Analysis of zinc binding sites in protein crystal structures

The geometrical properties of zinc binding sites in a dataset of high quality protein crystal structures deposited in the Protein Data Bank have been examined to identify important differences between zinc sites that are directly involved in catalysis and those that play a structural role. Coordination angles in the zinc primary coordination sphere are compared with ideal values for each coordination geometry, and zinc coordination distances are compared with those in small zinc complexes from the Cambridge Structural Database as a guide of expected trends. We find that distances and angles in the primary coordination sphere are in general close to the expected (or ideal) values. Deviations occur primarily for oxygen coordinating atoms and are found to be mainly due to H-bonding of the oxygen coordinating ligand to protein residues, bidentate binding arrangements, and multi-zinc sites. We find that H-bonding of oxygen containing residues (or water) to zinc bound histidines is almost universal in our dataset and defines the elec-His-Zn motif. Analysis of the stereochemistry shows that carboxyl elec-His-Zn motifs are geometrically rigid, while water elec-His-Zn motifs show the most geometrical variation. As catalytic motifs have a higher proportion of carboxyl elec atoms than structural motifs, they provide a more rigid framework for zinc binding. This is understood biologically, as a small distortion in the zinc position in an enzyme can have serious consequences on the enzymatic reaction. We also analyze the sequence pattern of the zinc ligands and residues that provide elecs, and identify conserved hydrophobic residues in the endopeptidases that also appear to contribute to stabilizing the catalytic zinc site. A zinc binding template in protein crystal structures is derived from these observations.     

Mutational and crystallographic analyses of the active site residues of the Bacillus circulans xylanase.

Using site-directed mutagenesis we have investigated the catalytic residues in a xylanase from Bacillus circulans. Analysis of the mutants E78D and E172D indicated that mutations in these conserved residues do not grossly alter the structure of the enzyme and that these residues participate in the catalytic mechanism. We have now determined the crystal structure of an enzyme-substrate complex to 108 A resolution using a catalytically incompetent mutant (E172C). In addition to the catalytic residues, Glu 78 and Glu 172, we have identified 2 tyrosine residues, Tyr 69 and Tyr 80, which likely function in substrate binding, and an arginine residue, Arg 112, which plays an important role in the active site of this enzyme. On the basis of our work we would propose that Glu 78 is the nucleophile and that Glu 172 is the acid-base catalyst in the reaction.     

木聚糖酶分子结构与重要酶学性质关系的研究进展

木聚糖是一种多聚五碳糖 ,是植物细胞中主要的半纤维素成分。木聚糖酶是可将木聚糖降解成低聚木糖和木糖的水解酶 ,它在饲料、造纸、食品、能源工业和环境科学上有着广阔的应用前景。随着分子生物学、结构生物学的发展及蛋白质工程的应用 ,对木聚糖酶结构和功能的研究不断深入。这里重点阐述与酶的活性、热稳定性、作用pH、等电点、底物亲和性及催化效率等重要性质相关的分子结构研究进展 ,讨论了其进一步的研究发展方向。研究木聚糖酶结构与功能的关系 ,对进一步加深木聚糖酶作用机制的了解、指导木聚糖酶的分子改良有重要意义。     

Locating the active sites of enzymes using mechanical properties

We have applied the calculation of mechanical properties to a dataset of almost 100 enzymes to determine the extent to which catalytic residues have distinct properties. Specifically, we have calculated force constants describing the ease of moving any given amino acid residue with respect to the other residues in the protein. The results show that catalytic residues are invariably associated with high force constants. Choosing an appropriate cutoff enables the detection of roughly 80% of catalytic residues with only 25% of false positives. It is shown that neither multidomain structures, nor the presence or absence of bound ligands hinder successful detections. It is however noted that active sites near the protein surface are more difficult to detect and that non-catalytic, but structurally key residues may also exhibit high force constants.     

Substrate binding and catalytic mechanism in phospholipase C from Bacillus Cereus: A molecular mechanics and molecular dynamics study

For the first time a consistent catalytic mechanism of phospholipase C from Bacillus cereus is reported based on molecular mechanics calculations. We have identified the position of the nucleophilic water molecule, which is directly involved in the hydrolysis of the natural substrate, phosphatidylcholine, in phospholipase C. This catalytically essential water molecule, after being activated by an acidic residue (Asp55), performs the nucleophilic attack on the phosphorus atom in the substrate, leading to a trigonal bipyramidal pentacoordinated intermediate (and structurally similar transition state). The subsequent collapse of the intermediate, regeneration of the enzyme, and release of the products has to involve a not yet identified second water molecule. The catalytic mechanism reported here is based on a series of molecular mechanics calculations. First, the x-ray structure of phospholipase C from B. cereus including a docked substrate molecule was subjected to a stepwise molecular mechanics energy minimization. Second, the location of the nucleophilic water molecule in the active site of the fully relaxed enzyme–substrate complex was determined by evaluation of nonbonded interaction energies between the complex and a water molecule. The nucleophilic water molecule is positioned at a distance (3.8 Å) from the phosphorus atom in the substrate, which is in good agreement with experimentally observed distances. Finally, the stability of the complex between phospholipase C, the substrate, and the nucleophilic water molecule was verified during a 100 ps molecular dynamics simulation. During the simulation the substrate undergoes a conformational change, but retains its localization in the active site. The contacts between the enzyme, the substrate, and the nucleophilic water molecule display some fluctuations, but remain within reasonable limits, thereby confirming the stability of the enzyme–substrate–water complex. The protocol developed for energy minimization of phospholipase C containing three zinc ions located closely together at the bottom of the active site cleft is reported in detail. In order to handle the strong electrostatic interactions in the active site realistically during energy minimization, delocalization of the charges from the three zinc ions was considered. Therefore, quantum mechanics calculations on the zinc ions and the zinc-coordinating residues were carried out prior to the molecular mechanics calculations, and two different sets of partial atomic charges (MNDO-Mulliken and AM1-ESP) were applied. After careful assignment of partial atomic charges, a complete energy minimization of the protein was carried out by a stepwise procedure without explicit solvent molecules. Energy minimization with either set of charges yielded structures, which were very similar both to the x-ray structure and to each other, although using AM1-ESP partial atomic charges and a dielectric constant of 4, yielded the best protein structure. © 1997 John Wiley and Sons, Inc. Biopoly 42: 319–336, 1997     

A novel combination of two classic catalytic schemes

The crystal structure of an alkaline Bacillus cellulase catalytic core, from glucoside hydrolase family 5, reveals a novel combination of the catalytic machinery of two classic textbook enzymes. The enzyme has the expected two glutamate residues in close proximity to one another in the active-site that are typical of retaining cellulases. However, the proton donor, glutamate 139 is also unexpectedly a member of a serine-histidine-glutamate catalytic triad, forming a novel combination of catalytic machineries. Structure and sequence analysis of glucoside hydrolase family 5 reveal that the triad is highly conserved, but with variations at the equivalent of the serine position. We speculate that the purpose of this novel catalytic triad is to control the protonation of the acid/base glutamate, facilitating the first step of the catalytic reaction, protonation of the substrate, by the proton donor glutamate. If correct, this will be a novel use for a catalytic triad.     

The catalytic mechanism of indole-3-glycerol phosphate synthase: crystal structures of complexes of the enzyme from Sulfolobus solfataricus with substrate analogue,substrate, and product

Indoleglycerol phosphate synthase catalyzes the ring closure of an N-alkylated anthranilate to a 3-alkyl indole derivative, a reaction requiring Lewis acid catalysis in vitro. Here, we investigated the enzymatic reaction mechanism through X-ray crystallography of complexes of the hyperthermostable enzyme from Sulfolobus solfataricus with the substrate 1-(o-carboxyphenylamino) 1-deoxyribulose 5-phosphate, a substrate analogue and the product indole-3-glycerol phosphate. The substrate and the substrate analogue are bound to the active site in a similar, extended conformation between the previously identified phosphate binding site and a hydrophobic pocket for the anthranilate moiety. This binding mode is unproductive, because the carbon atoms that are to be joined are too far apart. The indole ring of the bound product resides in a second hydrophobic pocket adjacent to that of the anthranilate moiety of the substrate. Although the hydrophobic moiety of the substrate moves during catalysis from one hydrophobic pocket to the other, the triosephosphate moiety remains rigidly bound to the same set of hydrogen-bonding residues. Simultaneously, the catalytically important residues Lys53, Lys110 and Glu159 maintain favourable distances to the atoms of the ligand undergoing covalent changes. On the basis of these data, the structures of two putative catalytic intermediates were modelled into the active site. This new structural information and the modelling studies provide further insight into the mechanism of enzyme-catalyzed indole synthesis. The charged epsilon-amino group of Lys110 is the general acid, and the carboxylate group of Glu159 is the general base. Lys53 guides the substrate undergoing conformational transitions during catalysis, by forming a salt-bridge to the carboxylate group of its anthranilate moiety.     

Structural insights into the human and avian IMP cyclohydrolase mechanism via crystal structures with the bound XMP inhibitor

Within de novo purine biosynthesis, the AICAR transformylase and IMP cyclohydrolase activities of the bifunctional enzyme ATIC convert the intermediate AICAR to the final product of the pathway, IMP. Identification of the AICAR transformylase active site and a proposed formyl transfer mechanism have already resulted from analysis of crystal structures of avian ATIC in complex with substrate and/or inhibitors. Herein, we focus on the IMPCH active site and the cyclohydrolase mechanism through comparison of crystal structures of XMP inhibitor complexes of human ATIC at 1.9 A resolution with the previously determined avian enzyme. This first human ATIC structure was also determined to ascertain whether any subtle structural differences, compared to the homologous avian enzyme, should be taken into account for structure-based inhibitor design. These structural comparisons, as well as comparative analyses with other IMP and XMP binding proteins, have enabled a catalytic mechanism to be formulated. The primary role of the IMPCH active site appears to be to induce a reconfiguration of the substrate FAICAR to a less energetically favorable, but more reactive, conformer. Backbone (Arg64 and Lys66) and side chain interactions (Thr67) in the IMPCH active site reorient the 4-carboxamide from the preferred conformer that binds to the AICAR Tfase active site to one that promotes intramolecular cyclization. Other backbone amides (Ile126 and Gly127) create an oxyanion hole that helps orient the formyl group for nucleophilic attack by the 4-carboxamide amine and then stabilize the anionic intermediate. Several other residues, including Lys66, Tyr104, Asp125, and Lys137', provide substrate specificity and likely enhance the catalytic rate through contributions to acid-base catalysis.     

Homology modeling and function of trehalose synthase from Pseudomonas putida P06

Trehalose is a non-reducing disaccharide that has wide applications in the food industry and pharmaceutical manufacturing. Trehalose synthase (TreS) from Pseudomonas putida P06 catalyzes the reversible interconversion of maltose and trehalose and may have applications in the food industry. However, the catalytic mechanism of TreS is not well understood. Here, we investigated the structural characteristics of this enzyme by homology modeling. The highly conserved Asp294 residue was identified to be critical for catalytic activity. In addition, flexible docking studies of the enzyme–substrate system were performed to predict the interactions between TreS and its substrate, maltose. Amino acids that interact extensively with the substrate and stabilize the substrate in an orientation suitable for enzyme catalysis were identified. The importance of these residues for catalytic activity was confirmed by the biochemical characterization of the relevant mutants generated by site-directed mutagenesis.     

Structural studies of Saccharomyces cerevesiae mitochondrial NADP-dependent isocitrate dehydrogenase in different enzymatic states reveal substantial conformational changes during the catalytic reaction

Isocitrate dehydrogenases (IDHs) catalyze oxidative decarboxylation of isocitrate (ICT) into alpha-ketoglutarate (AKG). We report here the crystal structures of Saccharomyces cerevesiae mitochondrial NADP-IDH Idp1p in binary complexes with coenzyme NADP, or substrate ICT, or product AKG, and in a quaternary complex with NADPH, AKG, and Ca(2+), which represent different enzymatic states during the catalytic reaction. Analyses of these structures identify key residues involved in the binding of these ligands. Comparisons among these structures and with the previously reported structures of other NADP-IDHs reveal that eukaryotic NADP-IDHs undergo substantial conformational changes during the catalytic reaction. Binding or release of the ligands can cause significant conformational changes of the structural elements composing the active site, leading to rotation of the large domain relative to the small and clasp domains along two hinge regions (residues 118-124 and residues 284-287) while maintaining the integrity of its secondary structural elements, and thus, formation of at least three distinct overall conformations. Specifically, the enzyme adopts an open conformation when bound to NADP, a quasi-closed conformation when bound to ICT or AKG, and a fully closed conformation when bound to NADP, ICT, and Ca(2+) in the pseudo-Michaelis complex or with NADPH, AKG, and Ca(2+) in the product state. The conformational changes of eukaryotic NADP-IDHs are quite different from those of Escherichia coli NADP-IDH, for which significant conformational changes are observed only between two forms of the apo enzyme, suggesting that the catalytic mechanism of eukaryotic NADP-IDHs is more complex than that of EcIDH, and involves more fine-tuned conformational changes.     

High-resolution crystal structure of the Fab-fragments of a family of mouse catalytic antibodies with esterase activity

The crystal structures of four related Fab fragments of a family of catalytic antibodies displaying differential levels of esterase activity have been solved in the presence and in the absence of the transition-state analogue (TSA) that was used to elicit the immune response. The electron density maps show that the TSA conformation is essentially identical, with limited changes on hapten binding. Interactions with the TSA explain the specificity for the D rather than the L-isomer of the substrate. Differences in the residues in the hapten-binding pocket, which increase hydrophobicity, appear to correlate with an increase in the affinity of the antibodies for their substrate. Analysis of the structures at the active site reveals a network of conserved hydrogen bond contacts between the TSA and the antibodies, and points to a critical role of two conserved residues, HisL91 and LysH95, in catalysis. However, these two key residues are set into very different contexts in their respective structures, with an apparent direct correlation between the catalytic power of the antibodies and the complexity of their interactions with the rest of the protein. This suggests that the catalytic efficiency may be controlled by contacts arising from a second sphere of residues at the periphery of the active site.     

The crystal structure of the ternary complex of staphylococcal nuclease, Ca2+, and the inhibitor pdTp, refined at 1.65 A

The structure of a complex of staphylococcal nuclease with Ca2+ and deoxythymidine 3',5'-bisphosphate (pdTp) has been refined by stereochemically restrained least-squares minimization to a crystallographic R value of 0.161 at 1.65 A resolution. The estimated root-mean-square (rms) error in the coordinates is 0.16 A. The final model comprises 1082 protein atoms, one calcium ion, the pdTp molecule, and 82 solvent water molecules; it displays an rms deviation from ideality of 0.017 A for bond distances and 1.8 degrees for bond angles. The mean distance between corresponding alpha carbons in the refined and unrefined structures is 0.6 A; we observe small but significant differences between the refined and unrefined models in the turn between residues 27 and 30, the loop between residues 44 and 50, the first helix, and the extended strand between residues 112 and 117 which forms part of the active site binding pocket. The details of the calcium liganding and solvent structure in the active site are clearly shown in the final electron density map. The structure of the catalytic site is consistent with the mechanism that has been proposed for this enzyme. However, we note that two lysines from a symmetry-related molecule in the crystal lattice may play an important role in determining the geometry of inhibitor binding, and that only one of the two required calcium ions is observed in the crystal structure; thus, caution is advised in extrapolating from the structure of the complex of enzyme and inhibitor to that of enzyme and substrate.     

Evolutionary variation and adaptation in a conserved protein kinase allosteric network: Implications for inhibitor design

The activation of protein kinases involves conformational changes in key functional regions of the kinase domain, a detailed understanding of which is essential for the design of selective protein kinase inhibitors. Through statistical analysis of protein kinase sequences and crystal structures from diverse organisms, we recently proposed that the activation of protein kinases involves a hidden strain switch in the catalytic loop. Specifically, we demonstrated that the backbone torsion-angles of residues in the catalytic loop switch from a “relaxed” to “strained” conformation upon kinase activation and the strained geometry results in a network of hydrogen bonds involving conserved non-catalytic residues in the ATP and substrate binding lobes. Here, we further explore this activation mechanism by analyzing families that lack the canonical hydrogen bonding interactions with the strained backbone. We find that alternative mechanisms have evolved to maintain catalytic loop strain. In PIM kinase, for example, two water molecules account for the lack of a conserved aspartate in the substrate binding by hydrogen bonds to the strained backbone. We discuss the relevance of these findings in the design of family-specific allosteric inhibitors, and in predicting the structural and functional impact of cancer mutations that alter the strain associated hydrogen bonding network. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).     

X-ray structure of papaya chitinase reveals the substrate binding mode of glycosyl hydrolase family 19 chitinases

The crystal structure of a chitinase from Carica papaya has been solved by the molecular replacement method and is reported to a resolution of 1.5 A. This enzyme belongs to family 19 of the glycosyl hydrolases. Crystals have been obtained in the presence of N-acetyl- d-glucosamine (GlcNAc) in the crystallization solution and two well-defined GlcNAc molecules have been identified in the catalytic cleft of the enzyme, at subsites -2 and +1. These GlcNAc moieties bind to the protein via an extensive network of interactions which also involves many hydrogen bonds mediated by water molecules, underlying their role in the catalytic mechanism. A complex of the enzyme with a tetra-GlcNAc molecule has been elaborated, using the experimental interactions observed for the bound GlcNAc saccharides. This model allows to define four major substrate interacting regions in the enzyme, comprising residues located around the catalytic Glu67 (His66 and Thr69), the short segment E89-R90 containing the second catalytic residue Glu89, the region 120-124 (residues Ser120, Trp121, Tyr123, and Asn124), and the alpha-helical segment 198-202 (residues Ile198, Asn199, Gly201, and Leu202). Water molecules from the crystal structure were introduced during the modeling procedure, allowing to pinpoint several additional residues involved in ligand binding that were not previously reported in studies of poly-GlcNAc/family 19 chitinase complexes. This work underlines the role played by water-mediated hydrogen bonding in substrate binding as well as in the catalytic mechanism of the GH family 19 chitinases. Finally, a new sequence motif for family 19 chitinases has been identified between residues Tyr111 and Tyr125.     

Model for Substrate Interactions in C5a Peptidase from Streptococcus pyogenes: A 1.9 Å Crystal Structure of the Active Form of ScpA

The crystal structure of an active form of ScpA has been solved to 1.9 Å resolution. ScpA is a multidomain cell-envelope subtilase from Streptococcus pyogenes that cleaves complement component C5a. The catalytic triad of ScpA is geometrically consistent with other subtilases, clearly demonstrating that the additional activation mechanism proposed for the Streptococcus agalactiae homologue (ScpB) is not required for ScpA. The ScpA structure revealed that access to the catalytic site is restricted by variable regions in the catalytic domain (vr7, vr9, and vr11) and by the presence of the inserted protease-associated (PA) domain and the second fibronectin type III domains (Fn2). Modeling of the ScpA-C5a complex indicates that the substrate binds with carboxyl-terminal residues (65-74) extended through the active site and core residues (1-64) forming exosite-type interactions with the Fn2 domain. This is reminiscent of the two-site mechanism proposed for C5a binding to its receptor. In the nonprime region of the active site, interactions with the substrate backbone are predicted to be more similar to those observed in kexins, involving a single β-strand in the peptidase. However, in contrast to kexins, there would be diminished emphasis on side-chain interactions, with little charged character in the S3-S1 and S6-S4 subsites occupied by the side chains of residues in vr7 and vr9. Substrate binding is anticipated to be dominated by ionic interactions in two distinct regions of ScpA. On the prime side of the active site, salt bridges are predicted between P1′, P2′, and P7′ residues, and residues in the catalytic and PA domains. Remote to the active site, a larger number of ionic interactions between residues in the C5a core and the Fn2 domain are observed in the model. Thus, both PA and Fn2 domains are expected to play significant roles in substrate recognition.     

X-ray structure of the R69D phosphatidylinositol-specific phospholipase C enzyme: insight into the role of calcium and surrounding amino acids in active site geometry and catalysis

Phosphatidylinositol-specific phospholipase Cs (PLCs) are a family of phosphodiesterases that catalyze the cleavage of the P-O bond via transesterification using the internal hydroxyl group of the substrate as a nucleophile, generating the five-membered cyclic inositol phosphate as an intermediate or product. To better understand the role of calcium in the catalytic mechanism of PLCs, we have determined the X-ray crystal structure of an engineered PLC enzyme from Bacillus thuringiensis to 2.1 A resolution. The active site of this enzyme has been altered by substituting the catalytic arginine with an aspartate at position 69 (R69D). This single-amino acid substitution converted a metal-independent, low-molecular weight enzyme into a metal ion-dependent bacterial PLC with an active site architecture similar to that of the larger metal ion-dependent mammalian PLC. The Ca(2+) ion shows a distorted square planar geometry in the active site that allows for efficient substrate binding and transition state stabilization during catalysis. Additional changes in the positions of the catalytic general acid/general base (GA/GB) were also observed, indicating the interrelation of the intricate hydrogen bonding network involved in stabilizing the active site amino acids. The functional information provided by this X-ray structure now allows for a better understanding of the catalytic mechanism, including stereochemical effects and substrate interactions, which facilitates better inhibitor design and sheds light on the possibilities of understanding how protein evolution might have occurred across this enzyme family.     

Crystal structure of RNase T, an exoribonuclease involved in tRNA maturation and end turnover

The 3' processing of most bacterial precursor tRNAs involves exonucleolytic trimming to yield a mature CCA end. This step is carried out by RNase T, a member of the large DEDD family of exonucleases. We report the crystal structures of RNase T from Escherichia coli and Pseudomonas aeruginosa, which show that this enzyme adopts an opposing dimeric arrangement, with the catalytic DEDD residues from one monomer closely juxtaposed with a large basic patch on the other monomer. This arrangement suggests that RNase T has to be dimeric for substrate specificity, and agrees very well with prior site-directed mutagenesis studies. The dimeric architecture of RNase T is very similar to the arrangement seen in oligoribonuclease, another bacterial DEDD family exoribonuclease. The catalytic residues in these two enzymes are organized very similarly to the catalytic domain of the third DEDD family exoribonuclease in E. coli, RNase D, which is monomeric.     

Investigations of the active site of Saccharomyces cerevisiae dolichyl-phosphate-mannose synthase using fluorescent labeled dolichyl-phosphate derivatives

Dolichol-phosphate mannose (Dol-P-Man) is a key mannosyl donor for the biosynthesis of N-linked oligosaccharides as well as for O-linked oligosaccharides on yeast glycoproteins, and for the synthesis of the glycosyl-phosphatidylinositol anchor found on many cell surface glycoproteins. It is synthesized by Dol-P-Man synthase which is the only glycosyltransferase in the dolichol pathway that has been expressed as an active protein, solubilized and purified in large enough quantities for structural investigations. Earlier studies showed that the enzyme is closely associated with membranes of endoplasmic reticulum with unique lipid requirements for its maximal activity. This potential target of antibiotic therapy is now being investigated at the molecular level to establish information about the structure of the enzyme as well as determine the nature and properties of the enzyme-phospholipid interactions. In this paper, we have determined the activities of the fluorescent labeled dolichyl-phosphate derivatives as well as the intramolecular distances between amino acid residues near the active site and/or the fluorophores of the substrate derivatives using fluorescence energy resonance transfer. These results also show that the conserved consensus sequence is not required by Dol-P-Man synthase neither for the recognition of Dol-P nor for the catalytic activity.     

Role of protein conformational dynamics in the catalysis by 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase

Enzymatic catalysis has conflicting structural requirements of the enzyme. In order for the enzyme to form a Michaelis complex, the enzyme must be in an open conformation so that the substrate can get into its active center. On the other hand, in order to maximize the stabilization of the transition state of the enzymatic reaction, the enzyme must be in a closed conformation to maximize its interactions with the transition state. The conflicting structural requirements can be resolved by a flexible active center that can sample both open and closed conformational states. For a bisubstrate enzyme, the Michaelis complex consists of two substrates in addition to the enzyme. The enzyme must remain flexible upon the binding of the first substrate so that the second substrate can get into the active center. The active center is fully assembled and stabilized only when both substrates bind to the enzyme. However, the side-chain positions of the catalytic residues in the Michaelis complex are still not optimally aligned for the stabilization of the transition state, which lasts only approximately 10(-13) s. The instantaneous and optimal alignment of catalytic groups for the transition state stabilization requires a dynamic enzyme, not an enzyme which undergoes a large scale of movements but an enzyme which permits at least a small scale of adjustment of catalytic group positions. This review will summarize the structure, catalytic mechanism, and dynamic properties of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase and examine the role of protein conformational dynamics in the catalysis of a bisubstrate enzymatic reaction.