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The type II transmembrane multidomain serine proteinase MT-SP1/matriptase is highly expressed in many human cancer-derived cell lines and has been implicated in extracellular matrix re-modeling, tumor growth, and metastasis. We have expressed the catalytic domain of MT-SP1 and solved the crystal structures of complexes with benzamidine at 1.3 A and bovine pancreatic trypsin inhibitor at 2.9 A. MT-SP1 exhibits a trypsin-like serine proteinase fold, featuring a unique nine-residue 60-insertion loop that influences interactions with protein substrates. The structure discloses a trypsin-like S1 pocket, a small hydrophobic S2 subsite, and an open negatively charged S4 cavity that favors the binding of basic P3/P4 residues. A complementary charge pattern on the surface opposite the active site cleft suggests a distinct docking of the preceding low density lipoprotein receptor class A domain. The benzamidine crystals possess a freely accessible active site and are hence well suited for soaking small molecules, facilitating the improvement of inhibitors. The crystal structure of the MT-SP1 complex with bovine pancreatic trypsin inhibitor serves as a model for hepatocyte growth factor activator inhibitor 1, the physiological inhibitor of MT-SP1, and suggests determinants for the substrate specificity.

Chymotrypsin family serine proteases play essential roles in key biological and pathological processes and are frequently targets of drug discovery efforts. This large enzyme family is also among the most advanced model systems for detailed studies of enzyme mechanism and structure/function relationships. Productive interactions between these enzymes and their substrates are widely believed to mimic the "canonical" interactions between serine proteases and "standard" inhibitors observed in numerous protease-inhibitor complexes. To test this central hypothesis we have synthesized and characterized a series of peptide analogs, based on model substrates and inhibitors of trypsin, that contain unnatural main chains. These results call into question a long accepted theory regarding the interaction of chymotrypsin family serine proteases with substrates and suggest that the canonical interactions observed between these enzymes and standard inhibitors may represent nonproductive rather than productive, substrate-like interactions.

Archetypal members of the chymotrypsin family of serine proteases, such as trypsin, chymotrypsin, and elastase, exhibit relatively broad substrate specificity. However, the successful development of efficient proteolytic cascades, such as the blood coagulation and fibrinolytic systems, required the evolution of proteases that displayed restricted specificity. Tissue-type plasminogen activator (t-PA), for example, possesses exquisitely stringent substrate specificity, and the molecular basis of this important biochemical property of t-PA remains obscure. Previous investigations of related serine proteases, which participate in the blood coagulation cascade, have focused attention on the residue that occupies position 192 (chymotrypsin numbering system), which plays a pivotal role in determining both the inhibitor and substrate specificity of these enzymes. Consequently, we created and characterized the kinetic properties of new variants of t-PA that contained point mutations at position 192. These studies demonstrated that, unlike in coagulation serine proteases, Gln-192 does not contribute significantly to the substrate or inhibitor specificity of t-PA in physiologically relevant reactions. Replacement of Gln-192 with a glutamic acid residue did, however, decrease the catalytic efficiency of mature, two-chain t-PA toward plasminogen in the absence of a fibrin co-factor.

We have previously used substrate phage display to identify peptide sequences that are efficiently and selectively cleaved by tissue-type plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA). We demonstrate that this information can be used to direct selective proteolysis to new protein targets. Sequences that were labile to selective cleavage by t-PA or u-PA when in the context of a peptide were introduced into the 43-52 (or Omega) loop of staphylococcal nuclease. Both t-PA and u-PA hydrolyze the engineered proteins at the inserted target sequences, and Km values for protein cleavage were reduced up to 200-fold relative to values for cleavage of analogous sequences within 15 residue peptides. Variation of loop size surrounding a target sequence affects the efficiency of t-PA approximately 5-fold more strongly than that of trypsin, suggesting that cleavage by t-PA is more dependent on target site mobility. Cleavage of proteins by t-PA and u-PA is sequence selective. u-PA is 47-fold more active than t-PA for cleavage of a sequence known to be u-PA selective within small peptide substrates, whereas t-PA is 230-fold more active toward a t-PA-selective sequence.

Human urokinase type plasminogen activator (u-PA) is a member of the chymotrypsin family of serine proteases that can play important roles in both health and disease. We have used substrate phage display techniques to characterize the specificity of this enzyme in detail and to identify peptides that are cleaved 840-5300 times more efficiently by u-PA than peptides containing the physiological target sequence of the enzyme. In addition, unlike peptides containing the physiological target sequence, the peptide substrates selected in this study were cleaved as much as 120 times more efficiently by u-PA than by tissue type plasminogen activator (t-PA), an intimately related enzyme. Analysis of the selected peptide substrates strongly suggested that the primary sequence SGRSA, from position P3 to P2', represents optimal subsite occupancy for substrates of u-PA. Insights gained in these investigations were used to design a variant of plasminogen activator inhibitor type 1, the primary physiological inhibitor of both u-PA and t-PA, that inhibited u-PA approximately 70 times more rapidly than it inhibited t-PA. These observations provide a solid foundation for the design of highly selective, high affinity inhibitors of u-PA and, consequently, may facilitate the development of novel therapeutic agents to inhibit the initiation and/or progression of selected human tumors.

Elucidating subtle specificity differences between closely related enzymes is a fundamental challenge for both enzymology and drug design. We have addressed this issue for two intimately related serine proteases, tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA), by modifying the technique of substrate phage display to create substrate subtraction libraries. Characterization of individual members of the substrate subtraction library accomplished the rapid, direct identification of small, highly selective substrates for t-PA. Comparison of the amino acid sequences of these selective substrates with the consensus sequence for optimal substrates for t-PA, derived using standard substrate phage display protocols, suggested that the P3 and P4 residues are the primary determinants of the ability of a substrate to discriminate between t-PA and u-PA. Mutagenesis of the P3 and P4 residues of plasminogen activator inhibitor type 1, the primary physiological inhibitor of both t-PA and u-PA, confirmed this prediction and indicated a predominant role for the P3 residue. Appropriate replacement of both the P3 and P4 residues enhanced the t-PA specificity of plasminogen activator inhibitor type 1 by a factor of 600, and mutation of the P3 residue alone increased this selectivity by a factor of 170. These results demonstrate that the combination of substrate phage display and substrate subtraction methods can be used to discover specificity differences between very closely related enzymes and that this information can be utilized to create highly selective inhibitors.

Fibrinolysis is regulated in part by the interaction of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1). Previous investigations suggest that three specific arginine residues, Arg-298, Arg-299, and Arg-304 of t-PA, play a critical role in this important regulatory interaction. Our earlier studies have demonstrated that conversion of any of these three residues to a glutamic acid residue reduced the rate of inhibition of t-PA by PAI-1 by factors varying from 58-64. In addition, we have reported that the second order rate constant for inhibition by PAI-1 of the variant t-PA/K296E,R298E,R299E is reduced by a factor of approximately 2800 compared with that of wild type t-PA. In this study, we have significantly extended our earlier observations by identifying t-PA variants that are substantially more resistant to inhibition by PAI-1 than any previously reported variants of t-PA or urokinase-type plasminogen activator. Single-chain t-PA/R275E,R298E, R299E,R304E, for example, is inhibited by PAI-1 approximately 120, 000 times less rapidly than single-chain, wild type t-PA. We also report the first direct comparison of the effects of charge reversal mutations of Arg-298, Arg-299, and/or Arg-304 on the properties of the single- and two-chain forms of t-PA. While these mutations confer extraordinary resistance to inhibition by PAI-1 to both forms of the enzyme, our observations reveal that the single-chain enzyme is affected to a greater extent than the two-chain enzyme. Two-chain, wild type t-PA is inhibited by PAI-1 approximately 1.4 times more rapidly than single-chain t-PA. The corresponding ratio increases to 7.6 or 6.7, respectively, for variants of t-PA containing the R298E, R299E or R298E,R299E,R304E mutations.

A wide variety of important biological processes, including both the formation and dissolution of blood clots, depend on specific cleavage of individual target proteins by serine proteases. For example, tissue type plasminogen activator (t-PA), a trypsin-like enzyme that catalyzes the rate-limiting step of the endogenous fibrinolytic cascade, has only one known substrate in vivo, a single peptide bond (Arg561-Val562) in the proenzyme plasminogen. We have previously suggested that the specificity of t-PA for plasminogen is mediated in part by direct protein-protein interactions between the protease domain of t-PA and plasminogen that are distinct from those occurring within t-PA's active site. We demonstrate in this study that residues 420-423 of t-PA, which form a fully solvent-exposed, hydrophobic region of a surface loop mapping near one edge of the active site of t-PA, form, or are essential for the integrity of, an important, secondary site of interaction between t-PA and plasminogen that significantly modulates the rate of plasminogen activation in the absence, but not the presence, of fibrin. Identification of this secondary site of interaction between t-PA and plasminogen provides new insight into molecular details of the evolution of stringent substrate specificity by t-PA and suggests a novel strategy to enhance the fibrin dependence of plasminogen activation by t-PA. While the activity of wild type t-PA is stimulated by fibrin by a factor of approximately 650, the activity of two variants characterized in this study, t-PA/R275E,P422G and t-PA/R275E,P422E, is stimulated by a factor of approximately 39,000 or 61,000, respectively. It is therefore possible that, compared with wild type t-PA, the two variants would display enhanced "clot selectivity" in vivo due to reduced activity in the circulation but full activity at a site of fibrin deposition.

In stark contrast to most other members of the chymotrypsin family of serine proteases, tissue type plasminogen activator (t-PA) is not synthesized and secreted as a true zymogen. Instead, single-chain t-PA exhibits very significant catalytic activity. Consequently, the zymogenicity, or ratio of the catalytic efficiencies of the mature, two-chain enzyme and the single-chain precursor, is only 3-9 for t-PA. Both we and others have previously proposed that Lys156 may contribute directly to this exceptional property of t-PA by forming interactions that selectively stabilize the active conformation of the single-chain enzyme. To test this hypothesis we created variants of t-PA in which Lys156 was replaced by a tyrosine residue. As predicted, the K156Y mutation selectively suppressed the activity of the single-chain enzyme and thereby substantially enhanced the enzyme's zymogenicity. In addition, however, this mutation produced a very dramatic increase in the ability of single-chain t-PA to discriminate among distinct fibrin co-factors. Compared with wild type t-PA, one of the variants characterized in this study, t-PA/R15E,K156Y, possessed substantially enhanced response to and selectivity among fibrin co-factors, resistance to inhibition by plasminogen activator inhibitor type 1, and significantly increased zymogenicity. The combination of these properties, and the maintenance of full activity in the presence of fibrin, suggest that the R15E,K156Y mutations may extend the therapeutic range of t-PA.

In striking contrast to most other members of the chymotrypsin family of serine proteases, tissue-type plasminogen activator (t-PA) is not synthesized and secreted as a true zymogen. The zymogenicity, or ratio of the catalytic efficiencies of the mature, two-chain enzyme and the single-chain precursor, is only 5-10 for t-PA. This exceptional property of t-PA, however, is not shared by urokinase (u-PA), a plasminogen activator that is very closely related to t-PA. The molecular basis of this important functional distinction between these two intimately related serine proteases has not been previously investigated. Based on observation of the recently described structures of the protease domains of two-chain t-PA and u-PA, and molecular modeling of the corresponding single-chain enzymes, we propose that the presence or absence of an acidic residue at position 144 (chymotrypsin numbering system) is the primary determinant of the distinct zymogenicities of the two enzymes. Consistent with this hypothesis, mutation of histidine 144 of t-PA to an acidic residue, as in u-PA, selectively suppressed the activity of single-chain t-PA and thereby significantly enhanced the enzyme's zymogenicity. A variant of t-PA containing an aspartate residue at position 144, for example, exhibited a zymogenicity of 150, compared to a value of 9 for wild type t-PA and 250 for u-PA.

Tissue-type plasminogen activator (t-PA) has evolved to optimize cleavage of plasminogen (Plg) while minimizing cleavage of other potential protein and peptide substrates. We find that the S2 and S2 subsites of t-PA are important determinants of specificity, and occupancy of the S3 subsite is essential for catalysis. t-PA efficiently hydrolyzes a protein substrate which incorporates an optimized substrate sequence, revealing the ability of the protease to participate in the highly selective cleavage of protein fusions. Surprisingly, t-PA cleaves this engineered protein substrate with a Km that is reduced 950-fold relative to the Km for hydrolysis of the same target sequence within a peptide. This reduction of Km suggests that binding is facilitated by interactions between protein substrate and protease that are distant from the P4-P2' residues. We use this kinetic data to derive a model in which several distinct mechanisms contribute to the remarkable specificity of t-PA.

Protein-protein interactions can be guided by contacts between surface loops within proteins. We therefore investigated the hypothesis that novel protein-protein interactions could be created using a strategy of "loop grafting" in which the amino acid sequence of a biologically active, flexible loop on one protein is used to replace a surface loop present on an unrelated protein. To test this hypothesis we replaced a surface loop within an epidermal growth factor module with the complementarity-determining region of a monoclonal antibody. Specifically, the HCDR3 from Fab-9, an antibody selected to bind the beta 3-integrins with nanomolar affinity (Smith, J. W., Hu, D., Satterthwait, A., Pinz-Sweeney, S., and Barbas, C. F., III (1994) J. Biol. Chem. 269, 32788-32795), was grafted into the epidermal growth factor-like module of human tissue-type plasminogen activator (t-PA). The resulting variant of t-PA bound to the platelet integrin alpha IIb beta 3 with nanomolar affinity, retained full enzymatic activity, and was stimulated normally by the physiological co-factor fibrin. Binding of the novel variant of t-PA to integrin alpha IIb beta 3 was dependent on the presence of divalent cations and was inhibited by an RGD-containing peptide, demonstrating that, like the donor antibody, the novel t-PA binds specifically to the ligand-binding site of the integrin. These findings suggest that surface loops within protein modules can, at least in some cases, be interchangeable and that phage display can be combined with loop grafting to direct proteins, at high affinity, to selected targets. In principle, these targets could include not only other proteins but also peptides, nucleic acids, carbohydrates, lipids, or even uncharacterized markers of specific cell types, tissues, or viruses.

Unlike most proteases, tissue-type plasminogen activator (t-PA) is not synthesized as an inactive precursor or zymogen. Instead, the single-chain "proenzyme" form of t-PA possesses very significant catalytic activity. Recent investigations of the molecular basis of the unusually high enzymatic activity of single-chain t-PA have focused attention upon Asp-194, a residue that is invariant among chymotrypsin-like enzymes. The critical role of this residue in securing the active conformation of mature chymotrypsin-like enzymes has been discussed extensively. Subsequent work, however, has indicated that this conserved residue can also form interactions that dramatically influence the catalytic activity of serine protease zymogens. While Asp-194 forms interactions that suppress the activity of the zymogen chymotrypsinogen, it may, by contrast, directly promote the catalytically active conformation of single-chain t-PA. To test the hypothesis that Asp-194 promotes the activity of both single- and two-chain t-PA and therefore plays opposing roles in single-chain t-PA and chymotrypsinogen, and also to examine whether this invariant residue plays an essential role in the stimulation of t-PA by fibrin, we used site-directed mutagenesis to construct the following variants of t-PA: t-PA/D194E, t-PA/D194N, t-PA/R15E,D194E, and t-PA/R15E,D194N. In the absence of fibrin, the activity of enzymes carrying a mutation at position 194 was reduced by factors of 1000-2000 compared to wild type t-PA. Similar reductions of activity were observed for both single- and two-chain variants, suggesting an important role for Asp-194 in both forms of the enzyme. The mutated enzymes, however, displayed a dramatically enhanced response to fibrin monomers. While the activity of wild type t-PA was stimulated by fibrin monomers by a factor of 960, the corresponding stimulation factor for the mutated enzymes varied from 498,000-1,050,000.

Unlike most proteases, tissue-type plasminogen activator (t-PA) is secreted from cells as an active, single chain "proenzyme" whose catalytic efficiency is comparable with that of the corresponding mature, two-chain enzyme. We have previously suggested that the absence of the "zymogen triad" (Asp194-His40-Ser32; chymotrypsin numbering) contributes to this unusually high enzymatic activity of single chain t-PA. Consistent with this prediction, the single chain form of a variant of t-PA containing the zymogen triad displayed dramatically reduced activity toward synthetic substrates. Activation cleavage of this variant, however, resulted in a mature, two-chain enzyme with full catalytic activity. To further examine the functional significance of the zymogen triad, we used site-specific mutagenesis to construct a variant of t-PA, t-PA/R275E,A292S,F305H, that contained this triad but could not be converted into its two-chain form by plasmin. Characterization of this variant demonstrated that the presence of the zymogen triad specifically suppressed plasminogen activation by single chain t-PA in the absence of fibrin. In addition, these studies indicated that, like wild type t-PA, zymogen activation of this variant could be accomplished by binding to the co-factor fibrin. The combination of full activity in the presence of fibrin and reduced activity in its absence resulted in novel variants of t-PA that displayed dramatically enhanced stimulation by fibrin. While the presence of fibrin increased the catalytic efficiency of t-PA toward plasminogen by a factor of approximately 520, this stimulation factor increased to 130,000 for t-PA/R275E,A292S,F305H. Plasmin-resistant, zymogen-like variants of t-PA, therefore, may represent thrombolytic enzymes with enhanced "clot selectivity."

Tissue-type plasminogen activator (t-PA) is a remarkably specific protease: the only known substrate of this enzyme in vivo is a single peptide bond (Arg560-Val561) within the proenzyme plasminogen. Part of the substrate specificity of t-PA is due to a ternary interaction between fibrin, t-PA and plasminogen which reduces the Km of t-PA for plasminogen by a factor of 440. However, even in the absence of fibrin, t-PA continues to hydrolyze plasminogen more rapidly than does trypsin, a homologous serine protease. We have measured the extent of the specificity of t-PA for plasminogen by assaying t-PA and trypsin toward substrates modeled after the peptide sequence in plasminogen surrounding Arg560-Val561. Surprisingly, t-PA hydrolyzes these substrates with kcat/Km values which are 28,000-210,000-fold lower than those obtained using trypsin. Both the high activity toward plasminogen and the low activity toward peptides are also exhibited by the isolated protease domain. This suggests that the protease domain, in spite of its high homology to the nonspecific enzyme trypsin, is inherently specific for recognition of one or more structural features displayed by native plasminogen.

Tissue-type plasminogen activator (t-PA) catalyzes the rate-limiting step in the fibrinolytic cascade: conversion of plasminogen to plasmin. Plasma contains several inhibitors of t-PA that limit its activity and prevent systemic activation of plasminogen. The most important of these is endothelial cell plasminogen activator inhibitor (PAI-1), a member of the serine protease inhibitor (serpin) gene family. We have previously demonstrated that mutation of arginine 304 of t-PA to a glutamic acid residue drastically reduces the rate of interaction between the enzyme and its suicide substrate, PAI-1, without affecting the reactivity of the enzyme toward its normal substrate, plasminogen (Madison, E. L., Goldsmith, E. J., Gerard, R.D., Gething, M.J., and Sambrook, J.F. (1989) Nature 339, 721-724). We report here the use of protein modeling to design a compensatory mutation in PAI-1 (glutamic acid 350 to arginine) and create a molecule that rapidly inhibits this "serpin-resistant" variant of t-PA.