Proteins: Structure, Function, Bioinformatics

Wiley Online Library : Proteins: Structure, Function, and Bioinformatics
  • Structural templates for comparative protein docking
    [Dec 2014]

    Abstract

    Structural characterization of protein-protein interactions is important for understanding life processes. Because of the inherent limitations of experimental techniques, such characterization requires computational approaches. Along with the traditional protein-protein docking (free search for a match between two proteins), comparative (template-based) modeling of protein-protein complexes has been gaining popularity. Its development puts an emphasis on full and partial structural similarity between the target protein monomers and the protein-protein complexes previously determined by experimental techniques (templates). The template-based docking relies on the quality and diversity of the template set. We present a carefully curated, non-redundant library of templates containing 4,950 full structures of binary complexes and 5,936 protein-protein interfaces extracted from the full structures at 12Å distance cut-off. Redundancy in the libraries was removed by clustering the PDB structures based on structural similarity. The value of the clustering threshold was determined from the analysis of the clusters and the docking performance on a benchmark set. High structural quality of the interfaces in the template and validation sets was achieved by automated procedures and manual curation. The library is included in the Dockground resource for molecular recognition studies at http://dockground.bioinformatics.ku.edu. This article is protected by copyright. All rights reserved.

    Categories: Journal Articles
  • The structure of S. lividans acetoacetyl-CoA synthetase shows a novel interaction between the C-terminal extension and the N-terminal domain
    [Dec 2014]

    Abstract

    The AMP-forming acyl-CoA synthetase enzymes catalyze a two-step reaction that involves the initial formation of an acyl adenylate that reacts in a second partial reaction to form a thioester between the acyl substrate and CoA. These enzymes utilize a Domain Alternation catalytic mechanism, whereby a ∼110 residue C-terminal domain rotates by 140° to form distinct catalytic conformations for the two partial reactions. The structure of an acetoacetyl-CoA synthetase (AacS) is presented that illustrates a novel aspect of this C-terminal domain. Specifically, several acetyl- and acetoacetyl-CoA synthetases contain a 30-residue extension on the C-terminus compared to other members of this family. Whereas residues from this extension are disordered in prior structures, the AacS structure shows that residues from this extension may interact with key catalytic residues from the N-terminal domain. This article is protected by copyright. All rights reserved.

    Categories: Journal Articles
  • Dynamics on human toll-like receptor 4 complexation to MD-2: The co-receptor stabilizing function
    [Dec 2014]

    Abstract

    The interaction between human Toll-Like Receptor 4 (hTLR4) and its co-receptor, myeloid differentiation factor 2 (MD-2), is important in Gram-negative bacteria lipopolysaccharide (LPS) recognition. In this process, MD-2 recognizes LPS and promotes the dimerization of the complex hTLR4 - MD-2 - LPS, triggering an intracellular immune signaling. In this study, we employed distinct computational methods to explore the dynamical properties of the hTLR4 - MD-2 complex and investigated the implications of the co-receptor complexation to the structural biology of hTLR4. We characterized both global and local dynamics of free and MD-2 complexed hTLR4, in both (hTLR4 - MD-2)1 and (hTLR4 - MD-2)2 states. Both molecular dynamics and normal mode analysis reveled a stabilization of the terminal regions of hTLR4 upon complexation to MD-2. We are able to identify conserved important residues involved on the hTLR4 - MD-2 interaction dynamics and disclose C-terminal motions that may be associated to the signaling process upon oligomerization. This article is protected by copyright. All rights reserved.

    Categories: Journal Articles
  • Modulation of HIV protease flexibility by the T80N mutation
    [Dec 2014]

    Abstract

    The flexibility of HIV protease plays a critical role in enabling enzymatic activity and is required for substrate access to the active site. While the importance of flexibility in the flaps that cover the active site is well known, flexibility in other parts of the enzyme is also critical for function. One key region is a loop containing Thr 80 which forms the walls of the active site. Although not situated within the active site, amino acid Thr80 is absolutely conserved. The mutation T80N preserves the structure of the enzyme but catalytic activity is completely lost. To investigate the potential influence of the T80N mutation on HIVp flexibility, wide-angle scattering (WAXS) data was measured for a series of HIV protease variants. Starting with a calculated WAXS pattern from a rigid atomic model, the modulations in the intensity distribution caused by structural fluctuations in the protein were predicted by simple analytic methods and compared to the experimental data. An analysis of T80N WAXS data shows that this variant is significantly more rigid than the WT across all length scales. The effects of this single point mutation extend throughout the protein, so as to alter the mobility of amino acids in the enzymatic core. These results support the contentions that significant protein flexibility extends throughout HIV protease and is critical to catalytic function. This article is protected by copyright. All rights reserved.

    Categories: Journal Articles
  • Conventional NMA as a better standard for evaluating elastic network models
    [Dec 2014]

    Abstract

    Normal mode analysis (NMA) is an important tool for studying protein dynamics. Due to the complexity of conventional NMA that employs an all-atom model and a semi-empirical force field, many simplified NMA models have been developed, some of which are known as elastic network models. The quality of these simplified NMA models was assessed mostly by evaluating their predictions against experimental B-factors, and rarely by comparing them with the original NMA. In this work, we take the effort to create a publicly-accessible dataset of proteins with their minimized structures, NMA modes, and mean-square fluctuations. Then, for the first time, we evaluate the quality of individual normal modes of several widely used elastic network models by comparing them with the conventional NMA. Our results demonstrate that the conventional NMA presents a better and more complete evaluation measure of the quality of elastic network models. This realization should be very helpful in improving current or designing new, higher-quality elastic network models. Moreover, by using the conventional NMA as the standard of evaluation, a number of interesting and significant insights into the elastic network models are gained. This article is protected by copyright. All rights reserved.

    Categories: Journal Articles
  • Electrostatic analysis of bacterial expansins
    [Nov 2014]

    ABSTRACT

    Expansins are a family of proteins with plant cell wall remodeling-activity, which bind cell wall components through hydrophobic and electrostatic interactions. A shallow area on the surface of the protein serves as the polysaccharide binding site (PBS) and it is composed of conserved residues. However, electric charge differences on the opposite face of the PBS produce basic, neutral, or acidic proteins. An analysis of forty-four bacterial expansins, homologues of BsEXLX1, revealed two main groups defined by: (a) the presence or absence of disulfide bonds; and (b) by the proteins isoelectric point (pI). We determined the location of the residues responsible for the pI on the structure of representative expansins. Our results suggest that the electric charge at the opposite site of the PBS may help in substrate differentiation among expansins from different species; in addition, electrostatic polarization between the front and the back of the molecule could affect expansin activity on cellulose. Proteins 2014; © 2014 Wiley Periodicals, Inc.

    Categories: Journal Articles
  • ω-Turn: A novel β-turn mimic in globular proteins stabilized by main-chain to side-chain CH···O interaction
    [Nov 2014]

    ABSTRACT

    Mimicry of structural motifs is a common feature in proteins. The 10-membered hydrogen-bonded ring involving the main-chain CO in a β-turn can be formed using a side-chain carbonyl group leading to Asx-turn. We show that the NH component of hydrogen bond can be replaced by a Cγ-H group in the side chain, culminating in a nonconventional CH···O interaction. Because of its shape this β-turn mimic is designated as ω-turn, which is found to occur ∼three times per 100 residues. Three residues (i to i + 2) constitute the turn with the CH···O interaction occurring between the terminal residues, constraining the torsion angles ϕi + 1, ψi + 1, ϕi + 2 and χ′1(i + 2) (using the interacting Cγ atom). Based on these angles there are two types of ω-turns, each of which can be further divided into two groups. Cβ-branched side-chains, and Met and Gln have high propensities to occur at i + 2; for the last two residues the carbonyl oxygen may participate in an additional interaction involving the S and amino group, respectively. With Cys occupying the i + 1 position, such turns are found in the metal-binding sites. N-linked glycosylation occurs at the consensus pattern Asn-Xaa-Ser/Thr; with Thr at i + 2, the sequence can adopt the secondary structure of a ω-turn, which may be the recognition site for protein modification. Location between two β-strands is the most common occurrence in protein tertiary structure, and being generally exposed ω-turn may constitute the antigenic determinant site. It is a stable scaffold and may be used in protein engineering and peptide design. Proteins 2014; © 2014 Wiley Periodicals, Inc.

    Categories: Journal Articles
  • Examination of polypeptide substrate specificity for Escherichia coli ClpB
    [Nov 2014]

    ABSTRACT

    Escherichia coli ClpB is a molecular chaperone that belongs to the Clp/Hsp100 family of AAA+ proteins. ClpB is able to form a hexameric ring structure to catalyze protein disaggregation with the assistance of the DnaK chaperone system. Our knowledge of the mechanism of how ClpB recognizes its substrates is still limited. In this study, we have quantitatively investigated ClpB binding to a number of unstructured polypeptides using steady-state anisotropy titrations. To precisely determine the binding affinity for the interaction between ClpB hexamers and polypeptide substrates the titration data were subjected to global non-linear least squares analysis incorporating the dynamic equilibrium of ClpB assembly. Our results show that ClpB hexamers bind tightly to unstructured polypeptides with binding affinities in the range of ∼3–16 nM. ClpB exhibits a modest preference of binding to Peptide B1 with a binding affinity of (1.7 ± 0.2) nM. Interestingly, we found that ClpB binds to an unstructured polypeptide substrate of 40 and 50 amino acids containing the SsrA sequence at the C-terminus with an affinity of (12 ± 3) nM and (4 ± 2) nM, respectively. Whereas, ClpB binds the 11-amino acid SsrA sequence with an affinity of (140 ± 20) nM, which is significantly weaker than other polypeptide substrates that we tested here. We hypothesize that ClpB, like ClpA, requires substrates with a minimum length for optimal binding. Finally, we present evidence showing that multiple ClpB hexamers are involved in binding to polypeptides ≥152 amino acids. Proteins 2014. © 2014 Wiley Periodicals, Inc.

    Categories: Journal Articles
  • Solution structure of a bacterial immunoglobulin-like domain of the outer membrane protein (LigB) from Leptospira
    [Nov 2014]

    ABSTRACT

    Leptospiral immunoglobulin-like (Lig) proteins are surface proteins expressed in pathogenic strains of Leptospira. LigB, an outer membrane protein containing tandem repeats of bacterial Ig-like (Big) domains and a no-repeat tail, has been identified as a virulence factor involved in adhesion of pathogenic Leptospira interrogans to host cells. A Big domain of LigB, LigBCen2R, was reported previously to bind the GBD domain of fibronectin, suggesting its important role in leptospiral infections. In this study, we determined the solution structure of LigBCen2R by nuclear magnetic resonance (NMR) spectroscopy. LigBCen2R adopts a canonical immunoglobulin-like fold which is comprised of a beta-sandwich of ten strands in three sheets. We indicated that LigBCen2R is able to bind to Ca2+ with a high affinity by isothermal titration calorimetry assay. NMR perturbation experiment identified a number of residues responsible for Ca2+ binding. Structural comparison of it with other Big domains demonstrates that they share a similar fold pattern, but vary in some structural characters. Since Lig proteins play a vital role in the infection to host cells, our study will contribute a structural basis to understand the interactions between Leptospira and host cells. Proteins 2014; © 2014 Wiley Periodicals, Inc.

    Categories: Journal Articles
  • Human α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD): A structural and mechanistic unveiling
    [Nov 2014]

    ABSTRACT

    Human α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase determines the fate of tryptophan metabolites in the kynurenine pathway by controlling the quinolinate levels for de novo nicotinamide adenine dinucleotide biosynthesis. The unstable nature of its substrate has made gaining insight into its reaction mechanism difficult. Our electron paramagnetic resonance (EPR) spectroscopic study on the Cu-substituted human enzyme suggests that the native substrate does not directly ligate to the metal ion. Substrate binding did not result in a change of either the hyperfine structure or the super-hyperfine structure of the EPR spectrum. We also determined the crystal structure of the human enzyme in its native catalytically active state (at 1.99 Å resolution), a substrate analogue-bound form (2.50 Å resolution), and a selected active site mutant form with one of the putative substrate binding residues altered (2.32 Å resolution). These structures illustrate that each asymmetric unit contains three pairs of dimers. Consistent with the EPR findings, the ligand-bound complex structure shows that the substrate analogue does not directly coordinate to the metal ion but is bound to the active site by two arginine residues through noncovalent interactions. Proteins 2014; © 2014 Wiley Periodicals, Inc.

    Categories: Journal Articles
  • Multiple proton relay routes in the reaction mechanism of RNAP II: Assessing the effect of structural model
    [Nov 2014]

    Abstract

    RNA polymerase II catalyzes the nucleotidyl transfer reaction for messenger RNA synthesis in eukaryotes. Two crystal structures of this system have been resolved, each with its own defects in the coordination sphere of Mg2+(A) resulting from chemical modifications. We have used both structures and also a novel hybrid of the two that allows a better exploration of the parts of configuration space that reflect substrate-enzyme interactions. MD and QM/MM calculations have been performed, the latter with the semiempirical AM1/d-PHOT method, calibrated against Density Functional Theory. Reaction path scans in 1-D provided insights about the role of Mg2+(A) which turns out to be more structural than catalytic. By contrast, 1-D scans of the incorporation of the nucleotidyl group yielded barriers that were much too high, necessitating the use of 2-D reaction coordinates. Three different proton acceptors for the initial reaction step were examined. For those models based on the two PDB structures the 2-D scans continued to yield very high barriers, indicating that the reaction is unlikely to proceed from these configurations. On the other hand, two hybrid models, chosen from the early and late parts of a 12ns molecular dynamics simulation yielded greatly reduced barriers in the range of ~17 to ~27 kcal/mol for the three proton acceptors, as compared to the experimental estimate of 18kcal/mol. The final step, release of pyrophosphate, was found to be facile. Our overall mechanism involves only active site residues or water without the need for external reactive agents such as the hydroxide ion previously proposed. This article is protected by copyright. All rights reserved.

    Categories: Journal Articles
  • Common functionally-important motions of the nucleotidebinding domain of Hsp70
    [Nov 2014]

    Abstract

    The 70 kDa Heat Shock Proteins (Hsp70) are a family of molecular chaperones involved in protein folding, aggregate prevention, and protein disaggregation. They consist of the substrate binding domain (SBD) that binds client substrates, and the nucleotide-binding domain (NBD), whose cycles of nucleotide hydrolysis and exchange underpin the activity of the chaperone. To characterize the structure-function relationships that link the binding state of the NBD to its conformational behavior, we analyzed the dynamics of the NBD of the Hsp70 chaperone from Bos taurus (pdb 3C7N:B) by all-atom canonical molecular dynamics simulations. It was found that essential motions within the NBD fall into three major classes: the mutual class, reflecting tendencies common to all binding states, and the ADP- and ATP-unique classes, which reflect conformational trends that are unique to either the ADP- or ATP-bound states, respectively. ‘Mutual’ class motions generally describe ‘in-plane’ and/or ‘out-of-plane’ (‘scissor-like’) rotation of the subdomains within the NBD. This result is consistent with experimental nuclear magnetic resonance data on the NBD. The ‘Unique’ class motions target specific regions on the NBD, usually surface loops or sites involved in nucleotide-binding and are, therefore, expected to be involved in allostery and signal transmission. For all classes, and especially for those of the ‘Unique’ type, regions of enhanced mobility can be identified; these are termed ‘hot-spots,’ and their locations generally parallel those found by NMR spectroscopy. The presence of magnesium and potassium cations in the nucleotide-binding pocket was also found to influence the dynamics of the NBD significantly. This article is protected by copyright. All rights reserved.

    Categories: Journal Articles
  • A QM/MM study of the reaction mechanism of (R)-hydroxynitrile lyases from Arabidopsis thaliana (AtHNL)
    [Nov 2014]

    ABSTRACT

    Hydroxynitrile lyases (HNLs) catalyze the conversion of chiral cyanohydrins to hydrocyanic acid (HCN) and aldehyde or ketone. Hydroxynitrile lyase from Arabidopsis thaliana (AtHNL) is the first R-selective HNL enzyme containing an α/β-hydrolases fold. In this article, the catalytic mechanism of AtHNL was theoretically studied by using QM/MM approach based on the recently obtained crystal structure in 2012. Two computational models were constructed, and two possible reaction pathways were considered. In Path A, the calculation results indicate that the proton transfer from the hydroxyl group of cyanohydrin occurs firstly, and then the cleavage of C1-C2 bond and the rotation of the generated cyanide ion (CN−) follow, afterwards, CN− abstracts a proton from His236 via Ser81. The C1-C2 bond cleavage and the protonation of CN− correspond to comparable free energy barriers (12.1 vs. 12.2 kcal mol−1), suggesting that both of the two processes contribute a lot to rate-limiting. In Path B, the deprotonation of the hydroxyl group of cyanohydrin and the cleavage of C1-C2 bond take place in a concerted manner, which corresponds to the highest free energy barrier of 13.2 kcal mol−1. The free energy barriers of Path A and B are very similar and basically agree well with the experimental value of HbHNL, a similar enzyme of AtHNL. Therefore, both of the two pathways are possible. In the reaction, the catalytic triad (His236, Ser81, and Asp208) acts as the general acid/base, and the generated CN− is stabilized by the hydroxyl group of Ser81 and the main-chain NH-groups of Ala13 and Phe82. Proteins 2014. © 2014 Wiley Periodicals, Inc.

    Categories: Journal Articles
  • Molecular Mechanisms and Design Principles for Promiscuous Inhibitors to Avoid Drug Resistance: Lessons Learned from HIV-1 Protease Inhibition
    [Nov 2014]

    Abstract

    Molecular recognition is central to biology and ranges from highly selective to broadly promiscuous. The ability to modulate specificity at will is particularly important for drug development, and discovery of mechanisms contributing to binding specificity is crucial for our basic understanding of biology and for applications in health care. In this study we used computational molecular design to create a large data set of diverse small molecules with a range of binding specificities. We then performed structural, energetic, and statistical analysis to the data set in order to study molecular mechanisms of achieving specificity goals. The work was done in the context of HIV-1 protease inhibition and the molecular designs targeted a panel of wild-type and drug-resistant mutant HIV-1 protease structures. The analysis focused on mechanisms for promiscuous binding to bind robustly even to resistance mutants. Broadly binding inhibitors tended to be smaller in size, more flexible in chemical structure, and more hydrophobic in nature compared to highly selective ones. Furthermore, structural and energetic analyses illustrated mechanisms by which flexible inhibitors achieved binding; we found ligand conformational adaptation near mutation sites and structural plasticity in targets through torsional flips of asymmetric functional groups to form alternative, compensatory packing interactions or hydrogen bonds. As no inhibitor bound to all variants, we designed small cocktails of inhibitors to do so and discovered that they often jointly covered the target set through mechanistic complementarity. Furthermore, utilizing structural plasticity observed in experiments, and potentially in simulations, is suggested to be a viable means of designing adaptive inhibitors that are promiscuous binders. This article is protected by copyright. All rights reserved.

    Categories: Journal Articles
  • Computational modeling of membrane proteins
    [Nov 2014]

    ABSTRACT

    The determination of membrane protein (MP) structures has always trailed that of soluble proteins due to difficulties in their overexpression, reconstitution into membrane mimetics, and subsequent structure determination. The percentage of MP structures in the protein databank (PDB) has been at a constant 1–2% for the last decade. In contrast, over half of all drugs target MPs, only highlighting how little we understand about drug-specific effects in the human body. To reduce this gap, researchers have attempted to predict structural features of MPs even before the first structure was experimentally elucidated. In this review, we present current computational methods to predict MP structure, starting with secondary structure prediction, prediction of trans-membrane spans, and topology. Even though these methods generate reliable predictions, challenges such as predicting kinks or precise beginnings and ends of secondary structure elements are still waiting to be addressed. We describe recent developments in the prediction of 3D structures of both α-helical MPs as well as β-barrels using comparative modeling techniques, de novo methods, and molecular dynamics (MD) simulations. The increase of MP structures has (1) facilitated comparative modeling due to availability of more and better templates, and (2) improved the statistics for knowledge-based scoring functions. Moreover, de novo methods have benefited from the use of correlated mutations as restraints. Finally, we outline current advances that will likely shape the field in the forthcoming decade. Proteins 2014. © 2014 Wiley Periodicals, Inc.

    Categories: Journal Articles
  • A data-driven approach to modeling the tripartite structure of multidrug resistance efflux pumps
    [Nov 2014]

    ABSTRACT

    Many bacterial pathogens are becoming increasingly resistant to antibiotic treatments, and a detailed understanding of the molecular basis of antibiotic resistance is critical for the development of next-generation approaches for combating bacterial infections. Studies focusing on pathogens have revealed the profile of resistance in these organisms to be due primarily to the presence of multidrug resistance efflux pumps: tripartite protein complexes which span the periplasm bridging the inner and outer membranes of Gram-negative bacteria. An atomic-level resolution tripartite structure remains imperative to advancing our understanding of the molecular mechanisms of pump function using both theoretical and experimental approaches. We develop a fast and consistent method for constructing tripartite structures which leverages existing data-driven models and provide molecular modeling approaches for constructing tripartite structures of multidrug resistance efflux pumps. Our modeling studies reveal that conformational changes in the inner membrane component responsible for drug translocation have limited impact on the conformations of the other pump components, and that two distinct models derived from conflicting experimental data are both consistent with all currently available measurements. Additionally, we investigate putative drug translocation pathways via geometric simulations based on the available crystal structures of the inner membrane pump component, AcrB, bound to two drugs which occupy distinct binding sites: doxorubicin and linezolid. These simulations suggest that smaller drugs may enter the pump through a channel from the cytoplasmic leaflet of the inner membrane, while both smaller and larger drug molecules may enter through a vestibule accessible from the periplasm. Proteins 2014. © 2014 Wiley Periodicals, Inc.

    Categories: Journal Articles
  • Crystal structures of ligand-bound octaprenyl pyrophosphate synthase from Escherichia coli reveal the catalytic and chain-length determining mechanisms
    [Nov 2014]

    ABSTRACT

    Octaprenyl pyrophosphate synthase (OPPs) catalyzes consecutive condensation reactions of one allylic substrate farnesyl pyrophosphate (FPP) and five homoallylic substrate isopentenyl pyrophosphate (IPP) molecules to form a C40 long-chain product OPP, which serves as a side chain of ubiquinone and menaquinone. OPPs belongs to the trans-prenyltransferase class of proteins. The structures of OPPs from Escherichia coli were solved in the apo-form as well as in complexes with IPP and a FPP thio-analog, FsPP, at resolutions of 2.2–2.6 Å, and revealed the detailed interactions between the ligands and enzyme. At the bottom of the active-site tunnel, M123 and M135 act in concert to form a wall which determines the final chain length. These results represent the first ligand-bound crystal structures of a long-chain trans-prenyltransferase and provide new information on the mechanisms of catalysis and product chain elongation. Proteins 2014. © 2014 Wiley Periodicals, Inc.

    Categories: Journal Articles
  • Structure and specificity of FEN-1 from Methanopyrus kandleri
    [Nov 2014]

    ABSTRACT

    DNA repair is fundamental to genome stability and is found in all three domains of life. However many archaeal species, such as Methanopyrus kandleri, contain only a subset of the eukaryotic nucleotide excision repair (NER) homologs, and those present often contain significant differences compared to their eukaryotic homologs. To clarify the role of the NER XPG-like protein Mk0566 from M. kandleri, its biochemical activity and three-dimensional structure were investigated. Both were found to be more similar to human FEN-1 than human XPG, suggesting a biological role in replication and long-patch base excision repair rather than in NER. Proteins 2014. © 2014 Wiley Periodicals, Inc.

    Categories: Journal Articles
  • Survey of phosphorylation near drug binding sites in the Protein Data Bank (PDB) and their effects
    [Nov 2014]

    ABSTRACT

    While it is currently estimated that 40 to 50% of eukaryotic proteins are phosphorylated, little is known about the frequency and local effects of phosphorylation near pharmaceutical inhibitor binding sites. In this study, we investigated how frequently phosphorylation may affect the binding of drug inhibitors to target proteins. We examined the 453 non-redundant structures of soluble mammalian drug target proteins bound to inhibitors currently available in the Protein Data Bank (PDB). We cross-referenced these structures with phosphorylation data available from the PhosphoSitePlus database. Three hundred twenty-two of 453 (71%) of drug targets have evidence of phosphorylation that has been validated by multiple methods or labs. For 132 of 453 (29%) of those, the phosphorylation site is within 12 Å of the small molecule-binding site, where it would likely alter small molecule binding affinity. We propose a framework for distinguishing between drug-phosphorylation site interactions that are likely to alter the efficacy of drugs versus those that are not. In addition we highlight examples of well-established drug targets, such as estrogen receptor alpha, for which phosphorylation may affect drug affinity and clinical efficacy. Our data suggest that phosphorylation may affect drug binding and efficacy for a significant fraction of drug target proteins. © 2014 Wiley Periodicals, Inc.

    Categories: Journal Articles
  • Role of active-site residues Tyr55 and Tyr114 in catalysis and substrate specificity of Corynebacterium diphtheriae C-S lyase
    [Nov 2014]

    ABSTRACT

    In recent years, there has been increased interest in bacterial methionine biosynthesis enzymes as antimicrobial targets because of their pivotal role in cell metabolism. C-S lyase from Corynebacterium diphtheriae is a pyridoxal 5′-phosphate-dependent enzyme in the transsulfuration pathway that catalyzes the α,β-elimination of sulfur-containing amino acids, such as l-cystathionine, to generate ammonia, pyruvate, and homocysteine, the immediate precursor of L-methionine. In order to gain deeper insight into the functional and dynamic properties of the enzyme, mutants of two highly conserved active-site residues, Y55F and Y114F, were characterized by UV-visible absorbance, fluorescence, and CD spectroscopy in the absence and presence of substrates and substrate analogs, as well as by steady-state kinetic studies. Substitution of Tyr55 with Phe apparently causes a 130-fold decrease in at pH 8.5 providing evidence that Tyr55 plays a role in cofactor binding. Moreover, spectral data show that the mutant accumulates the external aldimine intermediate suggesting that the absence of interaction between the hydroxyl moiety and PLP-binding residue Lys222 causes a decrease in the rate of substrate deprotonation. Mutation of Tyr114 with Phe slightly influences hydrolysis of l-cystathionine, and causes a change in substrate specificity towards l-serine and O-acetyl-l-serine compared to the wild type enzyme. These findings, together with computational data, provide useful insights in the substrate specificity of C-S lyase, which seems to be regulated by active-site architecture and by the specific conformation in which substrates are bound, and will aid in development of inhibitors. Proteins 2014. © 2014 Wiley Periodicals, Inc.

    Categories: Journal Articles