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Assembly line polyketide synthases (PKSs) are large multimodular enzymes responsible for the biosynthesis of diverse antibiotics in bacteria. Structural and mechanistic analysis of these megasynthases can benefit from the discovery of reagents that recognize individual domains or linkers in a site-specific manner. Monoclonal antibodies not only have proven themselves as premier tools in analogous applications but also have the added benefit of constraining the conformational flexibility of their targets in unpredictable but often useful ways. Here we have exploited a library based on the naïve human antibody repertoire to discover a F(ab) (3A6) that recognizes the terminal thioesterase (TE) domain of the 6-deoxyerythronolide B synthase with high specificity. Biochemical assays were used to verify that 3A6 binding does not inhibit enzyme turnover. The co-crystal structure of the TE–3A6 complex was determined at 2.45 Å resolution, resulting in atomic characterization of this protein–protein recognition mechanism. F(ab) binding had minimal effects on the structural integrity of the TE. In turn, these insights were used to interrogate via small-angle X-ray scattering the solution-phase conformation of 3A6 complexed to a catalytically competent PKS module and bimodule. Altogether, we have developed a high-affinity monoclonal antibody tool that recognizes the TE domain of the 6-deoxyerythronolide B synthase while maintaining its native function.

Herpesviruses rely on a homodimeric protease for viral capsid maturation. A small molecule, DD2, previously shown to disrupt dimerization of Kaposi’s sarcoma-associated herpesvirus protease (KSHV Pr) by trapping an inactive monomeric conformation and two analogues generated through carboxylate bioisosteric replacement (compounds 2 and 3) were shown to inhibit the associated proteases of all three human herpesvirus (HHV) subfamilies (α, β, and γ). Inhibition data reveal that compound 2 has potency comparable to or better than that of DD2 against the tested proteases. Nuclear magnetic resonance spectroscopy and a new application of the kinetic analysis developed by Zhang and Poorman [Zhang, Z. Y., Poorman, R. A., et al. (1991) J. Biol. Chem. 266, 15591–15594] show DD2, compound 2, and compound 3 inhibit HHV proteases by dimer disruption. All three compounds bind the dimer interface of other HHV proteases in a manner analogous to binding of DD2 to KSHV protease. The determination and analysis of cocrystal structures of both analogues with the KSHV Pr monomer verify and elaborate on the mode of binding for this chemical scaffold, explaining a newly observed critical structure–activity relationship. These results reveal a prototypical chemical scaffold for broad-spectrum allosteric inhibition of human herpesvirus proteases and an approach for the identification of small molecules that allosterically regulate protein activity by targeting protein–protein interactions.

Cruzain is a member of the papain/cathepsin L family of cysteine proteases, and the major cysteine protease of the protozoan Trypanosoma cruzi, the causative agent of Chagas disease. We report an autoinduction methodology that provides soluble cruzain in high yields (>30 mg/L in minimal medium). These increased yields provide sufficient quantities of active enzyme for use in nuclear magnetic resonance (NMR)-based ligand mapping. Using circular dichroism and NMR spectroscopy, we also examined the solution-state structural dynamics of the enzyme in complex with a covalently bound vinyl sulfone inhibitor (K777). We report the backbone amide and side chain carbon chemical shift assignments of cruzain in complex with K777. These resonance assignments were used to identify and map residues located in the substrate binding pocket, including the catalytic Cys25 and His162. Selective [(15)N]Cys, [(15)N]His, and [(13)C]Met labeling was performed to quickly assess cruzain-ligand interactions for a set of eight low-molecular weight compounds exhibiting micromolar binding or inhibition. Chemical shift perturbation mapping verified that six of the eight compounds bind to cruzain at the active site. Three different binding modes were delineated for the compounds, namely, covalent, noncovalent, and noninteracting. These results provide examples of how NMR spectroscopy can be used to screen compounds for fast evaluation of enzyme-inhibitor interactions to facilitate lead compound identification and subsequent structural studies.

The structure of HIV protease (HIV Pr) bound to JE-2147 (also named AG1776 or KNI-764) is determined here to 1.09 A resolution. This highest-resolution structure for HIV Pr allows refinement of anisotropic displacement parameters (ADPs) for all atoms. Clustering based on the directional information in ADPs defines two sets of subdomains such that within each set, subdomains undergo similar anisotropic motion. These sets are (a) the core of monomer A grouped with both substrate-binding flaps and (b) the core of monomer B coupled to both catalytic aspartates (25A/B). The four-stranded beta-sheet (1-4 A/B and 95-99 A/B) that forms a significant part of the dimer interface exhibits large anisotropic amplitudes that differ from those of the other sets of subdomains. JE-2147 is shown here to be a picomolar inhibitor (K(i) = 41 +/- 18 pM). The structure is used to interpret the mechanism of association of JE-2147, a second-generation inhibitor for which binding is enthalpically driven, with respect to first-generation inhibitors for which binding is predominantly entropically driven [Velazquez-Campoy, A., et al. (2001) Arch. Biochem. Biophys. 390, 169-175]. Relative to the entropically driven inhibitor complexes, the JE-2147-HIV Pr complex exhibits an approximately 0.5 A movement of the substrate flaps in toward the substrate, suggesting a more compatible enthalpically driven association. Domains of the protease identified by clustering of ADPs also suggest a model of enthalpy-entropy compensation for all HIV Pr inhibitors in which dynamic coupling of the flaps is offset by an increased level of motion of the beta-sheet domain of the dimer interface (1-4 A/B and 95-99 A/B).

The design and synthesis of a novel iodine-labile serine protease inhibitor was realized by the use of an ecotin analogue containing allylglycine at position 84 in lieu of methionine. Allylglycine- containing ecotins were synthesized by in vitro translation of the ecotin gene containing an engineered nonsense codon (TAG) at the positions of interest. A misacylated suppressor tRNA activated with the unnatural amino acid allylglycine was employed for the suppression of the nonsense codons in a cell-free protein biosynthesizing system, permitting the elaboration of ecotin analogues containing allyglycine at the desired sites. The derived ecotin analogues were capable of inhibiting bovine trypsin with inhibitory constants (Kis) comparable to that of wild-type ecotin. Iodine treatment of ecotin analogue Met84 A Gly resulted in the deactivation of ecotin, caused by peptide backbone cleavage at its P1 reactive site. Upon iodine treatment, active trypsin could be released from the protein complex with ecotin analogue Met84 A Gly. This constitutes a novel strategy for modulation of serine protease activity and more generally for alteration of protein-protein interaction by a simple chemical reagent. We have recently described a novel strategy for site- specific cleavage of proteins (1). This technique allows cleavage of the protein amide backbone at a single, pre- determined site with a simple chemical reagent. The strategy involves the use of protein analogues containing the non- native amino acid allylglycine ( A Gly) 1 at a specific site. This amino acid can be attached to suppressor tRNACUA as an activated ester by chemical misacylation (2-9); readthrough of a nonsense (UAG) codon (10-15) at a predetermined site by the use of a suitable protein synthesizing system afforded full-length protein containing A Gly at that site. Subsequent iodine treatment resulted in protein cleavage at the expected site through a presumed iodolactone intermediate. This strategy has been employed successfully to activate a trypsin from its zymogen form (trypsinogen) by chemical removal of the activation peptide, suggesting the compatibility of this technique with the integrity of functional proteins ( 16).

The crystal structure of fiddler crab collagenase complexed with the dimeric serine protease inhibitor ecotin at 2.5 A resolution reveals an extended cleft providing binding sites for at least 11 contiguous substrate residues. Comparison of the positions of nine intermolecular main chain hydrogen bonding interactions in the cleft, with the known sequences at the cleavage site of type I collagen, suggests that the protease binding loop of ecotin adopts a conformation mimicking that of the cleaved strand of collagen. A well-defined groove extending across the binding surface of the enzyme readily accommodates the two other polypeptide chains of the triple-helical substrate. These observations permit construction of a detailed molecular model for collagen recognition and cleavage by this invertebrate serine protease. Ecotin undergoes a pronounced internal structural rearrangement which permits binding in the observed conformation. The capacity for such rearrangement appears to be a key determinant of its ability to inhibit a wide range of serine proteases. Collagen is an essential component of the extracellular matrix in higher organisms, and its degradation by specific proteases is a key step in connective tissue remodeling (Woessner, 1991). In vertebrate species, the family of enzymes known as the matrix metalloproteases are respon- sible for collagen cleavage. These enzymes possess an N-terminal 80-amino acid propeptide which is cleaved to generate mature enzyme, a catalytic domain of some 180

Crab collagenolytic serine protease 1 efficiently cleaves peptide bonds directly C-terminal to basic, polar, and hydrophobic amino acids. The crystal structure of this enzyme complexed to the protein inhibitor ecotin at 2.5 A resolution reveals a large primary binding pocket punctuated on one wall by the side chain of aspartate-226. Removal or relocation of this negatively charged group by site-directed mutagenesis generates variant enzymes which retain very high activities toward selected substrates. Full retention of activity toward hydrophobic substrates in collagenase D226G is accompanied by a 10-100-fold reduction in k(cat)/Km toward basic residues. In contrast, restoration of the negative charge in a trypsin-like position in collagenase D226G/G189D regenerates nearly full activity toward basic substrates while introducing a 5-fold decrease in k(cat)/Km toward hydrophobic amino acids. These results imply that the collagenase S1 pocket has multiple distinct binding sites for different amino acid side chains, a suggestion supported by molecular modeling studies based on the crystal structure. The ease of specificity modification in the primary binding site of this serine protease parallels similar observations with the bacterial enzymes alpha-lytic protease and subtilisin, and stands in sharp distinction to the extensive mutagenesis required to alter specificity in trypsin.

Strain is eliminated as a factor in hydrolysis of the scissile peptide bond by human immunodeficiency virus (HIV)-1 and simian immunodeficiency virus (SIV), based on the first eight complexes of products of hydrolysis with the enzymes. The carboxyl group generated at the scissile bond interacts with both catalytic aspartic acids. The structures directly suggest the interactions of the gemdiol intermediate with the active site. Based on the structures, the nucleophilic water is displaced stereospecifically by substrate binding toward one catalytic aspartic acid, while the scissile carbonyl becomes hydrogen bonded to the other catalytic aspartic acid in position for hydrolysis. Crystal structures for two N-terminal (P) products and two C-terminal (Q) products provide unambiguous density for the ligands at 2.2-2.6 A resolution and 17-21% R factors. The N-terminal product, Ac-S-L-N-F/, overlaps closely with the N-terminal sequences of peptidomimetic inhibitors bound to the protease. Comparison of the two C-terminal products, /F-L-E-K and /F(NO2)-E-A-Nle-S, indicates that the P2' residue is highly constrained, while the positioning of the P1' and P3' residues are sequence dependent.

The three-dimensional structures of complexes of trypsin N143H, E151H bound to ecotin A86H are determined at 2.0 A resolution via X-ray crystallography in the absence and presence of the transition metals Zn2+, Ni2+, and Cu2+. The binding site for these transition metals was constructed by substitution of key amino acids with histidine at the trypsin-ecotin interface in the S2'/P2' pocket. Three histidine side chains, two on trypsin at positions 143 and 151 and one on ecotin at position 86, anchor the metals and provide extended catalytic recognition for substrates with His in the P2' pocket. Comparisons of the three-dimensional structures show the different geometries that result upon the binding of metal in the engineered tridentate site and suggest a structural basis for the kinetics of the metal-regulated catalysis. Of the three metals, the binding of zinc results in the most favorable binding geometry, not dissimilar to those observed in naturally occurring zinc binding proteins.

Much of the catalytic power of trypsin is derived from the unusual buried, charged side chain of Aspl02. A polar cave provides the stabilization for maintaining the buried charge, and it features the conserved amino acid Ser214 adjacent to Aspl02. Ser214 has been replaced with Ala, Glu, and Lys in rat anionic trypsin, and the consequences of these changes have been determined. Three-dimensional structures of the Glu and Lys variant trypsins reveal that the new 214 side chains are buried. The 2.2-A crystal structure (R = 0.150) of trypsin S214K shows that Lys214 occupies the position held by Ser214 and a buried water molecule in the buried polar cave. Lys214-Nf is solvent inaccessible and is less than 5 A from the catalytic Aspl02. The side chain of Glu214 (2.8 A, R = 0.168) in trypsin S214E shows two conformations. In the major one, the Glu carboxylate in S214E forms a hydrogen bond with Aspl02. Analytical isoelectrofocusing results show that trypsin S214K has a significantly different isoelectric point than trypsin, corresponding to an additional positive charge. The kinetic parameter kat demonstrates that, compared to trypsin, S214K has 1% of the catalytic activity on a tripeptide amide substrate and S214E is 44% as active. Electrostatic potential calculations provide corroboration of the charge on Lys214 and are consistent with the kinetic results, suggesting that the presence of Lys214 has disturbed the electrostatic potential of Aspl02. Catalysis in the serine proteases is attributed to a triad of amino acids which orchestrate an attack by serine (Ser195 in trypsin) on the substrate carbonyl carbon. The nucleophilicity of the serine is enhanced by an adjacent histidine (His57 in trypsin) which serves as a general base and subsequently mediates transfer of a proton to the leaving group. The buried aspartic acid (Asp102 in trypsin) forms a hydrogen bond to the histidine, and its negative charge likely stabilizes the charged imidazole of the histidine which is present in the transition state (Warshel & Russell, 1986). Replacement of this aspartic acid with a neutral amino acid in trypsin (Asn) (Craik et al., 1987) and subtilisin (Ala) (Carter & Wells, 1988) reduced kat by approximately 104 for both enzymes and provided evidence for the role of aspartic acid in the catalytic triad. In agreement with theory, these experiments suggested the importance of the negative charge on the aspartic acid. In the experiments reported here, we attempt to change the electric field at the active site of rat anionic trypsin (hereafter called trypsin) with minimal structural disruption of the

Severe acute respiratory syndrome (SARS) is an emerging infectious disease associated with a high rate of mortality. The SARS-associated coronavirus (SARS-CoV) has been identified as the etiological agent of the disease. Although public health procedures have been effective in combating the spread of SARS, concern remains about the possibility of a recurrence. Various approaches are being pursued for the development of efficacious therapeutics. One promising approach is to develop small molecule inhibitors of the essential major polyprotein processing protease 3Clpro. Here we report a complete description of the tetrapeptide substrate specificity of 3Clpro using fully degenerate peptide libraries consisting of all 160,000 possible naturally occurring tetrapeptides. The substrate specificity data show the expected P1-Gln P2-Leu specificity and elucidate a novel preference for P1-His containing substrates equal to the expected preference for P1-Gln. These data were then used to develop optimal substrates for a high-throughput screen of a 2000 compound small-molecule inhibitor library consisting of known cysteine protease inhibitor scaffolds. We also report the 1.8 A X-ray crystal structure of 3Clpro bound to an irreversible inhibitor. This inhibitor, an alpha,beta-epoxyketone, inhibits 3Clpro with a k3/Ki of 0.002 microM(-1) s(-1) in a mode consistent with the substrate specificity data. Finally, we report the successful rational improvement of this scaffold with second generation inhibitors. These data provide the foundation for a rational small-molecule inhibitor design effort based upon the inhibitor scaffold identified, the crystal structure of the complex, and a more complete understanding of P1-P4 substrate specificity.

Distinct mechanisms have evolved to regulate the function of proteolytic enzymes. Viral proteases in particular have developed novel regulatory mechanisms, presumably due to their comparatively rapid life cycles and responses to constant evolutionary pressure. Herpesviruses are a family of human pathogens that require a viral protease with a concentration-dependent zymogen activation involving folding of two alpha-helices and activation of the catalytic machinery, which results in formation of infectious virions. Kaposi's sarcoma-associated herpesvirus protease (KSHV Pr) is unique among the herpesvirus proteases in possessing an autolysis site in the dimer interface, which removes the carboxyl-terminal 27 amino acids comprising an alpha-helix adjacent to the active site. Truncation results in the irreversible loss of dimerization and concomitant inactivation. We characterized the conformational and functional differences between the active dimer, inactive monomer, and inactive truncated protease to determine the different protease regulatory mechanisms that control the KSHV lytic cycle. Circular dichroism revealed a loss of 31% alpha-helicity upon dimer dissociation. Comparison of the full-length and truncated monomers by NMR showed differences in 21% of the protein structure, mainly located adjacent to the dimer interface, with little perturbation of the overall protein upon truncation. Fluorescence polarization and active site labeling, with a transition state mimetic, characterized the functional effects of these conformational changes. Substrate turnover is abolished in both the full-length and truncated monomers; however, substrate binding remained intact. Disruption of the helix 6 interaction with the active site oxyanion loop is therefore used in two independent regulatory mechanisms of proteolytic activity.

The C[bond]N coupling constants centered at the C(epsilon 1) and C(delta 2) carbons in histidine residues depend on the protonation state and tautomeric form of the imidazole ring, making them excellent indicators of pH or pK(a), and the ratio of the tautomeric states. In this paper, we demonstrate that the intensity ratios for the C(epsilon 1)-H and C(delta 2)-H cross-peaks measured with a constant time HSQC experiment without and with J(C[bond]N) amplitude modulation are determined by the ratios of the protonated and deprotonated forms and tautomeric states. This allows one to investigate the tautomeric state of histidines as well as their pK(a) in situations where changing the pH value by titration is difficult, for example, for in-cell NMR experiments. We apply this technique to the investigation of the bacterial protein NmerA and determine that the intracellular pH in the Escherichia coli cytoplasm is 7.1 +/- 0.1.

The primary specificity of papain-like cysteine proteases (family C1, clan CA) is determined by S2-P2 interactions. Despite the high amino acid sequence identities and structural similarities between cathepsins K and L, only cathepsin K is capable of cleaving interstitial collagens in their triple helical domains. To investigate this specificity, we have engineered the S2 pocket of human cathepsin K into a cathepsin L-like subsite. Using combinatorial fluorogenic substrate libraries, the P1-P4 substrate specificity of the cathepsin K variant, Tyr67Leu/Leu205Ala, was determined and compared with those of cathepsins K and L. The introduction of the double mutation into the S2 subsite of cathepsin K rendered the unique S2 binding preference of the protease for proline and leucine residues into a cathepsin L-like preference for bulky aromatic residues. Homology modeling and docking calculations supported the experimental findings. The cathepsin L-like S2 specificity of the mutant protein and the integrity of its catalytic site were confirmed by kinetic analysis of synthetic di- and tripeptide substrates as well as pH stability and pH activity profile studies. The loss of the ability to accept proline in the S2 binding pocket by the mutant protease completely abolished the collagenolytic activity of cathepsin K whereas its overall gelatinolytic activity remained unaffected. These results indicate that Tyr67 and Leu205 play a key role in the binding of proline residues in the S2 pocket of cathepsin K and are required for its unique collagenase activity.

The mechanism of herpesviral protease activation upon dimerization was studied using two independent spectroscopic assays augmented by directed mutagenesis. Spectroscopic changes, attributable to dimer interface conformational plasticity, were observed upon dimerization of Kaposi's sarcoma-associated herpesvirus protease (KSHV Pr). KSHV Pr's dissociation constant of 585 +/- 135 nM at 37 degrees C was measured by a concentration-dependent, 100-fold increase in specific activity to a value of 0.275 +/- 0.023 microM product min(-1) (microM enzyme)(-1). A 4 nm blue-shifted fluorescence emission spectrum and a 25% increase in ellipticity at 222 nm were detected by circular dichroism upon dimer association. This suggested enhanced hydrophobic packing within the dimer interface and/or core, as well as altered secondary structures. To better understand the structure-activity relationship between the monomer and the dimer, KSHV Pr molecules were engineered to remain monomeric via substitution of two separate residues within the dimer interface, L196 and M197. These mutants were proteolytically inactive while exhibiting the spectroscopic signature and thermal stability of wild type, dissociated monomers (T(M) = 75 degrees C). KSHV Pr conformational changes were found to be relevant in vivo, as the autoproteolytic inactivation of KSHV Pr at its dimer disruption site [Pray et al. (1999) J. Mol. Biol. 289, 197-203] was detected in viral particles from KSHV-infected cells. This characterization of structural plasticity suggests that the structure of the KSHV Pr monomer is stable and significantly different from its structure in the dimer. This structural uniqueness should be considered in the development of compounds targeting the dimer interface of KSHV Pr monomers.

The structure of Kaposi's sarcoma-associated herpesvirus protease (KSHV Pr), at 2.2 A resolution, reveals the active-site geometry and defines multiple possible target sites for drug design against a human cancer-producing virus. The catalytic triad of KSHV Pr, (Ser114, His46, and His157) and transition-state stabilization site are arranged as in other structurally characterized herpesviral proteases. The distal histidine-histidine hydrogen bond is solvent accessible, unlike the situation in other classes of serine proteases. As in all herpesviral proteases, the enzyme is active only as a weakly associated dimer (K(d) approximately 2 microM), and inactive as a monomer. Therefore, both the active site and dimer interface are potential targets for antiviral drug design. The dimer interface in KSHV Pr is compared with the interface of other herpesviral proteases. Two conserved arginines (Arg209), one from each monomer, are buried within the same region of the dimer interface. We propose that this conserved arginine may provide a destabilizing element contributing to the tuned micromolar dissociation of herpesviral protease dimers.

The Ni(II) complex of the tripeptide NH2-glycine-glycine-histidine-COOH (GGH) mediates efficient protein-protein cross-linking in the presence of oxidants such as oxone and monoperoxyphthalic acid (MMPP). Here we demonstrate that GGH fused to the amino terminus of a protein can still support cross-linking. The tripeptide was expressed at the amino terminus of ecotin, a dimeric macromolecular serine protease inhibitor found in the periplasm of Escherichia coli. In the presence of Ni(OAc)2 and MMPP, GGH-ecotin is cross-linked to give a species that has an apparent molecular mass of a GGH-ecotin dimer with no observable protein degradation. The cross-linking reaction occurs between two ecotin proteins in a dimer complex. Furthermore, GGH-ecotin can be cross-linked to a serine protease target, trypsin, and the reaction is specific for proteins that interact with ecotin. The cross-linking reaction has been carried out on small peptides, and the reaction products have been analyzed by matrix-assisted laser desorption/ionization mass spectrometry. The target of the reaction is tyrosine, and the product is bityrosyl cross-links. The yield of the cross-linking is on the order of 15%. However, the reaction efficiency can be increased 4-fold by a single amino acid substitution in the carboxy terminus of ecotin that places an engineered tyrosine within 5 A of a naturally occurring tyrosine. This cross-linking methodology allows for the protein cross-linking reagent to be encoded for at the DNA level, thus circumventing the need for posttranslational modification.

Rigid body rotation of five domains and movements within their interfacial joints provide a rational context for understanding why HIV protease mutations that arise in drug resistant strains are often spatially removed from the drug or substrate binding sites. Domain motions associated with substrate binding in the retroviral HIV-1 and SIV proteases are identified and characterized. These motions are in addition to closure of the flaps and result from rotations of approximately 6-7 degrees at primarily hydrophobic interfaces. A crystal structure of unliganded SIV protease (incorporating the point mutation Ser 4 His to stabilize the protease against autolysis) was determined to 2.0 A resolution in a new space group, P3221. The structure is in the most "open" conformation of any retroviral protease so far examined, with six residues of the flaps disordered. Comparison of this and unliganded HIV structures, with their respective liganded structures by difference distance matrixes identifies five domains of the protease dimer that move as rigid bodies against one another: one terminal domain encompassing the N- and C-terminal beta sheet of the dimer, two core domains containing the catalytic aspartic acids, and two flap domains. The two core domains rotate toward each other on substrate binding, reshaping the binding pocket. We therefore show that, for enzymes, mutations at interdomain interfaces that favor the unliganded form of the target active site will increase the off-rate of the inhibitor, allowing the substrate greater access for catalysis. This offers a mechanism of resistance to competitive inhibitors, especially when the forward enzymatic reaction rate exceeds the rate of substrate dissociation.

Recognition for proteolysis by trypsin depends almost exclusively on tight binding of arginine or lysine side chains by the primary substrate specificity pocket. Although extended subsite interactions are important for catalysis, the majority of binding energy is localized in the P1 pocket. Analysis of the interactions of trypsin with the P1 residue of the bound inhibitors ecotin and bovine pancreatic trypsin inhibitor suggested that the mutation D189S would improve metal-assisted trypsin N143H, E151H specificity toward peptides that have a Tyr at P1 and a His at P2'. In the presence of transition metals, the catalytic efficiency of the triple mutant Tn N143H, E151H, D189S improved toward the tyrosine-containing peptide AGPYAHSS. Trypsin N143H, E151H, D189S exhibits a 25-fold increase in activity with nickel and a 150-fold increase in activity with zinc relative to trypsin N143H, E151H on this peptide. In addition, activity of trypsin N143H, E151H, D189S toward an arginine-containing peptide, YLVGPRGHFYDA, is enhanced by copper, nickel, and zinc. With this substrate, copper yields a 30-fold, nickel a 70-fold, and zinc a 350-fold increase in activity over background hydrolysis without metal. These results demonstrate that the engineering of multiple substrate binding subsites in trypsin can be used to delocalize protease specificity by increasing relative substrate binding contributions from alternate engineered subsites.

Histidine 57 of the catalytic triad of trypsin was replaced with alanine to determine whether the resulting variant would be capable of substrate-assisted catalysis [Carter, P., & Wells, J. A. (1987) Science 237, 394-9]. A 2.5-fold increase in kcat/Km was observed on tri- or tetrapeptide substrates containing p-nitroanilide leaving groups and histidine at P2. In contrast, hydrolysis of peptide substrates extending from P6 to P6' is improved 70-300-fold by histidine in the P2 or P1' position. This preference creates new protease specificities for sequences HR decreases, R decreases H, HK decreases, and K decreases H. The ability of histidine from either the P2 or the P1' position of substrate to participate in catalysis emphasizes the considerable variability of proteolytically active orientations which can be assumed by the catalytic triad. Trypsin H57A is able to hydrolyze fully folded ornithine decarboxylase with complete specificity at a site containing the sequence HRH. Trypsin H57A was compared to enteropeptidase in its ability to cleave a propeptide from trypsinogen. Trypsin H57A cleaved the propeptide of a variant trypsinogen containing an introduced FPVDDDHR cleavage site only 100-fold slower than enteropeptidase cleaved trypsinogen. The selective cleavage of folded proteins suggests that trypsin H57A can be used for specific peptide and protein cleavage. The extension of substrate-assisted catalysis to the chymotrypsin family of proteolytic enzymes indicates that it may be possible to apply this strategy to a wide range of serine proteases and thereby develop various unique specificities for peptide and protein hydrolysis.

Histidine substrate specificity has been engineered into trypsin by creating metal binding sites for Ni2+ and Zn2+ ions. The sites bridge the substrate and enzyme on the leaving-group side of the scissile bond. Application of simple steric and geometric criteria to a crystallographically derived enzyme-substrate model suggested that histidine specificity at the P2' position might be achieved by a tridentate site involving amino acid residues 143 and 151 of trypsin. Trypsin N143H/E151H hydrolyzes a P2'-His-containing peptide (AGPYAHSS) exclusively in the presence of nickel or zinc with a high level of catalytic efficiency. Since cleavage following the tyrosine residue is normally highly disfavored by trypsin, this result demonstrates that a metal cofactor can be used to modulate specificity in a designed fashion. The same geometric criteria applied in the primary S1 binding pocket suggested that the single-site mutation D189H might effect metal-dependent His specificity in trypsin. However, kinetic and crystallographic analysis of this variant showed that the design was unsuccessful because His189 rotates away from substrate causing a large perturbation in adjacent surface loops. This observation suggests that the reason specificity modification at the trypsin S1 site requires extensive mutagenesis is because the pocket cannot deform locally to accommodate alternate P1 side chains. By taking advantage of the extended subsites, an alternate substrate specificity has been engineered into trypsin.

The specificity of trypsin for Arg- and Lys-containing substrates depends upon the presence of Asp189 at the base of the primary binding pocket. The crystal structure of anionic rat trypsin D189S complexed with BPTI reveals that removal of the aspartate side chain permits the binding of a well-ordered acetate ion in a similar position. The acetate makes polar interactions with Gly226, Tyr228, and several water molecules and is further accommodated by rotation of the Ser189 side chain out of the binding pocket. The carboxylate group of the acetate anion is oriented toward the substrate in a manner similar to that of Asp189 and Asp226 in wild-type trypsin and trypsin D189G/G226D. Evaluation of kinetic parameters for amide substrate cleavage by trypsin D189S shows that high concentrations of acetate increase the catalytic efficiency of the enzyme by as much as 300-fold. Under these conditions, the rate of substrate turnover toward a peptidylarginine amide substrate equals that of wild-type trypsin. These data demonstrate that the well-established requirement for a negatively charged moiety at the base of the trypsin specificity pocket may be fulfilled by a noncovalently bound ligand. The binding pocket of this variant maintains a trypsin-like conformation, explaining the inability of the mutant enzyme to efficiently hydrolyze chymotrypsin substrates possessing Phe in the P1 position.

A variant of the simian immunodeficiency virus protease (SIV PR), covalently bound to the inhibitor 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP), was crystallized. The structure of the inhibited complex was determined by X-ray crystallography to a resolution of 2.4 A and refined to an R factor of 19%. The variant, SIV PR S4H, was shown to diminish the rate of autolysis by at least 4-fold without affecting enzymatic parameters. The overall root mean square (rms) deviation of the alpha-carbons from the structure of HIV-1PR complexed with a peptidomimetic inhibitor (7HVP) was 1.16 A. The major differences are concentrated in three surface loops with rms differences between 1.2 and 2.1 A. For 60% of the molecule the rms deviation was only 0.6 A. The structure reveals one molecule of EPNP bound per protease dimer, a stoichiometry confirmed by mass spectral analysis. The epoxide moiety forms a covalent bond with either of the active site aspartic acids of the dimer, and the phenyl moiety occupies the P1 binding site. The EPNP nitro group interacts with Arg 8. This structure suggests a starting template for the design of nonpeptide-based irreversible inhibitors of the SIV and related HIV-1 and HIV-2 PRs.

The X-ray crystal structure of the copper complex of the rat trypsin mutant Arg96 to His96 (trypsin R96H) has been determined in order to ascertain the nature of the engineered metal-binding site and to understand the structural basis for the metal-induced enzymatic inhibition. In the structure, the catalytically essential His57 residue is reoriented out of the active-site pocket and forms a chelating, metal-binding site with residue His96. The copper is bound to the N epsilon 2 atoms of both histidine residues with Cu-N epsilon 2 = 2.2 A and N epsilon 2-Cu-N epsilon 2 = 89 degrees. The metal is clearly bound to a third ligand leading to a distorted square planar geometry at Cu. The X-ray results do not unambiguously yield the identity of this third ligand, but chemical data suggest that it is a deprotonated, chelating Tris molecule which was used as a carrier to solubilize the copper in alkaline solution (pH 8.0). Upon reorientation of His57, a unique water molecule moves into the active site and engages in hydrogen-bonding with Asp102-O delta 2 and His57-N delta 1. Except for small movements of the peptide backbone near His96, the remainder of the trypsin molecule is isostructural with the native enzyme. These data support the notion that the effective inhibition of catalytic activity by metal ions observed in trypsin R96H is indeed caused by a specific and reversible reorganization of the active site in the enzyme.

The dimeric nature of the HIV protease has been exploited to devise a novel mode of inhibiting the enzyme. The use of defective monomers or nonidentical subunits to exchange with wild-type homodimers produces catalytically defective heterodimers. Incubation of the HIVl or HIV2 protease with a 4-fold molar exccss of an inactive mutant of HIVl leads to 80 and 95% inhibition of enzyme activity, respectively. Incubating HIVl and H1V2 proteases at a 15 ratio results in a 50% reduction of activity of the mixed enzymes. The HIV 1 /H1V2 heterodimer was identified by ion-exchange HPLC. The heterodimer may display a disordered dimer interface, thereby affecting the catalytic potential of the enzyme. This mechanism of inactivation is an example of a dominant negative mutation that can obliterate the activity of a naturally occurring multisubunit enzyme. Furthermore, it provides an alternative to active-site-directed inhibitors for the development of antiviral agents that target the dimeric interface of the HIV protease.

A recombinant trypsin was designed whose catalytic activity can be regulated by varying the concentration of Cu2+ in solution. Substitution of Arg-96 with a His in rat trypsin (trypsin R96H) places a new imidazole group on the surface of the enzyme near the essential active-site His-57. The unique spatial orientation of these His side chains results in the formation of a stable, metal-binding site that chelates divalent first-row transition-metal ions. Occupancy of this site by a metal ion prevents the imidazole group of His-57 from participating as a general base in catalysis. As a consequence, the primary effect of the transition metal ion is to inhibit the esterase and amidase activities of trypsin R96H. The apparent Ki for this inhibition is in the micromolar range for copper, nickel, and zinc, the tightest binding being to Cu2+ at 21 microM. Trypsin R96H activity can be fully restored by removing the bound Cu2+ ion with EDTA. Multiple cycles of inhibition by Cu2+ ions and reactivation by EDTA demonstrate that reversible regulatory control has been introduced into the enzyme. These results describe a novel mode of inhibition of serine protease activity that may also prove applicable to other proteins.

Active site serine 195 of rat anionic trypsin was replaced with a cysteine by site-specific mutagenesis in order to determine if a thiol group could function as the catalytic nucleophile in serine protease active site environment. Two genetically modified rat thiol trypsins were generated; the first variant contained a single substitution of Ser195 with Cys (trypsin S195C) while the second variant contained the Ser195 to Cys as well as an Asp102 to Asn substitution (trypsin D102N,S195C) that more fully mimics the putative catalytic triad of papain. Both variants were expressed as his J signal peptide-trypsin fusion proteins to high levels under the control of the tac promoter. The mature forms of both variants were secreted into the periplasmic space of Escherichia coli. Trypsin S195C shows a low level of activity toward the activated ester substrate Z-Lys-pNP, while both trypsin S195C and trypsin D102N,S195C were active toward the fluorogenic tripeptide substrate Z-GPR-AMC. Esterase and peptidase activities of both thiol trypsin variants were inhibited by known Cys protease inhibitors as well as by specific trypsin inhibitors. The kcat of trypsin S195C was reduced by a factor of 6.4 x 10(5) relative to that of trypsin while the kcat of trypsin D102N,S195C was lowered by a factor of 3.4 x 10(7) with Z-GPR-AMC as substrate. Km values were unaffected. The loss of activity of trypsin D102N,S195C was partially attributed to an inappropriate Asn102-His57 interaction that precludes the formation of the catalytically competent His57-Cys195 ion pair although loss of the negative charge of D102 at the active site probably contributes to diminished activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Mast cell tryptase is a secretory granule associated serine protease with trypsin-like specificity released extracellularly during mast cell degranulation. To determine the full primary structure of the catalytic domain and precursor forms of tryptase and to gain insight into its mode of activation, we cloned cDNAs coding for the complete amino acid sequence of dog mast cell tryptase and a second, possibly related, serine protease. Using RNA from dog mastocytoma cells, we constructed a cDNA library in lambda gt 10. Screening of the library with an oligonucleotide probe based on the N-terminal sequence of tryptase purified from the same cell source allowed us to isolate and sequence overlapping clones coding for dog mast cell tryptase. The tryptase sequence includes the essential residues of the catalytic triad and an aspartic acid at the base of the putative substrate binding pocket that confers P1 Arg and Lys specificity on tryptic serine proteases. The apparent N-terminal signal/activation peptide terminates in a glycine. A glycine in this position has not been observed previously in serine proteases and suggests a novel mode of activation. Additional screening of the library with a trypsinogen cDNA led to the isolation and sequencing of a full-length clone apparently coding for the complete sequence of a second tryptic serine protease (DMP) which is only 53.4% identical with the dog tryptase sequence but which contains an apparent signal/activation peptide also terminating in a glycine. Thus, the proteases encoded by these cloned cDNAs may share a common mode of activation from N-terminally extended precursors.

A cDNA encoding rat cationic trypsinogen has been isolated by immunoscreening from a rat pancreas cDNA library. The protein encoded by this cDNA is highly basic and contains all of the structural features observed in trypsinogens. The amino acid sequence of rat cationic trypsinogen is 75% and 77% homologous to the two anionic rat trypsinogens. The homology of rat cationic trypsinogen to these anionic trypsinogens is lower than its homology to other mammalian cationic trypsinogens, suggesting that anionic and cationic trypsins probably diverged prior to the divergence of rodents and ungulates. The most unusual feature of this trypsinogen is the presence of an activation peptide containing five aspartic acid residues, in contrast to all other reported trypsinogen activation peptides which contain four acidic amino acid residues. Comparisons of cationic and anionic trypsins reveal that the majority of the charge changes occur in the C-terminal portion of the protein, which forms the substrate binding site. Several regions of conserved charge differences between cationic and anionic trypsins have been identified in this region, which may influence the rate of hydrolysis of protein substrates.

To test the role of Asp-189 which is located at the base of the substrate binding pocket in determining the specificity of trypsin toward basic substrates, this residue was replaced with a lysine residue by site-directed mutagenesis. Both rat trypsinogen and Lys-189 trypsinogen were expressed and secreted into the periplasmic space of Escherichia coli. The proteins were purified to homogeneity and activated by porcine enterokinase, and their catalytic activities were determined on natural and synthetic substrates. Lys-189 trypsin displayed no catalytic activity toward arginyl and lysyl substrates. Further, there was no compensatory change in specificity toward acidic substrates; no cleavage of aspartyl or glutamyl bonds was detected. Additional studies of substrate specificity involving gas-phase sequence analyses of digested natural substrates revealed an inherent but low chymotrypsin-like activity of trypsin. This activity was retained but modified by the Asp to Lys change at position 189. In addition to hydrolyzing phenylalanyl and tyrosyl peptide bonds, the mutant enzyme has the unique property of cleaving leucyl bonds. On the basis of computer graphic modeling studies of the Lys-189 side chain, it appears that the positively charged NH2 group is directed outside the substrate binding pocket. The resulting hydrophobic cavity may explain the altered substrate specificity of the mutant enzyme. The relatively low chymotrypsin-like activity of both recombinant enzymes may be due to distorted positioning of the scissile bond with respect to the catalytic triad rather than to the lack of sufficient interaction between the hydrophobic side chains and the substrate binding pocket of the enzyme.

We have determined the three-dimensional structures of engineered rat trypsins which mimic the active sites of two classes of cysteine proteases. The catalytic serine was replaced with cysteine (S195C) to test the ability of sulfur to function as a nucleophile in a serine protease environment. This variant mimics the cysteine trypsin class of thiol proteases. An additional mutation of the active site aspartate to an asparagine (D102N) created the catalytic triad of the papain-type cysteine proteases. Rat trypsins S195C and D102N,S195C were solved to 2.5 and 2.0 A, respectively. The refined structures were analyzed to determine the structural basis for the 106-fold loss of activity of trypsin S195C and the 108-fold loss of activity of trypsin D102N,S195C, relative to rat trypsin. The active site thiols were found in a reduced state in contrast to the oxidized thiols found in previous thiol protease structures. These are the first reported structures of serine proteases with the catalytic centers of sulfhydryl proteases. Structure analysis revealed only subtle global changes in enzyme conformation. The substrate binding pocket is unaltered, and active site amino acid 102 forms hydrogen bonds to H57 and S214 as well as to the backbone amides of A56 and H57. In trypsin S195C, D102 is a hydrogen-bond acceptor for H57 which allows the other imidazole nitrogen to function as a base during catalysis. In trypsin D102N,S195C, the asparagine at position 102 is a hydro- gen-bond donor to H57 which places a proton on the imidazole nitrogen proximal to the nucleophile. This tautomer of H57 is unable to act as a base in catalysis. The 100-fold diminished activity of trypsin D102N,S195C compared to trypsin S195C is most likely due to stabilization of the incorrect tautomer of H57, which is unable to form the critical thiolate-imidazolium ion pair with C195-S?, and to altered electrostatics at the catalytic site due to removal of the negative charge from residue 102. H57 has shifted by 0.2 A but remains within hydrogen-bonding distance of D/N102 (2.7 A) and S195 (3.5 A). The sulfur nucleophile of C195 is larger than the oxygen it replaces but is not sterically hindered. In both structures, C195-Sy may also be able to form hydrogen bonds with D193-N (3.7 A), which is part of the oxyanion hole. The sulfur atom points away from the binding pocket compared to the oxygen atom in trypsin, and the strand of peptide chain which includes residue 193 is closer to the side chain of residue 195 in rat trypsin than it is in cow trypsin. This causes occlusion of the oxyanion hole which could account for the inefficacy of these enzymes. Other possible contributing factors to the reduced activity of the two enzymes are discussed.