Structural basis for the Zn2+ inhibition of the zymogen-like kallikrein-related peptidase 10.

Although kallikrein-related peptidase 10 (KLK10) is expressed in a variety of human tissues and body fluids, knowledge of its physiological functions is fragmentary. Similarly, the pathophysiology of KLK10 in cancer is not well understood. In some cancer types, a role as tumor suppressor has been suggested, while in others elevated expression is associated with poor patient prognosis. Active human KLK10 exhibits a unique, three residue longer N-terminus with respect to other serine proteases and an extended 99-loop nearly as long as in tissue kallikrein KLK1. Crystal structures of recombinant ligand-free KLK10 and a Zn2+ bound form explain to some extent the mixed trypsin- and chymotrypsin-like substrate specificity. Zn2+-inhibition of KLK10 appears to be based on a unique mechanism, which involves direct binding and blocking of the catalytic triad. Since the disordered N-terminus and several loops adopt a zymogen-like conformation, the active protease conformation is very likely induced by interaction with the substrate, in particular at the S1 subsite and at the unusual Ser193 as part of the oxyanion hole. The KLK10 structures indicate that the N-terminus, the nearby 75-, 148-, and the 99-loops are connected in an allosteric network, which is present in other trypsin-like serine proteases with several variations.

Structural basis for the sheddase function of human meprin ß metalloproteinase at the plasma membrane.

Ectodomain shedding at the cell surface is a major mechanism to regulate the extracellular and circulatory concentration or the activities of signaling proteins at the plasma membrane. Human meprin ß is a 145-kDa disulfide-linked homodimeric multidomain type-I membrane metallopeptidase that sheds membrane-bound cytokines and growth factors, thereby contributing to inflammatory diseases, angiogenesis, and tumor progression. In addition, it cleaves amyloid precursor protein (APP) at the ß-secretase site, giving rise to amyloidogenic peptides. We have solved the X-ray crystal structure of a major fragment of the meprin ß ectoprotein, the first of a multidomain oligomeric transmembrane sheddase, and of its zymogen. The meprin ß dimer displays a compact shape, whose catalytic domain undergoes major rearrangement upon activation, and reveals an exosite and a sugar-rich channel, both of which possibly engage in substrate binding. A plausible structure-derived working mechanism suggests that substrates such as APP are shed close to the plasma membrane surface following an "N-like" chain trace.

A standard orientation for metallopeptidases.

Visualization of three-dimensional structures is essential to the transmission of information to the general reader and the comparison of related structures. Therefore, it would be useful to provide a common framework. Based on the work of Schechter and Berger, and the finding that most peptidases bind their substrates in extended conformation, we suggest a "standard orientation" for the overall description of metallopeptidases (MPs) as done before for peptidases of other classes. This entails a frontal view of the horizontally-aligned active-site cleft. A substrate is bound N- to C-terminally from left (on the non-primed side of the cleft) to right (on the primed side), and the catalytic metal ion resides at the cleft bottom at roughly half width. This view enables us to see that most metalloendopeptidases are bifurcated into an upper and a lower sub-domain by the cleft, whose back is framed by a nearly horizontal "active-site helix." The latter comprises a short zinc-binding consensus sequence, either HEXXH or HXXEH, which provides two histidines to bind the single catalytic metal and the general-base/acid glutamate required for catalysis. In addition, an oblique "backing helix" is observed behind the active-site helix, and a mixed ß-sheet of at least three strands is positioned in the upper sub-domain paralleling the cleft. The lowermost "upper-rim" strand of the sheet runs antiparallel to the substrate bound in the cleft and therefore contributes both to delimitating the cleft top and to binding of the substrate main-chain on its non-primed side through ß-ribbon-like interactions. In contrast, in metalloexopeptidases, which chop off N- or C-terminal residues only, extensive binding on both sides of the cleft is not required and a different overall scaffold is generally observed. This consists of an αßα-sandwich, which is reminiscent of, but clearly distinct from, the archetypal α/ß-hydrolase fold. Metalloexopeptidases have their active sites at the C-terminal end of a central, eight-stranded twisted ß-sheet, and can contain one or two catalytic metal ions. As the zinc-binding site and the residues engaged in substrate binding and catalysis are mainly provided by loops connecting the ß-sheet strands and the helices on either side, the respective standard orientations vary with respect to the position of the sheets. The standard orientation of eight prototypic MP structures is presented and discussed. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.

The dimer interface of the membrane type 1 matrix metalloproteinase hemopexin domain: crystal structure and biological functions.

Homodimerization is an essential step for membrane type 1 matrix metalloproteinase (MT1-MMP) to activate proMMP-2 and to degrade collagen on the cell surface. To uncover the molecular basis of the hemopexin (Hpx) domain-driven dimerization of MT1-MMP, a crystal structure of the Hpx domain was solved at 1.7 Å resolution. Two interactions were identified as potential biological dimer interfaces in the crystal structure, and mutagenesis studies revealed that the biological dimer possesses a symmetrical interaction where blades II and III of molecule A interact with blades III and II of molecule B. The mutations of amino acids involved in the interaction weakened the dimer interaction of Hpx domains in solution, and incorporation of these mutations into the full-length enzyme significantly inhibited dimer-dependent functions on the cell surface, including proMMP-2 activation, collagen degradation, and invasion into the three-dimensional collagen matrix, whereas dimer-independent functions, including gelatin film degradation and two-dimensional cell migration, were not affected. These results shed light on the structural basis of MT1-MMP dimerization that is crucial to promote cellular invasion.

Pore-forming scissors? A first structural glimpse of gamma-secretase.

Amyloid plaques, which are composed of amyloid-beta peptide (Abeta), signify Alzheimer's disease pathology. Secretases generate Abeta by processing the beta-amyloid precursor protein. gamma-Secretase, a complex comprising four different proteins, liberates Abeta from its precursor by intramembrane proteolysis. The first impression of the shape of gamma-secretase has recently been revealed by electron microscopy. It indicates a spherical transmembrane particle with an interior chamber that, presumably, accommodates its catalytic residues, and two openings that might be exit sites for the cleavage products.

Staphylocoagulase is a prototype for the mechanism of cofactor-induced zymogen activation.

Many bacterial pathogens secrete proteins that activate host trypsinogen-like enzyme precursors, most notably the proenzymes of the blood coagulation and fibrinolysis systems. Staphylococcus aureus, an important human pathogen implicated in sepsis and endocarditis, secretes the cofactor staphylocoagulase, which activates prothrombin, without the usual proteolytic cleavages, to directly initiate blood clotting. Here we present the 2.2 A crystal structures of human alpha-thrombin and prethrombin-2 bound to a fully active staphylocoagulase variant. The cofactor consists of two domains, each with three-helix bundles; this is a novel fold that is distinct from known serine proteinase activators, particularly the streptococcal plasminogen activator streptokinase. The staphylocoagulase fold is conserved in other bacterial plasma-protein-binding factors and extracellular-matrix-binding factors. Kinetic studies confirm the importance of isoleucine 1 and valine 2 at the amino terminus of staphylocoagulase for zymogen activation. In addition to making contacts with the 148 loop and (pro)exosite I of prethrombin-2, staphylocoagulase inserts its N-terminal peptide into the activation pocket of bound prethrombin-2, allosterically inducing functional catalytic machinery. These investigations demonstrate unambiguously the validity of the zymogen-activation mechanism known as 'molecular sexuality'.

Structural basis of the zinc inhibition of human tissue kallikrein 5.

Human kallikrein 5 (hK5) is a member of the tissue kallikrein family of serine peptidases. It has trypsin-like substrate specificity, is inhibited by metal ions, and is abundantly expressed in human skin, where it is believed to play a central role in desquamation. To further understand the interaction of hK5 with substrates and metal ions, active recombinant hK5 was crystallized in complex with the tripeptidyl aldehyde inhibitor leupeptin, and structures at 2.3 A resolution were obtained with and without Zn2+. While the overall structure and the specificity of S1 pocket for basic side-chains were similar to that of hK4, a closely related family member, both differed in their interaction with Zn2+. Unlike hK4, the 75-loop of hK5 is not structured to bind a Zn2+. Instead, Zn2+ binds adjacent to the active site, becoming coordinated by the imidazole rings of His99 and His96 not present in hK4. This zinc binding is accompanied by a large shift in the backbone conformation of the 99-loop and by large movements of both His side-chains. Modeling studies show that in the absence of bound leupeptin, Zn2+ is likely further coordinated by the imidazolyl side-chain of the catalytic His57 which can, similar to equivalent His57 imidazole groups in the related rat kallikrein proteinase tonin and in an engineered metal-binding rat trypsin, rotate out of its triad position to provide the third co-ordination site of the bound Zn2+, rendering Zn2+-bound hK5 inactive. In solution, this mode of binding likely occurs in the presence of free and substrate saturated hK5, as kinetic analyses of Zn2+ inhibition indicate a non-competitive mechanism. Supporting the His57 re-orientation, Zn2+ does not fully inhibit hK5 hydrolysis of tripeptidyl substrates containing a P2-His residue. The P2 and His57 imidazole groups would lie next to each other in the enzyme-substrate complex, indicating that incomplete inhibition is due to competition between both imidazole groups for Zn2+. The His96-99-57 triad is thus suggested to be responsible for the Zn2+-mediated inhibition of hK5 catalysis.

Crystal structures of MMP-9 complexes with five inhibitors: contribution of the flexible Arg424 side-chain to selectivity.

Human matrix metalloproteinase 9 (MMP-9), also called gelatinase B, is particularly involved in inflammatory processes, bone remodelling and wound healing, but is also implicated in pathological processes such as rheumatoid arthritis, atherosclerosis, tumour growth, and metastasis. We have prepared the inactive E402Q mutant of the truncated catalytic domain of human MMP-9 and co-crystallized it with active site-directed synthetic inhibitors of different binding types. Here, we present the X-ray structures of five MMP-9 complexes with gelatinase-specific, tight binding inhibitors: a phosphinic acid (AM-409), a pyrimidine-2,4,6-trione (RO-206-0222), two carboxylate (An-1 and MJ-24), and a trifluoromethyl hydroxamic acid inhibitor (MS-560). These compounds bind by making a compromise between optimal coordination of the catalytic zinc, favourable hydrogen bond formation in the active-site cleft, and accommodation of their large hydrophobic P1' groups in the slightly flexible S1' cavity, which exhibits distinct rotational conformations of the Pro421 carbonyl group in each complex. In all these structures, the side-chain of Arg424 located at the bottom of the S1' cavity is not defined in the electron density beyond C(gamma), indicating its mobility. However, we suggest that the mobile Arg424 side-chain partially blocks the S1' cavity, which might explain the weaker binding of most inhibitors with a long P1' side-chain for MMP-9 compared with the closely related MMP-2 (gelatinase A), which exhibits a short threonine side-chain at the equivalent position. These novel structural details should facilitate the design of more selective MMP-9 inhibitors.

Crystal structure of the human carboxypeptidase N (kininase I) catalytic domain.

Human carboxypeptidase N (CPN), a member of the CPN/E subfamily of "regulatory" metallo-carboxypeptidases, is an extracellular glycoprotein synthesized in the liver and secreted into the blood, where it controls the activity of vasoactive peptide hormones, growth factors and cytokines by specifically removing C-terminal basic residues. Normally, CPN circulates in blood plasma as a hetero-tetramer consisting of two 83 kDa (CPN2) domains each flanked by a 48 to 55 kDa catalytic (CPN1) domain. We have prepared and crystallized the recombinant C-terminally truncated catalytic domain of human CPN1, and have determined and refined its 2.1 A crystal structure. The structural analysis reveals that CPN1 has a pear-like shape, consisting of a 319 residue N-terminal catalytic domain and an abutting, cylindrically shaped 79 residue C-terminal beta-sandwich transthyretin (TT) domain, more resembling CPD-2 than CPM. Like these other CPN/E members, two surface loops surrounding the active-site groove restrict access to the catalytic center, offering an explanation for why some larger protein carboxypeptidase inhibitors do not inhibit CPN. Modeling of the Pro-Phe-Arg C-terminal end of the natural substrate bradykinin into the active site shows that the S1' pocket of CPN1 might better accommodate P1'-Lys than Arg residues, in agreement with CPN's preference for cleaving off C-terminal Lys residues. Three Thr residues at the distal TT edge of CPN1 are O-linked to N-acetyl glucosamine sugars; equivalent sites in the membrane-anchored CPM are occupied by basic residues probably involved in membrane interaction. In tetrameric CPN, each CPN1 subunit might interact with the central leucine-rich repeat tandem of the cognate CPN2 subunit via a unique hydrophobic surface patch wrapping around the catalytic domain-TT interface, exposing the two active centers.

Structure of a novel thrombin inhibitor with an uncharged D-amino acid as P1 residue.

Thrombin, the ultimate proteinase of the coagulation cascade, is an attractive target for the treatment of a variety of cardiovascular diseases. Previously, a series of novel thrombin inhibitors, discovered by employing a powerful and new computer-assisted multiparameter optimization process (CADDIS), have been synthesized. We have now crystallized the complex of human alpha-thrombin with the most potent of these inhibitors, 8-5 (K(i)=3 nM), and have determined its 2.3A X-ray crystal structure. The Fourier map displayed clear electron density for the inhibitor. The central part of the inhibitor binds in an improved melagatran-like mode, while the structure identifies a d-tyrosine as P1 residue which forms a charged hydrogen bond with Asp 189 of thrombin. This is the first crystal structure of a thrombin-inhibitor complex, where an uncharged inhibitor residue makes hydrogen bonds within the thrombin S1 pocket. Additionally, novel favourable intermolecular hydrogen bonds of the inhibitor with the thrombin backbone become possible due to the d-configuration of the P1 residue. Two flanking voluminous side chains increase the strength of the subjacent hydrogen bonding system by shielding it from the bulk solvent.

Structural determinants of specificity and regulation of activity in the allosteric loop network of human KLK8/neuropsin.

Human KLK8/neuropsin, a kallikrein-related serine peptidase, is mostly expressed in skin and the hippocampus regions of the brain, where it regulates memory formation by synaptic remodeling. Substrate profiles of recombinant KLK8 were analyzed with positional scanning using fluorogenic tetrapeptides and the proteomic PICS approach, which revealed the prime side specificity. Enzyme kinetics with optimized substrates showed stimulation by Ca2+ and inhibition by Zn2+, which are physiological regulators. Crystal structures of KLK8 with a ligand-free active site and with the inhibitor leupeptin explain the subsite specificity and display Ca2+ bound to the 75-loop. The variants D70K and H99A confirmed the antagonistic role of the cation binding sites. Molecular docking and dynamics calculations provided insights in substrate binding and the dual regulation of activity by Ca2+ and Zn2+, which are important in neuron and skin physiology. Both cations participate in the allosteric surface loop network present in related serine proteases. A comparison of the positional scanning data with substrates from brain suggests an adaptive recognition by KLK8, based on the tertiary structures of its targets. These combined findings provide a comprehensive picture of the molecular mechanisms underlying the enzyme activity of KLK8.

Macrocyclic statine-based inhibitors of BACE-1.

Minimal sequence requirements for binding of substrate-derived statine peptides to the aspartyl enzyme were established on the basis of the X-ray cocrystal structure of the hydroxyethylene-octapeptide OM00-3 in complexation with BACE-1. With this information to hand, macrocyclic compounds that conformationally restrict and preorganize the peptide backbone for an entropically favoured binding to the enzyme active site cleft were designed. By means of a side chain-to-side chain ring closure between two aspartyl residues in the P2 and P3' positions through phenylene-1,3-dimethanamine, a 23-membered ring structure was obtained; this structure retained an extended conformation of the peptide backbone, including the transition state analogue statine for tight interactions with the two aspartyl residues of the active centre. The conformational preorganization of the inhibitor molecule was verified by NMR structural analysis and was then confirmed by the crystal structure of the BACE-1/inhibitor complex. Detailed insights into the binding mode of this macrocyclic inhibitor explained its moderate binding affinity in cell-free assays (K(i)=2.5 microM) and yielded precious information for possible structural optimization in view of the lack of steric clashes of the macrocycle with the flap domain of the enzyme.

Crystal structures of human tissue kallikrein 4: activity modulation by a specific zinc binding site.

Human tissue kallikrein 4 (hK4) belongs to a 15-member family of closely related serine proteinases. hK4 is predominantly expressed in prostate, activates hK3/PSA, and is up-regulated in prostate and ovarian cancer. We have identified active monomers of recombinant hK4 besides inactive oligomers in solution. hK4 crystallised in the presence of zinc, nickel, and cobalt ions in three crystal forms containing cyclic tetramers and octamers. These structures display a novel metal site between His25 and Glu77 that links the 70-80 loop with the N-terminal segment. Micromolar zinc as present in prostatic fluid inhibits the enzymatic activity of hK4 against fluorogenic substrates. In our measurements, wild-type hK4 exhibited a zinc inhibition constant (IC50) of 16 microM including a permanent residual activity, in contrast to the zinc-independent mutants H25A and E77A. Since the Ile16 N terminus of wild-type hK4 becomes more accessible for acetylating agents in the presence of zinc, we propose that zinc affects the hK4 active site via the salt-bridge formed between the N terminus and Asp194 required for a functional active site. hK4 possesses an unusual 99-loop that creates a groove-like acidic S2 subsite. These findings explain the observed specificity of hK4 for the P1 to P4 substrate residues. Moreover, hK4 shows a negatively charged surface patch, which may represent an exosite for prime-side substrate recognition.

X-ray structures of free and leupeptin-complexed human alphaI-tryptase mutants: indication for an alpha-->beta-tryptase transition.

Tryptases alpha and beta are trypsin-like serine proteinases expressed in large amounts by mast cells. Beta-tryptase is a tetramer that has enzymatic activity, but requires heparin binding to maintain functional and structural stability, whereas alpha-tryptase has little, if any, enzymatic activity but is a stable tetramer in the absence of heparin. As shown previously, these differences can be mainly attributed to the different conformations of the 214-220 segment. Interestingly, the replacement of Asp216 by Gly, which is present in beta-tryptase, results in enzymatically active but less stable alpha-tryptase mutants. We have solved the crystal structures of both the single (D216G) and the double (K192Q/D216G) mutant forms of recombinant human alphaI-tryptase in complex with the peptide inhibitor leupeptin, as well as the structure of the non-inhibited single mutant. The inhibited mutants exhibited an open functional substrate binding site, while in the absence of an inhibitor, the open (beta-tryptase-like) and the closed (alpha-tryptase-like) conformations were present simultaneously. This shows that both forms are in a two-state equilibrium, which is influenced by the residues in the vicinity of the active site and by inhibitor/substrate binding. Novel insights regarding the observed stability differences as well as a potential proteolytic activity of wild-type alpha-tryptase, which may possess a cryptic active site, are discussed.

Rigidity and flexibility of dipeptidyl peptidase IV: crystal structures of and docking experiments with DPIV.

Dipeptidyl peptidase IV (DPIV) is an alpha,beta-hydrolase-like serine exopeptidase, which removes dipeptides, preferentially with a C-terminal l-Pro residue, from the N terminus of longer peptide substrates. Previously, we determined the tetrameric 1.8A crystal structure of native porcine DPIV. Each monomer is composed of a beta-propeller and a catalytic domain, which together embrace an internal cavity housing the active centre. This cavity is connected to the bulk solvent by a "propeller opening" and a "side opening". Here, we analyse DPIV complexes with a t-butyl-Gly-Pro-Ile tripeptide, Pro-boroPro, a piperazine purine compound, and aminoethyl phenyl sulfonylfluoride. The latter two compounds bind to the active-site groove in a compact and a quite bulky manner, respectively, causing considerable shifts of the catalytic Ser630 side-chain and of the Tyr547 phenolic group, which forms the oxyanion hole. The tripeptide, mimicking a peptide substrate, is clamped to the active site through tight interactions via its N-terminal alpha-ammonium group, the P2 carbonyl group, the P1-l-Pro side-chain, the C-terminal carboxylate group, and the stable orthoacid ester amide formed between the scissile peptide carbonyl group and Ser630 O(gamma). This stable trapping of the tripeptide could be due to stabilization of the protonated His740 imidazolium cation by the adjacent negatively charged C-terminal carboxylate group, preventing proton transfer to the leaving group nitrogen atom. Docking experiments with the compact rigid 58 residue protein aprotinin, which had been shown to be processed by DPIV, indicate that the Arg1-Pro2 N terminus can access the DPIV active site only upon widening of its side openings, probably by separation of the first and the last propeller blades, and/or of the catalytic and the propeller domain.

Proprotein convertase models based on the crystal structures of furin and kexin: explanation of their specificity.

In eukaryotes, many secreted proteins and peptide hormones are excised from larger precursors by calcium-dependent serine proteinases, the proprotein/prohormone convertases (PCs). These PCs cleave their protein substrates very specifically following multiple basic residues. The seven mammalian PCs and their yeast orthologue kexin are multi-domain proteinases consisting of a subtilisin-related catalytic domain, a conserved P-domain and a variable, often cysteine-rich domain, which in some PCs is followed by an additional C-terminal trans-membrane domain and a short cytoplasmic domain. The recently published crystal structures of the soluble mouse furin and yeast kexin ectodomains have revealed the relative arrangement of catalytic and P domains, the exact domain fold and the detailed architecture of the substrate binding clefts. Based on these experimental structures, we now have modelled the structures of the other human/mouse PCs. According to topology and to structure-based sequence comparisons, these other PCs closely resemble furin, with PC4, PACE4 and PC5/6 being more similar, and PC1/3, PC2 and PC7 being less similar to furin. Except for PC1 and PC2, this order of similarity is valid for the catalytic as well as for the P domains, and is almost reversed using kexin as a reference molecule. A similar order results from the number and clustering of negative charges lining the non-prime subsites, explaining the gradually decreasing requirement for basic residues N-terminal to substrate cleavage sites. The preference of the different PCs for distinct substrates seems to be governed by overall charge compensation and matching of the detailed charge distribution pattern.

Secondary amides of sulfonylated 3-amidinophenylalanine. New potent and selective inhibitors of matriptase.

Matriptase is an epithelium-derived type II transmembrane serine protease and has been implicated in the activation of substrates such as pro-HGF/SF and pro-uPA, which are likely involved in tumor progression and metastasis. Through screening, we have identified bis-basic secondary amides of sulfonylated 3-amidinophenylalanine as matriptase inhibitors. X-ray analyses of analogues 8 and 31 in complex with matriptase revealed that these inhibitors occupy, in addition to part of the previously described S4-binding site, the cleft formed by the molecular surface and the unique 60 loop of matriptase. Therefore, optimization of the inhibitors included the incorporation of appropriate sulfonyl substituents that could improve binding of these inhibitors into both characteristic matriptase subsites. The most potent derivatives inhibit matriptase highly selective with K(i) values below 5 nM. Molecular modeling revealed that their improved affinity results from interaction with the S4 site of matriptase. Analogues 8 and 59 were studied in an orthotopic xenograft mouse model of prostate cancer. Compared to control, both inhibitors reduced tumor growth, as well as tumor dissemination.

1.2 A crystal structure of the serine carboxyl proteinase pro-kumamolisin; structure of an intact pro-subtilase.

Kumamolisin, an extracellular proteinase derived from an acido/thermophilic Bacillus, belongs to the sedolisin family of endopeptidases characterized by a subtilisin-like fold and a Ser-Glu-Asp catalytic triad. In kumamolisin, the Asp82 carboxylate hydrogen bonds to Glu32-Trp129, which might act as a proton sink stabilizing the catalytic residues. The 1.2/1.3 A crystal structures of the Glu32-->Ala and Trp129-->Ala mutants show that both mutations affect the active-site conformation, causing a 95% activity decrease. In addition, the 1.2 A crystal structure of the Ser278-->Ala mutant of pro-kumamolisin was determined. The prodomain exhibits a half-beta sandwich core docking to the catalytic domain similarly as the equivalent subtilisin prodomains in their catalytic-domain complexes. This pro-kumamolisin structure displays, for the first time, the uncleaved linker segment running across the active site and connecting the prodomain with the properly folded catalytic domain. The structure strongly points to an initial intramolecular activation cleavage in subtilases, as presumed for pro-subtilisin and pro-furin.

The 1.4 a crystal structure of kumamolysin: a thermostable serine-carboxyl-type proteinase.

Kumamolysin is a thermostable endopeptidase from Bacillus novosp. MN-32, exhibiting maximal proteolytic activity around pH 3. It belongs to the newly identified family of serine-carboxyl proteinases, which also includes CLN2, a human lysosomal homolog recently implicated in a fatal neurodegenerative disease. Kumamolysin and its complexes with two aldehyde inhibitors were crystallized, and their three-dimensional structures were solved and refined with X-ray data to 1.4 A resolution. As its Pseudomonas homolog, kumamolysin exhibits a Ser/Glu/Asp catalytic triad with particularly short interconnecting hydrogen bonds and an oxyanion hole enabling the reactive serine to attack substrate peptide bonds at quite acidic pH. An additional Glu/Trp pair, unique to kumamolysin, might further facilitate proton delocalization during nucleophilic attack, in particular at high temperature.

Crystal structure of the dinuclear zinc aminopeptidase PepV from Lactobacillus delbrueckii unravels its preference for dipeptides.

PepV from Lactobacillus delbrueckii, a dinuclear zinc peptidase, has been characterized as an unspecific amino dipeptidase. The crystal structure of PepV in complex with the phosphinic inhibitor AspPsi[PO(2)CH(2)]AlaOH, a dipeptide substrate mimetic, reveals a "catalytic domain" and a "lid domain," which together form an internal active site cavity that traps the inhibitor. The catalytic domain is topologically similar to catalytic domains from amino- and carboxypeptidases. However, the lid domain is unique among the related enzymes. In contrast to the other related exopeptidases, PepV recognizes and fixes the dipeptide backbone, while the side chains are not specifically probed and can vary, rendering it a nonspecific dipeptidase. The cocrystallized inhibitor illustrates the two roles of the two catalytic zinc ions, namely stabilization of the tetrahedral intermediate and activation of the catalytic water molecule.