Binding of nonphysiological protein and peptide substrates to proteases: differences between urokinase-type plasminogen activator and trypsin and contributions to the evolution of regulated proteolysis

Understanding the regulation of physiological processes requires detailed knowledge of the recognition of substrates by enzymes. One of the most productive model systems for the study of enzyme-substrate interactions is the serine protease family; however, most studies of protease action have used small substrates that contain an activated, non-natural scissile bond. Because few kinetic or structural studies have used protein substrates, the physiologically relevant target of most proteases, it seems likely that important mechanisms of substrate recognition and processing by proteases have not yet been fully elucidated. Consistent with this hypothesis, we have observed that K(m) values for protein substrates are reduced as much as 200-15000-fold relative to those of analogous peptide substrates. Here we examine the thermodynamic consequences of interactions between proteases and their substrates using staphylococcal nuclease (SNase) and SNase variants as model protein substrates. We have obtained values for enthalpy, entropy, and K(d) for binding of proteins and peptides by the nonspecific protease trypsin and the highly specific protease urokinase-type plasminogen activator (u-PA). To avoid cleavage of substrates during these measurements, we used inactive variants of trypsin and u-PA whose catalytic serine S195 had been replaced by alanine. Differences in the K(d) values for binding of protein and peptide substrates closely approximate the large differences observed in the corresponding K(m) values. Improved binding of protein substrates is due to decreased enthalpy, and this effect is pronounced for the selective protease u-PA. Fundamental differences in recognition of analogous protein and peptide substrates may have influenced the evolution of protease specificity.     

Identification of novel prostate-specific antigen-binding peptides modulating its enzyme activity.

Prostate-specific antigen (PSA) is a serine protease with highly prostate-specific expression. Measurement of PSA in serum is widely used for diagnosis and monitoring of prostate cancer. PSA dissolves the seminal gel forming after ejaculation. It has been suggested to mediate invasion and metastasis of prostate cancer but also to exert antiangiogenic activity. We have identified peptides specific for PSA by screening cyclic phage display peptide libraries. PSA-binding peptides were isolated from four different libraries and produced as a fusion protein with glutathione S-transferase (GST). The phage and fusion proteins were shown to bind to PSA specifically as indicated by lack of binding to other serine proteinases. A peptide with four cysteines showed the highest affinity for PSA. Zn2+, an inhibitor of PSA activity, increased the affinity of the peptides to PSA. The binding specificity was characterized by cross-inhibition using monoclonal anti-PSA antibodies of known epitope specificities. The peptides bound to the same region as mAbs specific for free PSA indicating that they bind close to the active site of the enzyme. The peptides enhanced the enzyme activity of PSA against a chromogenic substrate. These results show that peptides binding to PSA and modulating its enzyme activity can be developed by phage display technique. The peptides have the potential to be used for identification of PSA variants and for imaging and targeting of prostatic tumors.     

Mechanism of substrate recognition by drug-resistant human immunodeficiency virus type 1 protease variants revealed by a novel structural intermediate

Human immunodeficiency virus type 1 (HIV-1) protease processes and cleaves the Gag and Gag-Pol polyproteins, allowing viral maturation, and therefore is an important target for antiviral therapy. Ligand binding occurs when the flaps open, allowing access to the active site. This flexibility in flap geometry makes trapping and crystallizing structural intermediates in substrate binding challenging. In this study, we report two crystal structures of two HIV-1 protease variants bound with their corresponding nucleocapsid-p1 variant. One of the flaps in each of these structures exhibits an unusual "intermediate" conformation. Analysis of the flap-intermediate and flap-closed crystal structures reveals that the intermonomer flap movements may be asynchronous and that the flap which wraps over the P3 to P1 (P3-P1) residues of the substrate might close first. This is consistent with our hypothesis that the P3-P1 region is crucial for substrate recognition. The intermediate conformation is conserved in both the wild-type and drug-resistant variants. The structural differences between the variants are evident only when the flaps are closed. Thus, a plausible structural model for the adaptability of HIV-1 protease to recognize substrates in the presence of drug-resistant mutations has been proposed.     

Rat mast cell protease 4 is a beta-chymase with unusually stringent substrate recognition profile

Activated mast cells release a variety of potent inflammatory mediators including histamine, cytokines, proteoglycans, and serine proteases. The serine proteases belong to either the chymase (chymotrypsin-like substrate specificity) or tryptase (trypsin-like specificity) family. In this report we have investigated the substrate specificity of a recently identified mast cell protease, rat mast cell protease-4 (rMCP-4). Based on structural homology, rMCP-4 is predicted to belong to the chymase family, although rMCP-4 has previously not been characterized at the protein level. rMCP-4 was expressed with an N-terminal His tag followed by an enterokinase site substituting for the native activation peptide. The enterokinase-cleaved fusion protein was labeled by diisopropyl fluorophosphate, demonstrating that it is an active serine protease. Moreover, rMCP-4 hydrolyzed MeO-Suc-Arg-Ala-Tyr-pNA, thus verifying that this protease belongs to the chymase family. rMCP-4 bound to heparin, and the enzymatic activity toward MeO-Suc-Arg-Ala-Tyr-pNA was strongly enhanced in the presence of heparin. Detailed analysis of the substrate specificity was performed using peptide phage display technique. After six rounds of amplification a consensus sequence, Leu-Val-Trp-Phe-Arg-Gly, was obtained. The corresponding peptide was synthesized, and rMCP-4 was shown to cleave only the Phe-Arg bond in this peptide. This demonstrates that rMCP-4 displays a striking preference for bulky/aromatic amino acid residues in both the P1 and P2 positions.     

Enzymatic activity of the Staphylococcus aureus SplB serine protease is induced by substrates containing the sequence Trp-Glu-Leu-Gln

Proteases are of significant importance for the virulence of Staphylococcus aureus. Nevertheless, their subset, the serine protease-like proteins, remains poorly characterized. Here presented is an investigation of SplB protease catalytic activity revealing that the enzyme possesses exquisite specificity and only cleaves efficiently after the sequence Trp-Glu-Leu-Gln. To understand the molecular basis for such selectivity, we solved the three-dimensional structure of SplB to 1.8 Å. Modeling of substrate binding to the protease demonstrated that selectivity relies in part on a canonical specificity pockets-based mechanism. Significantly, the conformation of residues that ordinarily form the oxyanion hole, an essential structural element of the catalytic machinery of serine proteases, is not canonical in the SplB structure. We postulate that within SplB, the oxyanion hole is only formed upon docking of a substrate containing the consensus sequence motif. It is suggested that this unusual activation mechanism is used in parallel with classical determinants to further limit enzyme specificity. Finally, to guide future development, we attempt to point at likely physiological substrates and thus the role of SplB in staphylococcal physiology.     

Computational method for the design of enzymes with altered substrate specificity.

A combination of enzyme kinetics and X-ray crystallographic analysis of site-specific mutants has been used to probe the determinants of substrate specificity for the enzyme alpha-lytic protease. We now present a generalized model for understanding the effects of mutagenesis on enzyme substrate specificity. This algorithm uses a library of side-chain rotamers to sample conformation space within the binding site for the enzyme-substrate complex. The free energy of each conformation is evaluated with a standard molecular mechanics force field, modified to include a solvation energy term. This rapid energy calculation based on coarse conformation sampling quite accurately predicts the relative catalytic efficiency of over 40 different alpha-lytic protease-substrate combinations. Unlike other computational approaches, with this method it is feasible to evaluate all possible mutations within the binding site. Using this algorithm, we have successfully designed a protease that is both highly active and selective for a non-natural substrate. These encouraging results indicate that it is possible to design altered enzymes solely on the basis of empirical energy calculations.     

A survey of furin substrate specificity using substrate phage display.

The substrate specificity of furin, a mammalian enzyme involved in the cleavage of many constitutively expressed protein precursors, was studied using substrate phage display. In this method, a multitude of substrate sequences are displayed as fusion proteins on filamentous phage particles and ones that are cleaved can be purified by affinity chromatography. The cleaved phage are propagated and submitted to additional rounds of protease selection to further enrich for good substrates. DNA sequencing of the cleaved phage is used to identify the substrate sequence. After 6 rounds of sorting a substrate phage library comprising 5 randomized amino acids (xxxxx), virtually all clones had an RxxR motif and many had Lys, Arg, or Pro before the second Arg. Nine of the selected sequences were assayed using a substrate-alkaline phosphatase fusion protein system. All were cleaved after the RxxR, and some substrates with Pro or Thr in P2 were also found to be cleaved as efficiently as RxKR or RxRR. To further elaborate surrounding determinants, we constructed 2 secondary libraries (xxRx(K/R)Rx and xxRxPRx). Although no consensus developed for the latter library, many of the sequences in the the former library had the 7-residue motif (L/P)RRF(K/R)RP, suggesting that the furin recognition sequence may extend over more than 4 residues. These studies further clarify the substrate specificity of furin and suggest the substrate phage method may be useful for identifying consensus substrate motifs in other protein processing enzymes.     

Structural basis for the substrate specificity of tobacco etch virus protease

Because of its stringent sequence specificity, the 3C-type protease from tobacco etch virus (TEV) is frequently used to remove affinity tags from recombinant proteins. It is unclear, however, exactly how TEV protease recognizes its substrates with such high selectivity. The crystal structures of two TEV protease mutants, inactive C151A and autolysis-resistant S219D, have now been solved at 2.2- and 1.8-A resolution as complexes with a substrate and product peptide, respectively. The enzyme does not appear to have been perturbed by the mutations in either structure, and the modes of binding of the product and substrate are virtually identical. Analysis of the protein-ligand interactions helps to delineate the structural determinants of substrate specificity and provides guidance for reengineering the enzyme to further improve its utility for biotechnological applications.     

Molecular insights into substrate specificity of prostate specific antigen through structural modeling

Prostate Specific Antigen's (PSA) role as a biomarker for prostate cancer is well established but the physiological role of its serine protease activity in the pathobiology of normal prostate and prostate carcinogenesis remains largely unknown. In light of recent studies that implicate PSA's enzymatic activity in the initiation and/or progression of prostate cancer, we performed a molecular modeling study of substrate binding at the catalytic site of PSA wherein a PSA‐selective substrate (HSSKLQ) was docked in an acyl‐enzyme conformation to a three‐dimensional homology model of PSA. Additionally, virtual positional scanning studies were conducted to gain mechanistic insights into substrate recognition of PSA. Subsequently, 13 novel peptide substrates of 6‐aa length and four peptide substrates with varying length were synthesized and assayed for PSA hydrolysis to evaluate the experimental validity of docking insights. Additionally, six novel aldehyde‐containing transition state analog inhibitors were synthesized and tested for their inhibitory potencies. The experimental data on the hydrolysis rates of the newly synthesized substrates and inhibitory potencies of the aldehyde peptides agreed with the docking predictions, providing validation of the docking methodology and demonstrating its utility towards the design of substrate‐mimetic inhibitors that can be used to explore PSA's role in the pathobiology of prostate cancer. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Crystal structure of tobacco etch virus protease shows the protein C terminus bound within the active site

Tobacco etch virus (TEV) protease is a cysteine protease exhibiting stringent sequence specificity. The enzyme is widely used in biotechnology for the removal of the affinity tags from recombinant fusion proteins. Crystal structures of two TEV protease mutants as complexes with a substrate and a product peptide provided the first insight into the mechanism of substrate specificity of this enzyme. We now report a 2.7A crystal structure of a full-length inactive C151A mutant protein crystallised in the absence of peptide. The structure reveals the C terminus of the protease bound to the active site. In addition, we determined dissociation constants of TEV protease substrate and product peptides using isothermal titration calorimetry for various forms of this enzyme. Data suggest that TEV protease could be inhibited by the peptide product of autolysis. Separate modes of recognition for native substrates and the site of TEV protease self-cleavage are proposed.     

Circular dichroic evidence for an ordered sequence of ligand/binding site interactions in the catalytic reaction of the cAMP-dependent protein kinase

A limiting requirement for substrate specificity of the cAMP-dependent protein kinase is the presence of one or two basic residues located to the N-terminal side of the target substrate serine. Furthermore, circular dichroic (CD) studies have shown that binding of protein substrate involves a series of at least two independent conformational changes in the enzyme, each of which is initiated by a recognition signal on the substrate protein. The present study attempts to elucidate further the complete sequence of enzyme/ligand interactions by using the synthetic substrate peptide Kemptide and analogues differing from it at crucial points in the sequence: the Ala-peptide, where alanine is substituted for the target serine, and D-Ser-Kemptide, where the target serine is in the D rather than the L configuration. Examination of the effects of binding of these substrates on the intrinsic UV CD of the enzyme and the induced CD in the presence of Blue Dextran has revealed a third step in the substrate/enzyme binding interaction. Although sections of the conformational change at the active site are dependent on the basic subsite and the serine hydroxyl group on the peptide, respectively, the complete conformational change requires that the substrate be bound in random coil conformation. Where this does not occur, the kinetics show that the peptide will not act either as substrate or as inhibitor of the enzyme. Further, the interaction between the serine hydroxyl group and an enzyme tyrosine residue, previously observed, appears to be dependent on the correct orientation as well as the mere presence of the target -OH group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Active-Site Gating Regulates Substrate Selectivity in a Chymotrypsin-Like Serine Protease: The Structure of Haemophilus influenzae Immunoglobulin A1 Protease

We report here the first structure of a member of the immunoglobulin A protease (IgAP) family at 1.75-Å resolution. This protease is a founding member of the type V (autotransporter) secretion system and is considered a virulence determinant among the bacteria expressing the enzyme. The structure of the enzyme fits that of a classic autotransporter in which several unique domains necessary for protein function are appended to a central, 100-Å-long β-helical domain. The N-terminal domain of the IgAP is found to possess a chymotrypsin-like fold. However, this catalytic domain contains a unique loop D that extends over the active site acting as a lid, gating substrate access. The data presented provide a structural basis for the known ability of IgAPs to cleave only the proline/serine/threonine-rich hinge peptide unique to IgA1 (isotype 1) in the context of the intact fold of the immunoglobulin. Based upon the structural data, as well as molecular modeling, a model suggesting that the unique extended loop D in this IgAP sterically occludes the active-site binding cleft in the absence of immunoglobulin binding is presented. Only in the context of binding of the IgA1-Fc domain in a valley formed between the N-terminal protease domain and another domain appended to the β-helix spine (domain 2) is the lid stabilized in an open conformation. The stabilization of this open conformation through Fc association subsequently allows access of the hinge peptide to the active site, resulting in recognition and cleavage of the substrate.     

Reaction kinetics of protease with substrate phage. Kinetic model developed using stromelysin

Peptide libraries generated using phage display have been widely applied to proteolytic enzymes for substrate selection and optimization, but the reaction kinetics between the enzyme and substrate phage are not well understood. Using a quantitative ELISA assay to monitor the disappearance of substrate, we have been able to follow the course of reaction between stromelysin, a metalloprotease, and its substrate phage. We found that under the proteolytic conditions where the enzyme was present in nanomolar concentration or higher, in excess over the substrate, the proteolysis of substrate phage was a single exponential event and the observed rate linear with respect to enzyme concentration. The enzyme concentration dependence could be described by pseudo first-order kinetic equations. Our data suggest that substrate binding is slow relative to the subsequent hydrolysis step, implying that the phage display selection process enriches clones that have high binding affinity to the protease, and the selection may not discriminate those of different chemical reactivity toward the enzyme. Considering that multiple substrate molecules may be present on a single phage particle, we regard the substrate phage reaction kinetic model as empirical. The validity of the model was ascertained when we successfully applied it to determine the binding affinity of a competitive inhibitor of stromelysin.     

Elucidation of the substrate specificity of the C1s protease of the classical complement pathway

The complement system is a central component of host defense but can also contribute to the inflammation seen in pathological conditions. The C1s protease of the first complement component, the C1 complex, initiates the pathway. In this study we have elucidated the full specificity of the enzyme for the first time using a randomized phage display library. It was found that, aside from the crucial P(1) position, the S(3) and S(2) subsites (in that order) played the greatest role in determining specificity. C1s prefers Leu or Val at P(3) and Gly or Ala residues at P(2). Apart from the S(2)' position, which showed specificity for Leu, prime subsites did not greatly affect specificity. It was evident, however, that together they significantly contributed to the efficiency of cleavage of a peptide. A peptide substrate based on the top sequence obtained in the phage display validated these results and produced the best kinetics of any C1s substrate to date. The results allow an understanding of the active site specificity of the C1s protease for the first time and provide a basis for the development of specific inhibitors aimed at controlling inflammation associated with complement activation in adverse pathological situations.     

A norovirus protease structure provides insights into active and substrate binding site integrity

Norovirus 3C-like proteases are crucial to proteolytic processing of norovirus polyproteins. We determined the crystal structure of the 3C-like protease from Chiba virus, a norovirus, at 2.8-A resolution. An active site including Cys139 and His30 is present, as is a hydrogen bond network that stabilizes the active site conformation. In the oxyanion hole backbone, a structural difference was observed probably upon substrate binding. A peptide substrate/enzyme model shows that several interactions between the two components are critical for substrate binding and that the S1 and S2 sites appropriately accommodate the substrate P1 and P2 residues, respectively. Knowledge of the structure and a previous mutagenesis study allow us to correlate proteolysis and structure.     

Structural, kinetic, and thermodynamic studies of specificity designed HIV-1 protease

HIV-1 protease recognizes and cleaves more than 12 different substrates leading to viral maturation. While these substrates share no conserved motif, they are specifically selected for and cleaved by protease during viral life cycle. Drug resistant mutations evolve within the protease that compromise inhibitor binding but allow the continued recognition of all these substrates. While the substrate envelope defines a general shape for substrate recognition, successfully predicting the determinants of substrate binding specificity would provide additional insights into the mechanism of altered molecular recognition in resistant proteases. We designed a variant of HIV protease with altered specificity using positive computational design methods and validated the design using X-ray crystallography and enzyme biochemistry. The engineered variant, Pr3 (A28S/D30F/G48R), was designed to preferentially bind to one out of three of HIV protease's natural substrates; RT-RH over p2-NC and CA-p2. In kinetic assays, RT-RH binding specificity for Pr3 increased threefold compared to the wild-type (WT), which was further confirmed by isothermal titration calorimetry. Crystal structures of WT protease and the designed variant in complex with RT-RH, CA-p2, and p2-NC were determined. Structural analysis of the designed complexes revealed that one of the engineered substitutions (G48R) potentially stabilized heterogeneous flap conformations, thereby facilitating alternate modes of substrate binding. Our results demonstrate that while substrate specificity could be engineered in HIV protease, the structural pliability of protease restricted the propagation of interactions as predicted. These results offer new insights into the plasticity and structural determinants of substrate binding specificity of the HIV-1 protease.     

Improved substrate specificity of water-soluble pyrroloquinoline quinone glucose dehydrogenase by a peptide ligand

A new approach in altering the substrate specificity of enzyme is proposed using glucose dehydrogenase, with pyrroroquinoine quinone (PQQGDH) as co-factor, as the model. This approach is based on the selection of random peptide phage displayed library. Using an M13 phage-display random peptide library, we have selected peptide ligands. Among the peptide ligands, a 7-mer peptide, composed of Thr-Thr-Ala-Thr-Glu-Tyr-Ser, caused PQQGDH substrate specificity to decrease significantly toward disaccharides, such as maltose and lactose, while a smaller effect was observed toward glucose. Consequently, this peptide narrowed the substrate specificity of PQQGDH, without a significant loss of the enzyme activity.     

Design and efficient production of bovine enterokinase light chain with higher specificity in E. coli

Enterokinase light chain (EKL) is a serine protease that recognizes Asp-Asp-Asp-Asp-Lys (D(4)K) sequence and cleaves the C-terminal peptide bond of the lysine residue. The utility of EKL as a site-specific cleavage enzyme is hampered by sporadic cleavage at other sites than the canonical D(4)K recognition sequence. In order to produce more site-specific EKL, we have generated several EKL mutants in E. coli with substitutions at Tyr174 and Lys99 using PDI (protein disulfide isomerase) fusion system. Substitution of Tyr174 by basic residues confers higher specificity on EKL. The production of EKL with higher specificity could widen the utility of EKL as a site-specific cleavage enzyme to produce various recombinant proteins with therapeutic or industrial values.     

Studies of specificity and catalysis in trypsin by structural analysis of site-directed mutants

We are probing the determinants of catalytic function and substrate specificity in serine proteases by kinetic and crystallographic characterization of genetically engineered site-directed mutants of rat trypsin. The role of the aspartyl residue at position 102, common to all members of the serine protease family, has been tested by substitution with asparagine. In the native enzyme, Asp102 accepts a hydrogen bond from the catalytic base His57, which facilitates the transfer of a proton from the enzyme nucleophile Ser195 to the substrate leaving group. At neutral pH, the mutant is four orders of magnitude less active than the naturally occurring enzyme, but its binding affinity for model substrates is virtually undiminished. Crystallographic analysis reveals that Asn102 donates a hydrogen bond to His57, forcing it to act as donor to Ser195. Below pH 6, His57 becomes statistically disordered. Presumably, the di-protonated population of histidyl side chains are unable to hydrogen bond to Asn102. Steric conflict may cause His57 to rotate away from the catalytic site. These results suggest that Asp102 not only provides inductive and orientation effects, but also stabilizes the productive tautomer of His57. Three experiments were carried out to alter the substrate specificity of trypsin. Glycine residues at positions 216 and 226 in the substrate-binding cavity were replaced by alanine residues in order to differentially affect lysine and arginine substrate binding. While the rate of catalysis by the mutant enzymes was reduced in the mutant enzymes, their substrate specificity was enhanced relative to trypsin. The increased specificity was caused by differential effects on the catalytic activity towards arginine and lysine substrates. The Gly----Ala substitution at 226 resulted in an altered conformation of the enzyme which is converted to an active trypsin-like conformation upon binding of a substrate analog. In a third experiment, Lys189, at the bottom of the specificity pocket, was replaced with an aspartate with the expectation that specificity of the enzyme might shift to aspartate. The mutant enzyme is not capable of cleaving at Arg and Lys or Asp, but shows an enhanced chymotrypsin-like specificity. Structural investigations of these mutants are in progress.