Structure of the hirugen and hirulog 1 complexes of alpha-thrombin

The isomorphous structures of the hirugen (N-acetylhirudin 53'-64' with sulfato-Tyr63') and hirulog 1 (D-Phe-Pro-Arg-Pro-(Gly)4 desulfato-Tyr63'-hirugen) complexes of human alpha-thrombin have been determined and refined at 2.2 A resolution to crystallographic R-factors of 0.167 and 0.163, respectively. The binding of hirugen to thrombin is similar to that of the binding of the C-terminal dodecapeptide of hirudin, including that of the terminal 3(10) helical turn. The sulfato Tyr63', which, as a result of sulfation, increases the binding affinity by an order of magnitude, is involved in an extended hydrogen bonding network utilizing all three sulfato oxygen atoms. The hirugen-thrombin complex is the first thrombin structure determined to have an unobstructed active site; this site is practically identical in positioning of catalytic residues and in its hydrogen bonding pattern with that of other serine proteinases. Hirulog 1, which is a poor thrombin substrate, is cleaved at the Arg3'-Pro4' bond in the crystal structure. The Arg3' of hirulog 1 occupies the specificity site, the D-Phe-Pro-Arg tripeptide is positioned like that of D-Phe-Pro-Arg chloromethylketone in the active site and the Pro4'(Gly)4 spacer to hirugen is disordered in the structure, as is the 3(10) turn of hirugen. The latter must be related to the simultaneous absence both of sulfation and of the last residue of hirudin (Gln65'). In addition, the autolysis loop of thrombin (Lys145-Gly150) is disordered in both structures. Changes in circular dichroism upon hirugen binding are therefore most likely the result of the flexibility associated with this loop.     

The methyl group of N(alpha)(Me)Arg-containing peptides disturbs the active-site geometry of thrombin, impairing efficient cleavage

Bivalent peptidic thrombin inhibitors consisting of an N-terminal d-cyclohexylalanine-Pro-N(alpha)(Me)Arg active-site fragment, a flexible polyglycine linker, and a C-terminal hirugen-like segment directed towards the fibrinogen recognition exosite inhibit thrombin with K(i) values in the picomolar range, remaining stable in buffered solution at pH 7.8 for at least 15 hours. In order to investigate the structural basis of this increased stability, the most potent of these inhibitors, I-11 (K(i)=37pM), containing an N(alpha)(Me)Arg-Thr bond, was crystallized in complex with human alpha-thrombin. X-ray data were collected to 1.8A resolution and the crystal structure of this complex was determined. The Fourier map displays clear electron density for the N-terminal fragment and for the exosite binding segment. It indicates, however, that in agreement with Edman sequencing, the peptide had been cleaved in the crystal, presumably due to the long incubation time of 14 days needed for crystallization and data collection. The N(alpha)(Me) group is directed toward the carbonyl oxygen atom of Ser214, pushing the Ser195 O(gamma) atom out of its normal site. This structure suggests that upon thrombin binding, the scissile peptide bond of the intact peptide and the Ser195 O(gamma) are separated from each other, impairing the nucleophilic attack of the Ser195 O(gamma) toward the N(alpha)(Me)Arg carbonyl group. In the time-scale of two weeks, however, cleavage geometries favoured by the crystal allow catalysis at a slow rate.     

Refined structure of the hirudin-thrombin complex

The structure of a recombinant hirudin (variant 2, Lys47) human alpha-thrombin complex has been refined using restrained least-squares methods to a crystallographic R-factor of 0.173. The hirudin structure consists of an N-terminal domain folded into a globular unit and a long 17-peptide C-terminal in an extended chain conformation. The N-terminal domain binds at the active-site of thrombin where Ile1' to Tyr3' penetrates to the catalytic triad. The alpha-amino group of Ile1' of hirudin makes a hydrogen bond with OG of Ser195 of thrombin, the side-chains of Ile1' and Tyr3' occupy the apolar site, Thr2' is at the entrance to, but does not enter, the S1 specificity site and Ile1' to Tyr3' form a parallel beta-strand with Ser214 to Gly219. The latter interaction is antiparallel in all other serine proteinase-protein inhibitor complexes. The extended C-terminal segment of hirudin, which is abundant in acidic residues, makes many electrostatic interactions with the fibrinogen binding exosite while the last five residues are in a 3(10) helical turn residing in a hydrophobic patch on the thrombin surface. The precision of the complementarity displayed by these two molecules produces numerous interactions, which although independently generally weak, together are responsible for the high degree of affinity and specificity. Although hirudin-thrombin and D-Phe-Pro-Arg-chloromethyl ketone-thrombin differ in conformation in the autolysis loop (Lys145 to Gly150), this is most likely due to different crystal packing interactions and changes in circular dichroism between the two are probably due to the inherent flexibility of the loop. An RGD sequence, which is generally known to be involved in cell surface receptor interactions, occurs in thrombin and is associated with a long solvent channel filled with water molecules leading to the surface from the end of the S1 site. However, the RGD triplet does not appear to be able to interact in concert in a surface binding mode.     

Crystal structures of alpha-lytic protease complexes with irreversibly bound phosphonate esters

The structures of the complexes with alpha-lytic protease of both phosphorus stereoisomers of N-[(2S)-2-[[[(1R)-1-[N-[(tert-butyloxycarbonyl)-L-alanyl-L-alanyl- L-prolyl]amino]-2-methylpropyl]-phenoxyphosphinyl]oxy]propanoyl]- L-alanine methyl ester, an analogue of the peptide Boc-Ala-Ala-Pro-Val-Ala-Ala where Val is replaced with an analogous phosphonate phenyl ester and the subsequent Ala is replaced with lactate, have been determined to high resolution (1.9 A) by X-ray crystallography. Both stereoisomers inactivate the enzyme but differ by a factor of 2 in the second-order rate constant for inactivation [Sampson, N. S., & Bartlett, P. A. (1991) Biochemistry (preceding paper in this issue)]. One isomer (B) forms a tetrahedral adduct in which the phosphonate phenyl ester is displaced by the active site serine (S195) and interacts with the enzyme across seven substrate recognition sites that span both sides of the scissile bond. Seven hydrogen bonds are formed with the enzyme, and 510 A2 of hydrophobic surface area is buried when the inhibitor interacts with the enzyme. Although two hydrogen bonds are gained by incorporation of two residues on the C-terminal side of the scissile bond into the inhibitor, there is very little adjustment in the structure of the enzyme in this region. Surprisingly, the active site histidine (H57) does not interact with the phosphonate, apparently because the phosphonate lacks negative charge in or near the oxyanion hole, and instead, the side chain rotates out of the active site cleft and hydrogen bonds with solvent. The other isomer (A) forms a mixture of two different tetrahedral adducts in the active site, both covalently bonded to Ser 195. One adduct, at approximately 58% occupancy, is exactly the same in structure as the complex formed with isomer B, and the other adduct, at 42% occupancy, has lost the two residues C-terminal to the scissile bond by hydrolysis. In the lower occupancy structure, His 57 does not rotate out of the active site and forms a hydrogen bond with the phosphonate oxygen instead. The structures of both complexes were insensitive to pH. As very little change in structure accompanies the histidine rotation, the complex with isomer B provides an excellent mimic for the structure of the transition state (or high-energy reaction intermediate) that spans both sides of the scissile bond.     

On the sequential determinants of calpain cleavage

The structural clues of substrate recognition by calpain are incompletely understood. In this study, 106 cleavage sites in substrate proteins compiled from the literature have been analyzed to dissect the signal for calpain cleavage and also to enable the design of an ideal calpain substrate and interfere with calpain action via site-directed mutagenesis. In general, our data underline the importance of the primary structure of the substrate around the scissile bond in the recognition process. Significant amino acid preferences were found to extend over 11 residues around the scissile bond, from P(4) to P(7)'. In compliance with earlier data, preferred residues in the P(2) position are Leu, Thr, and Val, and in P(1) Lys, Tyr, and Arg. In position P(1) ', small hydrophilic residues, Ser and to a lesser extent Thr and Ala, occur most often. Pro dominates the region flanking the P(2)-P(1)' segment, i.e. positions P(3) and P(2)'-P(4)'; most notable is its occurrence 5.59 times above chance in P(3)'. Intriguingly, the segment C-terminal to the cleavage site resembles the consensus inhibitory region of calpastatin, the specific inhibitor of the enzyme. Further, the position of the scissile bond correlates with certain sequential attributes, such as secondary structure and PEST score, which, along with the amino acid preferences, suggests that calpain cleaves within rather disordered segments of proteins. The amino acid preferences were confirmed by site-directed mutagenesis of the autolysis sites of Drosophila calpain B; when amino acids at key positions were changed to less preferred ones, autolytic cleavage shifted to other, adjacent sites. Based on these preferences, a new fluorogenic calpain substrate, DABCYLTPLKSPPPSPR-EDANS, was designed and synthesized. In the case of micro- and m-calpain, this substrate is kinetically superior to commercially available ones, and it can be used for the in vivo assessment of the activity of these ubiquitous mammalian calpains.     

The function of calcium in protein C activation by thrombin and the thrombin-thrombomodulin complex can be distinguished by mutational analysis of protein C derivatives.

Protein C activation is catalyzed on endothelium by a complex between thrombin and thrombomodulin. Ca2+ stimulates protein C activation in the presence, and inhibits in the absence, of thrombomodulin. Protein C has Asp residues at the P3 and P3' positions relative to the scissile bond at Arg169-Leu. To determine the contribution of these residues to the Ca2+ effect on activation, we have expressed human 4-carboxyglutamic acid (Gla)-domainless protein C and 3 mutants with Asp-->Gly substitutions at P3, P3', and both positions. Ca2+ interaction with the protein C derivatives was monitored by changes in intrinsic fluorescence, and the Ca2+ dependence of activation by thrombin and a complex of thrombin-thrombomodulin with a soluble thrombomodulin derivative (the fourth through sixth epidermal growth factor domains). The affinity for Ca2+ of the mutants was reduced 3-6-fold, which was reflected by a comparable change in the Ca2+ concentration required for the half-maximal rate of activation by the thrombin-thrombomodulin complex. However, Ca2+ no longer effectively inhibited activation of the mutants by thrombin alone. We conclude that 1) the Asp residues play a specific role in the Ca(2+)-dependent inhibition of protein C activation by thrombin; 2) these mutations alter the affinity of Ca2+ for the high affinity binding site; and 3) the Asp residues in the P3 and P3' sites do not contribute in a positive fashion to rapid activation by the thrombin-thrombomodulin complex.     

Design and evaluation of novel bivalent thrombin inhibitors based on amidinophenylalanines.

Two bivalent thrombin inhibitors were synthesized, which consist of a benzamidine-based active-site-blocking segment, a fibrinogen recognition exosite inhibitor and a peptidic linker connecting these fragments. BZA-1 hirulog contains an Nalpha-(2-naphthylsulfonyl)-S-3-amidinophenylalanyl-is onipecotic acid residue connected via the carboxyl group to the linker segment. The active-site-directed moiety of BZA-2 hirulog [Nalpha-(2-naphthylsulfonyl-glutamyl)-R-4-amidinophenylal anyl-piperid ide] was coupled to the linker via the side chain of the glutamic acid. Both BZA-hirulogs contain almost identical linker-exo site inhibitor parts, except for the substitution of a glycine as the first linker residue in BZA-1 hirulog by a gamma-amino butyric acid in BZA-2 hirulog, thus increasing flexibility and linker length by two additional atoms. BZA-1 hirulog showed moderate potency (Ki = 0. 50 +/- 0.14 nM), while BZA-2 hirulog was characterized as a slow, tight binding inhibitor of thrombin (Ki = 0.29 +/- 0.08 pM). The stability in human plasma of both analogs was strongly improved compared with hirulog-1. For BZA-2 hirulog a significantly reduced plasma clearance was observed after intravenous injection in rats compared with BZA-1 hirulog and hirulog-1. The X-ray structure of the BZA-2 hirulog in complex with human alpha-thrombin was solved and confirmed the expected bivalent binding mode.     

Molecular dynamics simulations and functional characterization of the interactions of the PAR2 ectodomain with factor VIIa

Signaling of the tissue factor‐FVIIa complex regulates angiogenesis, tumor growth, and inflammation. TF‐FVIIa triggers cell signaling events by cleavage of protease activated receptor (PAR2) at the Arg36‐Ser37 scissile bond. The recognition of PAR2 by the FVIIa protease domain is poorly understood. We perform molecular modeling and dynamics simulations to derive the PAR2‐FVIIa interactions. Docking of the PAR2 Arg36‐Ser37 scissile bond to the S1 site and subsequent molecular dynamics leads to interactions of the PAR2 ectodomain with P and P′ sites of the FVIIa catalytic cleft as well as to electrostatic interactions between a stably folded region of PAR2 and a cluster of basic residues remote from the catalytic cleft of FVIIa. To address the functional significance of this interaction for PAR2 cleavage, we employed two antibodies with epitopes previously mapped to this cluster of basic residues. Although these antibodies do not block the catalytic cleft, both antibodies completely abrogated PAR2 activation by TF‐FVIIa. Our simulations indicate a conformation of the PAR2 ectodomain that limits the cleavage site to no more than 33 Å from its membrane proximal residue. Since the active site of FVIIa in the TF‐FVIIa complex is ～75 Å above the membrane, cleavage of the folded conformation of PAR2 would require tilting of the TF‐FVIIa complex toward the membrane, indicating that additional cellular factors may be required to properly align the scissile bond of PAR2 with TF‐FVIIa. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Discovery of amino acid motifs for thrombin cleavage and validation using a model substrate

Understanding the active site preferences of an enzyme is critical to the design of effective inhibitors and to gaining insights into its mechanisms of action on substrates. While the subsite specificity of thrombin is understood, it is not clear whether the enzyme prefers individual amino acids at each subsite in isolation or prefers to cleave combinations of amino acids as a motif. To investigate whether preferred peptide motifs for cleavage could be identified for thrombin, we exposed a phage-displayed peptide library to thrombin. The resulting preferentially cleaved substrates were analyzed using the technique of association rule discovery. The results revealed that thrombin selected for amino acid motifs in cleavage sites. The contribution of these hypothetical motifs to substrate cleavage efficiency was further investigated using the B1 IgG-binding domain of streptococcal protein G as a model substrate. Introduction of a P(2)-P(1)' LRS thrombin cleavage sequence within a major loop of the protein led to cleavage of the protein by thrombin, with the cleavage efficiency increasing with the length of the loop. Introduction of further P(3)-P(1) and P(1)-P(1)'-P(3)' amino acid motifs into the loop region yielded greater cleavage efficiencies, suggesting that the susceptibility of a protein substrate to cleavage by thrombin is influenced by these motifs, perhaps because of cooperative effects between subsites closest to the scissile peptide bond.     

Structural basis for the inhibition of porcine pepsin by Ascaris pepsin inhibitor-3

The three-dimensional structures of pepsin inhibitor-3 (PI-3) from Ascaris suum and of the complex between PI-3 and porcine pepsin at 1. 75 A and 2.45 A resolution, respectively, have revealed the mechanism of aspartic protease inhibition by this unique inhibitor. PI-3 has a new fold consisting of two domains, each comprising an antiparallel beta-sheet flanked by an alpha-helix. In the enzyme-inhibitor complex, the N-terminal beta-strand of PI-3 pairs with one strand of the 'active site flap' (residues 70-82) of pepsin, thus forming an eight-stranded beta-sheet that spans the two proteins. PI-3 has a novel mode of inhibition, using its N-terminal residues to occupy and therefore block the first three binding pockets in pepsin for substrate residues C-terminal to the scissile bond (S1'-S3'). The molecular structure of the pepsin-PI-3 complex suggests new avenues for the rational design of proteinaceous aspartic proteinase inhibitors.     

Crystal structures of the photosystem II D1 C-terminal processing protease

We report here the first three-dimensional structure of the D1 C-terminal processing protease (D1P), which is encoded by the ctpA gene. This enzyme removes the C-terminal extension of the D1 polypeptide of photosystem II of oxygenic photosynthesis. Proteolytic processing is necessary to allow the light driven assembly of the tetranuclear manganese cluster, which is responsible for photosynthetic water oxidation. The X-ray structure of the Scenedesmus obliquus enzyme has been determined at 1.8 A resolution using the multiwavelength anomalous dispersion method. The enzyme is monomeric and is composed of three folding domains. The middle domain is topologically homologous to known PDZ motifs and is proposed to be the site at which the substrate C-terminus binds. The remainder of the substrate likely extends across the face of the enzyme, interacting at its scissile bond with the enzyme active site Ser 372 / Lys 397 catalytic dyad, which lies at the center of the protein at the interface of the three domains.     

Crystal structure of human cathepsin S.

We have determined the 2.5 A structure (Rcryst = 20.5%, Rfree = 28.5%) of a complex between human cathepsin S and the potent, irreversible inhibitor 4-morpholinecarbonyl-Phe-hPhe-vinyl sulfone-phenyl. Noncrystallographic symmetry averaging and other density modification techniques were used to improve electron density maps which were nonoptimal due to systematically incomplete data. Methods that reduce the number of parameters were implemented for refinement. The refined structure shows cathepsin S to be similar to related cysteine proteases such as papain and cathepsins K and L. As expected, the covalently-bound inhibitor is attached to the enzyme at Cys 25, and enzyme binding subsites S3-S1' are occupied by the respective inhibitor substituents. A somewhat larger S2 pocket than what is found in similar enzymes is consistent with the broader specificity of cathepsin S at this site, while Lys 61 in the S3 site may offer opportunities for selective inhibition of this enzyme. The presence of Arg 137 in the S1' pocket, and proximal to Cys 25 may have implications not only for substrate specificity C-terminal to the scissile bond, but also for catalysis.     

Crystal structure of bacteriophage T4 5' nuclease in complex with a branched DNA reveals how flap endonuclease-1 family nucleases bind their substrates

Bacteriophage T4 RNase H, a flap endonuclease-1 family nuclease, removes RNA primers from lagging strand fragments. It has both 5' nuclease and flap endonuclease activities. Our previous structure of native T4 RNase H (PDB code 1TFR) revealed an active site composed of highly conserved Asp residues and two bound hydrated magnesium ions. Here, we report the crystal structure of T4 RNase H in complex with a fork DNA substrate bound in its active site. This is the first structure of a flap endonuclease-1 family protein with its complete branched substrate. The fork duplex interacts with an extended loop of the helix-hairpin-helix motif class 2. The 5' arm crosses over the active site, extending below the bridge (helical arch) region. Cleavage assays of this DNA substrate identify a primary cut site 7-bases in from the 5' arm. The scissile phosphate, the first bond in the duplex DNA adjacent to the 5' arm, lies above a magnesium binding site. The less ordered 3' arm reaches toward the C and N termini of the enzyme, which are binding sites for T4 32 protein and T4 45 clamp, respectively. In the crystal structure, the scissile bond is located within the double-stranded DNA, between the first two duplex nucleotides next to the 5' arm, and lies above a magnesium binding site. This complex provides important insight into substrate recognition and specificity of the flap endonuclease-1 enzymes.     

Serpins of oat (Avena sativa) grain with distinct reactive centres and inhibitory specificity

Most proteinase inhibitors from plant seeds are assumed to contribute to broad-spectrum protection against pests and pathogens. In oat (Avena sativa L.) grain the main serine proteinase inhibitors were found to be serpins, which utilize a unique mechanism of irreversible inhibition. Four distinct inhibitors of the serpin superfamily were detected by native PAGE as major seed albumins and purified by thiophilic adsorption and anion exchange chromatography. The four serpins OSZa-d are the first proteinase inhibitors characterized from this cereal. An amino acid sequence close to the blocked N-terminus, a reactive centre loop sequence, and the second order association rate constant (ka') for irreversible complex formation with pancreas serine proteinases at 24 degrees C were determined for each inhibitor. OSZa and OSZb, both with the reactive centre scissile bond P1-P1' Thr downward arrow Ser, were efficient inhibitors of pancreas elastase (ka' > 105M-1 s-1). Only OSZb was also an inhibitor of chymotrypsin at the same site (ka' = 0.9 x 105M-1 s-1). OSZc was a fast inhibitor of trypsin at P1-P1' Arg downward arrow Ser (ka' = 4 x 106M-1 s-1); however, the OSZc-trypsin complex was short-lived with a first order dissociation rate constant kd = 1.4 x 10-4 s-1. OSZc was also an inhibitor of chymotrypsin (ka' > 106M-1 s-1), presumably at the overlapping site P2-P1 Ala downward arrow Arg, but > 90% of the serpin was cleaved as substrate. OSZd was cleaved by chymotrypsin at the putative reactive centre bond P1-P1' Tyr downward arrow Ser, and no inhibition was detected. Together the oat grain serpins have a broader inhibitory specificity against digestive serine proteinases than represented by the major serpins of wheat, rye or barley grain. Presumably the serpins compensate for the low content of reversible inhibitors of serine proteinases in oats in protection of the grain against pests or pathogens.     

Refined crystal structure of the complex of subtilisin BPN' and Streptomyces subtilisin inhibitor at 1.8 A resolution

The crystal structure of subtilisin BPN' complexed with a proteinaceous inhibitor SSI (Streptomyces subtilisin inhibitor) was refined at 1.8 A resolution to an R-factor of 0.177 with a root-mean-square deviation from ideal bond lengths of 0.014 A. The work finally established that the SSI-subtilisin complex is a Michaelis complex with a distance between the O gamma of active Ser221 and the carbonyl carbon of the scissile peptide bond being an intermediate value between a covalent bond and a van der Waals' contact, 2.7 A. This feature, as well as the geometry of the catalytic triad and the oxyanion hole, is coincident with that found in other highly refined crystal structures of the complex of subtilisin Novo, subtilisin Carlsberg, bovine trypsin or Streptomyces griseus protease B with their proteinaceous inhibitors. The enzyme-inhibitor beta-sheet interaction is composed of two separate parts: that between the P1-P3 residues of SSI and the 125-127 chain segment (the "S1-3 site") of subtilisin and that between the P4-P6 residues of SSI and th 102-104 chain segment (the "S4-6 site") of subtilisin. The latter beta-interaction is unique to subtilisin. In contrast, the beta-sheet interaction previously found in the complex of subtilisin Novo and chymotrypsin inhibitor 2 or in the complex of subtilisin Carlsberg and Eglin C is distinct from the present complex in that the two types of beta-interactions are not separate. As for the flexibility of the molecules comprising the present complex, the following observations were made by comparing the B-factors for free and complexed SSI and comparing those for free and complexed subtilisin BPN'. The rigidification of the component molecules upon complex formation occurs in a very localized region: in SSI, the "primary" and "secondary" contact regions and the flanking region; in subtilisin BPN', the S1-3 and S4-6 sites and the flanking region.     

Human Ras converting enzyme endoproteolytic specificity at the P2' and P3' positions of K-Ras-derived peptides

The membrane associated endoprotease, hRCE1, is responsible for one step in Ras membrane localization. The "CAAX" sequence at the C-terminal of farnesylated Ras proteins is cleaved by hRCE1 to yield an AAX tri-peptide. We found that an 8-aa K-Ras-derived "CAA" peptide, KSKTKC(farnesyl)VI, was a better substrate for hRCE1 than a KSKTKC(f)VIM "CAAX" peptide. When we examined hRCE1 activity on the same K-Ras core peptide with H-Ras (VLS) or N-Ras (VVM) C-terminal AAX sequences, we also found that in each case, the CAA peptides were better hRCE1 substrates. For each peptide set we examined, the P2' (A) and P3' (X) positions appeared independent in influencing hRCE1 activity on peptide substrates. We found that at the P3' position, methionine was better than serine; while at the P2' position, isoleucine and valine were better than leucine. Additionally, we found that a similar noncleaved peptide (modified at P'2 with a nitrophenyl group) could act as a competitive inhibitor of the reaction. Thus, hRCE1 has important functional interaction with the P2' and P3' substrate positions in addition to the farnesylated cysteine at the scissile bond site. This data could be useful in design of peptidomimetic inhibitors of hRCE1. Such inhibitors may be useful in treatment of cancer and inflammatory disease.     

Structure-Function Analysis of Staphylococcus aureus Amidase Reveals the Determinants of Peptidoglycan Recognition and Cleavage

The bifunctional major autolysin AtlA of Staphylococcus aureus cleaves the bacterium''s peptidoglycan network (PGN) at two distinct sites during cell division. Deletion of the enzyme results in large cell clusters with disordered division patterns, indicating that AtlA could be a promising target for the development of new antibiotics. One of the two functions of AtlA is performed by the N-acetylmuramyl-l-alanine amidase AmiA, which cleaves the bond between the carbohydrate and the peptide moieties of PGN. To establish the structural requirements of PGN recognition and the enzymatic mechanism of cleavage, we solved the crystal structure of the catalytic domain of AmiA (AmiA-cat) in complex with a peptidoglycan-derived ligand at 1.55 Å resolution. The peptide stem is clearly visible in the structure, forming extensive contacts with protein residues by docking into an elongated groove. Less well defined electron density and the analysis of surface features indicate likely positions of the carbohydrate backbone and the pentaglycine bridge. Substrate specificity analysis supports the importance of the pentaglycine bridge for fitting into the binding cleft of AmiA-cat. PGN of S. aureus with l-lysine tethered with d-alanine via a pentaglycine bridge is completely hydrolyzed, whereas PGN of Bacillus subtilis with meso-diaminopimelic acid directly tethered with d-alanine is not hydrolyzed. An active site mutant, H370A, of AmiA-cat was completely inactive, providing further support for the proposed catalytic mechanism of AmiA. The structure reported here is not only the first of any bacterial amidase in which both the PGN component and the water molecule that carries out the nucleophilic attack on the carbonyl carbon of the scissile bond are present; it is also the first peptidoglycan amidase complex structure of an important human pathogen.     

Extended interactions with prothrombinase enforce affinity and specificity for its macromolecular substrate

The specific action of serine proteinases on protein substrates is a hallmark of blood coagulation and numerous other physiological processes. Enzymic recognition of substrate sequences preceding the scissile bond is considered to contribute dominantly to specificity and function. We have investigated the contribution of active site docking by unique substrate residues preceding the scissile bond to the function of prothrombinase. Mutagenesis of the authentic P(1)-P(3) sequence in prethrombin 2/fragment 1.2 yielded substrate variants that could be converted to thrombin by prothrombinase. Proteolytic activation was also observed with a substrate variant containing the P(1)-P(3) sequence found in a coagulation zymogen not known to be activated by prothrombinase. Lower rates of activation of the variants derived from a decrease in maximum catalytic rate but not in substrate affinity. Replacement of the P(1) residue with Gln yielded an uncleavable derivative that retained the affinity of the wild type substrate for prothrombinase but did not engage the active site of the enzyme. Thus, active site docking of the substrate contributes to catalytic efficiency, but it is does not determine substrate affinity nor does it fully explain the specificity of prothrombinase. Therefore, extended interactions between prothrombinase and substrate regions removed from the cleavage site drive substrate affinity and enforce the substrate specificity of this enzyme complex.     

Crystal structures of thrombin complexed to a novel series of synthetic inhibitors containing a 5,5-trans-lactone template.

The binding modes of four active site-directed, acylating inhibitors of human alpha-thrombin have been determined using X-ray crystallography. These inhibitors (GR157368, GR166081, GR167088, and GR179849) are representatives of a series utilizing a novel 5, 5-trans-lactone template to specifically acylate Ser195 of thrombin, resulting in an acyl complex. In each case the crystal structure of the complex reveals a binding mode which is consistent with the formation of a covalent bond between the ring-opened lactone of the inhibitor and residue Ser195. Improvements in potency and selectivity of these inhibitors for thrombin are rationalized on the basis of the observed protein/inhibitor interactions identified in these complexes. Occupation of the thrombin S2 and S3 pockets is shown to be directly correlated with improved binding and a degree of selectivity. The binding mode of GR179849 to thrombin is compared with the thrombin/PPACK complex [Bode, W., Turk, D., and Karshikov, A. (1992) Protein Sci. 1, 426-471] as this represents the archetypal binding mode for a thrombin inhibitor. This series of crystal structures is the first to be reported of synthetic, nonpeptidic acylating inhibitors bound to thrombin and provides details of the molecular recognition features that resulted in nanomolar potency.     

Proteome-derived, database-searchable peptide libraries for identifying protease cleavage sites

We introduce human proteome-derived, database-searchable peptide libraries for characterizing sequence-specific protein interactions. To identify endoprotease cleavage sites, we used peptides in such libraries with protected primary amines to simultaneously determine sequence preferences on the N-terminal (nonprime P) and C-terminal (prime P') sides of the scissile bond. Prime-side cleavage products were tagged with biotin, isolated and identified by tandem mass spectrometry, and the corresponding nonprime-side sequences were derived from human proteome databases using bioinformatics. Identification of hundreds to over 1,000 individual cleaved peptides allows the consensus protease cleavage site and subsite cooperativity to be readily determined from P6 to P6'. For the highly specific GluC protease, >95% of the 558 cleavage sites identified displayed the canonical selectivity. For the broad-specificity matrix metalloproteinase 2, >1,200 peptidic cleavage sites were identified. Profiling of HIV protease 1, caspase 3, caspase 7, cathepsins K and G, elastase and thrombin showed that this approach is broadly applicable to all mechanistic classes of endoproteases.