3 ubiquitin ligases that includes the Human Homologue of Drosophila Ariadne (HHARI). These E3 ligases are proposed to use a hybrid RING/HECT mechanism whereby the enzyme uses facets of both the RING and HECT enzymes to transfer ubiquitin to a substrate. We now present the solution structure of the HHARI RING2 domain, the key portion of this E3 ligase required for the RING/HECT hybrid mechanism. The structure shows the domain possesses two Zn2+-binding sites and a single exposed cysteine used for ubiquitin catalysis. A structural comparison of the RING2 domain with the HECT E3 ligase NEDD4 reveals a near mirror image of the cysteine and histidine residues in the catalytic site. Further, a tandem pair of aromatic residues exists near the C-terminus of the HHARI RING2 domain that is conserved in other RBR E3 ligases. One of these aromatic residues is remotely located from the catalytic site that is reminiscent of the location found in HECT E3 enzymes where it is used for ubiquitin catalysis. These observations provide an initial structural rationale for the RING/HECT hybrid mechanism for ubiquitination used by the RBR E3 ligases. PMID:24058416

  9. Assay and Inhibition of the Purified Catalytic Domain of Diacylglycerol Lipase Beta.

    PubMed

    Singh, Praveen K; Markwick, Rachel; Lu, Leanne; Howell, Fiona V; Williams, Gareth; Doherty, Patrick

    2016-05-17

    The diacylglycerol lipases (DAGLα and DAGLβ) hydrolyze DAG to generate 2-arachidonoylglycerol (2-AG), the principal endocannabinoid and main precursor of arachidonic acid (AA). The DAGLs make distinct tissue specific contributions toward 2-AG and AA levels, and therefore, selective modulators for these enzymes could play crucial roles toward harnessing their therapeutic potential. Relatively high-throughput assays have recently been reported for DAGLα and have proven useful toward the characterization of inhibitors of this enzyme. Similar assays are also warranted for DAGLβ which was the aim of this study. We first adapted previously reported DAGLα membrane assays (using PNPB and DiFMUO as substrates) to measure recombinant DAGLβ activity in membranes. In contrast to results with DAGLα, both substrates provided a relatively limited signal window for measuring DAGLβ activity, however, an improved window was obtained when employing a third commercially available substrate, EnzChek. In order to further improve on the assay parameters, we successfully purified the glutathione S-transferase (GST) tagged catalytic domain of DAGLβ. Activity of the enzyme was confirmed using EnzChek as well as two DAGL inhibitors (THL and OMDM-188). The purified DAGLβ catalytic domain assay described here provides the basis for a relatively clean and convenient assay with the potential to be adapted for high-throughput drug discovery efforts. PMID:27115711

  10. A Novel Catalytic Function of Synthetic IgG-Binding Domain (Z Domain) from Staphylococcal Protein A: Light Emission with Coelenterazine.

    PubMed

    Inouye, Satoshi; Sahara-Miura, Yuiko

    2014-01-01

    The synthetic IgG-binding domain (Z domain) of staphylococcal protein A catalyzes the oxidation of coelenterazine to emit light like a coelenterazine-utilizing luciferase. The Z domain derivatives (ZZ-gCys, Z-gCys and Z-domain) were purified and the luminescence properties were characterized by comparing with coelenterazine-utilizing luciferases, including Renilla luciferase, Gaussia luciferase and the catalytic 19 kDa protein of Oplophorus luciferase. Three Z domain derivatives showed luminescence activity with coelenterazine and the order of the initial maximum intensity of luminescence was ZZ-gCys (100%) > Z-gCys (36.8%) > Z-domain (1.1%) > bovine serum albumin (BSA; 0.9%) > staphylococcal protein A (0.1%) and the background value of coelenterazine (0.1%) in our conditions. The luminescence properties of ZZ-gCys showed the similarity to that of Gaussia luciferase, including the luminescence pattern, the emission spectrum, the stimulation by halogen ions and nonionic detergents and the substrate specificity for coelenterazine analogues. In contrast, the luminescence properties of Z-gCys were close to the catalytic 19 kDa protein of Oplophorus luciferase. The catalytic region of the Z domain for the luminescence reaction might be different from the IgG-binding region of the Z domain. PMID:24138575

  11. The Structure of the Catalytic Domain of a Plant Cellulose Synthase and Its Assembly into Dimers[C][W][OPEN

    PubMed Central

    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.

    2014-01-01

    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. PMID:25012190

  12. 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

  13. 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.

  14. 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. PMID:27208174

  15. 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. PMID:22303012

  16. Alternating Access to the Transmembrane Domain of the ATP-binding Cassette Protein Cystic Fibrosis Transmembrane Conductance Regulator (ABCC7)*

    PubMed Central

    Wang, Wuyang; Linsdell, Paul

    2012-01-01

    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. PMID:22303012

  17. 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

  18. 1.92 Angstrom Zinc-Free APOBEC3F Catalytic Domain Crystal Structure.

    PubMed

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

    2016-06-01

    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. PMID:27139641

  19. Conformational selection in the recognition of phosphorylated substrates by the catalytic domain of human Pin1.

    PubMed

    Velazquez, Hector A; Hamelberg, Donald

    2011-11-01

    Post-translational phosphorylation and the related conformational changes in signaling proteins are responsible for regulating a wide range of subcellular processes. Human Pin1 is central to many of these cell signaling pathways in normal and aberrant subcellular processes, catalyzing cis-trans isomerization of the peptide ω-bond in phosphorylated serine/threonine-proline motifs in many proteins. Pin1 has therefore been identified as a possible drug target in many diseases, including cancer and Alzheimer's. The effects of phosphorylation on Pin1 substrates, and the atomistic basis for Pin1 recognition and catalysis, are not well understood. Here, we determine the conformational consequences of phosphorylation on Pin1 substrate analogues and the mechanism of recognition by the catalytic domain of Pin1 using all-atom molecular dynamics simulations. We show that phosphorylation induces backbone conformational changes on the peptide substrate analogues. We also show that Pin1 recognizes specific conformations of its substrate by conformational selection. Furthermore, dynamical correlated motions in the free Pin1 enzyme are present in the enzyme of the enzyme-substrate complex when the substrate is in the transition state configuration, suggesting that these motions play significant roles during catalytic turnover. These results provide a detailed atomistic picture of the mechanism of Pin1 recognition that can be exploited for drug design purposes and further our understanding of the synergistic complexities of post-translational phosphorylation and cis-trans isomerization. PMID:21967280

  20. 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.

  1. 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

  2. Essential lysine residues within transmembrane helix 1 of diphtheria toxin facilitate COPI binding and catalytic domain entry

    PubMed Central

    Trujillo, Carolina; Taylor-Parker, Julian; Harrison, Robert; Murphy, John R.

    2014-01-01

    The translocation of the diphtheria toxin catalytic domain from the lumen of early endosomes into the cytosol of eukaryotic cells is an essential step in the intoxication process. We have previously shown that the in vitro translocation of the catalytic domain from the lumen of toxin pre-loaded endosomal vesicles to the external medium requires the addition of cytosolic proteins including coatomer protein complex I (COPI) to the reaction mixture. Further, we have shown that transmembrane helix 1 plays an essential, but as yet undefined role in the entry process. We have used both site-directed mutagenesis and a COPI complex precipitation assay to demonstrate that interaction(s) between at least three lysine residues in transmembrane helix 1 are essential for both COPI complex binding and the delivery of the catalytic domain into the target cell cytosol. Finally, a COPI binding domain swap was used to demonstrate that substitution of the lysine-rich transmembrane helix 1with the COPI binding portion of the p23 adaptor cytoplasmic tail results in a mutant that displays full wild type activity. Thus, irrespective of sequence, the ability of transmembrane helix 1 to bind to COPI complex appears to be the essential feature for catalytic domain delivery to the cytosol. PMID:20398220

  3. Redox-Linked Domain Movements in the Catalytic Cycle of Cytochrome P450 Reductase

    PubMed Central

    Huang, Wei-Cheng; Ellis, Jacqueline; Moody, Peter C.E.; Raven, Emma L.; Roberts, Gordon C.K.

    2013-01-01

    Summary NADPH-cytochrome P450 reductase is a key component of the P450 mono-oxygenase drug-metabolizing system. There is evidence for a conformational equilibrium involving large-scale domain motions in this enzyme. We now show, using small-angle X-ray scattering (SAXS) and small-angle neutron scattering, that delivery of two electrons to cytochrome P450 reductase leads to a shift in this equilibrium from a compact form, similar to the crystal structure, toward an extended form, while coenzyme binding favors the compact form. We present a model for the extended form of the enzyme based on nuclear magnetic resonance and SAXS data. Using the effects of changes in solution conditions and of site-directed mutagenesis, we demonstrate that the conversion to the extended form leads to an enhanced ability to transfer electrons to cytochrome c. This structural evidence shows that domain motion is linked closely to the individual steps of the catalytic cycle of cytochrome P450 reductase, and we propose a mechanism for this. PMID:23911089

  4. Diversity between mammalian tolloid proteinases: Oligomerisation and non-catalytic domains influence activity and specificity

    PubMed Central

    Bayley, Christopher P.; Ruiz Nivia, Hilda D.; Dajani, Rana; Jowitt, Thomas A.; Collins, Richard F.; Rada, Heather; Bird, Louise E.; Baldock, Clair

    2016-01-01

    The mammalian tolloid family of metalloproteinases is essential for tissue patterning and extracellular matrix assembly. The four members of the family: bone morphogenetic protein-1 (BMP-1), mammalian tolloid (mTLD), tolloid-like (TLL)-1 and TLL-2 differ in their substrate specificity and activity levels, despite sharing similar domain organization. We have previously described a model of substrate exclusion by dimerisation to explain differences in the activities of monomeric BMP-1 and dimers of mTLD and TLL-1. Here we show that TLL-2, the least active member of the tolloid family, is predominantly monomeric in solution, therefore it appears unlikely that substrate exclusion via dimerisation is a mechanism for regulating TLL-2 activity. X-ray scattering and electron microscopy structural and biophysical analyses reveal an elongated shape for the monomer and flexibility in the absence of calcium. Furthermore, we show that TLL-2 can cleave chordin in vitro, similar to other mammalian tolloids, but truncated forms of TLL-2 mimicking BMP-1 are unable to cleave chordin. However, both the N- and C-terminal non-catalytic domains from all mammalian tolloids bind chordin with high affinity. The mechanisms underlying substrate specificity and activity in the tolloid family are complex with variation between family members and depend on both multimerisation and substrate interaction. PMID:26902455

  5. Explorations of Catalytic Domains in Non-Ribosomal Peptide Synthetase Enzymology

    PubMed Central

    Hur, Gene H.; Vickery, Christopher R.; Burkart, Michael D.

    2016-01-01

    Many pharmaceuticals on the market today belong to a large class of natural products called nonribosomal peptides (NRPs). Originating from bacteria and fungi, these peptide-based natural products consist not only of the 20 canonical L-amino acids, but also non-proteinogenic amino acids, heterocyclic rings, sugars, and fatty acids, generating tremendous chemical diversity. As a result, these secondary metabolites exhibit a broad array of bioactivity ranging from antimicrobial to anticancer. The biosynthesis of these complex compounds is carried out by large multimodular megaenzymes called nonribosomal peptide synthetases (NRPSs). Each module is responsible for incorporation of a monomeric unit into the natural product peptide and is composed of individual domains that perform different catalytic reactions. Biochemical and bioinformatic investigations of these enzymes have uncovered the key principles of NRP synthesis, expanding the pharmaceutical potential of their enzymatic processes. Progress has been made in the manipulation of this biosynthetic machinery to develop new chemoenzymatic approaches for synthesizing novel pharmaceutical agents with increased potency. This review focuses on the recent discoveries and breakthroughs in the structural elucidation, molecular mechanism, and chemical biology underlying the discrete domains within NRPSs. PMID:22802156

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  8. 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

  9. 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.

  10. 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.

  11. 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.

  12. 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

  13. 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

  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

    2016-01-01

    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. PMID:26725083

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

    PubMed Central

    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

    2016-01-01

    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. DOI: http://dx.doi.org/10.7554/eLife.12095.001 PMID:26725083

  16. Exchange of ADP with ATP in the CII ATPase domain promotes autophosphorylation of cyanobacterial clock protein KaiC

    PubMed Central

    Nishiwaki-Ohkawa, Taeko; Kitayama, Yohko; Ochiai, Erika; Kondo, Takao

    2014-01-01

    The cyanobacterial circadian oscillator can be reconstituted in vitro. In the presence of KaiA and KaiB, the phosphorylation state of KaiC oscillates with a periodicity of ∼24 h. KaiC is a hexameric P-loop ATPase with autophosphorylation and autodephosphorylation activities. Recently, we found that dephosphorylation of KaiC occurs via reversal of the phosphorylation reaction: a phosphate group attached to Ser431/Thr432 is transferred to KaiC-bound ADP to generate ATP, which is subsequently hydrolyzed. This unusual reaction mechanism suggests that the KaiC phosphorylation rhythm is sustained by periodic shifts in the equilibrium of the reversible autophosphorylation reaction, the molecular basis of which has never been elucidated. Because KaiC-bound ATP and ADP serve as substrates for the forward and reverse reactions, respectively, we investigated the regulation of the nucleotide-bound state of KaiC. In the absence of KaiA, the condition in which the reverse reaction proceeds, KaiC favored the ADP-bound state. KaiA increased the ratio of ATP to total KaiC-bound nucleotides by facilitating the release of bound ADP and the incorporation of exogenous ATP, allowing the forward reaction to proceed. When both KaiA and KaiB were present, the ratio of ATP to total bound nucleotides exhibited a circadian rhythm, whose phase was advanced by several hours relative to that of the phosphorylation rhythm. Based on these findings, we propose that the direction of the reversible autophosphorylation reaction is regulated by KaiA and KaiB at the level of substrate availability and that this regulation sustains the oscillation of the phosphorylation state of KaiC. PMID:24616498

  17. Exchange of ADP with ATP in the CII ATPase domain promotes autophosphorylation of cyanobacterial clock protein KaiC.

    PubMed

    Nishiwaki-Ohkawa, Taeko; Kitayama, Yohko; Ochiai, Erika; Kondo, Takao

    2014-03-25

    The cyanobacterial circadian oscillator can be reconstituted in vitro. In the presence of KaiA and KaiB, the phosphorylation state of KaiC oscillates with a periodicity of ∼24 h. KaiC is a hexameric P-loop ATPase with autophosphorylation and autodephosphorylation activities. Recently, we found that dephosphorylation of KaiC occurs via reversal of the phosphorylation reaction: a phosphate group attached to Ser431/Thr432 is transferred to KaiC-bound ADP to generate ATP, which is subsequently hydrolyzed. This unusual reaction mechanism suggests that the KaiC phosphorylation rhythm is sustained by periodic shifts in the equilibrium of the reversible autophosphorylation reaction, the molecular basis of which has never been elucidated. Because KaiC-bound ATP and ADP serve as substrates for the forward and reverse reactions, respectively, we investigated the regulation of the nucleotide-bound state of KaiC. In the absence of KaiA, the condition in which the reverse reaction proceeds, KaiC favored the ADP-bound state. KaiA increased the ratio of ATP to total KaiC-bound nucleotides by facilitating the release of bound ADP and the incorporation of exogenous ATP, allowing the forward reaction to proceed. When both KaiA and KaiB were present, the ratio of ATP to total bound nucleotides exhibited a circadian rhythm, whose phase was advanced by several hours relative to that of the phosphorylation rhythm. Based on these findings, we propose that the direction of the reversible autophosphorylation reaction is regulated by KaiA and KaiB at the level of substrate availability and that this regulation sustains the oscillation of the phosphorylation state of KaiC. PMID:24616498

  18. Phosphatidylserine-containing membranes alter the thermal stability of prothrombin's catalytic domain: a differential scanning calorimetric study.

    PubMed

    Lentz, B R; Zhou, C M; Wu, J R

    1994-05-10

    Denaturation profiles of bovine prothrombin and its isolated fragments were examined in the presence of Na2EDTA, 5 mM CaCl2, and CaCl2 plus membranes containing 1-palmitoyl-2-oleoyl-3-sn-phosphatidylcholine (POPC) in combination with bovine brain phosphatidylserine (PS). We have shown previously [Lentz, B. R., Wu, J. R., Sorrentino, A. M., & Carleton, J. A. (1991) Biophys. J. 60, 70] that binding to PS/POPC (25/75) large unilamellar vesicles resulted in an enthalpy loss in the main endotherm of prothrombin denaturation (Tm approximately 57-58 degrees C) and a comparable enthalpy gain in a minor endotherm (Tm approximately 59 degrees C) accompanying an upward shift in peak temperature (Tm approximately 73 degrees C). This minor endotherm was also responsive to Ca2+ binding and, in the absence of PS/POPC membranes, corresponded to melting of the N-terminal, Ca2+ and membrane binding domain (fragment 1). Peak deconvolution analysis of the prothrombin denaturation profile and extensive studies of the denaturation of isolated prothrombin domains in the presence and absence of PS/POPC vesicles suggested that membrane binding induced changes in the C-terminal catalytic domain of prothrombin (prethrombin 2) and in a domain that links fragment 1 with the catalytic domain (fragment 2). Specifically, the results have confirmed that the fragment 2 domain interacts with the stabilizes the prethrombin 2 domain and also have shown that fragment 2 interacts directly with the membrane. In addition, the results have demonstrated a heretofore unrecognized interaction between the catalytic and membrane binding domains. This interaction can account for another portion of the denaturation enthalpy that appears at high temperatures in the presence of membranes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8180168

  19. 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

  20. Small-angle X-ray scattering analysis reveals the ATP-bound monomeric state of the ATPase domain from the homodimeric MutL endonuclease, a GHKL phosphotransferase superfamily protein.

    PubMed

    Iino, Hitoshi; Hikima, Takaaki; Nishida, Yuya; Yamamoto, Masaki; Kuramitsu, Seiki; Fukui, Kenji

    2015-05-01

    DNA mismatch repair is an excision system that removes mismatched bases chiefly generated by replication errors. In this system, MutL endonucleases direct the excision reaction to the error-containing strand of the duplex by specifically incising the newly synthesized strand. Both bacterial homodimeric and eukaryotic heterodimeric MutL proteins belong to the GHKL ATPase/kinase superfamily that comprises the N-terminal ATPase and C-terminal dimerization regions. Generally, the GHKL proteins show large ATPase cycle-dependent conformational changes, including dimerization-coupled ATP binding of the N-terminal domain. Interestingly, the ATPase domain of human PMS2, a subunit of the MutL heterodimer, binds ATP without dimerization. The monomeric ATP-bound state of the domain has been thought to be characteristic of heterodimeric GHKL proteins. In this study, we characterized the ATP-bound state of the ATPase domain from the Aquifex aeolicus MutL endonuclease, which is a homodimeric GHKL protein unlike the eukaryotic MutL. Gel filtration, dynamic light scattering, and small-angle X-ray scattering analyses clearly showed that the domain binds ATP in a monomeric form despite its homodimeric nature. This indicates that the uncoupling of dimerization and ATP binding is a common feature among bacterial and eukaryotic MutL endonucleases, which we suggest is closely related to the molecular mechanisms underlying mismatch repair. PMID:25809295

  1. Involvement of tryptophans at the catalytic and subunit-binding domains of transcarboxylase.

    PubMed

    Kumar, G K; Beegen, H; Wood, H G

    1988-08-01

    Transcarboxylase from Propionibacterium shermanii is a multisubunit enzyme. It consists of one central hexameric subunit to which six outer dimeric subunits are attached through twelve biotinyl subunits. Both the central and the outer subunits are multi-tryptophan (Trp) proteins, and each contains 5 Trps per monomer. The roles of the Trps during catalysis and assembly of the enzyme have been studied by using N-bromosuccinimide (NBS) oxidation as a probe. Modification of approximately 10 Trps of the total 90 Trps of the intact enzyme results in loss of activity. Both the substrates, viz., methylmalonyl-CoA and pyruvate, afford protection (approximately 50%) against inactivation caused by NBS. Analyses of tryptic peptide maps and intrinsic fluorescence studies have indicated that modification of 10 Trps of the whole enzyme does not cause extensive conformational changes. Therefore, the Trps appear to be essential for catalytic activity. NBS modification of the individual subunits at pH 6.5 has demonstrated differential reactivity of their Trps. Modification of the exposed/reactive Trps of either one of the subunits significantly affects the subunit assembly with the complementary unmodified subunits to form active enzyme. It is proposed that Trps are involved at the subunit-binding domains of either the central or the outer subunit of transcarboxylase, in addition to those critical for catalysis. PMID:3191102

  2. 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.

  3. Expression and characterization of catalytic domain of Plasmodium falciparum subtilisin-like protease 3.

    PubMed

    Alam, Asrar; Bhatnagar, Raj K; Chauhan, Virander S

    2012-05-01

    PfSUB3 is the third subtilisin-like protease annotated in Plasmodium genome database "PlasmoDB". The other two members, PfSUB1 and PfSUB2 have been implicated in merozoite egress and invasion in asexual blood stages. In this study, we recombinantly expressed a region of PfSUB3 spanning from Asn(334) to Glu(769) (PfSUB3c) which encompassed the predicted catalytic domain with all the active site residues and predicted mature region spanning from Thr(516) to Glu(769) (PfSUB3m) in E. coli. PfSUB3m showed PMSF-sensitive proteolytic activity in in vitro assays. Replacement of active site serine with alanine in PfSUB3m resulted in inactive protein. We found that PfSUB3c and PfSUB3m undergo truncation to produce a 25-kDa species which was sufficient for proteolytic activity. Quantitative real-time PCR, immnufluorescence assay and Western blot analyses revealed that PfSUB3 is expressed at late asexual blood stages. Serine protease activity of PfSUB3 and its expression in the late stages of erythrocytic schizogony are indicative of some possible role of the protease in merozoite egress and/or invasion processes. PMID:22285468

  4. Biochemical and spectroscopic characterization of the catalytic domain of MMP16 (cdMMP16).

    PubMed

    Meng, Fan; Yang, Hao; Aitha, Mahesh; George, Sam; Tierney, David L; Crowder, Michael W

    2016-07-01

    Membrane-bound matrix metalloproteinase 16 (MMP16/MT3-MMP) is considered a drug target due to its role(s) in disease processes such as cancer and inflammation. Biochemical characterization of MMP16 is critical for developing new generation MMP inhibitors (MMPi), which exhibit high efficacies and selectivities. Herein, a modified over-expression and purification protocol was used to prepare the catalytic domain of MMP16 (cdMMP16). The resulting recombinant enzyme exhibited steady-state kinetic constants of K m = 10.6 ± 0.7 μM and k cat = 1.14 ± 0.02 s(-1), when using FS-6 as substrate, and the enzyme bound 1.8 ± 0.1 eq of Zn(II). The enzymatic activity of cdMMP16 is salt concentration-dependent, and cdMMP16 exhibits autoproteolytic activity under certain conditions, which may be related to an in vivo regulatory mechanism of MMP16 and of other membrane-type MMPs (MT-MMPs). Co(II)-substituted analogs (Co2- and ZnCo) of cdMMP16 were prepared and characterized using several spectroscopic techniques, such as UV-Vis, (1)H NMR, and EXAFS spectroscopies. A well-characterized cdMMP16 is now available for future inhibitor screening efforts. PMID:27229514

  5. 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+). PMID:27338011

  6. The Role of Catalytic Substrate Morphology on the Shape and Domain Size of Two-Dimensional Boron Nitride Sheets

    NASA Astrophysics Data System (ADS)

    Griep, Mark; Tay, Roland; Tumlin, Travis; Teo, Edwin; Mallick, Govind; Karna, Shashi

    2014-03-01

    Two-dimensional (2D) nanomaterials, including graphene and boron nitride (BN), has been of intense interest in recent years due to their exceptional electronic, thermal, and mechanical properties. Tailoring these novel properties to their maximum potential requires precise control of the atomic layer growth process. In recent years, catalytic growth of 2-D nanomaterials using chemical vapor deposition (CVD) process has emerged as an attractive approach due to their low-cost, scalalibility, and ability totransfer the grown materials on various substrates. In this approach, The the morphology and purity of the catalytic surface plays a critical role on the shape, size, and growth kintectics of the 2D nanomaterial. In this work, we present the results of our systematic studies of the role of catalytic surface morphology on the shape and domain size of CVD grown hexagonal boron nitride (hBN) films. The present work clearly demonstrates that that the presence of surface roghness in the form of ridges leads to a preferential growth of small-domain triangular BN sheets. A 10 to 100-fold reduction in the surfcae roughness leads to increased domain BN triangles, eventually transitioning to large-domain hexagonal shaped hBN sheets.

  7. Mutational analysis of predicted interactions between the catalytic and P domains of prohormone convertase 3 (PC3/PC1)

    PubMed Central

    Ueda, Kazuya; Lipkind, Gregory M.; Zhou, An; Zhu, Xiaorong; Kuznetsov, Andrey; Philipson, Louis; Gardner, Paul; Zhang, Chunling; Steiner, Donald F.

    2003-01-01

    The subtilisin-like prohormone convertases (PCs) contain an essential downstream domain (P domain), which has been predicted to have a β-barrel structure that interacts with and stabilizes the catalytic domain (CAT). To assess possible sites of hydrophobic interaction, a series of mutant PC3–enhanced GFP constructs were prepared in which selected nonpolar residues on the surface of CAT were substituted by the corresponding polar residues in subtilisin Carlsberg. To investigate the folding potential of the isolated P domain, signal peptide–P domain–enhanced GFP constructs with mutated and/or truncated P domains were also made. All mutants were expressed in βTC3 cells, and their subcellular localization and secretion were determined. The mutants fell into three main groups: (i) Golgi/secreted, (ii) ER/nonsecreted, and (iii) apoptosis inducing. The destabilizing CAT mutations indicate that the side chains of V292, T328, L351, Q408, H409, V412, and F441 and nonpolar fragments of the side chains of R405 and W413 form a hydrophobic patch on CAT that interacts with the P domain. We also have found that the P domain can fold independently, as indicated by its secretion. Interestingly, T594, which is near the P domain C terminus, was not essential for P domain secretion but is crucial for the stability of intact PC3. T594V produced a stable enzyme, but T594D did not, which suggests that T594 participates in important hydrophobic interactions within PC3. These findings support our conclusion that the catalytic and P domains contribute to the folding and thermodynamic stability of the convertases through reciprocal hydrophobic interactions. PMID:12721373

  8. The xynC gene from Fibrobacter succinogenes S85 codes for a xylanase with two similar catalytic domains.

    PubMed Central

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

    1993-01-01

    The xynC gene of Fibrobacter succinogenes S85 codes for a 66.4-kDa xylanase which consists of three distinct domains separated by two flexible regions rich in serine residues. Domains A and B of XynC code for catalytic domains with 56.5% identity and 9.6% similarity with each other, and both domains share homology with xylanases of Ruminococcus flavefaciens, Neocallimastix patriciarum, Clostridium acetobutylicum, Bacillus pumilus, Bacillus subtilis, and Bacillus circulans. More than 88% of the xylanase activity of Escherichia coli cells carrying the original 13-kb recombinant plasmid was released from intact cells by cold water washes. The major products of hydrolysis of xylan by both domains were xylose and xylobiose, indicating that the xynC gene product exhibits catalytic properties similar to those of the XynA xylanases from R. flavefaciens and N. patriciarum. So far, these features are not shared broadly with bacteria from other environments and may indicate specific selection for this domain structure in the highly competitive environment of the rumen. Images PMID:8244936

  9. 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

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

    PubMed

    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

    2014-03-11

    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

  11. 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.

  12. Roles of the {beta} subunit hinge domain in ATP synthase F{sub 1} sector: Hydrophobic network formed by introduced {beta}Phe174 inhibits subunit rotation

    SciTech Connect

    Nakanishi-Matsui, Mayumi; Kashiwagi, Sachiko; Kojima, Masaki; Nonaka, Takamasa; Futai, Masamitsu

    2010-04-30

    The ATP synthase {beta} subunit hinge domain ({beta}Phe148 {approx} {beta}Gly186, P-loop/{alpha}-helixB/loop/{beta}-sheet4, Escherichia coli residue numbering) dramatically changes in conformation upon nucleotide binding. We previously reported that F{sub 1} with the {beta}Ser174 to Phe mutation in the domain lowered the {gamma} subunit rotation speed, and thus decreased the ATPase activity [M. Nakanishi-Matsui, S. Kashiwagi, T. Ubukata, A. Iwamoto-Kihara, Y. Wada, M. Futai, Rotational catalysis of Escherichia coli ATP synthase F{sub 1} sector. Stochastic fluctuation and a key domain of the {beta} subunit, J. Biol. Chem. 282 (2007) 20698-20704.]. Homology modeling indicates that the amino acid replacement induces a hydrophobic network, in which the {beta}Met159, {beta}Ile163, and {beta}Ala167 residues of the {beta} subunit are involved together with the mutant {beta}Phe174. The network is expected to stabilize the conformation of {beta}{sub DP} (nucleotide-bound form of the {beta} subunit), resulting in increased activation energy for transition to {beta}{sub E} (empty {beta} subunit). The modeling further predicts that replacement of {beta}Met159 with Ala or Ile weakens the hydrophobic network. As expected, these two mutations experimentally suppressed the ATPase activities as well as subunit rotation of {beta}S174F. Furthermore, the rotation rate decreased with the increase of the strength in the hydrophobic network. These results indicate that the smooth conformational change of the {beta} subunit hinge domain is pertinent for the rotational catalysis.

  13. The type II pullulanase of Thermococcus hydrothermalis: molecular characterization of the gene and expression of the catalytic domain.

    PubMed

    Erra-Pujada, M; Debeire, P; Duchiron, F; O'Donohue, M J

    1999-05-01

    The gene encoding a hyperthermostable type II pullulanase produced by Thermococcus hydrothermalis (Th-Apu) has been isolated. Analysis of a total of 5.2 kb of genomic DNA has revealed the presence of three open reading frames, one of which (apuA) encodes the pullulanase. This enzyme is composed of 1,339 amino acid residues and exhibits a multidomain structure. In addition to a typical N-terminal signal peptide, Th-Apu possesses a catalytic domain, a domain bearing S-layer homology-like motifs, a Thr-rich region, and a potential C-terminal transmembrane domain. The presence of these noncatalytic domains suggests that Th-Apu may be anchored to the cell surface and be O glycosylated. PMID:10322035

  14. 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. PMID:22320201

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

    PubMed Central

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

    2016-01-01

    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. PMID:26668325

  16. The second metal-binding site of 70 kDa heat-shock protein is essential for ADP binding, ATP hydrolysis and ATP synthesis.

    PubMed Central

    Wu, Xueji; Yano, Mihiro; Washida, Hiroyo; Kido, Hiroshi

    2004-01-01

    The chaperone activity of Hsp70 (70 kDa heat-shock protein) in protein folding and its conformational switch, including oligomeric and monomeric interconversion, are regulated by the hydrolysis of ATP and the ATP-ADP exchange cycle. The crystal structure of human ATPase domain shows two metal-binding sites, the first for ATP binding and a second, in close proximity to the first, whose function remains unknown [Sriram, Osipiuk, Freeman, Morimoto and Joachimiak (1997) Structure 5, 403-414]. In this study, we have characterized the second metal-binding motif by site-directed mutagenesis and the kinetics of ATP and ADP binding, and found that the second metal-binding site, comprising a loop co-ordinated by His-227, Glu-231 and Asp-232, participates both in ATP hydrolysis and ATP-synthetic activities, in co-operation with the first metal-binding site. The first metal-binding site, a catalytic centre, is essential for ATP binding and the second site for ADP binding in the reactions of ATP hydrolysis and ATP synthesis. PMID:14664695

  17. Effects of mutations in the {beta} subunit hinge domain on ATP synthase F{sub 1} sector rotation: Interaction between Ser 174 and Ile 163

    SciTech Connect

    Kashiwagi, Sachiko; Iwamoto-Kihara, Atsuko; Kojima, Masaki; Nonaka, Takamasa; Futai, Masamitsu Nakanishi-Matsui, Mayumi

    2008-01-11

    A complex of {gamma}, {epsilon}, and c subunits rotates in ATP synthase (F{sub o}F{sub 1}) coupling with proton transport. Replacement of {beta}Ser174 by Phe in {beta}-sheet4 of the {beta} subunit ({beta}S174F) caused slow {gamma} subunit revolution of the F{sub 1} sector, consistent with the decreased ATPase activity [M. Nakanishi-Matsui, S. Kashiwagi, T. Ubukata, A. Iwamoto-Kihara, Y. Wada, M. Futai, Rotational catalysis of Escherichia coli ATP synthase F1 sector. Stochastic fluctuation and a key domain of the {beta} subunit, J. Biol. Chem. 282 (2007) 20698-20704]. Modeling of the domain including {beta}-sheet4 and {alpha}-helixB predicted that the mutant {beta}Phe174 residue undergoes strong and weak hydrophobic interactions with {beta}Ile163 and {beta}Ile166, respectively. Supporting this prediction, the replacement of {beta}Ile163 in {alpha}-helixB by Ala partially suppressed the {beta}S174F mutation: in the double mutant, the revolution speed and ATPase activity recovered to about half of the levels in the wild-type. Replacement of {beta}Ile166 by Ala lowered the revolution speed and ATPase activity to the same levels as in {beta}S174F. Consistent with the weak hydrophobic interaction, {beta}Ile166 to Ala mutation did not suppress {beta}S174F. Importance of the hinge domain [phosphate-binding loop (P-loop)/{alpha}-helixB/loop/{beta}-sheet4, {beta}Phe148-{beta}Gly186] as to driving rotational catalysis is discussed.

  18. 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

  19. Allosteric regulation of focal adhesion kinase by PIP₂ and ATP.

    PubMed

    Zhou, Jing; Bronowska, Agnieszka; Le Coq, Johanne; Lietha, Daniel; Gräter, Frauke

    2015-02-01

    Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that regulates cell signaling, proliferation, migration, and development. A major mechanism of regulation of FAK activity is an intramolecular autoinhibitory interaction between two of its domains--the catalytic and FERM domains. Upon cell adhesion to the extracellular matrix, FAK is being translocated toward focal adhesion sites and activated. Interactions of FAK with phosphoinositide phosphatidylinsositol-4,5-bis-phosphate (PIP₂) are required to activate FAK. However, the molecular mechanism of the activation remains poorly understood. Recent fluorescence resonance energy transfer experiments revealed a closure of the FERM-kinase interface upon ATP binding, which is reversed upon additional binding of PIP₂. Here, we addressed the allosteric regulation of FAK by performing all-atom molecular-dynamics simulations of a FAK fragment containing the catalytic and FERM domains, and comparing the dynamics in the absence or presence of ATP and PIP₂. As a major conformational change, we observe a closing and opening motion upon ATP and additional PIP₂ binding, respectively, in good agreement with the fluorescence resonance energy transfer experiments. To reveal how the binding of the regulatory PIP₂ to the FERM F2 lobe is transduced to the very distant F1/N-lobe interface, we employed force distribution analysis. We identified a network of mainly charged residue-residue interactions spanning from the PIP₂ binding site to the distant interface between the kinase and FERM domains, comprising candidate residues for mutagenesis to validate the predicted mechanism of FAK activation. PMID:25650936

  20. Linking structural features from mitochondrial and bacterial F-type ATP synthases to their distinct mechanisms of ATPase inhibition.

    PubMed

    Krah, Alexander

    2015-10-01

    ATP synthases are molecular motors, which synthesize ATP, the ubiquitous energy source in all living cells. They use an electrochemical gradient to drive a rotation in the membrane embedded Fo domain, namely the c-ring, causing a conformational change in the soluble F1 domain which leads to the catalytic event. In the opposite fashion, they can also hydrolyse ATP to maintain the ion gradient across the membrane. To prevent wasteful ATP hydrolysis, bacteria and mammals have developed peculiar mechanistic features in addition to a common one, namely MgADP inhibition. Here I discuss the distinct ATPase inhibition mechanism in mitochondrial (IF1) and bacterial (subunits ε and ζ) F-type ATP synthases, based on available structural, biophysical and biochemical data. PMID:26140992

  1. 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

  2. N-Terminal Region of the Catalytic Domain of Human N-Myristoyltransferase 1 Acts as an Inhibitory Module

    PubMed Central

    Kumar, Sujeet; Sharma, Rajendra K.

    2015-01-01

    N-myristoyltransferase (NMT) plays critical roles in the modulation of various signaling molecules, however, the regulation of this enzyme in diverse cellular states remains poorly understood. We provide experimental evidence to show for the first time that for the isoform 1 of human NMT (hNMT1), the regulatory roles extend into the catalytic core. In our present study, we expressed, purified, and characterized a truncation mutant devoid of 28 N-terminal amino acids from the catalytic module (Δ28-hNMT1s) and compared its properties to the full-length catalytic domain of hNMT1. The deletion of the N-terminal peptide had no effect on the enzyme stability. Our findings suggest that the N-terminal region in the catalytic module of hNMT1 functions serves as a regulatory control element. The observations of an ~3 fold increase in enzymatic efficiency following removal of the N-terminal peptide of hNMT1s indicates that N-terminal amino acids acts as an inhibitory segment and negatively regulate the enzyme activity. Our findings that the N-terminal region confers control over activity, taken together with the earlier observations that the N-terminal of hNMT1 is differentially processed in diverse cellular states, suggests that the proteolytic processing of the peptide segment containing the inhibitory region provides a molecular mechanism for physiological up-regulation of myristoyltransferase activity. PMID:26000639

  3. Cobinamides Are Novel Coactivators of Nitric Oxide Receptor That Target Soluble Guanylyl Cyclase Catalytic Domain

    PubMed Central

    Sharina, Iraida; Sobolevsky, Michael; Doursout, Marie-Francoise; Gryko, Dorota

    2012-01-01

    Soluble guanylyl cyclase (sGC), a ubiquitously expressed heme-containing receptor for nitric oxide (NO), is a key mediator of NO-dependent processes. In addition to NO, a number of synthetic compounds that target the heme-binding region of sGC and activate it in a NO-independent fashion have been described. We report here that dicyanocobinamide (CN2-Cbi), a naturally occurring intermediate of vitamin B12 synthesis, acts as a sGC coactivator both in vitro and in intact cells. Heme depletion or heme oxidation does not affect CN2-Cbi-dependent activation. Deletion mutagenesis demonstrates that CN2-Cbi targets a new regulatory site and functions though a novel mechanism of sGC activation. Unlike all known sGC regulators that target the N-terminal regulatory regions, CN2-Cbi directly targets the catalytic domain of sGC, resembling the effect of forskolin on adenylyl cyclases. CN2-Cbi synergistically enhances sGC activation by NO-independent regulators 3-(4-amino-5-cyclopropylpyrimidine-2-yl)-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine (BAY41-2272), 4-[((4-carboxybutyl){2-[(4-phenethylbenzyl)oxy]phenethyl}amino) methyl [benzoic]-acid (cinaciguat or BAY58-2667), and 5-chloro-2-(5-chloro-thiophene-2-sulfonylamino-N-(4-(morpholine-4-sulfonyl)-phenyl)-benzamide sodium salt (ataciguat or HMR-1766). BAY41-2272 and CN2-Cbi act reciprocally by decreasing the EC50 values. CN2-Cbi increases intracellular cGMP levels and displays vasorelaxing activity in phenylephrine-constricted rat aortic rings in an endothelium-independent manner. Both effects are synergistically potentiated by BAY41-2272. These studies uncover a new mode of sGC regulation and provide a new tool for understanding the mechanism of sGC activation and function. CN2-Cbi also offers new possibilities for its therapeutic applications in augmenting the effect of other sGC-targeting drugs. PMID:22171090

  4. The specialized Hsp70 (HscA) interdomain linker binds to its nucleotide-binding domain and stimulates ATP hydrolysis in both cis and trans configurations.

    PubMed

    Alderson, T Reid; Kim, Jin Hae; Cai, Kai; Frederick, Ronnie O; Tonelli, Marco; Markley, John L

    2014-11-25

    Proteins from the isc operon of Escherichia coli constitute the machinery used to synthesize iron-sulfur (Fe-S) clusters for delivery to recipient apoproteins. Efficient and rapid [2Fe-2S] cluster transfer from the holo-scaffold protein IscU depends on ATP hydrolysis in the nucleotide-binding domain (NBD) of HscA, a specialized Hsp70-type molecular chaperone with low intrinsic ATPase activity (0.02 min(-1) at 25 °C, henceforth reported in units of min(-1)). HscB, an Hsp40-type cochaperone, binds to HscA and stimulates ATP hydrolysis to promote cluster transfer, yet while the interactions between HscA and HscB have been investigated, the role of HscA's interdomain linker in modulating ATPase activity has not been explored. To address this issue, we created three variants of the 40 kDa NBD of HscA: NBD alone (HscA386), NBD with a partial linker (HscA389), and NBD with the full linker (HscA395). We found that the rate of ATP hydrolysis of HscA395 (0.45 min(-1)) is nearly 15-fold higher than that of HscA386 (0.035 min(-1)), although their apparent affinities for ATP are equivalent. HscA395, which contains the full covalently linked linker peptide, exhibited intrinsic tryptophan fluorescence emission and basal thermostability that were higher than those of HscA386. Furthermore, HscA395 displayed narrower (1)H(N) line widths in its two-dimensional (1)H-(15)N TROSY-HSQC spectrum in comparison to HscA386, indicating that the peptide in the cis configuration binds to and stabilizes the structure of the NBD. The addition to HscA386 of a synthetic peptide with a sequence identical to that of the interdomain linker (L(387)LLDVIPLS(395)) stimulated its ATPase activity and induced widespread NMR chemical shift perturbations indicative of a binding interaction in the trans configuration. PMID:25372495

  5. Calpain-Mediated Processing of Adenylate Cyclase Toxin Generates a Cytosolic Soluble Catalytically Active N-Terminal Domain

    PubMed Central

    Ostolaza, Helena

    2013-01-01

    Bordetella pertussis, the whooping cough pathogen, secretes several virulence factors among which adenylate cyclase toxin (ACT) is essential for establishment of the disease in the respiratory tract. ACT weakens host defenses by suppressing important bactericidal activities of the phagocytic cells. Up to now, it was believed that cell intoxication by ACT was a consequence of the accumulation of abnormally high levels of cAMP, generated exclusively beneath the host plasma membrane by the toxin N-terminal catalytic adenylate cyclase (AC) domain, upon its direct translocation across the lipid bilayer. Here we show that host calpain, a calcium-dependent Cys-protease, is activated into the phagocytes by a toxin-triggered calcium rise, resulting in the proteolytic cleavage of the toxin N-terminal domain that releases a catalytically active “soluble AC”. The calpain-mediated ACT processing allows trafficking of the “soluble AC” domain into subcellular organella. At least two strategic advantages arise from this singular toxin cleavage, enhancing the specificity of action, and simultaneously preventing an indiscriminate activation of cAMP effectors throughout the cell. The present study provides novel insights into the toxin mechanism of action, as the calpain-mediated toxin processing would confer ACT the capacity for a space- and time-coordinated production of different cAMP “pools”, which would play different roles in the cell pathophysiology. PMID:23840759

  6. Diverse Functional Properties of Wilson Disease ATP7B Variants

    PubMed Central

    Huster, Dominik; Kühne, Angelika; Bhattacharjee, Ashima; Raines, Lily; Jantsch, Vanessa; Noe, Johannes; Schirrmeister, Wiebke; Sommerer, Ines; Sabri, Osama; Berr, Frieder; Mössner, Joachim; Stieger, Bruno; Caca, Karel; Lutsenko, Svetlana

    2012-01-01

    BACKGROUND & AIMS Wilson disease is a severe disorder of copper metabolism caused by mutations in ATP7B, which encodes a copper-transporting adenosine triphosphatase. The disease presents with a variable phenotype that complicates the diagnostic process and treatment. Little is known about the mechanisms that contribute to the different phenotypes of the disease. METHODS We analyzed 28 variants of ATP7B from patients with Wilson disease that affected different functional domains; the gene products were expressed using the baculovirus expression system in Sf9 cells. Protein function was analyzed by measuring catalytic activity and copper (64Cu) transport into vesicles. We studied intracellular localization of variants of ATP7B that had measurable transport activities and were tagged with green fluorescent protein in mammalian cells using confocal laser scanning microscopy. RESULTS Properties of ATP7B variants with pathogenic amino-acid substitution varied greatly even if substitutions were in the same functional domain. Some variants had complete loss of catalytic and transport activity, whereas others lost transport activity but retained phosphor-intermediate formation or had partial losses of activity. In mammalian cells, transport-competent variants differed in stability and subcellular localization. CONCLUSIONS Variants in ATP7B associated with Wilson disease disrupt the protein’s transport activity, result in its mislocalization, and reduce its stability. Single assays are insufficient to accurately predict the effects of ATP7B variants the function of its product and development of Wilson disease. These findings will contribute to our understanding of genotype–phenotype correlation and mechanisms of disease pathogenesis. PMID:22240481

  7. Regulation of Aerobic Energy Metabolism in Podospora anserina by Two Paralogous Genes Encoding Structurally Different c-Subunits of ATP Synthase

    PubMed Central

    Sellem, Carole H.; di Rago, Jean-Paul; Lasserre, Jean-Paul; Ackerman, Sharon H.; Sainsard-Chanet, Annie

    2016-01-01

    Most of the ATP in living cells is produced by an F-type ATP synthase. This enzyme uses the energy of a transmembrane electrochemical proton gradient to synthesize ATP from ADP and inorganic phosphate. Proton movements across the membrane domain (FO) of the ATP synthase drive the rotation of a ring of 8–15 c-subunits, which induces conformational changes in the catalytic part (F1) of the enzyme that ultimately promote ATP synthesis. Two paralogous nuclear genes, called Atp9-5 and Atp9-7, encode structurally different c-subunits in the filamentous fungus Podospora anserina. We have in this study identified differences in the expression pattern for the two genes that correlate with the mitotic activity of cells in vegetative mycelia: Atp9-7 is transcriptionally active in non-proliferating (stationary) cells while Atp9-5 is expressed in the cells at the extremity (apex) of filaments that divide and are responsible for mycelium growth. When active, the Atp9-5 gene sustains a much higher rate of c-subunit synthesis than Atp9-7. We further show that the ATP9-7 and ATP9-5 proteins have antagonist effects on the longevity of P. anserina. Finally, we provide evidence that the ATP9-5 protein sustains a higher rate of mitochondrial ATP synthesis and yield in ATP molecules per electron transferred to oxygen than the c-subunit encoded by Atp9-7. These findings reveal that the c-subunit genes play a key role in the modulation of ATP synthase production and activity along the life cycle of P. anserina. Such a degree of sophistication for regulating aerobic energy metabolism has not been described before. PMID:27442014

  8. Crystallization and preliminary X-ray analysis of the ATPase domain of the σ(54)-dependent transcription activator NtrC1 from Aquifex aeolicus bound to the ATP analog ADP-BeFx.

    PubMed

    Sysoeva, Tatyana A; Yennawar, Neela; Allaire, Marc; Nixon, B Tracy

    2013-12-01

    One way that bacteria regulate the transcription of specific genes to adapt to environmental challenges is to use different σ factors that direct the RNA polymerase holoenzyme to distinct promoters. Unlike σ(70) RNA polymerase (RNAP), σ(54) RNAP is unable to initiate transcription without an activator: enhancer-binding protein (EBP). All EBPs contain one ATPase domain that belongs to the family of ATPases associated with various cellular activities (AAA+ ATPases). AAA+ ATPases use the energy of ATP hydrolysis to remodel different target macromolecules to perform distinct functions. These mechanochemical enzymes are known to form ring-shaped oligomers whose conformations strongly depend upon nucleotide status. Here, the crystallization of the AAA+ ATPase domain of an EBP from Aquifex aeolicus, NtrC1, in the presence of the non-hydrolyzable ATP analog ADP-BeFx is reported. X-ray diffraction data were collected from two crystals from two different protein fractions of the NtrC1 ATPase domain. Previously, this domain was co-crystallized with ADP and ATP, but the latter crystals were grown from the Walker B substitution variant E239A. Therefore, the new data sets are the first for a wild-type EBP ATPase domain co-crystallized with an ATP analog and they reveal a new crystal form. The resulting structure(s) will shed light on the mechanism of EBP-type transcription activators. PMID:24316836

  9. Purification, crystallization and preliminary crystallographic analysis of the catalytic domain of the extracellular cellulase CBHI from Trichoderma harzianum

    PubMed Central

    Colussi, Francieli; Textor, Larissa C.; Serpa, Viviane; Maeda, Roberto Nobuyuki; Pereira, Nei; Polikarpov, Igor

    2010-01-01

    The filamentous fungus Trichoderma harzianum has a considerable cellulolytic activity that is mediated by a complex of enzymes which are essential for the hydrolysis of microcrystalline cellulose. These enzymes were produced by the induction of T. harzianum with microcrystalline cellulose (Avicel) under submerged fermentation in a bioreactor. The catalytic core domain (CCD) of cellobiohydrolase I (CBHI) was purified from the extracellular extracts and submitted to robotic crystallization. Diffraction-quality CBHI CCD crystals were grown and an X-ray diffraction data set was collected under cryogenic conditions using a synchrotron-radiation source. PMID:20823521

  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 F0F1 ATP Synthase Complex Localizes to Membrane Rafts in Gonadotrope Cells.

    PubMed

    Allen-Worthington, Krystal; Xie, Jianjun; Brown, Jessica L; Edmunson, Alexa M; Dowling, Abigail; Navratil, Amy M; Scavelli, Kurt; Yoon, Hojean; Kim, Do-Geun; Bynoe, Margaret S; Clarke, Iain; Roberson, Mark S

    2016-09-01

    Fertility in mammals requires appropriate communication within the hypothalamic-pituitary-gonadal axis and the GnRH receptor (GnRHR) is a central conduit for this communication. The GnRHR resides in discrete membrane rafts and raft occupancy is required for signaling by GnRH. The present studies use immunoprecipitation and mass spectrometry to define peptides present within the raft associated with the GnRHR and flotillin-1, a key raft marker. These studies revealed peptides from the F0F1 ATP synthase complex. The catalytic subunits of the F1 domain were validated by immunoprecipitation, flow cytometry, and cell surface biotinylation studies demonstrating that this complex was present at the plasma membrane associated with the GnRHR. The F1 catalytic domain faces the extracellular space and catalyzes ATP synthesis when presented with ADP in normal mouse pituitary explants and a gonadotrope cell line. Steady-state extracellular ATP accumulation was blunted by coadministration of inhibitory factor 1, limiting inorganic phosphate in the media, and by chronic stimulation of the GnRHR. Steady-state extracellular ATP accumulation was enhanced by pharmacological inhibition of ecto-nucleoside triphosphate diphosphohydrolases. Kisspeptin administration induced coincident GnRH and ATP release from the median eminence into the hypophyseal-portal vasculature in ovariectomized sheep. Elevated levels of extracellular ATP augmented GnRH-induced secretion of LH from pituitary cells in primary culture, which was blocked in media containing low inorganic phosphate supporting the importance of extracellular ATP levels to gonadotrope cell function. These studies indicate that gonadotropes have intrinsic ability to metabolize ATP in the extracellular space and extracellular ATP may serve as a modulator of GnRH-induced LH secretion. PMID:27482602

  12. 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.

  13. Cancer associated missense mutations in BAP1 catalytic domain induce amyloidogenic aggregation: A new insight in enzymatic inactivation

    PubMed Central

    Bhattacharya, Sushmita; Hanpude, Pranita; Maiti, Tushar Kanti

    2015-01-01

    BRCA1 associated protein 1 (BAP1) is a nuclear deubiquitinase that regulates tumor suppressor activity and widely involves many cellular processes ranging from cell cycle regulation to gluconeogenesis. Impairment of enzymatic activity and nuclear localization induce abnormal cell proliferation. It is considered to be an important driver gene, which undergoes frequent mutations in several cancers. However the role of mutation and oncogenic gain of function of BAP1 are poorly understood. Here, we investigated cellular localization, enzymatic activity and structural changes for four missense mutants of the catalytic domain of BAP1, which are prevalent in different types of cancer. These mutations triggered cytoplasmic/perinuclear accumulation in BAP1 deficient cells, which has been observed in proteins that undergo aggregation in cellular condition. Amyloidogenic activity of mutant BAP1 was revealed from its reactivity towards anti oligomeric antibody in HEK293T cells. We have also noted structural destabilization in the catalytic domain mutants, which eventually produced beta amyloid structure as indicated in atomic force microscopy study. The cancer associated mutants up-regulate heat shock response and activates transcription of genes normally co-repressed by BAP1. Overall, our results unambiguously demonstrate that structural destabilization and subsequent aggregation abrogate its cellular mechanism leading to adverse outcome. PMID:26680512

  14. 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(-). PMID:26183108

  15. 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

  16. 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. PMID:25681694

  17. Recombinant expression of the precursor of the hemorrhagic metalloproteinase HF3 and its non-catalytic domains using a cell-free synthesis system.

    PubMed

    Menezes, Milene C; Imbert, Lionel; Kitano, Eduardo S; Vernet, Thierry; Serrano, Solange M T

    2016-09-01

    Snake venom metalloproteinases (SVMPs) participate in snakebite pathology such as hemorrhage, inflammation, and necrosis. They are synthesized as latent multi-domain precursors whose processing generates either catalytically active enzymes or free non-enzymatic domains. Recombinant expression of the precursor of P-III class SVMPs has failed due to the instability of the multi-domain polypeptide structure. Conversely, functional recombinant non-catalytic domains were obtained by prokaryotic expression systems. Here, we show for the first time the recombinant expression of the precursor of HF3, a highly hemorrhagic SVMP from Bothrops jararaca, and its non-catalytic domains, using an E. coli-based cell-free synthesis system. The precursor of HF3, composed of pro-, metalloproteinase-, disintegrin-like-, and cysteine-rich domains, and containing 38 Cys residues, was successfully expressed and purified. A protein composed of the disintegrin-like and cysteine-rich domains (DC protein) and the cysteine-rich domain alone (C protein) were expressed in vitro individually and purified. Both proteins were shown to be functional in assays monitoring the interaction with matrix proteins and in modulating the cleavage of fibrinogen by HF3. These data indicate that recombinant expression using prokaryotic-based cell-free synthesis emerges as an attractive alternative for the study of the structure and function of multi-domain proteins with a high content of Cys residues. PMID:27209197

  18. A conserved motif in transmembrane helix 1 of diphtheria toxin mediates catalytic domain delivery to the cytosol

    PubMed Central

    Ratts, Ryan; Trujillo, Carolina; Bharti, Ajit; vanderSpek, Johanna; Harrison, Robert; Murphy, John R.

    2005-01-01

    A 10-aa motif in transmembrane helix 1 of diphtheria toxin that is conserved in anthrax edema factor, anthrax lethal factor, and botulinum neurotoxin serotypes A, C, and D was identified by blast, clustal w, and meme computational analysis. Using the diphtheria toxin-related fusion protein toxin DAB389IL-2, we demonstrate that introduction of the L221E mutation into a highly conserved residue within this motif results in a nontoxic catalytic domain translocation deficient phenotype. To further probe the function of this motif in the process by which the catalytic domain is delivered from the lumen of early endosomes to the cytosol, we constructed a gene encoding a portion of diphtheria toxin transmembrane helix 1, T1, which carries the motif and is expressed from a CMV promoter. We then isolated stable transfectants of Hut102/6TG cells that express the T1 peptide, Hut102/6TG-T1. In contrast to the parental cell line, Hut102/6TG-T1 cells are ca. 104-fold more resistant to the fusion protein toxin. This resistance is completely reversed by coexpression of small interfering RNA directed against the gene encoding the T1 peptide in Hut102/6TG-T1 cells. We further demonstrate by GST-DT140-271 pull-down experiments in the presence and absence of synthetic T1 peptides the specific binding of coatomer protein complex subunit β to this region of the diphtheria toxin transmembrane domain. PMID:16230620

  19. Structural biology of Rad50 ATPase: ATP-driven conformational control in DNA double-strand break repair and the ABC-ATPase superfamily.

    PubMed

    Hopfner, K P; Karcher, A; Shin, D S; Craig, L; Arthur, L M; Carney, J P; Tainer, J A

    2000-06-23

    To clarify the key role of Rad50 in DNA double-strand break repair (DSBR), we biochemically and structurally characterized ATP-bound and ATP-free Rad50 catalytic domain (Rad50cd) from Pyrococcus furiosus. Rad50cd displays ATPase activity plus ATP-controlled dimerization and DNA binding activities. Rad50cd crystal structures identify probable protein and DNA interfaces and reveal an ABC-ATPase fold, linking Rad50 molecular mechanisms to ABC transporters, including P glycoprotein and cystic fibrosis transmembrane conductance regulator. Binding of ATP gamma-phosphates to conserved signature motifs in two opposing Rad50cd molecules promotes dimerization that likely couples ATP hydrolysis to dimer dissociation and DNA release. These results, validated by mutations, suggest unified molecular mechanisms for ATP-driven cooperativity and allosteric control of ABC-ATPases in DSBR, membrane transport, and chromosome condensation by SMC proteins. PMID:10892749

  20. 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%.

  1. Characterization of recombinant HPV6 and 11 E1 helicases: effect of ATP on the interaction of E1 with E2 and mapping of a minimal helicase domain.

    PubMed

    White, P W; Pelletier, A; Brault, K; Titolo, S; Welchner, E; Thauvette, L; Fazekas, M; Cordingley, M G; Archambault, J

    2001-06-22

    To better characterize the enzymatic activities required for human papillomavirus (HPV) DNA replication, the E1 helicases of HPV types 6 and 11 were produced using a baculovirus expression system. The purified wild type proteins and a version of HPV11 E1 lacking the N-terminal 71 amino acids, which was better expressed, were found to be hexameric over a wide range of concentrations and to have helicase and ATPase activities with relatively low values for K(m)(ATP) of 12 microm for HPV6 E1 and 6 microm for HPV11 E1. Interestingly, the value of K(m)(ATP) was increased 7-fold in the presence of the E2 transactivation domain. In turn, ATP was found to perturb the co-operative binding of E1 and E2 to DNA. Mutant and truncated versions of in vitro translated E1 were used to identify a minimal ATPase domain composed of the C-terminal 297 amino acids. This fragment was expressed, purified, and found to be fully active in ATP hydrolysis, single-stranded DNA binding, and unwinding assays, despite lacking the minimal origin-binding domain. PMID:11304544

  2. Phosphate exchange and ATP synthesis by DMSO-pretreated purified bovine mitochondrial ATP synthase.

    PubMed Central

    Beharry, S; Bragg, P D

    2001-01-01

    Purified soluble bovine mitochondrial F(1)F(o)-ATP synthase contained 2 mol of ATP, 2 mol of ADP and 6 mol of P(i)/mol. Incubation of this enzyme with 1 mM [(32)P]P(i) caused the exchange of 2 mol of P(i)/mol of F(1)F(o)-ATP synthase. The labelled phosphates were not displaced by ATP. Transfer of F(1)F(o)-ATP synthase to a buffer containing 30% (v/v) DMSO and 1 mM [(32)P]P(i) resulted in the loss of bound nucleotides with the retention of 1 mol of ATP/mol of F(1)F(o)-ATP synthase. Six molecules of [(32)P]P(i) were incorporated by exchange with the existing bound phosphate. Removal of the DMSO by passage of the enzyme through a centrifuged column of Sephadex G-50 resulted in the exchange of one molecule of bound [(32)P]P(i) into the bound ATP. Azide did not prevent this [(32)P]P(i)<-->ATP exchange reaction. The bound labelled ATP could be displaced from the enzyme by exogenous ATP. Addition of ADP to the DMSO-pretreated F(1)F(o)-ATP synthase in the original DMSO-free buffer resulted in the formation of an additional molecule of bound ATP. It was concluded that following pretreatment with and subsequent removal of DMSO the F(1)F(o)-ATP synthase contained one molecule of ATP at a catalytic site which was competent to carry out a phosphate-ATP exchange reaction using enzyme-bound inorganic radiolabelled phosphate. In the presence of ADP an additional molecule of labelled ATP was formed from enzyme-bound P(i) at a second catalytic site. The bound phosphate-ATP exchange reaction is not readily accommodated by current mechanisms for the ATP synthase. PMID:11139383

  3. The catalytic region and PEST domain of PTPN18 distinctly regulate the HER2 phosphorylation and ubiquitination barcodes.

    PubMed

    Wang, Hong-Mei; Xu, Yun-Fei; Ning, Shang-Lei; Yang, Du-Xiao; Li, Yi; Du, Yu-Jie; Yang, Fan; Zhang, Ya; Liang, Nan; Yao, Wei; Zhang, Ling-Li; Gu, Li-Chuan; Gao, Cheng-Jiang; Pang, Qi; Chen, Yu-Xin; Xiao, Kun-Hong; Ma, Rong; Yu, Xiao; Sun, Jin-Peng

    2014-09-01

    The tyrosine phosphorylation barcode encoded in C-terminus of HER2 and its ubiquitination regulate diverse HER2 functions. PTPN18 was reported as a HER2 phosphatase; however, the exact mechanism by which it defines HER2 signaling is not fully understood. Here, we demonstrate that PTPN18 regulates HER2-mediated cellular functions through defining both its phosphorylation and ubiquitination barcodes. Enzymologic characterization and three crystal structures of PTPN18 in complex with HER2 phospho-peptides revealed the molecular basis for the recognition between PTPN18 and specific HER2 phosphorylation sites, which assumes two distinct conformations. Unique structural properties of PTPN18 contribute to the regulation of sub-cellular phosphorylation networks downstream of HER2, which are required for inhibition of HER2-mediated cell growth and migration. Whereas the catalytic domain of PTPN18 blocks lysosomal routing and delays the degradation of HER2 by dephosphorylation of HER2 on pY(1112), the PEST domain of PTPN18 promotes K48-linked HER2 ubiquitination and its rapid destruction via the proteasome pathway and an HER2 negative feedback loop. In agreement with the negative regulatory role of PTPN18 in HER2 signaling, the HER2/PTPN18 ratio was correlated with breast cancer stage. Taken together, our study presents a structural basis for selective HER2 dephosphorylation, a previously uncharacterized mechanism for HER2 degradation and a novel function for the PTPN18 PEST domain. The new regulatory role of the PEST domain in the ubiquitination pathway will broaden our understanding of the functions of other important PEST domain-containing phosphatases, such as LYP and PTPN12. PMID:25081058

  4. The catalytic region and PEST domain of PTPN18 distinctly regulate the HER2 phosphorylation and ubiquitination barcodes

    PubMed Central

    Wang, Hong-Mei; Xu, Yun-Fei; Ning, Shang-Lei; Yang, Du-Xiao; Li, Yi; Du, Yu-Jie; Yang, Fan; Zhang, Ya; Liang, Nan; Yao, Wei; Zhang, Ling-Li; Gu, Li-Chuan; Gao, Cheng-Jiang; Pang, Qi; Chen, Yu-Xin; Xiao, Kun-Hong; Ma, Rong; Yu, Xiao; Sun, Jin-Peng

    2014-01-01

    The tyrosine phosphorylation barcode encoded in C-terminus of HER2 and its ubiquitination regulate diverse HER2 functions. PTPN18 was reported as a HER2 phosphatase; however, the exact mechanism by which it defines HER2 signaling is not fully understood. Here, we demonstrate that PTPN18 regulates HER2-mediated cellular functions through defining both its phosphorylation and ubiquitination barcodes. Enzymologic characterization and three crystal structures of PTPN18 in complex with HER2 phospho-peptides revealed the molecular basis for the recognition between PTPN18 and specific HER2 phosphorylation sites, which assumes two distinct conformations. Unique structural properties of PTPN18 contribute to the regulation of sub-cellular phosphorylation networks downstream of HER2, which are required for inhibition of HER2-mediated cell growth and migration. Whereas the catalytic domain of PTPN18 blocks lysosomal routing and delays the degradation of HER2 by dephosphorylation of HER2 on pY1112, the PEST domain of PTPN18 promotes K48-linked HER2 ubiquitination and its rapid destruction via the proteasome pathway and an HER2 negative feedback loop. In agreement with the negative regulatory role of PTPN18 in HER2 signaling, the HER2/PTPN18 ratio was correlated with breast cancer stage. Taken together, our study presents a structural basis for selective HER2 dephosphorylation, a previously uncharacterized mechanism for HER2 degradation and a novel function for the PTPN18 PEST domain. The new regulatory role of the PEST domain in the ubiquitination pathway will broaden our understanding of the functions of other important PEST domain-containing phosphatases, such as LYP and PTPN12. PMID:25081058

  5. Crystal structures of the catalytic domains of pseudouridine synthases RluC and RluD from Escherichia coli.

    PubMed

    Mizutani, Kenji; Machida, Yoshitaka; Unzai, Satoru; Park, Sam-Yong; Tame, Jeremy R H

    2004-04-20

    The most frequent modification of RNA, the conversion of uridine bases to pseudouridines, is found in all living organisms and often in highly conserved locations in ribosomal and transfer RNA. RluC and RluD are homologous enzymes which each convert three specific uridine bases in Escherichia coli ribosomal 23S RNA to pseudouridine: bases 955, 2504, and 2580 in the case of RluC and 1911, 1915, and 1917 in the case of RluD. Both have an N-terminal S4 RNA binding domain. While the loss of RluC has little phenotypic effect, loss of RluD results in a much reduced growth rate. We have determined the crystal structures of the catalytic domain of RluC, and full-length RluD. The S4 domain of RluD appears to be highly flexible or unfolded and is completely invisible in the electron density map. Despite the conserved topology shared by the two proteins, the surface shape and charge distribution are very different. The models suggest significant differences in substrate binding by different pseudouridine synthases. PMID:15078091

  6. 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). PMID:24742683

  7. Structure and Function of the Catalytic Domain of the Dihydrolipoyl Acetyltransferase Component in Escherichia coli Pyruvate Dehydrogenase Complex*

    PubMed Central

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

    2014-01-01

    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). PMID:24742683

  8. 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

  9. Identification of three critical acidic residues of poly(ADP-ribose) glycohydrolase involved in catalysis: determining the PARG catalytic domain

    PubMed Central

    Patel, Chandra N.; Koh, David W.; Jacobson, Myron K.; Oliveira, Marcos A.

    2005-01-01

    PARG [poly(ADP-ribose) glycohydrolase] catalyses the hydrolysis of α(1″→2′) or α(1‴→2″) O-glycosidic linkages of ADP-ribose polymers to produce free ADP-ribose. We investigated possible mechanistic similarities between PARG and glycosidases, which also cleave O-glycosidic linkages. Glycosidases typically utilize two acidic residues for catalysis, thus we targeted acidic residues within a conserved region of bovine PARG that has been shown to contain an inhibitor-binding site. The targeted glutamate and aspartate residues were changed to asparagine in order to minimize structural alterations. Mutants were purified and assayed for catalytic activity, as well as binding, to an immobilized PARG inhibitor to determine ability to recognize substrate. Our investigation revealed residues essential for PARG catalytic activity. Two adjacent glutamic acid residues are found in the conserved sequence Gln755-Glu-Glu757, and a third residue found in the conserved sequence Val737-Asp-Phe-Ala-Asn741. Our functional characterization of PARG residues, along with recent identification of an inhibitor-binding residue Tyr796 and a glycine-rich region Gly745-Gly-Gly747 important for PARG function, allowed us to define a PARG ‘signature sequence’ [vDFA-X3-GGg-X6–8-vQEEIRF-X3-PE-X14-E-X12-YTGYa], which we used to identify putative PARG sequences across a range of organisms. Sequence alignments, along with our mapping of PARG functional residues, suggest the presence of a conserved catalytic domain of approx. 185 residues which spans residues 610–795 in bovine PARG. PMID:15658938

  10. Structure of the catalytic domain of avian sarcoma virus integrase with a bound HIV-1 integrase-targeted inhibitor

    PubMed Central

    Lubkowski, Jacek; Yang, Fan; Alexandratos, Jerry; Wlodawer, Alexander; Zhao, He; Burke, Terrence R.; Neamati, Nouri; Pommier, Yves; Merkel, George; Skalka, Anna Marie

    1998-01-01

    The x-ray structures of an inhibitor complex of the catalytic core domain of avian sarcoma virus integrase (ASV IN) were solved at 1.9- to 2.0-Å resolution at two pH values, with and without Mn2+ cations. This inhibitor (Y-3), originally identified in a screen for inhibitors of the catalytic activity of HIV type 1 integrase (HIV-1 IN), was found in the present study to be active against ASV IN as well as HIV-1 IN. The Y-3 molecule is located in close proximity to the enzyme active site, interacts with the flexible loop, alters loop conformation, and affects the conformations of active site residues. As crystallized, a Y-3 molecule stacks against its symmetry-related mate. Preincubation of IN with metal cations does not prevent inhibition, and Y-3 binding does not prevent binding of divalent cations to IN. Three compounds chemically related to Y-3 also were investigated, but no binding was observed in the crystals. Our results identify the structural elements of the inhibitor that likely determine its binding properties. PMID:9560188

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

    PubMed Central

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

    2002-01-01

    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. PMID:11997452

  12. 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.

  13. 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

  14. 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.

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

    DOE PAGESBeta

    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; et al

    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

  16. A Novel Family of Soluble Minimal Scaffolds Provides Structural Insight into the Catalytic Domains of Integral Membrane Metallopeptidases*

    PubMed Central

    López-Pelegrín, Mar; Cerdà-Costa, Núria; Martínez-Jiménez, Francisco; Cintas-Pedrola, Anna; Canals, Albert; Peinado, Juan R.; Marti-Renom, Marc A.; López-Otín, Carlos; Arolas, Joan L.; Gomis-Rüth, F. Xavier

    2013-01-01

    In the search for structural models of integral-membrane metallopeptidases (MPs), we discovered three related proteins from thermophilic prokaryotes, which we grouped into a novel family called “minigluzincins.” We determined the crystal structures of the zymogens of two of these (Pyrococcus abyssi proabylysin and Methanocaldococcus jannaschii projannalysin), which are soluble and, with ∼100 residues, constitute the shortest structurally characterized MPs to date. Despite relevant sequence and structural similarity, the structures revealed two unique mechanisms of latency maintenance through the C-terminal segments previously unseen in MPs as follows: intramolecular, through an extended tail, in proabylysin, and crosswise intermolecular, through a helix swap, in projannalysin. In addition, structural and sequence comparisons revealed large similarity with MPs of the gluzincin tribe such as thermolysin, leukotriene A4 hydrolase relatives, and cowrins. Noteworthy, gluzincins mostly contain a glutamate as third characteristic zinc ligand, whereas minigluzincins have a histidine. Sequence and structural similarity further allowed us to ascertain that minigluzincins are very similar to the catalytic domains of integral membrane MPs of the MEROPS database families M48 and M56, such as FACE1, HtpX, Oma1, and BlaR1/MecR1, which are provided with trans-membrane helices flanking or inserted into a minigluzincin-like catalytic domain. In a time where structural biochemistry of integral-membrane proteins in general still faces formidable challenges, the minigluzincin soluble minimal scaffold may contribute to our understanding of the working mechanisms of these membrane MPs and to the design of novel inhibitors through structure-aided rational drug design approaches. PMID:23733187

  17. 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. PMID:26477500

  18. Allosteric Modification, the Primary ATP Activation Mechanism of ANF-RGC

    PubMed Central

    Duda, Teresa; Yadav, Prem; Sharma, Rameshwar K.

    2011-01-01

    ANF-RGC is the prototype receptor membrane guanylate cyclase being both the receptor and the signal transducer of the most hypotensive hormones, ANF and BNP. It is a single transmembrane-spanning protein. After binding these hormones at the extracellular domain it at its intracellular domain signals activation of the C-terminal catalytic module and accelerates the production of its second messenger, cyclic GMP, which controls blood pressure, cardiac vasculature and fluid secretion. ATP is obligatory for the post-transmembrane dynamic events leading to ANF-RGC activation. It functions through the ATP regulated module, ARM (KHD) domain, of ANF-RGC. In the current over-a-decade-held-model “phosphorylation of the KHD is absolutely required for hormone-dependent activation of NPR-A” (Potter, L.R., and T. Hunter. 1998. Mol. Cell. Biol. 18: 2164–2172). The presented study challenges this concept. It demonstrates that, instead, ATP allosteric modification of ARM is the primary signaling step of ANF-GC activation. In this 2-step new dynamic model, ATP in the first step binds ARM. This triggers in it a chain of transduction events, which cause its allosteric modification. The modification partially activates (about 50%) ANF-RGC; and concomitantly also prepares the ARM for the second successive step. In this second step, ARM is phosphorylated and ANF-RGC achieves additional (~50%) full catalytic activation. The study defines a new paradigm of ANF-RGC signaling mechanism. PMID:21222471

  19. Poliovirus Replication Requires the N-terminus but not the Catalytic Sec7 Domain of ArfGEF GBF1

    PubMed Central

    Belov, George A.; Kovtunovych, Gennadiy; Jackson, Catherine L.; Ehrenfeld, Ellie

    2010-01-01

    Viruses are intracellular parasites whose reproduction relies on factors provided by the host. The cellular protein GBF1 is critical for poliovirus replication. Here we show that the contribution of GBF1 to virus replication is different from its known activities in uninfected cells. Normally GBF1 activates the Arf GTPases necessary for formation of COPI transport vesicles. GBF1 function is modulated by p115 and Rab1b. However, in polio-infected cells, p115 is degraded and neither p115 nor Rab1b knock-down affects virus replication. Poliovirus infection is very sensitive to BFA, an inhibitor of Arf activation by GBF1. BFA targets the catalytic Sec7 domain of GBF1. Nevertheless the BFA block of polio replication is rescued by expression of only the N-terminal region of GBF1 lacking the Sec7 domain. Replication of BFA-resistant poliovirus in the presence of BFA is uncoupled from Arf activation but is dependent on GBF1. Thus the function(s) of this protein essential for viral replication can be separated from those required for cellular metabolism. PMID:20497182

  20. Stability and solubility engineering of the EphB4 tyrosine kinase catalytic domain using a rationally designed synthetic library.

    PubMed

    Overman, R C; Green, I; Truman, C M; Read, J A; Embrey, K J; McAlister, M S B; Attwood, T K

    2013-10-01

    The inability to generate soluble, correctly folded recombinant protein is often a barrier to successful structural and functional studies. Access to affordable synthetic genes has, however, made it possible to design, make and test many more variants of a target protein to identify suitable constructs. We have used rational design and gene synthesis to create a controlled randomised library of the EphB4 receptor tyrosine kinase, with the aim of obtaining soluble, purifiable and active catalytic domain material at multi-milligram levels in Escherichia coli. Three main parameters were tested in designing the library--construct length, functional mutations and stability grafting. These variables were combined to generate a total of 9720 possible variants. The screening of 480 clones generated a 3% hit rate, with a purifiable solubility of up to 15 mg/L for some EphB4 constructs that was largely independent of construct length. Sequencing of the positive clones revealed a pair of hydrophobic core mutations that were key to obtaining soluble material. A minimal kinase domain construct containing these two mutations exhibited a +4.5°C increase in thermal stability over the wild-type protein. These approaches will be broadly applicable for solubility engineering of many different protein target classes. Atomic coordinates and structural factors have been deposited in PDB under the accession 2yn8 (EphB4 HP + staurosporine). PMID:23840071

  1. In vitro mutagenesis and functional expression in Escherichia coli of a cDNA encoding the catalytic domain of human DNA ligase I.

    PubMed Central

    Kodama, K; Barnes, D E; Lindahl, T

    1991-01-01

    Human cDNAs encoding fragments of DNA ligase I, the major replicative DNA ligase in mammalian cells, have been expressed as lacZ fusion proteins in Escherichia coli. A cDNA encoding the carboxyl-terminal catalytic domain of human DNA ligase I was able to complement a conditional-lethal DNA ligase mutation in E. coli as measured by growth of the mutant strain at the non-permissive temperature. Targeted deletions of the amino and carboxyl termini of the catalytic domain identified a minimum size necessary for catalytic function and a maximum size for optimal complementing activity in E. coli. The human cDNA was subjected to systematic site-directed mutagenesis in vitro and mutant polypeptides assayed for functional expression in the E. coli DNA ligase mutant. Such functional analysis of the active site of DNA ligase I identified specific residues required for the formation of an enzyme-adenylate reaction intermediate. Images PMID:1956768

  2. Mechanistic studies on full length and the catalytic domain of the tandem SH2 domain-containing protein tyrosine phosphatase: analysis of phosphoenzyme levels and Vmax stimulatory effects of glycerol and of a phosphotyrosyl peptide ligand.

    PubMed

    Wang, J; Walsh, C T

    1997-03-11

    SHP-1, a protein tyrosine phosphatase containing two tandem SH2 domains, is autoinhibited at rest by its N-terminal SH2 (N-SH2) domain. Relief from autoinhibition and a subsequent 10-60-fold increase in V(max) have been observed upon N-SH2 domain engagement by a specific phosphotyrosyl ligand or upon deletion of the SH2 domains to yield the catalytic PTPase domain. In this study, we observed that glycerol and propane-1,2-diols, at concentrations of 4-6 M, accelerated the k(cat) of the full length enzyme by 47-fold and of the PTPase domain by 8-fold. Glycerol also increases the rate of proteolytic cleavage between the SH2 and catalytic PTPase domains. In stopped-flow studies using p-nitrophenyl phosphate (pNPP) as a substrate, a burst of p-nitrophenolate in the full length enzyme was not observed; however, a 50-70% stoichiometric burst was observed with the PTPase domain. Rapid quench studies using [32P]pNPP as a substrate showed a very low level of covalent [32P]phosphocysteinyl enzyme intermediate accumulation: 0.06% in the full length enzyme and 1% in the PTP domain. Stimulation by glycerol reduced the accumulating levels of phosphocysteinyl enzyme in both cases of full length SHP-1 and the PTPase domain; however, glycerol is not acting as a cosubstrate since no glycerophosphate product was detectable. It is likely that, for full length SHP-1, with pNPP as a model substrate, enzyme-substrate complex (ES) accumulates in its basal autoinhibited state, whereas enzyme-product complex (EP(i)) accumulates in its pY ligand-bound activated state. Glycerol probably relaxes the compact structure of SHP-1 and the PTP domain, thereby accelerating the catalytic rates in both cases by increasing forward reaction rates of ES and EP(i). PMID:9062130

  3. 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. PMID:25839341

  4. 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

  5. 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.

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

    DOE PAGESBeta

    Qiu, Wei; Lam, Robert; Voytyuk, Oleksandr; Romanov, Vladimir; Gordon, Roni; Gebremeskel, Simon; Vodsedalek, Jakub; Thompson, Christine; Beletskaya, Irina; Battaile, Kevin P.; et al

    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

  7. Calpain cleavage of the B isoform of Ins(1,4,5)P3 3-kinase separates the catalytic domain from the membrane anchoring domain.

    PubMed Central

    Pattni, Krupa; Millard, Thomas H; Banting, George

    2003-01-01

    Inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3] is one of the key intracellular second messengers in cells and mobilizes Ca2+ stores in the ER (endoplasmic reticulum). Ins(1,4,5)P3 has a short half-life within the cell, and is rapidly metabolized through one of two pathways, one of which involves further phosphorylation of the inositol ring: Ins(1,4,5)P3 3-kinase (IP3-3K) phosphorylates Ins(1,4,5)P3, resulting in the formation of inositol (1,3,4,5)-tetrakisphosphate [Ins(1,3,4,5)P4]. There are three known isoforms of IP3-3K, designated IP3-3KA, IP3-3KB and IP3-3KC. These have differing N-termini, but highly conserved C-termini harbouring the catalytic domain. The three IP3-3K isoforms have different subcellular locations and the B-kinase is uniquely present in both cytosolic and membrane-bound pools. As it is the N-terminus of the B-kinase that differs most from the A- and C-kinases, we have hypothesized that this portion of the protein may be responsible for membrane localization. Although there are no known membrane-targeting protein motifs within the sequence of IP3-3KB, it is found to be tightly associated with the ER membrane. Here, we show that specific regions of the N-terminus of IP3-3KB are necessary and sufficient for efficient membrane localization of the protein. We also report that, in the presence of Ca2+, the kinase domain of IP3-3KB is cleaved from the membrane-anchoring region by calpain. PMID:12906709

  8. 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

  9. A single domain of human prostatic acid phosphatase shows antibody-mediated restoration of catalytic activity.

    PubMed Central

    Choe, B K; Dong, M K; Walz, D; Gleason, S; Rose, N R

    1982-01-01

    By limited proteolysis with mouse submaxillaris protease, human prostatic acid phosphatase (EC 3.1.3.2) was cleaved into three fragments, Sp1, Sp2, and Sp3, which individually had no enzymatic activity. One of the fragments, Sp3, regained enzymatic activity after interaction with rabbit antibody to prostatic acid phosphatase. The Sp3 fragment was purified and characterized as to its molecular weight, amino acid composition, and carbohydrate content. The Sp3 fragment behaved like the parent molecule in L(+)-tartrate affinity and in trapping of a phosphoryl intermediate. The same Sp3 fragment also bears the most prominent antigenic determinants. This evidence suggest that Sp3 is the enzymatically active domain of prostatic acid phosphatase. Images PMID:6193513

  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. The core histone N-terminal domains are required for multiple rounds of catalytic chromatin remodeling by the SWI/SNF and RSC complexes.

    PubMed

    Logie, C; Tse, C; Hansen, J C; Peterson, C L

    1999-02-23

    SWI/SNF and RSC are large, distinct multi-subunit complexes that use the energy of ATP hydrolysis to disrupt nucleosome structure, facilitating the binding of transcription factors or restriction enzymes to nucleosomes [Cote, J., Quinn, J., Workman, J. L., and Peterson, C. L. (1994) Science 265, 53-60 (1); Lorch, Y., Cairns, B. R., Zhang, M., and Kornberg, R. D. (1998) Cell 94, 29-34 (2)]. Here we have used a quantitative assay to measure the activities of these ATP-dependent chromatin remodeling complexes using nucleosomal arrays reconstituted with hypoacetylated, hyperacetylated, or partially trypsinized histones. This assay is based on measuring the kinetics of restriction enzyme digestion of a site located within the central nucleosome of a positioned 11-mer array [Logie, C., and Peterson, C. L. (1997) EMBO J. 16, 6772-6782 (3)]. We find that the DNA-stimulated ATPase activities of SWI/SNF and RSC are not altered by the absence of the histone N-termini. Furthermore, ATP-dependent nucleosome remodeling is also equivalent on all three substrate arrays under reaction conditions where the concentrations of nucleosomal array and either SWI/SNF or RSC are equivalent. However, SWI/SNF and RSC cannot catalytically remodel multiple nucleosomal arrays in the absence of the histone termini, and this catalytic activity of SWI/SNF is decreased by histone hyperacetylation. These results indicate that the histone termini are important for SWI/SNF and RSC function; and, furthermore, our data defines a step in the remodeling cycle where the core histone termini exert their influence. This step appears to be after remodeling, but prior to intermolecular transfer of the remodelers to new arrays. PMID:10029546

  12. 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

  13. 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.

  14. 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.

  15. Truncation of the Catalytic Domain of the Cylindromatosis Tumor Suppressor Impairs Lung Maturation1

    PubMed Central

    Trompouki, Eirini; Tsagaratou, Ageliki; Kosmidis, Stylianos K; Dollé, Pascal; Qian, Jun; Kontoyiannis, Dimitris L; Cardoso, Wellington V; Mosialos, George

    2009-01-01

    Cyld encodes a 956-amino acid deubiquitinating enzyme (CYLD), which is a negative regulator of nuclear factor κB and mitogen-activated protein kinase pathways. Mutations that truncate and inactivate the carboxyl-terminal deubiquitinating domain of CYLD underlie the development of skin appendage tumors in humans, whereas down-regulation of Cyld expression has been associated with the development of various types of human malignancies including lung cancer. To establish an animal model of human CYLD inactivation and characterize the biological role of CYLD in vivo, we generated mice carrying a homozygous deletion of Cyld exon 9 (CyldΔ9/Δ9 mice) using a conditional approach. Deletion of exon 9 would cause a carboxyl-terminal truncation of CYLD and inactivation of its deubiquitinating activity. In accordance with previous studies, fibroblasts from CyldΔ9/Δ9 embryos had hyperactive nuclear factor κB and c-Jun kinase pathways compared with control fibroblasts. CyldΔ9/Δ9 newborn mice were smaller than wild-type littermates with a short and kinky tail and nomajor developmental defects. However, CyldΔ9/Δ9 mice died shortly after birth from apparent respiratory dysfunction. Histological examination of E18.5 CyldΔ9/Δ9 lungs demonstrated an immature phenotype characterized by hyperplasic mesenchyme but apparently normal epithelial, smooth muscle. and endothelial structures. Our study identifies an important role of CYLD in lung maturation, which may underlie the development of many cases of lung cancer. PMID:19412431

  16. Antibodies against recombinant catalytic domain of lethal toxin of Clostridium sordellii neutralize lethal toxin toxicity in HeLa cells.

    PubMed

    Arya, Preetika; Ponmariappan, S; Singh, Lokendra; Prasad, G B K S

    2013-02-01

    Lethal toxin of Clostridium sordellii (MLD 150 ng/kg) is one of the most potent Clostridial toxins and is responsible for most of the diseases including sudden death syndrome in cattle, sheep and toxic shock syndrome, necrotizing faciitis, neonatal omphalitis and gangrene in humans. Lethal toxin (TcsL) is a single chain protein of about 270 kDa. In the present study, 1.6 kb DNA fragment encoding for the catalytic domain of TcsL was PCR amplified, cloned in pQE30 UA vector and expressed in E. coli SG 13009. The expression of recombinant lethal toxin protein (rTcsL) was optimized and it was purified under native conditions using a single step Ni-NTA affinity chromatography. The purified recombinant protein was used for the production of polyclonal antibodies in mice and rabbit. The raised antibodies reacted specifically with the purified rTcsL and intact native lethal toxin on Western blot. The biological activity of the recombinant protein was tested in HeLa cells where it showed the cytotoxicity. Further, the polyclonal antibodies were used for in-vitro neutralization of purified rTcsL, acid precipitated C. sordellii and C. difficile native toxins in HeLa cells. Mice and rabbit anti-rTcsL sera effectively neutralized the cytotoxicity of rTcsL and C. sordellii native toxin but it did not neutralize the cytotoxicity of C. difficile toxin in HeLa cells. PMID:22894159

  17. 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.

  18. Structure of the nucleotide-binding subunit B of the energy producer A1A0 ATP synthase in complex with adenosine diphosphate.

    PubMed

    Kumar, Anil; Manimekalai, Malathy Sony Subramanian; Grüber, Gerhard

    2008-11-01

    A1A0 ATP synthases are the major energy producers in archaea. Like the related prokaryotic and eukaryotic F1F0 ATP synthases, they are responsible for most of the synthesis of adenosine triphosphate. The catalytic events of A1A0 ATP synthases take place inside the A3B3 hexamer of the A1 domain. Recently, the crystallographic structure of the nucleotide-free subunit B of Methanosarcina mazei Gö1 A1A0 ATP synthase has been determined at 1.5 A resolution. To understand more about the nucleotide-binding mechanism, a protocol has been developed to crystallize the subunit B-ADP complex. The crystallographic structure of this complex has been solved at 2.7 A resolution. The ADP occupies a position between the essential phosphate-binding loop and amino-acid residue Phe149, which are involved in the binding of the antibiotic efrapeptin in the related F1F0 ATP synthases. This trapped ADP location is about 13 A distant from its final binding site and is therefore called the transition ADP-binding position. In the trapped ADP position the structure of subunit B adopts a different conformation, mainly in its C-terminal domain and also in the final nucleotide-binding site of the central alphabeta-domain. This atomic model provides insight into how the substrate enters into the nucleotide-binding protein and thereby into the catalytic A3B3 domain. PMID:19020348

  19. Inhibition of protein kinase C catalytic activity by additional regions within the human protein kinase Calpha-regulatory domain lying outside of the pseudosubstrate sequence.

    PubMed

    Kirwan, Angie F; Bibby, Ashley C; Mvilongo, Thierry; Riedel, Heimo; Burke, Thomas; Millis, Sherri Z; Parissenti, Amadeo M

    2003-07-15

    The N-terminal pseudosubstrate site within the protein kinase Calpha (PKCalpha)-regulatory domain has long been regarded as the major determinant for autoinhibition of catalytic domain activity. Previously, we observed that the PKC-inhibitory capacity of the human PKCalpha-regulatory domain was only reduced partially on removal of the pseudosubstrate sequence [Parissenti, Kirwan, Kim, Colantonio and Schimmer (1998) J. Biol. Chem. 273, 8940-8945]. This finding suggested that one or more additional region(s) contributes to the inhibition of catalytic domain activity. To assess this hypothesis, we first examined the PKC-inhibitory capacity of a smaller fragment of the PKCalpha-regulatory domain consisting of the C1a, C1b and V2 regions [GST-Ralpha(39-177): this protein contained the full regulatory domain of human PKCalpha fused to glutathione S-transferase (GST), but lacked amino acids 1-38 (including the pseudosubstrate sequence) and amino acids 178-270 (including the C2 region)]. GST-Ralpha(39-177) significantly inhibited PKC in a phorbol-independent manner and could not bind the peptide substrate used in our assays. These results suggested that a region within C1/V2 directly inhibits catalytic domain activity. Providing further in vivo support for this hypothesis, we found that expression of N-terminally truncated pseudosubstrate-less bovine PKCalpha holoenzymes in yeast was capable of inhibiting cell growth in a phorbol-dependent manner. This suggested that additional autoinhibitory force(s) remained within the truncated holoenzymes that could be relieved by phorbol ester. Using tandem PCR-mediated mutagenesis, we observed that mutation of amino acids 33-86 within GST-Ralpha(39-177) dramatically reduced its PKC-inhibitory capacity when protamine was used as substrate. Mutagenesis of a broad range of sequences within C2 (amino acids 159-242) also significantly reduced PKC-inhibitory capacity. Taken together, these observations support strongly the existence of

  20. 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. PMID:25771858

  1. The crystal structure of the catalytic domain of the ser/thr kinase PknA from M. tuberculosis shows an Src-like autoinhibited conformation.

    PubMed

    Wagner, Tristan; Alexandre, Matthieu; Duran, Rosario; Barilone, Nathalie; Wehenkel, Annemarie; Alzari, Pedro M; Bellinzoni, Marco

    2015-05-01

    Signal transduction mediated by Ser/Thr phosphorylation in Mycobacterium tuberculosis has been intensively studied in the last years, as its genome harbors eleven genes coding for eukaryotic-like Ser/Thr kinases. Here we describe the crystal structure and the autophosphorylation sites of the catalytic domain of PknA, one of two protein kinases essential for pathogen's survival. The structure of the ligand-free kinase domain shows an auto-inhibited conformation similar to that observed in human Tyr kinases of the Src-family. These results reinforce the high conservation of structural hallmarks and regulation mechanisms between prokaryotic and eukaryotic protein kinases. PMID:25586004

  2. A diverse superfamily of enzymes with ATP-dependent carboxylate-amine/thiol ligase activity.

    PubMed Central

    Galperin, M. Y.; Koonin, E. V.

    1997-01-01

    The recently developed PSI-BLAST method for sequence database search and methods for motif analysis were used to define and expand a superfamily of enzymes with an unusual nucleotide-binding fold, referred to as palmate, or ATP-grasp fold. In addition to D-alanine-D-alanine ligase, glutathione synthetase, biotin carboxylase, and carbamoyl phosphate synthetase, enzymes with known three-dimensional structures, the ATP-grasp domain is predicted in the ribosomal protein S6 modification enzyme (RimK), urea amidolyase, tubulin-tyrosine ligase, and three enzymes of purine biosynthesis. All these enzymes possess ATP-dependent carboxylate-amine ligase activity, and their catalytic mechanisms are likely to include acylphosphate intermediates. The ATP-grasp superfamily also includes succinate-CoA ligase (both ADP-forming and GDP-forming variants), malate-CoA ligase, and ATP-citrate lyase, enzymes with a carboxylate-thiol ligase activity, and several uncharacterized proteins. These findings significantly extend the variety of the substrates of ATP-grasp enzymes and the range of biochemical pathways in which they are involved, and demonstrate the complementarity between structural comparison and powerful methods for sequence analysis. PMID:9416615

  3. Improved catalytic efficiency, thermophilicity, anti-salt and detergent tolerance of keratinase KerSMD by partially truncation of PPC domain.

    PubMed

    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

  4. 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

  5. A single catalytic domain of the junction-resolving enzyme T7 endonuclease I is a non-specific nicking endonuclease

    PubMed Central

    Guan, Chudi; Kumar, Sanjay

    2005-01-01

    A stable heterodimeric protein containing a single correctly folded catalytic domain (SCD) of T7 endonuclease I was produced by means of a trans-splicing intein system. As predicted by a model presented earlier, purified SCD protein acts a non-specific nicking endonuclease on normal linear DNA. The SCD retains some ability to recognize and cleave a deviated DNA double-helix near a nick or a strand-crossing site. Thus, we infer that the non-specific and nicked-site cleavage activities observed for the native T7 endonuclease I (as distinct from the resolution activity) are due to uncoordinated actions of the catalytic domains. The positively charged C-terminus of T7 Endo I is essential for the enzymatic activity of SCD, as it is for the native enzyme. We propose that the preference of the native enzyme for the resolution reaction is achieved by cooperativity in the binding of its two catalytic domains when presented with two of the arms across a four-way junction or cruciform structure. PMID:16264086

  6. Recombinant expression and biochemical characterization of the catalytic domain of acetylcholinesterase-1 from the African malaria mosquito, Anopheles gambiae

    PubMed Central

    Jiang, Haobo; Liu, Siwei; Zhao, Picheng; Pope, Carey

    2009-01-01

    Acetylcholinesterases (AChEs) and their genes from susceptible and resistant insects have been extensively studied to understand the molecular basis of target site insensitivity. Due to the existence of other resistance mechanisms, however, it can be problematic to correlate directly a mutation with the resistant phenotype. An alternative approach involves recombinant expression and characterization of highly purified wild-type and mutant AChEs, which serves as a reliable platform for studying structure-function relationships. We expressed the catalytic domain of Anopheles gambiae AChE1 (r-AgAChE1) using the baculovirus system and purified it 26,000-fold from the conditioned medium to near homogeneity. While KM's of r-AgAChE1 were comparable for ATC, AβMTC, PTC, and BTC, Vmax's were substantially different. The IC50's for eserine, carbaryl, paraoxon, BW284C51, malaoxon, and ethopropazine were 8.3, 72.5, 83.6, 199, 328, and 6.59×104 nM, respectively. We determined kinetic constants for inhibition of r-AgAChE1 by four of these compounds. The enzyme bound eserine or paraoxon stronger than carbaryl or malaoxon. Because the covalent modification of r-AgAChE1 by eserine occurred faster than that by the other compounds, eserine is more potent than paraoxon, carbaryl, and malaoxon. Furthermore, we found that choline inhibited r-AgAChE1, a phenomenon related to the enzyme activity decrease at high concentrations of acetylcholine. PMID:19607916

  7. Structure-guided studies of the SHP-1/JAK1 interaction provide new insights into phosphatase catalytic domain substrate recognition

    PubMed Central

    Alicea-Velázquez, Nilda L.; Jakoncic, Jean; Boggon, Titus J.

    2013-01-01

    SHP-1 (PTPN6) is a member of the SHP sub-family of protein tyrosine phosphatases and plays a critical role in the regulation of the JAK/STAT signaling pathway. Previous studies suggested that SHP-1 contains a PTP1B-like second phosphotyrosine pocket that allows for binding of tandem phosphotyrosine residues, such as those found in the activation loop of JAK kinases. To discover the structural nature of the interaction between SHP-1 and the JAK family member, JAK1, we determined the 1.8 Å co-crystal structure of the SHP-1 catalytic domain and a JAK1-derived substrate peptide. This structure reveals electron density for only one bound phosphotyrosine residue. To investigate the role of the predicted second site pocket we determined the structures of SHP-1 in complex with phosphate and sulfate to 1.37 Å and 1.7 Å, respectively, and performed anomalous scattering experiments for a selenate-soaked crystal. These crystallographic data suggest that SHP-1 does not contain a PTP1B-like second site pocket. This conclusion is further supported by analysis of the relative dephosphorylation and binding affinities of mono-and tandem-phosphorylated peptide substrates. The crystal structures instead indicate that SHP-1 contains an extended C-terminal helix α2′ incompatible with the predicted second phosphotyrosine binding site. This study suggests that SHP-1 defines a new category of PTP1B-like protein tyrosine phosphatases with a hindered second phosphotyrosine pocket. PMID:23296072

  8. Crystal structure of the catalytic domain of UCHL5, a proteasome-associated human deubiquitinating enzyme, reveals an unproductive form of the enzyme

    PubMed Central

    Maiti, Tushar K.; Permaul, Michelle; Boudreaux, David A.; Mahanic, Christina; Mauney, Sarah; Das, Chittaranjan

    2012-01-01

    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 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 are 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, Kim SW, Kawai K, Mizushima T, Yamane T, Hamazaki J, Murata S, Tanaka K and Morimoto Y (2009) Biochem Biophys Res Commun 390, 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. PMID:21995438

  9. Rates of various reactions catalyzed by ATP synthase as related to the mechanism of ATP synthesis

    SciTech Connect

    Berkich, D.A.; Williams, G.D.; Masiakos, P.T.; Smith, M.B.; Boyer, P.D.; LaNoue, K.F. )

    1991-01-05

    The forward and reverse rates of the overall reaction catalyzed by the ATP synthase in intact rat heart mitochondria, as measured with 32P, were compared with the rates of two partial steps, as measured with 18O. Such rates have been measured previously, but their relationship to one another has not been determined, nor have the partial reactions been measured in intact mitochondria. The partial steps measured were the rate of medium Pi formation from bound ATP (in state 4 this also equals the rate of medium Pi into bound ATP) and the rate of formation of bound ATP from bound Pi within the catalytic site. The rates of both partial reactions can be measured by 31P NMR analysis of the 18O distribution in Pi and ATP released from the enzyme during incubation of intact mitochondria with highly labeled (18O)Pi. Data were obtained in state 3 and 4 conditions with variation in substrate concentrations, temperature, and mitochondrial membrane electrical potential gradient (delta psi m). Although neither binding nor release of ATP is necessary for phosphate/H2O exchange, in state 4 the rate of incorporation of at least one water oxygen atom into phosphate is approximately twice the rate of the overall reaction rate under a variety of conditions. This can be explained if the release of Pi or ATP at one catalytic site does not occur, unless ATP or Pi is bound at another catalytic site. Such coupling provides strong support for the previously proposed alternating site mechanism. In state 3 slow reversal of ATP synthesis occurs within the mitochondrial matrix and can be detected as incorporation of water oxygen atoms into medium Pi even though medium (32P)ATP does not give rise to 32Pi in state 3. These data can be explained by lack of translocation of ATP from the medium to the mitochondrial matrix.

  10. 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

  11. Changes in Protein Domains outside the Catalytic Site of the Bacteriophage Qβ Replicase Reduce the Mutagenic Effect of 5-Azacytidine

    PubMed Central

    Cabanillas, Laura; Sanjuán, Rafael

    2014-01-01

    ABSTRACT The high genetic heterogeneity and great adaptability of RNA viruses are ultimately caused by the low replication fidelity of their polymerases. However, single amino acid substitutions that modify replication fidelity can evolve in response to mutagenic treatments with nucleoside analogues. Here, we investigated how two independent mutants of the bacteriophage Qβ replicase (Thr210Ala and Tyr410His) reduce sensitivity to the nucleoside analogue 5-azacytidine (AZC). Despite being located outside the catalytic site, both mutants reduced the mutation frequency in the presence of the drug. However, they did not modify the type of AZC-induced substitutions, which was mediated mainly by ambiguous base pairing of the analogue with purines. Furthermore, the Thr210Ala and Tyr410His substitutions had little or no effect on replication fidelity in untreated viruses. Also, both substitutions were costly in the absence of AZC or when the action of the drug was suppressed by adding an excess of natural pyrimidines (uridine or cytosine). Overall, the phenotypic properties of these two mutants were highly convergent, despite the mutations being located in different domains of the Qβ replicase. This suggests that treatment with a given nucleoside analogue tends to select for a unique functional response in the viral replicase. IMPORTANCE In the last years, artificial increase of the replication error rate has been proposed as an antiviral therapy. In this study, we investigated the mechanisms by which two substitutions in the Qβ replicase confer partial resistance to the mutagenic nucleoside analogue AZC. As opposed to previous work with animal viruses, where different mutations selected sequentially conferred nucleoside analogue resistance through different mechanisms, our results suggest that there are few or no alternative AZC resistance phenotypes in Qβ. Also, despite resistance mutations being highly costly in the absence of the drug, there was no sequential

  12. Multiple Steps to Activate FAK’s Kinase Domain: Adaptation to Confined Environments?

    PubMed Central

    Herzog, Florian A.; Vogel, Viola

    2013-01-01

    Protein kinases regulate cell signaling by phosphorylating their substrates in response to environment-specific stimuli. Using molecular dynamics, we studied the catalytically active and inactive conformations of the kinase domain of the focal adhesion kinase (FAK), which are distinguished by displaying a structured or unstructured activation loop, respectively. Upon removal of an ATP analog, we show that the nucleotide-binding pocket in the catalytically active conformation is structurally unstable and fluctuates between an open and closed configuration. In contrast, the pocket remains open in the catalytically inactive form upon removal of an inhibitor from the pocket. Because temporal pocket closures will slow the ATP on-rate, these simulations suggest a multistep process in which the kinase domain is more likely to bind ATP in the catalytically inactive than in the active form. Transient closures of the ATP-binding pocket might allow FAK to slow down its catalytic cycle. These short cat naps could be adaptions to crowded or confined environments by giving the substrate sufficient time to diffuse away. The simulations show further how either the phosphorylation of the activation loop or the activating mutations of the so-called SuperFAK influence the electrostatic switch that controls kinase activity. PMID:23746525

  13. Theoretical models of catalytic domains of protein phosphatases 1 and 2A with Zn2+ and Mn2+ metal dications and putative bioligands in their catalytic centers.

    PubMed

    Woźniak-Celmer, E; Ołdziej, S; Ciarkowski, J

    2001-01-01

    The oligomeric metalloenzymes protein phosphatases dephosphorylate OH groups of Ser/Thr or Tyr residues of proteins whose actions depend on the phosphorus signal. The catalytic units of Ser/Thr protein phosphatases 1, 2A and 2B (PP1c, PP2Ac and PP2Bc, respectively), which exhibit about 45% sequence similarity, have their active centers practically identical. This feature strongly suggests that the unknown structure of PP2Ac could be successfully homology-modeled from the known structures of PP1c and/or PP2Bc. Initially, a theoretical model of PP1c was built, including a phosphate and a metal dication in its catalytic site. The latter was modeled, together with a structural hydroxyl anion, as a triangular pseudo-molecule (Zno or Mno), composed of two metal cations (double Zn2+ or Mn2+, respectively) and the OH- group. To the free PP1c two inhibitor sequences R29RRRPpTPAMLFR40 of DARPP-32 and R30RRRPpTPATLVLT42 of Inhibitor-1, and two putative substrate sequences LRRApSVA and QRRQRKpRRTI were subsequently docked. In the next step, a free PP2Ac model was built via homology re-modeling of the PP1c template and the same four sequences were docked to it. Thus, together, 20 starting model complexes were built, allowing for combination of the Zno and Mno pseudo-molecules, free enzymes and the peptide ligands docked in the catalytic sites of PP1c and PP2Ac. All models were subsequently subjected to 250-300 ps molecular dynamics using the AMBER 5.0 program. The equilibrated trajectories of the final 50 ps were taken for further analyses. The theoretical models of PP1c complexes, irrespective of the dication type, exhibited increased mobilities in the following residue ranges: 195-200, 273-278, 287-209 for the inhibitor sequences and 21-25, 194-200, 222-227, 261, 299-302 for the substrate sequences. Paradoxically, the analogous PP2Ac models appeared much more stable in similar simulations, since only their "prosegment" residues 6-10 and 14-18 exhibited an increased mobility

  14. Crystal Structures of the p21-Activated Kinases PAK4, PAK5, and PAK6 Reveal Catalytic Domain Plasticity of Active Group II PAKs

    PubMed Central

    Eswaran, Jeyanthy; Lee, Wen Hwa; Debreczeni, Judit É.; Filippakopoulos, Panagis; Turnbull, Andrew; Fedorov, Oleg; Deacon, Sean W.; Peterson, Jeffrey R.; Knapp, Stefan

    2007-01-01

    Summary p21-activated kinases have been classified into two groups based on their domain architecture. Group II PAKs (PAK4–6) regulate a wide variety of cellular functions, and PAK deregulation has been linked to tumor development. Structural comparison of five high-resolution structures comprising all active, monophosphorylated group II catalytic domains revealed a surprising degree of domain plasticity, including a number of catalytically productive and nonproductive conformers. Rearrangements of helix αC, a key regulatory element of kinase function, resulted in an additional helical turn at the αC N terminus and a distortion of its C terminus, a movement hitherto unseen in protein kinases. The observed structural changes led to the formation of interactions between conserved residues that structurally link the glycine-rich loop, αC, and the activation segment and firmly anchor αC in an active conformation. Inhibitor screening identified six potent PAK inhibitors from which a tri-substituted purine inhibitor was cocrystallized with PAK4 and PAK5. PMID:17292838

  15. The rad50 signature motif: essential to ATP binding and biological function.

    PubMed

    Moncalian, Gabriel; Lengsfeld, Bettina; Bhaskara, Venugopal; Hopfner, Karl-Peter; Karcher, Annette; Alden, Erinn; Tainer, John A; Paull, Tanya T

    2004-01-23

    The repair of double-strand breaks in DNA is an essential process in all organisms, and requires the coordinated activities of evolutionarily conserved protein assemblies. One of the most critical of these is the Mre11/Rad50 (M/R) complex, which is present in all three biological kingdoms, but is not well-understood at the biochemical level. Previous structural analysis of a Rad50 homolog from archaebacteria illuminated the catalytic core of the enzyme, an ATP-binding domain related to the ABC transporter family of ATPases. Here, we present the crystallographic structure of the Rad50 mutant S793R. This missense signature motif mutation changes the key serine residue in the signature motif that is conserved among Rad50 homologs and ABC ATPases. The S793R mutation is analogous to the mutation S549R in the cystic fibrosis transmembrane conductance regulator (CFTR) that results in cystic fibrosis. We show here that the serine to arginine change in the Rad50 protein prevents ATP binding and disrupts the communication among the other ATP-binding loops. This structural change, in turn, alters the communication between Rad50 monomers and thus prevents Rad50 dimerization. The equivalent mutation was made in the human Rad50 gene, and the resulting mutant protein did form a complex with Mre11 and Nbs1, but was specifically deficient in all ATP-dependent enzymatic activities. This signature motif structure-function homology extends to yeast, because the same mutation introduced into the Saccharomyces cerevisiae RAD50 gene generated an allele that failed to complement a rad50 deletion strain in DNA repair assays in vivo. These structural and biochemical results extend our understanding of the Rad50 catalytic domain and validate the use of the signature motif mutant to test the role of Rad50 ATP binding in diverse organisms. PMID:14698290

  16. Demonstration of a transitory tight binding of ATP and of committed Pi and ADP during ATP synthesis by chloroplasts

    PubMed Central

    Smith, Daniel J.; Boyer, Paul D.

    1976-01-01

    Rapid mixing, quenching, and filtration experiments with chloroplast thylakoid membranes, with energization by acid-base transition, demonstrate that an ATP tightly bound to the isolated membranes is a transient intermediate in the catalytic sequence for ATP synthesis. The experiments also show that most of the Pi and ADP bound at a catalytic site is committed to ATP formation without interchange with medium Pi or ADP. Other results give evidence that upon energization, the tightly bound ADP that is detectable in isolated thylakoid membranes or coupling factor ATPase is rapidly released to the medium from a catalytic site. These findings support an alternating site model in which an energy-requiring conformational transition loosens ATP binding at one site and simultaneously promotes Pi and ADP binding at the other site in a manner favoring ATP formation. PMID:16592374

  17. 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

  18. Domain motions of glucosamine-6P synthase: Comparison of the anisotropic displacements in the crystals and the catalytic hinge-bending rotation

    PubMed Central

    Mouilleron, Stéphane; Golinelli-Pimpaneau, Béatrice

    2007-01-01

    Glucosamine-6-phosphate synthase channels ammonia over 18 Å from glutamine at the glutaminase site to fructose-6P at the synthase site. We have modeled the anisotropic displacements of the glutaminase and synthase domains from the two crystallized states, the enzyme in complex with fructose-6P or in complex with glucose-6P and a glutamine affinity analog, using TLS (rigid-body motion in terms of translation, libration, and screw motions) refinement implemented in REFMAC. The domains displacements in the crystal lattices are compared to the movement of the glutaminase domain relative to the synthase domain that occurs during the catalytic cycle upon glutamine binding, which was visualized by comparing the two structures. This movement was analyzed by the program DYNDOM as a 22.8° rotation around an effective hinge axis running approximately parallel to helix 300–317 of the synthase domain, the glutaminase loop that covers the glutaminase site upon glutamine binding acting as the mechanical hinge. PMID:17322533

  19. ATP hydrolysis-driven gating in cystic fibrosis transmembrane conductance regulator

    PubMed Central

    Muallem, Daniella; Vergani, Paola

    2008-01-01

    Proteins belonging to the ATP-binding cassette superfamily couple ATP binding and hydrolysis at conserved nucleotide-binding domains (NBDs) to diverse cellular functions. Most superfamily members are transporters, while cystic fibrosis transmembrane conductance regulator (CFTR), alone, is an ion channel. Despite this functional difference, recent results have suggested that CFTR shares a common molecular mechanism with other members. ATP binds to partial binding sites on the surface of the two NBDs, which then associate to form a NBD dimer, with complete composite catalytic sites now buried at the interface. ATP hydrolysis and γ-phosphate dissociation, with the loss of molecular contacts linking the two sides of the composite site, trigger dimer dissociation. The conformational signals generated by NBD dimer formation and dissociation are transmitted to the transmembrane domains where, in transporters, they drive the cycle of conformational changes that translocate the substrate across the membrane; in CFTR, they result in opening and closing (gating) of the ion-permeation pathway. PMID:18957373

  20. 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. PMID:22253434

  1. 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. PMID:26018081

  2. 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.

  3. Catalytic domains of the LAR and CD45 protein tyrosine phosphatases from Escherichia coli expression systems: Purification and characterization for specificity and mechanism

    SciTech Connect

    Cho, Hyeongjin; Ramer, S.E.; Itoh, Michiyasu; Saito, Haruo; Walsh, C.T. ); Kitas, E.; Bannwarth, W.; Burn, P. )

    1992-01-14

    The cytoplasmic domains of two human transmembrane protein tyrosine phosphatases (PTPases), LAR and CD45, have been expressed in Escherichia coli, purified to near-homogeneity, and compared for catalytic efficiency toward several phosphotyrosine-containing peptide substrates. A 615-residue LAR fragment (LAR-D1D2) containing both tandemly repeated PTPase domains shows almost identical specific activity and high catalytic efficiency as the 40-kDa single-domain LAR-D1 fragment, consistent with a single functional active site in the 70-kDa LAR-D1D2 enzyme. A 90-kDa fragment of the human leukocyte CD45 PTPase, containing two similar tandemly repeated PTPase domains, shows parallel specificity to LAR-D1 and LAR-D1D2 with a high k{sub cat}/K{sub M} value for a phosphotyrosyl undecapeptide. Sufficient purified LAR-D1 and LAR-D1D2 PTPases were available to demonstrate enzymatic exchange of {sup 18}O from {sup 18}O{sub 4} inorganic phosphate into H{sub 2} {sup 16}O at rates of {approximately}1 {times} 10{sup {minus}2} s{sup {minus}1}. The oxygen-18 exchange probably proceeds via a phosphoenzyme intermediate. Brief incubation of all three PTPase fragments with a ({sup 32}P)phosphotyrosyl peptide substrate prior to quench with SDS sample buffer and gel electrophoresis led to autoradiographic detection of {sup 32}P-labeled enzymes. Pulse/chase studies on the LAR {sup 32}P-enzyme showed turnover of the labeled phosphoryl group.

  4. Crystal structure of the catalytic domain of Pseudomonas exotoxin A complexed with a nicotinamide adenine dinucleotide analog: implications for the activation process and for ADP ribosylation.

    PubMed Central

    Li, M; Dyda, F; Benhar, I; Pastan, I; Davies, D R

    1996-01-01

    The catalytic, or third domain of Pseudomonas exotoxin A (PEIII) catalyzes the transfer of ADP ribose from nicotinamide adenine dinucleotide (NAD) to elongation factor-2 in eukaryotic cells, inhibiting protein synthesis. We have determined the structure of PEIII crystallized in the presence of NAD to define the site of binding and mechanism of activation. However, NAD undergoes a slow hydrolysis and the crystal structure revealed only the hydrolysis products, AMP and nicotinamide, bound to the enzyme. To better define the site of NAD binding, we have now crystallized PEIII in the presence of a less hydrolyzable NAD analog, beta-methylene-thiazole-4-carboxamide adenine dinucleotide (beta-TAD), and refined the complex structure at 2.3 angstroms resolution. There are two independent molecules of PEIII in the crystal, and the conformations of beta-TAD show some differences in the two binding sites. The beta-TAD attached to molecule 2 appears to have been hydrolyzed between the pyrophosphate and the nicotinamide ribose. However molecule 1 binds to an intact beta-TAD and has no crystal packing contacts in the vicinity of the binding site, so that the observed conformation and interaction with the PEIII most likely resembles that of NAD bound to PEIII in solution. We have compared this complex with the catalytic domains of diphtheria toxin, heat labile enterotoxin, and pertussis toxin, all three of which it closely resembles. Images Fig. 1 Fig. 3 PMID:8692916

  5. Processing of the yellow fever virus nonstructural polyprotein: a catalytically active NS3 proteinase domain and NS2B are required for cleavages at dibasic sites.

    PubMed Central

    Chambers, T J; Grakoui, A; Rice, C M

    1991-01-01

    The vaccinia virus-T7 transient expression system was used to further examine the role of the NS3 proteinase in processing of the yellow fever (YF) virus nonstructural polyprotein in BHK cells. YF virus-specific polyproteins and cleavage products were identified by immunoprecipitation with region-specific antisera, by size, and by comparison with authentic YF virus polypeptides. A YF virus polyprotein initiating with a signal sequence derived from the E protein fused to the N terminus of NS2A and extending through the N-terminal 356 amino acids of NS5 exhibited processing at the 2A-2B, 2B-3, 3-4A, 4A-4B, and 4B-5 cleavage sites. Similar results were obtained with polyproteins whose N termini began within NS2A (position 110) or with NS2B. When the NS3 proteinase domain was inactivated by replacing the proposed catalytic Ser-138 with Ala, processing at all sites was abolished. The results suggest that an active NS3 proteinase domain is necessary for cleavage at the diabasic nonstructural cleavage sites and that cleavage at the proposed 4A-4B signalase site requires prior cleavage at the 4B-5 site. Cleavages were not observed with a polyprotein whose N terminus began with NS3, but cleavage at the 4B-5 site could be restored by supplying the the NS2B protein in trans. Several experimental results suggested that trans cleavage at the 4B-5 site requires association of NS2B and the NS3 proteinase domain. Coexpression of different proteinases and catalytically inactive polyprotein substrates revealed that trans cleavage at the 2B-3 and 4B-5 sites was relatively efficient when compared with trans cleavage at the 2A-2B and 3-4A sites. Images PMID:1833562

  6. Thermodynamics of proton transport coupled ATP synthesis.

    PubMed

    Turina, Paola; Petersen, Jan; Gräber, Peter

    2016-06-01

    The thermodynamic H(+)/ATP ratio of the H(+)-ATP synthase from chloroplasts was measured in proteoliposomes after energization of the membrane by an acid base transition (Turina et al. 2003 [13], 418-422). The method is discussed, and all published data obtained with this system are combined and analyzed as a single dataset. This meta-analysis led to the following results. 1) At equilibrium, the transmembrane ΔpH is energetically equivalent to the transmembrane electric potential difference. 2) The standard free energy for ATP synthesis (reference reaction) is ΔG°ref=33.8±1.3kJ/mol. 3) The thermodynamic H(+)/ATP ratio, as obtained from the shift of the ATP synthesis equilibrium induced by changing the transmembrane ΔpH (varying either pHin or pHout) is 4.0±0.1. The structural H(+)/ATP ratio, calculated from the ratio of proton binding sites on the c-subunit-ring in F0 to the catalytic nucleotide binding sites on the β-subunits in F1, is c/β=14/3=4.7. We infer that the energy of 0.7 protons per ATP that flow through the enzyme, but do not contribute to shifting the ATP/(ADP·Pi) ratio, is used for additional processes within the enzyme, such as activation, and/or energy dissipation, due e.g. to internal uncoupling. The ratio between the thermodynamic and the structural H(+)/ATP values is 0.85, and we conclude that this value represents the efficiency of the chemiosmotic energy conversion within the chloroplast H(+)-ATP synthase. PMID:26940516

  7. The INA complex facilitates assembly of the peripheral stalk of the mitochondrial F1Fo-ATP synthase

    PubMed Central

    Lytovchenko, Oleksandr; Naumenko, Nataliia; Oeljeklaus, Silke; Schmidt, Bernhard; von der Malsburg, Karina; Deckers, Markus; Warscheid, Bettina; van der Laan, Martin; Rehling, Peter

    2014-01-01

    Mitochondrial F1Fo-ATP synthase generates the bulk of cellular ATP. This molecular machine assembles from nuclear- and mitochondria-encoded subunits. Whereas chaperones for formation of the matrix-exposed hexameric F1-ATPase core domain have been identified, insight into how the nuclear-encoded F1-domain assembles with the membrane-embedded Fo-region is lacking. Here we identified the INA complex (INAC) in the inner membrane of mitochondria as an assembly factor involved in this process. Ina22 and Ina17 are INAC constituents that physically associate with the F1-module and peripheral stalk, but not with the assembled F1Fo-ATP synthase. Our analyses show that loss of Ina22 and Ina17 specifically impairs formation of the peripheral stalk that connects the catalytic F1-module to the membrane embedded Fo-domain. We conclude that INAC represents a matrix-exposed inner membrane protein complex that facilitates peripheral stalk assembly and thus promotes a key step in the biogenesis of mitochondrial F1Fo-ATP synthase. PMID:24942160

  8. Dimerization of cAMP phosphodiesterase-4 (PDE4) in living cells requires interfaces located in both the UCR1 and catalytic unit domains

    PubMed Central

    Bolger, Graeme B.; Dunlop, Allan J.; Meng, Dong; Day, Jon P.; Klussmann, Enno; Baillie, George S.; Adams, David R.; Houslay, Miles D.

    2015-01-01

    PDE4 family cAMP phosphodiesterases play a pivotal role in determining compartmentalised cAMP signalling through targeted cAMP breakdown. Expressing the widely found PDE4D5 isoform, as both bait and prey in a yeast 2-hybrid system, we demonstrated interaction consistent with the notion that long PDE4 isoforms form dimers. Four potential dimerization sites were uncovered using a scanning peptide array approach, where a recombinant purified PDE4D5 fusion protein was used to probe a 25-mer library of overlapping peptides covering the entire PDE4D5 sequence. Key residues involved in PDE4D5 dimerization were defined using a site-directed mutagenesis programme directed by an alanine scanning peptide array approach. Critical residues stabilising PDE4D5 dimerization were defined within the regulatory UCR1 region found in long, but not short, PDE4 isoforms, namely the Arg173, Asn174 and Asn175 (DD1) cluster. Disruption of the DD1 cluster was not sufficient, in itself, to destabilise PDE4D5 homodimers. Instead, disruption of an additional interface, located on the PDE4 catalytic unit, was also required to convert PDE4D5 into a monomeric form. This second dimerization site on the conserved PDE4 catalytic unit is dependent upon a critical ion pair interaction. This involves Asp463 and Arg499 in PDE4D5, which interact in a trans fashion involving the two PDE4D5 molecules participating in the homodimer. PDE4 long isoforms adopt a dimeric state in living cells that is underpinned by two key contributory interactions, one involving the UCR modules and one involving an interface on the core catalytic domain. We propose that short forms do not adopt a dimeric configuration because, in the absence of the UCR1 module, residual engagement of the remaining core catalytic domain interface provides insufficient free energy to drive dimerization. The functioning of PDE4 long and short forms is thus poised to be inherently distinct due to this difference in quaternary structure. PMID

  9. 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

  10. Giant protein kinases: domain interactions and structural basis of autoregulation.

    PubMed Central

    Kobe, B; Heierhorst, J; Feil, S C; Parker, M W; Benian, G M; Weiss, K R; Kemp, B E

    1996-01-01

    The myosin-associated giant protein kinases twitchin and titin are composed predominantly of fibronectin- and immunoglobulin-like modules. We report the crystal structures of two autoinhibited twitchin kinase fragments, one from Aplysia and a larger fragment from Caenorhabditis elegans containing an additional C-terminal immunoglobulin-like domain. The structure of the longer fragment shows that the immunoglobulin domain contacts the protein kinase domain on the opposite side from the catalytic cleft, laterally exposing potential myosin binding residues. Together, the structures reveal the cooperative interactions between the autoregulatory region and the residues from the catalytic domain involved in protein substrate binding, ATP binding, catalysis and the activation loop, and explain the differences between the observed autoinhibitory mechanism and the one found in the structure of calmodulin-dependent kinase I. Images PMID:9003756

  11. Cell cycle-dependent nuclear accumulation of the p94fer tyrosine kinase is regulated by its NH2 terminus and is affected by kinase domain integrity and ATP binding.

    PubMed

    Ben-Dor, I; Bern, O; Tennenbaum, T; Nir, U

    1999-02-01

    p94fer and p51ferT are two tyrosine kinases that are encoded by differentially spliced transcripts of the FER locus in the mouse. The two tyrosine kinases share identical SH2 and kinase domains but differ in their NH2-terminal amino acid sequence. Unlike p94fer, the presence of which has been demonstrated in most mammalian cell lines analyzed, the expression of p51ferT is restricted to meiotic cells. Here, we show that the two related tyrosine kinases also differ in their subcellular localization profiles. Although p51ferT accumulates constitutively in the cell nucleus, p94fer is cytoplasmic in quiescent cells and enters the nucleus concomitantly with the onset of S phase. The nuclear translocation of the FER proteins is driven by a nuclear localization signal (NLS), which is located within the kinase domain of these enzymes. The functioning of that NLS depends on the integrity of the kinase domain but was not affected by inactivation of the kinase activity. The NH2 terminus of p94fer dictated the cell cycle-dependent functioning of the NLS of FER kinase. This process was governed by coiled-coil forming sequences that are present in the NH2 terminus of the kinase. The regulatory effect of the p94fer NH2-terminal sequences was not affected by kinase activity but was perturbed by mutations in the kinase domain ATP binding site. Ectopic expression of the constitutively nuclear p51ferT in CHO cells interfered with S-phase progression in these cells. This was not seen in p94fer-overexpressing cells. The FER tyrosine kinases seem, thus, to be regulated by novel mechanisms that direct their different subcellular distribution profiles and may, consequently, control their cellular functioning. PMID:10074905

  12. Synthesis and Evaluation of a Novel Deguelin Derivative, L80, which Disrupts ATP Binding to the C-terminal Domain of Heat Shock Protein 90.

    PubMed

    Lee, Su-Chan; Min, Hye-Young; Choi, Hoon; Kim, Ho Shin; Kim, Kyong-Cheol; Park, So-Jung; Seong, Myeong A; Seo, Ji Hae; Park, Hyun-Ju; Suh, Young-Ger; Kim, Kyu-Won; Hong, Hyun-Seok; Kim, Hee; Lee, Min-Young; Lee, Jeewoo; Lee, Ho-Young

    2015-08-01

    The clinical benefit of current anticancer regimens for lung cancer therapy is still limited due to moderate efficacy, drug resistance, and recurrence. Therefore, the development of effective anticancer drugs for first-line therapy and for optimal second-line treatment is necessary. Because the 90-kDa molecular chaperone heat shock protein (Hsp90) contributes to the maturation of numerous mutated or overexpressed oncogenic proteins, targeting Hsp90 may offer an effective anticancer therapy. Here, we investigated antitumor activities and toxicity of a novel deguelin-derived C-terminal Hsp90 inhibitor, designated L80. L80 displayed significant inhibitory effects on the viability, colony formation, angiogenesis-stimulating activity, migration, and invasion of a panel of non-small cell lung cancer cell lines and their sublines with acquired resistance to paclitaxel with minimal toxicity to normal lung epithelial cells, hippocampal cells, vascular endothelial cells, and ocular cells. Biochemical analyses and molecular docking simulation revealed that L80 disrupted Hsp90 function by binding to the C-terminal ATP-binding pocket of Hsp90, leading to the disruption of the interaction between hypoxia-inducible factor (HIF)-1α and Hsp90, downregulation of HIF-1α and its target genes, including vascular endothelial growth factor (VEGF) and insulin-like growth factor 2 (IGF2), and decreased the expression of various Hsp90 client proteins. Consistent with these in vitro findings, L80 exhibited significant antitumor and antiangiogenic activities in H1299 xenograft tumors. These results suggest that L80 represents a novel C-terminal Hsp90 inhibitor with effective anticancer activities with minimal toxicities. PMID:25976766

  13. In vitro selection of RNase P RNA reveals optimized catalytic activity in a highly conserved structural domain.

    PubMed

    Frank, D N; Ellington, A E; Pace, N R

    1996-12-01

    In vitro selection techniques are useful means of dissecting the functions of both natural and artificial ribozymes. Using a self-cleaving conjugate containing the Escherichia coli ribonuclease P RNA and its substrate, pre-tRNA (Frank DN, Harris ME, Pace NR, 1994, Biochemistry 33:10800-10808), we have devised a method to select for catalytically active variants of the RNase P ribozyme. A selection experiment was performed to probe the structural and sequence constraints that operate on a highly conserved region of RNase P: the J3/4-P4-J2/4 region, which lies within the core of RNase P and is thought to bind catalytically essential magnesium ions (Harris ME et al., 1994, EMBO J 13:3953-3963; Hardt WD et al., 1995, EMBO J 14:2935-2944; Harris ME, Pace NR, 1995, RNA 1:210-218). We sought to determine which, if any, of the nearly invariant nucleotides within J3/4-P4-J2/4 are required for ribozyme-mediated catalysis. Twenty-two residues in the J3/4-P4-J2/4 component of RNase P RNA were randomized and, surprisingly, after only 10 generations, each of the randomized positions returned to the wild-type sequence. This indicates that every position in J3/4-P4-J2/4 contributes to optimal catalytic activity. These results contrast sharply with selections involving other large ribozymes, which evolve improved catalytic function readily in vitro (Chapman KB, Szostak JW, 1994, Curr Opin Struct Biol 4:618-622; Joyce GF, 1994, Curr Opin Struct Biol 4:331-336; Kumar PKR, Ellington AE, 1995, FASEB J 9:1183-1195). The phylogenetic conservation of J3/4-P4-J2/4, coupled with the results reported here, suggests that the contribution of this structure to RNA-mediated catalysis was optimized very early in evolution, before the last common ancestor of all life. PMID:8972768

  14. The type II and X cellulose-binding domains of Pseudomonas xylanase A potentiate catalytic activity against complex substrates by a common mechanism.

    PubMed Central

    Gill, J; Rixon, J E; Bolam, D N; McQueen-Mason, S; Simpson, P J; Williamson, M P; Hazlewood, G P; Gilbert, H J

    1999-01-01

    Xylanase A (Pf Xyn10A), in common with several other Pseudomonas fluorescens subsp. cellulosa polysaccharidases, consists of a Type II cellulose-binding domain (CBD), a catalytic domain (Pf Xyn10A(CD)) and an internal domain that exhibits homology to Type X CBDs. The Type X CBD of Pf Xyn10A, expressed as a discrete entity (CBD(X)) or fused to the catalytic domain (Pf Xyn10A'), bound to amorphous and bacterial microcrystalline cellulose with a K(a) of 2.5 x 10(5) M(-1). CBD(X) exhibited no affinity for soluble forms of cellulose or cello-oligosaccharides, suggesting that the domain interacts with multiple cellulose chains in the insoluble forms of the polysaccharide. Pf Xyn10A' was 2-3 times more active against cellulose-hemicellulose complexes than Pf Xyn10A(CD); however, Pf Xyn10A' and Pf Xyn10A(CD) exhibited the same activity against soluble substrates. CBD(X) did not disrupt the structure of plant-cell-wall material or bacterial microcrystalline cellulose, and did not potentiate Pf Xyn10A(CD) when not covalently linked to the enzyme. There was no substantial difference in the affinity of full-length Pf Xyn10A and the enzyme's Type II CBD for cellulose. The activity of Pf Xyn10A against cellulose-hemicellulose complexes was similar to that of Pf Xyn10A', and a derivative of Pf Xyn10A in which the Type II CBD is linked to the Pf Xyn10A(CD) via a serine-rich linker sequence [Bolam, Cireula, McQueen-Mason, Simpson, Williamson, Rixon, Boraston, Hazlewood and Gilbert (1998) Biochem J. 331, 775-781]. These data indicate that CBD(X) is functional in Pf Xyn10A and that no synergy, either in ligand binding or in the potentiation of catalysis, is evident between the Type II and X CBDs of the xylanase. PMID:10455036

  15. In vitro catalytic activity of N-terminal and C-terminal domains in NukM, the post-translational modification enzyme of nukacin ISK-1.

    PubMed

    Shimafuji, Chinatsu; Noguchi, Megumi; Nishie, Mami; Nagao, Jun-Ichi; Shioya, Kouki; Zendo, Takeshi; Nakayama, Jiro; Sonomoto, Kenji

    2015-12-01

    Lantibiotics are antibacterial peptides containing unique thioether cross-links termed lanthionine and methyllanthionine. NukM, the modifying enzyme of nukacin ISK-1, which is produced by Staphylococcus warneri ISK-1, catalyzes the dehydration of specific Ser/Thr residues in a precursor peptide, followed by conjugative addition of intramolecular Cys to dehydrated residues to generate a cyclic structure. By contrast, the precursor peptide of nisin is modified by 2 enzymes, NisB and NisC, which mediate dehydration and cyclization, respectively. While the C-terminal domain of NukM is homologous to NisC, the N-terminal domain has no homology with other known proteins. We expressed and characterized the N- and C-terminal domains of NukM, NukMN, and NukMC, separately. In vitro reconstitution revealed that full-length NukM fully modified the substrate peptide NukA. NukMN partially phosphorylated, dehydrated, and cyclized NukA. By contrast, NukMC did not catalyze dehydration, phosphorylation, or cyclization reactions. Interaction studies using surface plasmon resonance analysis indicated that NukM and NukMN can bind NukA with high affinity, whereas NukMC has low substrate-recognition activity. These results suggest that NukMN is mainly responsible for substrate recognition and dehydration and that the whole NukM structure, including the C-terminal domain, is required for the complete modification of NukA. To the best of our knowledge, this is the first report providing insights into the in vitro catalytic activity of individual domains of a LanM-type modification enzyme. PMID:25971839

  16. 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. PMID:23663131

  17. 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. PMID:18774801

  18. Relationship of tightly bound ADP and ATP to control and catalysis by chloroplast ATP synthase

    SciTech Connect

    Zhou, J.; Xue, Z.; Du, Z.; Melese, T.; Boyer, P.D.

    1988-07-12

    Whether the tightly bound ADP that can cause a pronounced inhibition of ATP hydrolysis by the chloroplast ATP synthase and F/sub 1/ ATPase (CF/sub 1/) is bound at catalytic sites or at noncatalytic regulatory sites or both has been uncertain. The authors have used photolabeling by 2-azido-ATP and 2-azido-ADP to ascertain the location, with Mg/sup 2 +/ activation, of tightly bound ADP (a) that inhibits the hydrolysis of ATP by chloroplast ATP synthase, (b) that can result in an inhibited form of CF/sub 1/ that slowly regains activity during ATP hydrolysis, and (c) that arises when low concentrations of ADP markedly inhibit the hydrolysis of GTP by CF/sub 1/. The data show that in all instances the inhibition is associated with ADP binding without inorganic phosphate (P/sub i/) at catalytic sites. After photophosphorylation of ADP or 2-azido-ADP with (/sup 32/P)P/sub i/, similar amounts of the corresponding triphosphates are present on washed thylakoid membranes. Trials with appropriately labeled substrates show that a small portion of the tightly bound 2-azido-ATP gives rise to covalent labeling with an ATP moiety at noncatalytic sites but that most of the bound 2-azido-ATP gives rise to covalent labeling with an ATP moiety at noncatalytic sites but that most of the bound 2-azido-ATP gives rise to covalent labeling by an ADP moiety at a catalytic site. They also report the occurrence of a 1-2-min delay in the onset of the Mg/sup 2 +/-induced inhibition after addition of CF/sub 1/ to solutions containing Mg/sup 2 +/ and ATP, and that this delay is not associated with the filling of noncatalytic sites. A rapid burst of P/sub i/ formation is followed by a much lower, constant steady-state rate. The burst is not observed with GTP as a substrate or with Ca/sup 2 +/ as the activating cation.

  19. Chemical modification with dihydro-4,4'-diisothiocyanostilbene-2,2'-disulfonate reveals the distance between K480 and K501 in the ATP-binding domain of the Na,K-ATPase.

    PubMed

    Gatto, C; Lutsenko, S; Kaplan, J H

    1997-04-01

    Dihydro-4,4'-diisothiocyanostilbene-2,2'-disulfonate (H2DIDS) inactivates the renal Na,K-ATPase in an ATP- and K-preventable fashion; inactivation results in the covalent incorporation of a single [3H2]DIDS molecule into the Na pump alpha-subunit. K+ protection is observed at low concentrations (< 2 mM) and reversed at higher concentrations. The biphasic effect is also seen with Rb+, to a lesser extent by Cs+, and not at all by Na+ or choline. After extensive tryptic digestion of 3H2DIDS-inactivated enzyme, a single radiolabeled peptide is seen in 16.5% Tricine gels. N-terminal amino acid sequencing revealed two sequences 470IVEIPFNSTNxYQLS and 495HLLVMxGAPER, the unidentified residues were K480 and K501, respectively. These data provide suggestive evidence of cross-linking by H2DIDS between the two lysines. CNBr digestion of 3H2DIDS-labeled alpha-subunit produced a single radioactive band of the predicted 15-kDa mass for cross-linking between K480 an K501 produced by cleavage at known methione residues. The 15-kDa band combined two N-terminal sequences 464RDRYAKIVEI and 501xGAPERILDR which include K480 and K501. Thus K480 and K501 are within approximately 14 A of each other in the Na-bound form of the enzyme and information about the occupancy of the cation binding domain is transmitted to the ATP binding loop of the Na,K-ATPase. PMID:9126281

  20. Roles of the propeptide and metal ions in the folding and stability of the catalytic domain of stromelysin (matrix metalloproteinase 3).

    PubMed

    Wetmore, D R; Hardman, K D

    1996-05-28

    Matrix metalloproteinases (MMPs) are an unique class of zinc metalloproteases in that 12 A from the catalytic zinc site is a second zinc site, the function of which has yet to be determined. In the pro form, the protease is inactive. Here we show that the heat-induced autocatalytic activation of pro to mature MMP3 is bimolecular. Further, the process is modulated by a low-affinity zinc. A mechanism is proposed by which the second zinc site may act as an enzymatic activator for the mature protease. A method for preparing completely metal-free protein is described. Surprisingly, there is a much more dramatic structural change between the apo and holo forms of the mature protein than there is between apo and holo proprotein. Apo mature MMP3 appears to form a native-like stable intermediate structure in which one or more of the tryptophan side chains is more solvent-exposed than in the holo form. Apo MMP3 is remarkably stable to thermal unfolding as monitored by CD; thus the metal ions do not appear to significantly stabilize the secondary structure of the catalytic domain. The apo mature MMP3 intermediate can be unfolded with heat, subsequently refolded, and reactivated by addition of zinc and calcium. Thus for MMP3, unlike subtilisin or alpha-lytic protease, the propeptide is not required for protein folding in a timely fashion and the role of intramolecular chaperone is not a universal one for the propeptides of proteases. PMID:8639603

  1. BIOGENESIS FACTOR REQUIRED FOR ATP SYNTHASE 3 Facilitates Assembly of the Chloroplast ATP Synthase Complex.

    PubMed

    Zhang, Lin; Duan, Zhikun; Zhang, Jiao; Peng, Lianwei

    2016-06-01

    Thylakoid membrane-localized chloroplast ATP synthases use the proton motive force generated by photosynthetic electron transport to produce ATP from ADP. Although it is well known that the chloroplast ATP synthase is composed of more than 20 proteins with α3β3γ1ε1δ1I1II1III14IV1 stoichiometry, its biogenesis process is currently unclear. To unravel the molecular mechanisms underlying the biogenesis of chloroplast ATP synthase, we performed extensive screening for isolating ATP synthase mutants in Arabidopsis (Arabidopsis thaliana). In the recently identified bfa3 (biogenesis factors required for ATP synthase 3) mutant, the levels of chloroplast ATP synthase subunits were reduced to approximately 25% of wild-type levels. In vivo labeling analysis showed that assembly of the CF1 component of chloroplast ATP synthase was less efficient in bfa3 than in the wild type, indicating that BFA3 is required for CF1 assembly. BFA3 encodes a chloroplast stromal protein that is conserved in higher plants, green algae, and a few species of other eukaryotic algae, and specifically interacts with the CF1β subunit. The BFA3 binding site was mapped to a region in the catalytic site of CF1β. Several residues highly conserved in eukaryotic CF1β are crucial for the BFA3-CF1β interaction, suggesting a coevolutionary relationship between BFA3 and CF1β. BFA3 appears to function as a molecular chaperone that transiently associates with unassembled CF1β at its catalytic site and facilitates subsequent association with CF1α during assembly of the CF1 subcomplex of chloroplast ATP synthase. PMID:27208269

  2. 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

  3. Mutations in the Catalytic Domain of Human Matrix Metalloproteinase-1 (MMP-1) That Allow for Regulated Activity through the Use of Ca2+

    PubMed Central

    Paladini, Rudolph D.; Wei, Ge; Kundu, Anirban; Zhao, Qiping; Bookbinder, Louis H.; Keller, Gilbert A.; Shepard, H. Michael; Frost, Gregory I.

    2013-01-01

    Conditionally active proteins regulated by a physiological parameter represent a potential new class of protein therapeutics. By systematically creating point mutations in the catalytic and linker domains of human MMP-1, we generated a protein library amenable to physiological parameter-based screening. Mutants screened for temperature-sensitive activity had mutations clustered at or near amino acids critical for metal binding. One mutant, GVSK (Gly159 to Val, Ser208 to Lys), contains mutations in regions of the catalytic domain involved in calcium and zinc binding. The in vitro activity of GVSK at 37 °C in high Ca2+ (10 mm) was comparable with MMP-1 (wild type), but in low Ca2+ (1 mm), there was an over 10-fold loss in activity despite having similar kinetic parameters. Activity decreased over 50% within 15 min and correlated with the degradation of the activated protein, suggesting that GVSK was unstable in low Ca2+. Varying the concentration of Zn2+ had no effect on GVSK activity in vitro. As compared with MMP-1, GVSK degraded soluble collagen I at the high but not the low Ca2+ concentration. In vivo, MMP-1 and GVSK degraded collagen I when perfused in Zucker rat ventral skin and formed higher molecular weight complexes with α2-macroglobulin, an inhibitor of MMPs. In vitro and in vivo complex formation and subsequent enzyme inactivation occurred faster with GVSK, especially at the low Ca2+ concentration. These data suggest that the activity of the human MMP-1 mutant GVSK can be regulated by Ca2+ both in vitro and in vivo and may represent a novel approach to engineering matrix-remodeling enzymes for therapeutic applications. PMID:23322779

  4. Crystal Structure of DNA Cytidine Deaminase ABOBEC3G Catalytic Deamination Domain Suggests a Binding Mode of Full-length Enzyme to Single-stranded DNA*

    PubMed Central

    Lu, Xiuxiu; Zhang, Tianlong; Xu, Zeng; Liu, Shanshan; Zhao, Bin; Lan, Wenxian; Wang, Chunxi; Ding, Jianping; Cao, Chunyang

    2015-01-01

    APOBEC3G (A3G) is a DNA cytidine deaminase (CD) that demonstrates antiviral activity against human immunodeficiency virus 1 (HIV-1) and other pathogenic virus. It has an inactive N-terminal CD1 virus infectivity factor (Vif) protein binding domain (A3G-CD1) and an actively catalytic C-terminal CD2 deamination domain (A3G-CD2). Although many studies on the structure of A3G-CD2 and enzymatic properties of full-length A3G have been reported, the mechanism of how A3G interacts with HIV-1 single-stranded DNA (ssDNA) is still not well characterized. Here, we reported a crystal structure of a novel A3G-CD2 head-to-tail dimer (in which the N terminus of the monomer H (head) interacts with the C terminus of monomer T (tail)), where a continuous DNA binding groove was observed. By constructing the A3G-CD1 structural model, we found that its overall fold was almost identical to that of A3G-CD2. We mutated the residues located in or along the groove in monomer H and the residues in A3G-CD1 that correspond to those seated in or along the groove in monomer T. Then, by performing enzymatic assays, we confirmed the reported key elements and the residues in A3G necessary to the catalytic deamination. Moreover, we identified more than 10 residues in A3G essential to DNA binding and deamination reaction. Therefore, this dimer structure may represent a structural model of full-length A3G, which indicates a possible binding mode of A3G to HIV-1 ssDNA. PMID:25542899

  5. Crystal structure of DNA cytidine deaminase ABOBEC3G catalytic deamination domain suggests a binding mode of full-length enzyme to single-stranded DNA.

    PubMed

    Lu, Xiuxiu; Zhang, Tianlong; Xu, Zeng; Liu, Shanshan; Zhao, Bin; Lan, Wenxian; Wang, Chunxi; Ding, Jianping; Cao, Chunyang

    2015-02-13

    APOBEC3G (A3G) is a DNA cytidine deaminase (CD) that demonstrates antiviral activity against human immunodeficiency virus 1 (HIV-1) and other pathogenic virus. It has an inactive N-terminal CD1 virus infectivity factor (Vif) protein binding domain (A3G-CD1) and an actively catalytic C-terminal CD2 deamination domain (A3G-CD2). Although many studies on the structure of A3G-CD2 and enzymatic properties of full-length A3G have been reported, the mechanism of how A3G interacts with HIV-1 single-stranded DNA (ssDNA) is still not well characterized. Here, we reported a crystal structure of a novel A3G-CD2 head-to-tail dimer (in which the N terminus of the monomer H (head) interacts with the C terminus of monomer T (tail)), where a continuous DNA binding groove was observed. By constructing the A3G-CD1 structural model, we found that its overall fold was almost identical to that of A3G-CD2. We mutated the residues located in or along the groove in monomer H and the residues in A3G-CD1 that correspond to those seated in or along the groove in monomer T. Then, by performing enzymatic assays, we confirmed the reported key elements and the residues in A3G necessary to the catalytic deamination. Moreover, we identified more than 10 residues in A3G essential to DNA binding and deamination reaction. Therefore, this dimer structure may represent a structural model of full-length A3G, which indicates a possible binding mode of A3G to HIV-1 ssDNA. PMID:25542899

  6. 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

  7. 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. PMID:26621635

  8. 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.

  9. The catalytic subunit of shiga-like toxin 1 interacts with ribosomal stalk proteins and is inhibited by their conserved C-terminal domain.

    PubMed

    McCluskey, Andrew J; Poon, Gregory M K; Bolewska-Pedyczak, Eleonora; Srikumar, Tharan; Jeram, Stanley M; Raught, Brian; Gariépy, Jean

    2008-04-25

    Shiga-like toxin 1 (SLT-1) is a type II ribosome-inactivating protein; its A(1) domain blocks protein synthesis in eukaryotic cells by catalyzing the depurination of a single adenine base in 28 S rRNA. The molecular mechanism leading to this site-specific depurination event is thought to involve interactions with eukaryotic ribosomal proteins. Here, we present evidence that the A(1) chain of SLT-1 binds to the ribosomal proteins P0, P1, and P2. These proteins were identified from a HeLa cell lysate by tandem mass spectrometry, and subsequently confirmed to bind to SLT-1 A(1) chain by yeast-two-hybrid and pull-down experiments using candidate full-length proteins. Moreover, the removal of the last 17 amino acids of either protein P1 or P2 abolishes the interaction with the A(1) chain, whereas P0, lacking this common C terminus, still binds to the A(1) domain. In vitro pull-down experiments using fusion protein-tagged C-terminal peptides corresponding to the common 7, 11, and 17 terminal residues of P1 and P2 confirmed that the A(1) chain of SLT-1 as well as the A chain of ricin bind to this shared C-terminal peptide motif. More importantly, a synthetic peptide corresponding to the 17 amino acid C terminus of P1 and P2 was shown to inhibit the ribosome-inactivating function of the A(1) chain of SLT-1 in an in vitro transcription and translation-coupled assay. These results suggest a role for the ribosomal stalk in aiding the A(1) chain of SLT-1 and other type II ribosome-inactivating proteins in localizing its catalytic domain near the site of depurination in the 28 S rRNA. PMID:18358491

  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.