Protein clefts in molecular recognition and function.

One of the primary factors determining how proteins interact with other molecules is the size of clefts in the protein's surface. In enzymes, for example, the active site is often characterized by a particularly large and deep cleft, while interactions between the molecules of a protein dimer tend to involve approximately planar surfaces. Here we present an analysis of how cleft volumes in proteins relate to their molecular interactions and functions. Three separate datasets are used, representing enzyme-ligand binding, protein-protein dimerization and antibody-antigen complexes. We find that, in single-chain enzymes, the ligand is bound in the largest cleft in over 83% of the proteins. Usually the largest cleft is considerably larger than the others, suggesting that size is a functional requirement. Thus, in many cases, the likely active sites of an enzyme can be identified using purely geometrical criteria alone. In other cases, where there is no predominantly large cleft, chemical interactions are required for pinpointing the correct location. In antibody-antigen interactions the antibody usually presents a large cleft for antigen binding. In contrast, protein-protein interactions in homodimers are characterized by approximately planar interfaces with several clefts involved. However, the largest cleft in each subunit still tends to be involved.     

Characterization of Human gamma-glutamyl hydrolase in solution demonstrates that the enzyme is a non-dissociating homodimer

Human gamma-glutamyl hydrolase (hGH) is a key enzyme in the metabolism of folic acid and in the pharmacology of many antifolate drugs. hGH catalyzes removal of the poly-gamma-glutamate chains of intracellular folic acid and antifolates. hGH crystallized as a homodimer with two putative active sites. However, the quaternary structure and the number of species of the enzyme in solution have not been determined. hGH has now been characterized using analytical ultracentrifugation and dynamic light scattering. HisTag fusion proteins of wild-type hGH, rat GH, and hGH expressed as a glycosylated protein were studied. Analyses of HisTag wild-type hGH were conducted over a range of protein concentrations (1.4-200 microM), ionic strengths (0-1 M NaCl), and pH (4.5-8.5). A single species with a molecular mass consistent with a homodimer was observed. Glycosylated hGH and HisTag rat gamma-glutamyl hydrolase also formed very stable homodimers. The lack of dissociation of the dimer, the large monomer-monomer interface, and the presence of catalytically essential Tyr-36 in the homodimer interface sequences suggest that homodimer formation is required for the hGH monomer to fold into an active conformation. The conservation of hGH monomer-monomer interface sequences in other mammalian and plant gamma-glutamyl hydrolase molecules suggests that they also exist as stable homodimers.     

Rescue of the orphan enzyme isoguanine deaminase

Cytosine deaminase (CDA) from Escherichia coli was shown to catalyze the deamination of isoguanine (2-oxoadenine) to xanthine. Isoguanine is an oxidation product of adenine in DNA that is mutagenic to the cell. The isoguanine deaminase activity in E. coli was partially purified by ammonium sulfate fractionation, gel filtration, and anion exchange chromatography. The active protein was identified by peptide mass fingerprint analysis as cytosine deaminase. The kinetic constants for the deamination of isoguanine at pH 7.7 are as follows: k(cat) = 49 s(-1), K(m) = 72 μM, and k(cat)/K(m) = 6.7 × 10(5) M(-1) s(-1). The kinetic constants for the deamination of cytosine are as follows: k(cat) = 45 s(-1), K(m) = 302 μM, and k(cat)/K(m) = 1.5 × 10(5) M(-1) s(-1). Under these reaction conditions, isoguanine is the better substrate for cytosine deaminase. The three-dimensional structure of CDA was determined with isoguanine in the active site.     

A switch in the mechanism of communication between the two DNA-binding sites in the SfiI restriction endonuclease

While many Type II restriction enzymes are dimers with a single DNA-binding cleft between the subunits, SfiI is a tetramer of identical subunits. Two of its subunits (a dimeric unit) create one DNA-binding cleft, and the other two create a second cleft on the opposite side of the protein. The two clefts bind specific DNA cooperatively to give a complex of SfiI with two recognition sites. This complex is responsible for essentially all of the DNA-cleavage reactions by SfiI: virtually none is due to the complex with one site. The communication between the DNA-binding clefts was examined by disrupting one of the very few polar interactions in the otherwise hydrophobic interface between the dimeric units: a tyrosine hydroxyl was removed by mutation to phenylalanine. The mutant protein remained tetrameric in solution and could bind two DNA sites. But instead of being activated by binding two sites, like wild-type SfiI, it showed maximal activity when bound to a single site and had a lower activity when bound to two sites. This interaction across the dimer interface thus enforces in wild-type SfiI a cooperative transition between inactive and active states in both dimers, but without this interaction as in the mutant protein, a single dimer can undergo the transition to give a stable intermediate with one inactive dimer and one active dimer.     

A rat monoclonal antibody that catalyses the hydrolysis of a nitrophenyl-beta-lactam

We report the first example of a monoclonal antibody-catalysed hydrolysis of a beta-lactam where the antibodies were generated by a simple transition-state analogue. A rat monoclonal antibody (1/91c/4d/26) generated by using an acyclic 4-nitrophenylphosphate immunogen catalysed the hydrolysis of corresponding 4-nitrophenyl carbonates but, more importantly, also catalysed the hydrolysis of N-(4-nitrophenyl)-azetidinone at pH 8 with k(cat)=8.7 x 10(-6)s(-1) and K(M)=35 microM. This is the first example of a rat monoclonal catalytic antibody.     

Structure of the Q237W mutant of HhaI DNA methyltransferase: an insight into protein-protein interactions

We have determined the structure of a mutant (Q237W) of HhaI DNA methyltransferase, complexed with the methyl-donor product AdoHcy. The Q237W mutant proteins were crystallized in the monoclinic space group C2 with two molecules in the crystallographic asymmetric unit. Protein-protein interface calculations in the crystal lattices suggest that the dimer interface has the specific characteristics for homodimer protein-protein interactions, while the two active sites are spatially independent on the outer surface of the dimer. The solution behavior suggests the formation of HhaI dimers as well. The same HhaI dimer interface is also observed in the previously characterized binary (M.HhaI-AdoMet) and ternary (M.HhaI-DNA-AdoHcy) complex structures, crystallized in different space groups. The dimer is characterized either by a non-crystallographic two-fold symmetry or a crystallographic symmetry. The dimer interface involves three segments: the amino-terminal residues 2-8, the carboxy-terminal residues 313-327, and the linker (amino acids 179-184) between the two functional domains--the catalytic methylation domain and the DNA target recognition domain. Both the amino- and carboxy-terminal segments are part of the methylation domain. We also examined protein-protein interactions of other structurally characterized DNA MTases, which are often found as a 2-fold related 'dimer' with the largest dimer interface area for the group-beta MTases. A possible evolutionary link between the Type I and Type II restriction-modification systems is discussed.     

The family 6 carbohydrate binding module CmCBM6-2 contains two ligand-binding sites with distinct specificities

The microbial degradation of the plant cell wall is an important biological process, representing a major component of the carbon cycle. Enzymes that mediate the hydrolysis of this composite structure are modular proteins that contain non-catalytic carbohydrate binding modules (CBMs) that enhance catalytic activity. CBMs are grouped into sequence-based families, and in a previous study we showed that a family 6 CBM (CBM6) that interacts with xylan contains two potential ligand binding clefts, designated cleft A and cleft B. Mutagenesis and NMR studies showed that only cleft A in this protein binds to xylan. Family 6 CBMs bind to a range of polysaccharides, and it was proposed that the variation in ligand specificity observed in these proteins reflects the specific cleft that interacts with the target carbohydrate. Here the biochemical properties of the C-terminal cellulose binding CBM6 (CmCBM6-2) from Cellvibrio mixtus endoglucanase 5A were investigated. The CBM binds to the beta1,4-beta1,3-mixed linked glucans lichenan and barley beta-glucan, cello-oligosaccharides, insoluble forms of cellulose, the beta1,3-glucan laminarin, and xylooligosaccharides. Mutagenesis studies, informed by the crystal structure of the protein (presented in the accompanying paper, Pires, V. M. R., Henshaw, J. L., Prates, J. A. M., Bolam, D., Ferreira, L. M. A. Fontes, C. M. G. A., Henrissat, B., Planas, A., Gilbert, H. J., Czjzek, M. (2004) J. Biol. Chem. 279, 21560-21568), show that both cleft A and B can accommodate cello-oligosaccharides and laminarin displays a preference for cleft A, whereas xylooligosaccharides exhibit absolute specificity for this site, and the beta1,4,-beta1,3-mixed linked glucans interact only with cleft B. The binding of CmCBM6-2 to insoluble cellulose involves synergistic interactions between cleft A and cleft B. These data show that CmCBM6-2 contains two binding sites that display differences in ligand specificity, supporting the view that distinct binding clefts with different specificities can contribute to the variation in ligand recognition displayed by family 6 CBMs. This is in sharp contrast to other CBM families, where variation in ligand binding is a result of changes in the topology of a single carbohydrate-binding site.     

High level production of the Magnaporthe grisea fructose 1,6-bisphosphate aldolase enzyme in Escherichia coli using a small volume bench-top fermentor

The Class II fructose 1,6-bisphosphate aldolase from the Rice Blast causative agent Magnaporthe grisea was subcloned in the Escherichia coli vector pT7-7. The enzyme was overexpressed using fed-batch fermentation in a small bench-top reactor. A total of 275 g of cells and 1.3 g of highly purified enzyme with a specific activity of 70 U/mg were obtained from a 1.5L culture. The purified enzyme is a homodimer of 39.6 kDa subunits with a zinc ion at the active site. Kinetic characterization indicates that the enzyme has a K(m) of 51 microM, a k(cat) of 46 s(-1), and a pH optimum of 7.8 for fructose 1,6-bisphosphate cleavage. The fermentation system procedure reported exemplifies the potential of using a lab-scale bioreactor for the large scale production of recombinant enzymes.     

A comparative biochemical and structural analysis of the intracellular chorismate mutase (Rv0948c) from Mycobacterium tuberculosis H(37)R(v) and the secreted chorismate mutase (y2828) from Yersinia pestis

The Rv0948c gene from Mycobacterium tuberculosis H(37)R(v) encodes a 90 amino acid protein as the natural gene product with chorismate mutase (CM) activity. The protein, 90-MtCM, exhibits Michaelis-Menten kinetics with a k(cat) of 5.5+/-0.2s(-1) and a K(m) of 1500+/-100microm at 37 degrees C and pH7.5. The 2.0A X-ray structure shows that 90-MtCM is an all alpha-helical homodimer (Protein Data Bank ID: 2QBV) with the topology of Escherichia coli CM (EcCM), and that both protomers contribute to each catalytic site. Superimposition onto the structure of EcCM and the sequence alignment shows that the C-terminus helix3 is shortened. The absence of two residues in the active site of 90-MtCM corresponding to Ser84 and Gln88 of EcCM appears to be one reason for the low k(cat). Hence, 90-MtCM belongs to a subfamily of alpha-helical AroQ CMs termed AroQ(delta.) The CM gene (y2828) from Yersinia pestis encodes a 186 amino acid protein with an N-terminal signal peptide that directs the protein to the periplasm. The mature protein, *YpCM, exhibits Michaelis-Menten kinetics with a k(cat) of 70+/-5s(-1) and K(m) of 500+/-50microm at 37 degrees C and pH7.5. The 2.1A X-ray structure shows that *YpCM is an all alpha-helical protein, and functions as a homodimer, and that each protomer has an independent catalytic unit (Protein Data Bank ID: 2GBB). *YpCM belongs to the AroQ(gamma) class of CMs, and is similar to the secreted CM (Rv1885c, *MtCM) from M.tuberculosis.     

Residue centrality, functionally important residues, and active site shape: analysis of enzyme and non-enzyme families

The representation of protein structures as small-world networks facilitates the search for topological determinants, which may relate to functionally important residues. Here, we aimed to investigate the performance of residue centrality, viewed as a family fold characteristic, in identifying functionally important residues in protein families. Our study is based on 46 families, including 29 enzyme and 17 non-enzyme families. A total of 80% of these central positions corresponded to active site residues or residues in direct contact with these sites. For enzyme families, this percentage increased to 91%, while for non-enzyme families the percentage decreased substantially to 48%. A total of 70% of these central positions are located in catalytic sites in the enzyme families, 64% are in hetero-atom binding sites in those families binding hetero-atoms, and only 16% belong to protein-protein interfaces in families with protein-protein interaction data. These differences reflect the active site shape: enzyme active sites locate in surface clefts, hetero-atom binding residues are in deep cavities, while protein-protein interactions involve a more planar configuration. On the other hand, not all surface cavities or clefts are comprised of central residues. Thus, closeness centrality identifies functionally important residues in enzymes. While here we focus on binding sites, we expect to identify key residues for the integration and transmission of the information to the rest of the protein, reflecting the relationship between fold and function. Residue centrality is more conserved than the protein sequence, emphasizing the robustness of protein structures.     

Protein-protein interfaces: analysis of amino acid conservation in homodimers

Evolutionary information derived from the large number of available protein sequences and structures could powerfully guide both analysis and prediction of protein-protein interfaces. To test the relevance of this information, we assess the conservation of residues at protein-protein interfaces compared with other residues on the protein surface. Six homodimer families are analyzed: alkaline phosphatase, enolase, glutathione S-transferase, copper-zinc superoxide dismutase, Streptomyces subtilisin inhibitor, and triose phosphate isomerase. For each family, random simulation is used to calculate the probability (P value) that the level of conservation observed at the interface occurred by chance. The results show that interface conservation is higher than expected by chance and usually statistically significant at the 5% level or better. The effect on the P values of using different definitions of the interface and of excluding active site residues is discussed.     

The crystal structure of an eukaryotic iron superoxide dismutase suggests intersubunit cooperation during catalysis

Superoxide dismutases (SODs) are a family of metalloenzymes that catalyze the dismutation of superoxide anion radicals into molecular oxygen and hydrogen peroxide. Iron superoxide dismutases (FeSODs) are only expressed in some prokaryotes and plants. A new and highly active FeSOD with an unusual subcellular localization has recently been isolated from the plant Vigna unguiculata (cowpea). This protein functions as a homodimer and, in contrast to the other members of the SOD family, is localized to the cytosol. The crystal structure of the recombinant enzyme has been solved and the model refined to 1.97 A resolution. The superoxide anion binding site is located in a cleft close to the dimer interface. The coordination geometry of the Fe site is a distorted trigonal bipyramidal arrangement, whose axial ligands are His43 and a solvent molecule, and whose in-plane ligands are His95, Asp195, and His199. A comparison of the structural features of cowpea FeSOD with those of homologous SODs reveals subtle differences in regard to the metal-protein interactions, and confirms the existence of two regions that may control the traffic of substrate and product: one located near the Fe binding site, and another in the dimer interface. The evolutionary conservation of reciprocal interactions of both monomers in neighboring active sites suggests possible subunit cooperation during catalysis.     

Amino acid discrimination by a highly differentiated metal center of an aminoacyl-tRNA synthetase

Escherichia coli cysteinyl-tRNA synthetase (CysRS) achieves high amino acid specificity without the need for an editing reaction. Crystallographic and spectroscopic studies have previously demonstrated that a major determinant of the specificity is an active site zinc ion that recognizes the substrate cysteine through a strong zinc-thiolate interaction. The active site cleft of CysRS is composed of highly or strictly conserved amino acids, including four inner-sphere zinc ligands, five histidine imidazoles at the base of the cleft, and a tryptophan that flips down upon cysteine binding to complete formation of the binding pocket. Here we establish the significance of each of these major features of the active site cleft by mutational analysis. Substitutions generally lead to substantially deleterious effects on K(m) and k(cat) parameters with respect to each of the cysteine, ATP, and tRNA(Cys) substrates. These findings emphasize the importance of the highly differentiated nature of the active site and provide new insights into the origins of selectivity without editing. Most mutants are less attenuated in tRNA aminoacylation than in adenylate synthesis, suggesting that tRNA binding drives a conformational change to help assemble the active site.     

Enzyme/non-enzyme discrimination and prediction of enzyme active site location using charge-based methods

Calculations of charge interactions complement analysis of a characterised active site, rationalising pH-dependence of activity and transition state stabilisation. Prediction of active site location through large DeltapK(a)s or electrostatic strain is relevant for structural genomics. We report a study of ionisable groups in a set of 20 enzymes, finding that false positives obscure predictive potential. In a larger set of 156 enzymes, peaks in solvent-space electrostatic properties are calculated. Both electric field and potential match well to active site location. The best correlation is found with electrostatic potential calculated from uniform charge density over enzyme volume, rather than from assignment of a standard atom-specific charge set. Studying a shell around each molecule, for 77% of enzymes the potential peak is within that 5% of the shell closest to the active site centre, and 86% within 10%. Active site identification by largest cleft, also with projection onto a shell, gives 58% of enzymes for which the centre of the largest cleft lies within 5% of the active site, and 70% within 10%. Dielectric boundary conditions emphasise clefts in the uniform charge density method, which is suited to recognition of binding pockets embedded within larger clefts. The variation of peak potential with distance from active site, and comparison between enzyme and non-enzyme sets, gives an optimal threshold distinguishing enzyme from non-enzyme. We find that 87% of the enzyme set exceeds the threshold as compared to 29% of the non-enzyme set. Enzyme/non-enzyme homologues, "structural genomics" annotated proteins and catalytic/non-catalytic RNAs are studied in this context.     

Histidine triad-like motif of the rotavirus NSP2 octamer mediates both RTPase and NTPase activities

Rotavirus NSP2 is an abundant non-structural RNA-binding protein essential for forming the viral factories that support replication of the double-stranded RNA genome. NSP2 exists as stable doughnut-shaped octamers within the infected cell, representing the tail-to-tail interaction of two tetramers. Extending diagonally across the surface of each octamer are four highly basic grooves that function as binding sites for single-stranded RNA. Between the N and C-terminal domains of each monomer is a deep electropositive cleft containing a catalytic site that hydrolyzes the gamma-beta phosphoanhydride bond of any NTP. The catalytic site has similarity to those of the histidine triad (HIT) family of nucleotide-binding proteins. Due to the close proximity of the grooves and clefts, we investigated the possibility that the RNA-binding activity of the groove promoted the insertion of the 5'-triphosphate moiety of the RNA into the cleft, and the subsequent hydrolysis of its gamma-beta phosphoanhydride bond. Our results show that NSP2 hydrolyzes the gammaP from RNAs and NTPs through Mg(2+)-dependent activities that proceed with similar reaction velocities, that require the catalytic His225 residue, and that produce a phosphorylated intermediate. Competition assays indicate that although both substrates enter the active site, RNA is the preferred substrate due to its higher affinity for the octamer. The RNA triphosphatase (RTPase) activity of NSP2 may account for the absence of the 5'-terminal gammaP on the (-) strands of the double-stranded RNA genome segments. This is the first report of a HIT-like protein with a multifunctional catalytic site, capable of accommodating both NTPs and RNAs during gammaP hydrolysis.     

Familial mutations and zinc stoichiometry determine the rate-limiting step of nitrocefin hydrolysis by metallo-beta-lactamase from Bacteroides fragilis

The diverse members of the metallo-beta-lactamase family are a growing clinical threat evolving under considerable selective pressure. The enzyme from Bacillus cereus differs from the Bacteroides fragilis enzyme in sequence, zinc stoichiometry, and mechanism. To chart the evolution of the more reactive B. fragilis enzyme, we have made changes in an active site cysteine residue as well as in zinc content to mimic that which occurs in the B. cereus enzyme. Specifically, by introducing a C104R mutation into the B. fragilis enzyme, binding of two zinc ions is maintained, but the k(cat) value for nitrocefin hydrolysis is decreased from 226 to 14 s(-)(1). Removal of 1 equiv of zinc from this mutant further decreases k(cat) to 4.4 s(-)(1). In both cases, the observed k(cat) closely approximates that found in the di- and monozinc forms of the B. cereus enzyme (12 and 6 s(-)(1), respectively). Pre-steady-state stopped-flow studies using nitrocefin as a substrate indicate that these enzyme forms share a similar mechanism featuring an anionic intermediate but that the rate-limiting step changes from protonation of that species to the C-N bond cleavage leading to the intermediate. Overall, features that contribute 3.7 kcal/mol toward the acceleration of the C-N bond cleavage step have been uncovered although some of the total acceleration is masked in the steady-state by a change in rate-limiting step. These experiments illustrate one step in the evolution of a catalytic mechanism and, in a larger perspective, one step in the evolution of antibiotic resistance mechanisms.     

Essential roles of zinc ligation and enzyme dimerization for catalysis in the aminoacylase-1/M20 family

Members of the aminoacylase-1 (Acy1)/M20 family of aminoacylases and exopeptidases exist as either monomers or homodimers. They contain a zinc-binding domain and a second domain mediating dimerization in the latter case. The roles that both domains play in catalysis have been investigated for human Acy1 (hAcy1) by x-ray crystallography and by site-directed mutagenesis. Structure comparison of the dinuclear zinc center in a mutant of hAcy1 reported here with dizinc centers in related enzymes points to a difference in zinc ligation in the Acy1/M20 family. Mutational analysis supports catalytic roles of zinc ions, a vicinal glutamate, and a histidine from the dimerization domain. By complementing different active site mutants of hAcy1, we show that catalysis occurs at the dimer interface. Reinterpretation of the structure of a monomeric homolog, peptidase V, reveals that a domain insertion mimics dimerization. We conclude that monomeric and dimeric Acy1/M20 family members share a unique active site architecture involving both enzyme domains. The study may provide means to improve homologous carboxypeptidase G2 toward application in antibody-directed enzyme prodrug therapy.     

A new method for computing the macromolecular electric potential

A general methodology is developed for the rigorous computation of the electrostatic potential for a protein of arbitrary shape, assuming the presence of linear dielectric media. The theory proceeds by considering the distribution of induced polarization charge at the dielectric interface, rather than by attempting a direct solution of Poisson's equation (as in the finite-difference approach of Warwicker & Watson). The method is applied to a study of two-dimensional model proteins, where it is shown that the presence of a cleft is associated with a region of relatively high potential in the solvent medium. The results of a preliminary calculation in three dimensions for the protein lysozyme are also discussed; again, a region of enhanced potential is observed near the cleft at the active site. Our computational evidence supports the suggestion of Warwicker & Watson that clefts are associated with important electrostatic effects.     

Corynebacterium glutamicum survives arsenic stress with arsenate reductases coupled to two distinct redox mechanisms

Arsenate reductases (ArsCs) evolved independently as a defence mechanism against toxic arsenate. In the genome of Corynebacterium glutamicum, there are two arsenic resistance operons (ars1 and ars2) and four potential genes coding for arsenate reductases (Cg_ArsC1, Cg_ArsC2, Cg_ArsC1' and Cg_ArsC4). Using knockout mutants, in vitro reconstitution of redox pathways, arsenic measurements and enzyme kinetics, we show that a single organism has two different classes of arsenate reductases. Cg_ArsC1 and Cg_ArsC2 are single-cysteine monomeric enzymes coupled to the mycothiol/mycoredoxin redox pathway using a mycothiol transferase mechanism. In contrast, Cg_ArsC1' is a three-cysteine containing homodimer that uses a reduction mechanism linked to the thioredoxin pathway with a k(cat)/K(M) value which is 10(3) times higher than the one of Cg_ArsC1 or Cg_ArsC2. Cg_ArsC1' is constitutively expressed at low levels using its own promoter site. It reduces arsenate to arsenite that can then induce the expression of Cg_ArsC1 and Cg_ArsC2. We also solved the X-ray structures of Cg_ArsC1' and Cg_ArsC2. Both enzymes have a typical low-molecular-weight protein tyrosine phosphatases-I fold with a conserved oxyanion binding site. Moreover, Cg_ArsC1' is unique in bearing an N-terminal three-helical bundle that interacts with the active site of the other chain in the dimeric interface.