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.

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 Lys 156 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 Lys 156 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.Proteases are normally synthesized as inactive precursors or zymogens that must either be proteolytically processed or bind to a specific co-factor to develop substantial catalytic activity. The increase in catalytic efficiency after zymogen activation, or zymogenicity, varies widely among individual members of the (chymo)trypsin family but, in almost all cases, is dramatic. For example, strong zymogens such as trypsinogen, chymotrypsinogen, or plasminogen are almost completely inactive with measured zymogenicities of 10 4 to 10 6 (1, 2). Other serine proteases exhibit intermediate zymogenicity. The enzymatic activity of Factor XIIa is 4000-fold greater than that of Factor XII (3), and the catalytic efficiency of urokinase is 250-fold greater than that of prourokinase (4). By contrast, the catalytic activities of single-and two-chain t-PA 1 vary by a factor of only 3-9 (5-9).We have suggested previously that the unusually high catalytic activity of single-chain t-PA results both from the absence of interactions, present in typical zymogens, that stabilize (an) inactive conformation(s) of the zymogen and the presence of interactions, absent in typical zymogens, that stabilize an active conformation of the single-chain enzyme (8 -10). Recent studies have provided substantial support for this hypothesis. We demonstrated that the absence of the zymogen triad contributes to the enzymatic activity of single-chain t-PA (8, 9), and two groups have suggested that Lys 156 2 stabilizes an active conformation of single-chain t-PA (...

The methanol-insoluble, heat-stable enterotoxin of Escherichia coli synthesized by clinical strains or strains that harbor the cloned gene was shown to be an extraceilular polypeptide. The toxin (STB) was first detected as an 8,100-M, precursor (pre-STB) that was converted to a transiently cell-associated 5,200-Mr form.Proteolytic conversion of pre-STB to STB was shown to be inhibited by the proton motive force uncoupler carbonyl cyanide m-chlorophenylhydrazone and did not occur in a secA background. After STB was detected as a cell-associated molecule, an extracellular form with identical electrophoretic mobility became apparent. The results suggest that there is no proteolytic processing during the mobilization of STB from the periplasm to the culture supernatant. The determined amino acid sequence of STB coincides fully with the 48 carboxy-terminal amino acids inferred from the DNA sequence. The 23 amino-terminal residues inferred from the DNA sequence were absent in the mature toxin.Enterotoxigenic Escherichia coli synthesizes the heatlabile and the heat-stable (ST) families of enterotoxins; these toxins have been shown to be responsible for secretory diarrhea in humans and animals (reviewed in references 1 and 30). STs have been classified as methanol soluble (STA) and methanol insoluble (STB) (3, 36), and these subdivisions correlate well with the inferred or known amino acid compositions of the toxins (18,21,27). The toxic activity of STA is resistant to proteases (6, 33), while STB is inactivated upon trypsin treatment (35). STAs are 18-or 19-amino-acid extracellular enterotoxins that result from two independent proteolytic cleavages on a 72-amino-acid precursor (prepro-STA); the first cleavage yields a periplasmic 53-aminoacid pro-STA that is extracellularly processed to mature STA (29a). The three disulfide bridges formed by the six cysteine residues of STA are sine qua non for toxic activity (6,10,33). A structural model based on proton nuclear magnetic resonance has been proposed for this toxin (11,24). It is also known that STA interacts with an enterocyte receptor that activates guanylate cyclase and results in increased intestinal secretion (8); in contrast to STA, very little is known about the mechanism of action and the export-secretion pathway of methanol-insoluble STB; its gene (estB) has been sequenced (21, 27), and the 71-codon open reading frame, when translated, is very different from the 72 residues of the precursor of STA (reviewed in reference 18). The first 23 amino-terminal amino acids inferred from the estB sequence have properties compatible with a signal peptide (21, 27), and it has been proposed that mature STB is a 48-residue molecule; it was unclear, however, whether STB is an extracellular polypeptide secreted into the medium like STA (16) or whether it is a periplasmic enterotoxin like heat-labile enterotoxin (26). In this communication, we show that mature STB is a 48-amino-acid extracellular polypeptide that corresponds to the previously inferred carboxy-terminal * Correspo...

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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.Many critical biological processes depend on specific cleavage of individual target proteins by serine proteases (1-3). One important example is the dissolution of blood clots in which the initiating and rate-limiting step is activation of the circulating zymogen plasminogen (4, 5). In mammalian systems, activation of plasminogen is accomplished by two closely related enzymes, tissue-type plasminogen activator (t-PA) 1 and urokinase (u-PA) (4 -7). t-PA and u-PA possess an extremely high degree of structural similarity (8, 9), share the same primary endogenous substrate and inhibitors (4), and exhibit remarkably stringent substrate specificity (5). In spite of these striking similarities, however, there are clear functional distinctions between the two enzymes. One particularly intriguing distinction is that, by contrast to single-chain u-PA, single-chain t-PA possesses unusually high catalytic activity and is therefore not a true zymogen (10 -13).Proteases are normally synthesized as inactive precursors or zymogens that must either be proteolytically processed or bind to a specific co-factor to develop substantial catalytic activity. The increase in catalytic efficiency after zymogen activation, or zymogenicity, varies widely among individual members of the (chymo)trypsin family but, in almost all cases, is dramatic. For example, strong zymogens such as trypsinogen, chymotrypsinogen, or plasminogen are almost completely inactive with measured zymogenicities of 10 4 to 10 6 (14, 15). Other serine proteases exhibit intermediate zymogenicity. The enzymatic activity of Factor XIIa is 4000-fold greater than that of Factor XII (16), and the catalytic efficiency of ur...

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 urokinasetype 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.The chymotrypsin family of serine proteases has evolved to include members with both widely divergent and intimately related substrate specificities (1). We chose two members of this family, tissue-type plasminogen activator (t-PA) 1 and urokinase (u-PA), to test the hypothesis that small molecule libraries could be used to identify substrates that discriminate between closely related enzymes. This choice of enzymes assured a rigorous test of the hypothesis because t-PA and u-PA possess an extremely high degree of structural similarity (2, 3), share the same primary physiological substrate (plasminogen) and inhibitors (plasminogen activator inhibitor types 1 and 2) (4, 5), and exhibit restricted substrate specificity (6 -8).Despite their striking similarities, the physiological roles of t-PA and u-PA are distinct (9), and many studies (10 -16), including several that utilize transgenic mice (9, 11, 16), suggest that selective inhibition of either enzyme might have beneficial therapeutic effects. Mice lacking t-PA, for example, are resistant to specific excitotoxins that cause extensive neurodegeneration in wild type mice (11), and mice lacking u-PA exhibit defects in the proliferation and/or migration of smooth muscle cells in a model of restenosis following vascular injury (9). u-PA-deficient mice are also resistant to the induction and/or progression of several tumor types in a two-stage, chemical carcinogenesis model (16).Be...

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 ␤ 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 factorlike module of human tissue-type plasminogen activator (t-PA). The resulting variant of t-PA bound to the platelet integrin ␣ IIb ␤ 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 ␣ IIb ␤ 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.Development of the ability to create novel protein-protein interactions promises to provide important new therapeutic agents as well as unique tools and reagents for the study of key biological processes. Such advances in protein engineering may also provide seminal information about how proteins interact.One strategy for manipulating protein-protein interactions is to employ random or nearly random (e.g. alanine scanning) (1) site-directed mutagenesis to identify amino acid residues critical for binding affinity and specificity. Cumbersome, large scale mutagenesis efforts followed by laborious, time-consuming assays of individual, mutated proteins can sometimes extend this approach to create new molecular interactions. For example, every residue in human growth hormone was scanned for activity prior to re-engineering the molecule to bind the prolactin receptor (2), and a similar strategy facilitated the construction of a variant of interleukin-3 that binds the monomeric ␣ receptor with higher affinity than it binds the interleukin-3 ␣␤ receptor (3). An important current challenge, therefore, is to create more rapid and efficient strategies to engineer proteins with new binding properties. A recent approach that can obviate the need to perform large numbers ...

The methanol-insoluble heat-stable enterotoxin of Escherichia coli (STB) was purified and characterized by automated Edman degradation and tryptic peptide analysis. The amino-terminal residue, Ser-24, confirmed that the first 23 amino acids inferred from the gene sequence were removed during translocation through the E. coli inner membrane. Tryptic peptide analysis coupled with automated Edman degradation revealed that disulphide bonds are formed between residues Cys-33 and Cys-71 and between Cys-44 and Cys-59. Oligonucleotide-directed mutagenesis performed on the STB gene demonstrated that disulphide bond formation does not precede translocation of the polypeptide through the inner membrane and that disulphide bridge formation is a periplasmic event; apparently, elimination of either of two disulphides of STB renders the molecule susceptible to periplasmic proteolysis. In addition, a loop defined by the Cys-44-Cys-59 bond contains at least two amino acids (Arg-52 and Asp-53) required for STB toxic activity.