Diverse stability and catalytic properties of human tryptase alpha and beta isoforms are mediated by residue differences at the S1 pocket

Recombinant human tryptases (rHTs) corresponding to alpha and beta isoforms were characterized. rHTbeta was similar to tryptase isolated from skin (HST); it was a tetramer, hydrolyzed model substrates efficiently, and was functionally unstable when incubated under physiological conditions. Activity was lost rapidly (t(1/2) approximately 1 min) by a reversible process similar to that observed for the spontaneous inactivation of HST. Circular dichroism (CD) and intrinsic fluorescence emission (IFE) spectra of active rHTbeta corresponded to those of active HST and upon spontaneous inactivation IFE decreased in parallel to activity loss. rHTalpha differed from HST in catalytic ability and stability. rHTalpha did not react with model substrates, an active site titrant, or a competitive inhibitor of HST/rHTbeta. IFE and CD spectra were similar to those of the active and not the spontaneously inactivated form of HST. Under physiological conditions, rHTalpha IFE decreased at a rate 900-fold slower than that observed for HST, and rHTalpha remained tetrameric when examined by size exclusion chromatography at physiological salt concentration. Thus, rHTalpha is a stable "inactive" form of HT. Three active site variants of rHTalpha, K192Q, D216G, and K192Q-D216G were characterized. Residues 192 and 216 (chymotrypsinogen numbers for residues 191 and 215 of rHTalpha) lie at the entrance to the primary specificity (S1) pocket, and the mutations converted them to the residues of HTbeta. While K192Q displayed the same properties as rHTalpha, the catalytic and stability characteristics of D216G and K192Q-D216G progressively approached those of HST. Thus, the contrasting stability/activity properties of rHTalpha and rHTbeta are largely related to differences at the S1 pocket. On the basis of the properties of the variants, we suggest that the side chain of Asp216 is blocking and stabilizing the S1 pocket and that this stabilization is sufficient to prevent spontaneous inactivation.     

Structural analysis of papain-like NlpC/P60 superfamily enzymes with a circularly permuted topology reveals potential lipid binding sites

NlpC/P60 superfamily papain-like enzymes play important roles in all kingdoms of life. Two members of this superfamily, LRAT-like and YaeF/YiiX-like families, were predicted to contain a catalytic domain that is circularly permuted such that the catalytic cysteine is located near the C-terminus, instead of at the N-terminus. These permuted enzymes are widespread in virus, pathogenic bacteria, and eukaryotes. We determined the crystal structure of a member of the YaeF/YiiX-like family from Bacillus cereus in complex with lysine. The structure, which adopts a ligand-induced, "closed" conformation, confirms the circular permutation of catalytic residues. A comparative analysis of other related protein structures within the NlpC/P60 superfamily is presented. Permutated NlpC/P60 enzymes contain a similar conserved core and arrangement of catalytic residues, including a Cys/His-containing triad and an additional conserved tyrosine. More surprisingly, permuted enzymes have a hydrophobic S1 binding pocket that is distinct from previously characterized enzymes in the family, indicative of novel substrate specificity. Further analysis of a structural homolog, YiiX (PDB 2if6) identified a fatty acid in the conserved hydrophobic pocket, thus providing additional insights into possible function of these novel enzymes.     

Structure Motivator: A tool for exploring small three-dimensional elements in proteins

Backround

Aspartyl aminopeptidase (DNPEP), with specificity towards an acidic amino acid at the N-terminus, is the only mammalian member among the poorly understood M18 peptidases. DNPEP has implicated roles in protein and peptide metabolism, as well as the renin-angiotensin system in blood pressure regulation. Despite previous enzyme and substrate characterization, structural details of DNPEP regarding ligand recognition and catalytic mechanism remain to be delineated.

Results

The crystal structure of human DNPEP complexed with zinc and a substrate analogue aspartate-??-hydroxamate reveals a dodecameric machinery built by domain-swapped dimers, in agreement with electron microscopy data. A structural comparison with bacterial homologues identifies unifying catalytic features among the poorly understood M18 enzymes. The bound ligands in the active site also reveal the coordination mode of the binuclear zinc centre and a substrate specificity pocket for acidic amino acids.

Conclusions

The DNPEP structure provides a molecular framework to understand its catalysis that is mediated by active site loop swapping, a mechanism likely adopted in other M18 and M42 metallopeptidases that form dodecameric complexes as a self-compartmentalization strategy. Small differences in the substrate binding pocket such as shape and positive charges, the latter conferred by a basic lysine residue, further provide the key to distinguishing substrate preference. Together, the structural knowledge will aid in the development of enzyme-/family-specific aminopeptidase inhibitors.     

The 1.8 A crystal structure of human cathepsin G in complex with Suc-Val-Pro-PheP-(OPh)2: a Janus-faced proteinase with two opposite specificities.

The crystal structure of human neutrophil cathepsin G, complexed with the peptidyl phosphonate inhibitor Suc-Val-Pro-PheP-(OPh)2, has been determined to a resolution of 1.8 A using Patterson search techniques. The cathepsin G structure shows the polypeptide fold characteristic of trypsin-like serine proteinases and is especially similar to rat mast cell proteinase II. Unique to cathepsin G, however, is the presence of Glu226 (chymotrypsinogen numbering), which is situated at the bottom of the S1 specificity pocket, dividing it into two compartments. For this reason, the benzyl side chain of the inhibitor PheP residue does not fully occupy the pocket but is, instead, located at its entrance. Its positively charged equatorial edge is involved in a favourable electrostatic interaction with the negatively charged carboxylate group of Glu226. Arrangement of this Glu226 carboxylate would also allow accommodation of a Lys side chain in this S1 pocket, in agreement with the recently observed cathepsin G preference for Lys and Phe at P1. The cathepsin G complex with the covalently bound phosphonate inhibitor mimics a tetrahedral substrate intermediate. A comparison of the Arg surface distributions of cathepsin G, leukocyte elastase and rat mast cell protease II shows no simple common recognition pattern for a mannose-6-phosphate receptor-independent targeting mechanism for sorting of these granular proteinases.     

Crystal structure of Saccharomyces cerevisiae 3'-phosphoadenosine-5'-phosphosulfate reductase complexed with adenosine 3',5'-bisphosphate

Most assimilatory bacteria, fungi, and plants species reduce sulfate (in the activated form of APS or PAPS) to produce reduced sulfur. In yeast, PAPS reductase reduces PAPS to sulfite and PAP. Despite the difference in substrate specificity and catalytic cofactor, PAPS reductase is homologous to APS reductase in both sequence and structure, and they are suggested to share the same catalytic mechanism. Metazoans do not possess the sulfate reduction pathway, which makes APS/PAPS reductases potential drug targets for human pathogens. Here, we present the 2.05 A resolution crystal structure of the yeast PAPS reductase binary complex with product PAP bound. The N-terminal region mediates dimeric interactions resulting in a unique homodimer assembly not seen in previous APS/PAPS reductase structures. The "pyrophosphate-binding" sequence (47)TTAFGLTG(54) defines the substrate 3'-phosphate binding pocket. In yeast, Gly54 replaces a conserved aspartate found in APS reductases vacating space and charge to accommodate the 3'-phosphate of PAPS, thus regulating substrate specificity. Also, for the first time, the complete C-terminal catalytic motif (244)ECGIH(248) is revealed in the active site. The catalytic residue Cys245 is ideally positioned for an in-line attack on the beta-sulfate of PAPS. In addition, the side chain of His248 is only 4.2 A from the Sgamma of Cys245 and may serve as a catalytic base to deprotonate the active site cysteine. A hydrophobic sequence (252)RFAQFL(257) at the end of the C-terminus may provide anchoring interactions preventing the tail from swinging away from the active site as seen in other APS/PAPS reductases.     

Crystal structure of human macrophage elastase (MMP-12) in complex with a hydroxamic acid inhibitor

Human macrophage elastase (MMP-12) is a member of the family of matrix metalloproteinases (MMPs) that plays, like other members of the family, an important role in inflammatory processes contributing to tissue remodelling and destruction. In particular, a prominent role of MMP-12 in the destruction of elastin in the lung alveolar wall and the pathogenesis of emphysema has been suggested. It is therefore an attractive therapeutic target. We describe here the crystal structure of the catalytic domain of MMP-12 in complex with a hydroxamic acid inhibitor, CGS27023A. MMP-12 adopts the typical MMP fold and binds a structural zinc ion and three calcium ions in addition to the catalytic zinc ion. The enzyme structure shows an ordered N terminus close to the active site that is identical in conformation with the superactivated form of MMP-8. The S1'-specificity pocket is large and extends into a channel through the protein, which puts MMP-12 into the class of MMPs 3, 8 and 13 with large and open specificity pockets. The two crystallographically independent molecules adopt different conformations of the S1'-loop and its neighbouring loop due to differing crystal packing environments, suggesting that flexibility or the possibility of structural adjustments of these loop segments are intrinsic features of the MMP-12 structure and probably a common feature for all MMPs. The inhibitor binds in a bidentate fashion to the catalytic zinc ion. Its polar groups form hydrogen bonds in a substrate-like manner with beta-strand sIV of the enzyme, while the hydrophobic substituents are either positioned on the protein surface and are solvent-exposed or fill the upper part of the specificity pocket. The present structure enables us to aid the design of potent and selective inhibitors for MMP-12.     

Mutational analysis of the primary substrate specificity pocket of complement factor B. Asp(226) is a major structural determinant for p(1)-Arg binding

Factor B is a serine protease, which despite its trypsin-like specificity has Asn instead of the typical Asp at the bottom of the S(1) pocket (position 189, chymotrypsinogen numbering). Asp residues are present at positions 187 and 226 and either one could conceivably provide the negative charge for binding the P(1)-Arg of the substrate. Determination of the crystal structure of the factor B serine protease domain has revealed that the side chain of Asp(226) is within the S(1) pocket, whereas Asp(187) is located outside the pocket. To investigate the possible role of these atypical structural features in substrate binding and catalysis, we constructed a panel of mutants of these residues. Replacement of Asp(187) caused moderate (50-60%) decrease in hemolytic activity, compared with wild type factor B, whereas replacement of Asn(189) resulted in more profound reductions (71-95%). Substitutions at these two positions did not significantly affect assembly of the alternative pathway C3 convertase. In contrast, elimination of the negative charge from Asp(226) completely abrogated hemolytic activity and also affected formation of the C3 convertase. Kinetic analyses of the hydrolysis of a P(1)-Arg containing thioester by selected mutants confirmed that residue Asp(226) is a primary structural determinant for P(1)-Arg binding and catalysis.     

Crystal structures reveal an induced-fit binding of a substrate-like Aza-peptide epoxide to SARS coronavirus main peptidase

The SARS coronavirus main peptidase (SARS-CoV M(pro)) plays an essential role in the life-cycle of the virus and is a primary target for the development of anti-SARS agents. Here, we report the crystal structure of M(pro) at a resolution of 1.82 Angstroms, in space group P2(1) at pH 6.0. In contrast to the previously reported structure of M(pro) in the same space group at the same pH, the active sites and the S1 specificity pockets of both protomers in the structure of M(pro) reported here are in the catalytically competent conformation, suggesting their conformational flexibility. We report two crystal structures of M(pro) having an additional Ala at the N terminus of each protomer (M(+A(-1))(pro)), both at a resolution of 2.00 Angstroms, in space group P4(3)2(1)2: one unbound and one bound by a substrate-like aza-peptide epoxide (APE). In the unbound form, the active sites and the S1 specificity pockets of both protomers of M(+A(-1))(pro) are observed in a collapsed (catalytically incompetent) conformation; whereas they are in an open (catalytically competent) conformation in the APE-bound form. The observed conformational flexibility of the active sites and the S1 specificity pockets suggests that these parts of M(pro) exist in dynamic equilibrium. The structural data further suggest that the binding of APE to M(pro) follows an induced-fit model. The substrate likely also binds in an induced-fit manner in a process that may help drive the catalytic cycle.     

Activation of chymotrypsinogen-A. An hypothesis based upon comparison of the crystal structures of chymotrypsinogen-A and alpha-chymotrypsin

An hypothesis is proposed for the genesis of catalytic activity in the activation of chymotrypsinogen. This hypothesis attributes most of the observed difference in enzymatic activity between chymotrypsinogen and α-chymotrypsin to the inability of the former to bind specific substrates and to form an important hydrogen bond with the acyl group of the substrate. Secondary effects resulting from the binding of substrate to enzyme may also contribute to the difference in activity between zymogen and enzyme. Reasonable estimates for the rateenhancement due to structural elements of chymotrypsin which are missing in chymotrypsinogen lead to a difference in activity between them which approximates to that observed.Besides the nature of the catalytic mechanism itself, the structure of chymotrypsinogen provides clues to the means by which the two critical carboxylates of Asp(102) and Asp(194) are stabilized in their interior positions.     

Engineering the primary substrate specificity of Streptomyces griseus trypsin

Streptomyces griseus trypsin (SGT) was chosen as a model scaffold for the development of serine proteases with enhanced substrate specificity. Recombinant SGT has been produced in a Bacillus subtilis expression system in a soluble active form and purified to homogeneity. The recombinant and native proteases have nearly identical enzymatic properties and structures. Four SGT mutants with alterations in the S1 substrate binding pocket (T190A, T190P, T190S, and T190V) were also expressed. The T190P mutant demonstrated the largest shift to a preference for Arg versus Lys in the P1 site. This was shown by a minor reduction in catalytic activity toward an Arg-containing substrate (k(cat) reduction of 25%). The crystal structures of the recombinant SGT and the T190P mutant in a complex with the inhibitor benzamidine were obtained at high resolution (approximately 1.9 A). The increase in P1 specificity, achieved with minimal effect on the catalytic efficiency, demonstrates that the T190P mutant is an ideal candidate for the design of additional substrate specificity engineered into the S2 to S4 binding pockets.     

Structure of Streptomyces maltosyltransferase GlgE, a homologue of a genetically validated anti-tuberculosis target

GlgE is a recently identified (1→4)-α-d-glucan:phosphate α-d-maltosyltransferase involved in α-glucan biosynthesis in bacteria and is a genetically validated anti-tuberculosis drug target. It is a member of the GH13_3 CAZy subfamily for which no structures were previously known. We have solved the structure of GlgE isoform I from Streptomyces coelicolor and shown that this enzyme has the same catalytic and very similar kinetic properties to GlgE from Mycobacterium tuberculosis. The S. coelicolor enzyme forms a homodimer with each subunit comprising five domains, including a core catalytic α-amylase-type domain A with a (β/α)(8) fold. This domain is elaborated with domain B and two inserts that are specifically configured to define a well conserved donor pocket capable of binding maltose. Domain A, together with domain N from the neighboring subunit, forms a hydrophobic patch that is close to the maltose-binding site and capable of binding cyclodextrins. Cyclodextrins competitively inhibit the binding of maltooligosaccharides to the S. coelicolor enzyme, showing that the hydrophobic patch overlaps with the acceptor binding site. This patch is incompletely conserved in the M. tuberculosis enzyme such that cyclodextrins do not inhibit this enzyme, despite acceptor length specificity being conserved. The crystal structure reveals two further domains, C and S, the latter being a helix bundle not previously reported in GH13 members. The structure provides a framework for understanding how GlgE functions and will help guide the development of inhibitors with therapeutic potential.     

Crystal structure of the anticoagulant slow form of thrombin

Using the thrombin mutant R77aA devoid of the site of autoproteolytic degradation at exosite I, we have solved for the first time the structure of thrombin free of any inhibitors and effector molecules and stabilized in the Na(+)-free slow form. The slow form shows subtle differences compared with the currently available structures of the Na(+)-bound fast form that carry inhibitors at the active site or exosite I. The most notable differences are the displacement of Asp-189 in the S1 specificity pocket, a downward shift of the 190-193 strand, a rearrangement of the side chain of Glu-192, and a significant shift in the position of the catalytic Ser-195 that is no longer within H-bonding distance from His-57. The structure of the slow form explains the reduced specificity toward synthetic and natural substrates and suggests a molecular basis for its anticoagulant properties.     

The crystal structure of the ring-hydroxylating dioxygenase from Sphingomonas CHY-1

The ring-hydroxylating dioxygenase (RHD) from Sphingomonas CHY-1 is remarkable due to its ability to initiate the oxidation of a wide range of polycyclic aromatic hydrocarbons (PAHs), including PAHs containing four- and five-fused rings, known pollutants for their toxic nature. Although the terminal oxygenase from CHY-1 exhibits limited sequence similarity with well characterized RHDs from the naphthalene dioxygenase family, the crystal structure determined to 1.85 A by molecular replacement revealed the enzyme to share the same global alpha(3)beta(3) structural pattern. The catalytic domain distinguishes itself from other bacterial non-heme Rieske iron oxygenases by a substantially larger hydrophobic substrate binding pocket, the largest ever reported for this type of enzyme. While residues in the proximal region close to the mononuclear iron atom are conserved, the central region of the catalytic pocket is shaped mainly by the side chains of three amino acids, Phe350, Phe404 and Leu356, which contribute to the rather uniform trapezoidal shape of the pocket. Two flexible loops, LI and LII, exposed to the solvent seem to control the substrate access to the catalytic pocket and control the pocket length. Compared with other naphthalene dioxygenases residues Leu223 and Leu226, on loop LI, are moved towards the solvent, thus elongating the catalytic pocket by at least 2 A. An 11 A long water channel extends from the interface between the alpha and beta subunits to the catalytic site. The comparison of these structures with other known oxygenases suggests that the broad substrate specificity presented by the CHY-1 oxygenase is primarily due to the large size and particular topology of its catalytic pocket and provided the basis for the study of its reaction mechanism.     

Bovine chymotrypsinogen A X-ray crystal structure analysis and refinement of a new crystal form at 1.8 A resolution

The X-ray structure of a new crystal form of chymotrypsinogen A grown from ethanol/water has been determined at 1.8 A resolution using Patterson search techniques. The crystals are of orthorhombic space group P212121 and contain two molecules in the asymmetric unit. Both independent molecules (referred to as A and B) have been crystallographically refined to a final R value of 0.173 with reflection data to 1.8 A resolution. Owing to different crystal contacts, both independent molecules show at various sites conformational differences, especially in segments 33-38, 142-153 and 215-222. If these three loops are omitted in a comparison, the root-mean-square (r.m.s.) deviation of the main-chain atoms of molecules A and B is 0.32 A. If segments 70-79, 143-152 and 215-221 are omitted, a comparison of either molecule A or molecule B with the chymotrypsinogen model of Freer et al. (1970) reveals an r.m.s. deviation of the alpha-carbon atoms of about 0.7 A. Compared with the active enzyme, four spatially adjacent peptide segments, in particular, are differently organized in the zymogen: the amino-terminal segment 11-19 runs in a rigid but strained conformation along the molecular surface due to the covalent linkage through Cys1; also segment 184-194 is in a rigid unique conformation due to several mutually stabilizing interactions with the amino-terminal segment; segment 216-222, which also lines the specificity pocket, adapts to different crystal contacts and exists in both chymotrypsinogen molecules in different, but defined conformations; in particular, disulfide bridge 191-220, which covalently links both latter segments, has opposite handedness in molecules A and B; finally, the autolysis loop 142 to 153 is organized in a variety of ways and in its terminal part is completely disordered. Thus, the allosteric activation domain (Huber & Bode, 1978) is organized in defined although different conformations in chymotrypsinogen molecules A and B, in contrast to trypsinogen, where all four homologous segments of the activation domain are disordered. This reflects the structural variability and deformability of the activation domain in serine proteinase proenzymes. If the aforementioned peptide segments are omitted, a comparison of our chymotrypsinogen models with gamma-chymotrypsin (Cohen et al., 1981) yields an r.m.s. deviation for alpha-carbon atoms of about 0.5 A. The residues of the "active site triad" are arranged similarly, but the oxyanion hole is lacking in chymotrypsinogen.(ABSTRACT TRUNCATED AT 400 WORDS)     

牙鲆弹性蛋白酶cDNA全长和蛋白质结构分析

为研究牙鲆丝氨酸蛋白酶家族的功能和及其家族的分子进化规律,从本实验室已构建的牙鲆肝胰脏cDNA文库进行了部分测序,从而筛选出一个弹性蛋白酶新成员：弹性蛋白酶5。在此基础上,结合Genbank数据库中已经提交的胰凝乳蛋白酶和胰蛋白酶,对三者蛋白质进行了序列分析和三维结构的比较。牙鲆弹性蛋白酶cDNA包含一个完整的读码框（提交Genbank的登录号为EU873084）。其编码区平均GC含量为54％,推测编码的蛋白质包含296个氨基酸,分子量为29．04KD,等电点为6．14。蛋白序列比较表明它和牙鲆弹性蛋白酶3相似性最高。通过同源建模得到弹性蛋白酶5的三维结构和牛胰凝乳蛋白酶结构相似,包含了2个α螺旋、β个8折叠和13个转角结构。牙鲆弹性蛋白酶、胰凝乳蛋白酶和胰蛋白酶中底物结合区的3个关键氨基酸有明显的区别,这些氨基酸的变化改变了底物结合位点开口的大小,胰凝乳蛋白酶2的三个关键氨基酸和牛胰凝乳蛋白酶相同,该区域能接受结构较大的芳香族氨基酸;胰蛋白酶3能更好的结合阳性氨基酸Lys或Arg;而弹性蛋白酶开口很小,只能结合小的残基。上述结果证明了牙鲆丝氨酸蛋白酶家族中的弹性蛋白酶、胰凝乳蛋白酶和胰蛋白酶底物结合位点的结构差异决定了其对底物选择的特异性。     

Structural and Biochemical Analysis of Human Pathogenic Astrovirus Serine Protease at 2.0 Å Resolution

Astroviruses are single-stranded RNA viruses with a replication strategy based on the proteolytic processing of a polyprotein precursor and subsequent release of the viral enzymes of replication. So far, the catalytic properties of the astrovirus protease as well as its structure have remained uncharacterized. In this study, the three-dimensional crystal structure of the predicted protease of human pathogenic astrovirus has been solved to 2.0 Å resolution. The protein displays the typical properties of trypsin-like enzymes but also several characteristic features: (i) a catalytic Asp-His-Ser triad in which the aspartate side chain is oriented away from the histidine, being replaced by a water molecule; (ii) a non-common conformation and composition of the S1 pocket; and (iii) the lack of the typical surface β-ribbons together with a “featureless” shape of the substrate-binding site. Hydrolytic activity assays indicate that the S1 pocket recognises Glu and Asp side chains specifically, which, therefore, are predicted to occupy the P1 position on the substrate cleavage site. The positive electrostatic potential featured by the S1 region underlies this specificity. The comparative structural analysis highlights the peculiarity of the astrovirus protease, and differentiates it from the human and viral serine proteases.     

The three-dimensional structure of the native ternary complex of bovine pancreatic procarboxypeptidase A with proproteinase E and chymotrypsinogen C.

The metalloexozymogen procarboxypeptidase A is mainly secreted in ruminants as a ternary complex with zymogens of two serine endoproteinases, chymotrypsinogen C and proproteinase E. The bovine complex has been crystallized, and its molecular structure analysed and refined at 2.6 A resolution to an R factor of 0.198. In this heterotrimer, the activation segment of procarboxypeptidase A essentially clamps the other two subunits, which shield the activation sites of the former from tryptic attack. In contrast, the propeptides of both serine proproteinases are freely accessible to trypsin. This arrangement explains the sequential and delayed activation of the constituent zymogens. Procarboxypeptidase A is virtually identical to the homologous monomeric porcine form. Chymotrypsinogen C displays structural features characteristic for chymotrypsins as well as elastases, except for its activation domain; similar to bovine chymotrypsinogen A, its binding site is not properly formed, while its surface located activation segment is disordered. The proproteinase E structure is fully ordered and strikingly similar to active porcine elastase; its specificity pocket is occluded, while the activation segment is fixed to the molecular surface. This first structure of a native zymogen from the proteinase E/elastase family does not fundamentally differ from the serine proproteinases known so far.     

Crystal structure of Bacillus subtilis signal peptide peptidase A

Signal peptide peptidase A (SppA) is a membrane-bound self-compartmentalized serine protease that functions to cleave the remnant signal peptides left behind after protein secretion and cleavage by signal peptidases. SppA is found in plants, archaea and bacteria. Here, we report the first crystal structure of a Gram-positive bacterial SppA. The 2.4-Å-resolution structure of Bacillus subtilis SppA (SppA_BS) catalytic domain reveals eight SppA_BS molecules in the asymmetric unit, forming a dome-shaped octameric complex. The octameric state of SppA_BS is supported by analytical size-exclusion chromatography and multi-angle light scattering analysis. Our sequence analysis, mutagenesis and activity assays are consistent with Ser147 serving as the nucleophile and Lys199 serving as the general base; however, they are located in different region of the protein, more than 29 Å apart. Only upon assembling the octamer do the serine and lysine come within close proximity, with neighboring protomers each providing one-half of the catalytic dyad, thus producing eight separate active sites within the complex, twice the number seen within Escherichia coli SppA (SppA_EC). The SppA_BS S1 substrate specificity pocket is deep, narrow and hydrophobic, but with a polar bottom. The S3 pocket, which is constructed from two neighboring proteins, is shallower, wider and more polar than the S1 pocket. A comparison of these pockets to those seen in SppA_EC reveals a significant difference in the size and shape of the S1 pocket, which we show is reflected in the repertoire of peptides the enzymes are capable of cleaving.     

Amino acid sequence and disulfide bridges of subunit III, a defective endopeptidase present in the bovine pancreatic 6 S procarboxypeptidase A complex

The sequence of the 240 amino acids and the position of the five S-S bridges of subunit III of the bovine pancreatic 6 S procarboxypeptidase A complex have been determined thus confirming its phylogenetic filiation with the pancreatic serine endopeptidase group. The subunit contains at equivalent positions all the elements of the catalytic site of these enzymes. The elements of a binding pocket very similar to that of porcine elastase I are also present in the protein thus accounting for its zymogen-like activity. The most obvious difference is the absence in the subunit of the two strongly hydrophobic amino acids (16 and 17 in the chymotrypsinogen numbering), which are known to participate in the stabilization of a fully functional binding pocket in active endopeptidases. Four of the five disulfide bridges of subunit III are homologous with those common to all pancreatic endopeptidases. In contrast the fifth bridge forms a very small loop of only four amino acids, which is not encountered in active endopeptidases. Other potentially lethal modifications in the structure of the subunit are not excluded.     

The crystal structure of pyroglutamyl peptidase I from Bacillus amyloliquefaciens reveals a new structure for a cysteine protease

BACKGROUND: The N-terminal pyroglutamyl (pGlu) residue of peptide hormones, such as thyrotropin-releasing hormone (TRH) and luteinizing hormone releasing hormone (LH-RH), confers resistance to proteolysis by conventional aminopeptidases. Specialized pyroglutamyl peptidases (PGPs) are able to cleave an N-terminal pyroglutamyl residue and thus control hormonal signals. Until now, no direct or homology-based three-dimensional structure was available for any PGP. RESULTS: The crystal structure of pyroglutamyl peptidase I (PGP-I) from Bacillus amyloliquefaciens has been determined to 1.6 A resolution. The crystallographic asymmetric unit of PGP-I is a tetramer of four identical monomers related by noncrystallographic 222 symmetry. The protein folds into an alpha/beta globular domain with a hydrophobic core consisting of a twisted beta sheet surrounded by five alpha helices. The structure allows the function of most of the conserved residues in the PGP-I family to be identified. The catalytic triad comprises Cys144, His168 and Glu81. CONCLUSIONS: The catalytic site does not have a conventional oxyanion hole, although Cys144, the sidechain of Arg91 and the dipole of an alpha helix could all stabilize a negative charge. The catalytic site has an S1 pocket lined with conserved hydrophobic residues to accommodate the pyroglutamyl residue. Aside from the S1 pocket, there is no clearly defined mainchain substrate-binding region, consistent with the lack of substrate specificity. Although the overall structure of PGP-I resembles some other alpha/beta twisted open-sheet structures, such as purine nucleoside phosphorylase and cutinase, there are important differences in the location and organization of the active-site residues. Thus, PGP-I belongs to a new family of cysteine proteases.