Directing Sequence-specific Proteolysis to New Targets
THE INFLUENCE OF LOOP SIZE AND TARGET SEQUENCE ON SELECTIVE PROTEOLYSIS BY TISSUE-TYPE PLASMINOGEN ACTIVATOR AND UROKINASE-TYPE PLASMINOGEN ACTIVATOR*

Gary S. CoombsDagger §, Robert C. BergstromDagger , Edwin L. Madisonpar , and David R. CoreyDagger **

From the Dagger  Department of Pharmacology and Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas 75235 and  Department of Vascular Biology, The Scripps Research Institute, La Jolla, California 92037

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

To regulate biological processes proteases must efficiently hydrolyze selected peptide bonds in target proteins while leaving other proteins intact. Many members of the chymotrypsin family of serine proteases, including those involved in blood clotting (1), fibrinolysis (2), complement activation (3), and growth and development (4-7), possess such specificity. Understanding mechanisms by which such proteases restrict their specificities in vivo may aid identification of physiologically relevant substrates and facilitate design of proteases with novel, highly restricted specificities. Such engineered proteases would be useful additions to the repertoire of biological research tools and might have wide-ranging therapeutic applications.

Tissue-type plasminogen activator (t-PA)1 is an attractive model for the study of mechanisms that restrict proteolysis by highly specific serine proteases (2). t-PA is stringently selective for its physiologic substrate plasminogen (Plg), even though its protease domain has high homology to the nonselective protease trypsin (43% overall and 87% within residues conserved in all known trypsins) (8). In previous reports we have shown that much of this specificity is inherent in the protease domain (9) and have used substrate phage display (10, 11) to define consensus sequences for optimal cleavage by t-PA (12, 13) and urokinase-type plasminogen activator (u-PA), a related protease that also targets Plg (14). The consensus sequence for t-PA cleavage was X(Y/F/R)GRdown-arrow (X')A, where X is a large hydrophobic residue and X' can be several different residues but is most often arginine, whereas the optimal sequence for u-PA was GSGRdown-arrow SA. We then obtained 6-14 amino acid peptides containing these sequences and demonstrated that hydrolysis by either t-PA or u-PA occurred with the selectivity predicted by the consensus sequences derived from the substrate phage display.

We now examine whether highly selective proteolysis can be targeted to engineered protein substrates. Proteolytic cleavage within internal surface loops is likely to be more challenging than hydrolysis of peptides because target sequences are constrained within a structured scaffold at both their amino and carboxyl termini. Little information has been reported regarding the interaction of plasminogen activators with any protein substrate except their physiologic target, Plg. However, data that does exist suggests significant differences in the behavior of t-PA toward peptides and proteins. First, peptides containing the amino acid sequence of the physiological cleavage site within Plg are poor substrates, but Plg is efficiently hydrolyzed (9). Second, introduction of a peptide sequence identified by phage display as being highly t-PA labile into an amino-terminal extension of a protein resulted in a 950-fold reduction in Km relative to the same target in a peptide (15). Finally, Thornton and co-workers (16, 17) have shown that the crystallographically determined conformations of 9 known sites of proteolysis within unrelated proteins are not similar to the crystallographically determined conformations of the reactive site loops of proteinaceous protease inhibitors in protease-inhibitor complexes. These authors conclude that up to 12 residues surrounding the scissile bonds would have to be deformed for these target sites to form interactions with proteases that are similar to those observed in trypsin-inhibitor complexes (interactions presumed to be similar to those formed within protease-substrate complexes). These observations suggest that the context of a target sequence can determine its ability to be a substrate and that interactions separate from those of the primary subsites in the substrate binding cleft may make significant contributions to proteolysis.

In this report, we investigate the importance of target site mobility and primary sequence for selective cleavage of engineered protein substrates by t-PA and u-PA. We replaced residues 44-51 within the 43-52 (or Omega ) loop of staphylococcal nuclease (SNase) with optimal t-PA or u-PA cleavage sites and measured kinetic parameters for proteolysis of these engineered protein substrates. t-PA and u-PA efficiently cleave the engineered SNase variants despite substrate loop constraint due to amino- and carboxyl-terminal attachment to core-forming structural elements of the protein, and both enzymes exhibit the same relative sequence selectivity observed for cleavage of peptide substrates, confirming that proteins can be engineered to be selectively labile to t-PA or u-PA. These studies indicate that highly selective proteases can hydrolyze introduced sequences within proteins.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Enzyme Preparations-- Purified t-PA (ActivaseTM) was provided by Genentech (San Francisco, CA). Bovine trypsin was purchased from Sigma. Purified u-PA isolated from human urine was purchased from Calbiochem. Enzyme concentrations were determined by titration with 4-methylumbelliferyl p-guanidinobenzoate (Sigma) using a Perkin-Elmer LS 50B luminescence fluorometer (9). Titrations of trypsin were performed in 100 mM NaCl, 20 mM CaCl2, 50 mM Tris-HCl (pH 8.0). Titrations of t-PA and u-PA were performed in 150 mM NaCl, 10 mM Tris-HCl (pH 7.5).

Mutagenesis and Expression of Protein Substrates-- The plasmid pONF1 (18), which expresses wild-type SNase, under control of the lac promoter was the expression vector for all of the mutants described. The wild-type sequence was initially altered by deleting the coding region for residues 45-50, and altering the codons for residues 44 and 51 to incorporate a SmaI site. This mutagenesis was accomplished by polymerase chain reaction (19) using universal primers 5'-AGGCCTCTAGATAACGAGGCG-3' and 5'-ACTCAAGCTTCGTTTACCATT-3' situated at unique XbaI and HinDIII sites respectively, and the oligonucleotides 5'-GTTGATACACCTGAACCCGGGGAGAAATATGGTCCTG-3' and 5'-AGGACCATATTTCTCCCCGGGTTCAGGTGTATCAACC-3' as mutagenic primers. All mutations, with one exception, were introduced by synthesizing oligonucleotides coding for a desired loop sequence, annealing and ligating them into the introduced SmaI site. The exception is a mutant in which the codon for residue 51 is deleted by the polymerase chain reaction method used to create the SmaI site. Escherichia coli strains HB101 and DH5alpha were used for expression of the mutants.

SNase was prepared as described (20). Cultures were grown to A600 = 0.9 and induced by addition of 2 mM lactose. Cultures were harvested 3-4 h after induction, and purification of SNase mutants was performed as described previously using BioRex 70 cation exchange resin (Bio-Rad). Purified nuclease mutants were dialyzed against 50 mM NaCl, 2 mM HEPES, pH 6.8, at 4 °C. Where necessary, nuclease solutions were concentrated by centrifugation at 1,000 rpm in 3-ml microconcentrators (Filtron; Northborough, MA). A280 of each purified SNase mutant was measured in a 1-ml quartz cuvette using a Hewlett-Packard 8452 diode array spectrophotometer. Concentrations were determined using the relation [SNase] = A280/varepsilon , where the molar absorption coefficient varepsilon  is 18280 M-1 cm-1 (corresponds to 0.93 mg/A280) (19). Purity was confirmed by polyacrylamide gel electrophoresis.

Analysis of Nucleolytic Activity of SNase Mutants-- Calf thymus DNA was purchased from Sigma and dissolved in 10 mM CaCl2, 40 mM Tris-Cl, pH 7.4, to a stock concentration of 1.86 mg/ml as determined by measuring A260 and using the molar absorption coefficient varepsilon  = 0.025 ml/µg-1 cm-1. This stock was denatured by heating to 100 °C for approximately 40 min after which remaining solid was removed by filtration through Whatman #4 filter paper (Whatman International, Maidstone, United Kingdom). Dilutions were then made in 10 mM CaCl2, 40 mM Tris-Cl, pH 7.4, in concentrations ranging from 5-60 µg/ml. DNA solutions of varying concentrations were then added to 1-ml quartz cuvettes and rapidly mixed with nuclease to final concentrations between 6 and 350 nM. Hydrolysis of the DNA was continuously monitored by following the increase in absorbance at 260 nm in a Hewlett-Packard 8452 diode array spectrophotometer over 40 min to 1 h for each digestion. Data was interpreted by Eadie-Hofstee analysis to obtain Km and Vmax for DNA hydrolysis. Errors were calculated as described (21). Vmax is usually reported rather than kcat because of the heterogeneous nature of the chromosomal DNA substrate, but for purposes of comparison with the uncatalyzed pseudo-first order rate we assumed an average molecular weight for tetranucleotide substrate of 1400 and a change in absorbance of 0.3 OD for complete hydrolysis of 50 mg/ml DNA (22, 23).

Analysis of Proteolytic Activity Toward Protein Substrates-- Potential protein substrates were incubated for various time periods at 37 °C with 10-50 nM trypsin or at concentrations of t-PA and u-PA ranging from 10 to 320 nM. Substrate protein concentrations varied from 1 to 20 µM. For kinetic assays, t-PA and u-PA concentrations were 50 nM, and 5-9 substrate concentrations were used within the range listed above. Proteolytic digests were terminated between 10 and 20% completion by addition of loading buffer and heating to 100 °C for 10-20 min. Samples of each digest were separated on 15% SDS-polyacrylamide gels and stained with Coomassie Brilliant Blue. After destaining, substrate to proteolytic product ratios were determined by densitometric scanning on a model 300A scanning densitometer (Molecular Dynamics, Sunnyvale, CA) operating with ImageQuant 3.0 software. The ratios obtained were used to determine initial velocities of cleavage. The site of cleavage was confirmed by amino-terminal amino acid sequencing of the carboxyl-terminal proteolytic product. Values for kcat and Km were derived from Eadie-Hofstee analysis.

For some protein substrates, it was not possible to achieve substrate concentrations that approached Km. For these proteins, individual parameters kcat and Km could not be determined. However, for [S] <<  Km, the Michaelis-Menten equation,
V<SUB>0</SUB>=<FR><NU>V<SUB><UP>max</UP></SUB>[<UP>S</UP>]</NU><DE>K<SUB>m</SUB>+[<UP>S</UP>]</DE></FR> (Eq. 1)
simplifies to the following.
V<SUB>0</SUB>=<FR><NU>V<SUB><UP>max</UP></SUB></NU><DE>K<SUB>m</SUB></DE></FR>[<UP>S</UP>] (Eq. 2)
Thus Vmax/Km can be obtained from the slope of linear plots of V0 (initial velocity) versus [S] (substrate concentration). Data were fit to Equation 2 by linear regression and in all cases R2 > 0.9. Under these conditions Km was estimated to be at least 5-fold higher than the highest substrate concentration used in the assay, because if [S] was any larger than 20% of Km, it would significantly affect the denominator of the above Michaelis-Menten equation, and the resultant systematic deviation from linearity would be about 2-fold greater than the errors we observed. This assumption allowed estimation of Vmax and Km as described (24, 25).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

SNase as a Protein Substrate-- We used SNase to study the activity of t-PA and u-PA toward protein substrates. SNase contains one of the trypsin cleavage sites examined by Thornton and co-workers (16, 17), carboxyl-terminal to lysine 48 within a surface loop formed by residues 43-52, and NMR studies (26, 27) and x-ray crystallography (28) conclude that this loop is highly flexible. These observations suggested that this site would be accessible to u-PA or t-PA. We prepared SNase variants in which residues 44-51 within the 43-52 loop were replaced by sequences that are selective for cleavage by t-PA or u-PA. NMR and kinetic studies have demonstrated that variants containing deletions within the 43-52 loop have essentially the same structure as wild type (29) and are catalytically active (30), but to provide experimental evidence that this was also true for our variants we obtained kinetic parameters for their hydrolysis of calf thymus DNA. Vmax/Km for the variants was decreased from 2000-20,000-fold relative to wild type (Table I). These substantial reductions were expected since residues 44-51 play a significant role in the rate-limiting processes of substrate binding and product release (31) and are adjacent to glutamate 43, a residue critical for efficient catalysis (32).

                              
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Table I
Kinetic constants for hydrolysis of denatured calf thymus DNA by wild type and nine SNase variants containing amino acid substitutions for the sequences TKHPKKGV at residues 44-51

Vmax is used to characterize the kinetics of SNase catalysis because of the heterogeneous nature of the DNA substrate, but it can be converted into an approximate kcat value assuming the molecular weight of an average substrate tetranucleotide to be 1400 and a change in absorbance of 0.3 A260 for complete hydrolysis of 50 µg/µl DNA (22, 23). Conversion of Vmax into kcat allows direct comparison with the background rate of phosphodiester hydrolysis. The lowest kcat we calculate for any variant, 0.1 s-1, was over 12 orders of magnitude greater than the pseudo-first order rate constant determined for the uncatalyzed reaction, 5.7 (10)-14 s-1 (23, 33-35). Retention of substantial catalytic activity suggests that most of the structural features necessary for catalysis are present in the variants and that their overall structure is similar to the wild-type enzyme. Structural similarity between variants is likely because Vmax/Km values varied by only 10-fold between the most and the least active variants and because 7 of the 9 mutants exhibited differences in Km of less than 5-fold.

Characterization of Cleavage of Engineered Protein Substrates-- We separately incubated t-PA, u-PA, or trypsin with the engineered SNase variants. Product separation by polyacrylamide gel electrophoresis demonstrated site-specific proteolysis by t-PA and u-PA of every variant (results not shown), and amino-terminal sequencing confirmed that this cleavage occurred within the inserted sequences at the predicted P1 arginine. Trypsin readily cleaved the variants at other sequences after the initial cleavage within the target loop, whereas u-PA and t-PA did not cleave other sequences even when digestions were allowed to run to completion.

To confirm that kinetic data could be obtained using SNase as a substrate we determined the range of concentrations of protease for which cleavage of SNase was linearly dependent on amount of the protease present and used protease concentrations within this range for all subsequent assays. We then determined the range of times in which appearance of cleavage products increased linearly and in which the only measurable cleavage occurred at the engineered target site (Fig. 1, A-D). For trypsin, appearance of product correlates linearly with time for digestions that were stopped prior to cleavage of 8% or less of total substrate (Fig. 2, A and C). For t-PA and u-PA, linear initial rates were maintained until cleavage of 20% of total substrate had occurred (Fig. 1, B and D for t-PA; u-PA data not shown). The linear range for trypsin-mediated hydrolysis is less than that for t-PA or u-PA because, as noted, trypsin has a much greater propensity to cut at other sites after initial hydrolysis within the 44-51 insertion.


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Fig. 1.   Dependence of site-selective hydrolysis of SNase variant (IV) by trypsin (20 nM) or t-PA (50 nM) as a function of time. A, cleavage of SNase variant (IV) (38 µM) by trypsin for 6-192 s; B, cleavage of SNase variant (IV) (28 µM) by t-PA for 0.77-12.3 h; C, percentage of cleavage of SNase variant (IV) by trypsin plotted as a function of time; D, percentage cleavage of SNase variant (IV) by t-PA plotted as a function of time. Data points in plots in C and D are an average of at least two separate assays.


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Fig. 2.   Selective cleavage of SNase variants by t-PA and u-PA at engineered target sequences. Each lane was loaded with 20-µl volumes containing 15 µM wild-type SNase, variant (VII), or variant (VIII). Concentrations of t-PA or u-PA were 75 nM. Each reaction was incubated at 37 °C for 4 h.

Influence of Insert Size on Catalytic Efficiency-- As noted above, Thornton and co-workers (16, 17) have shown that sequences within proteins must deform from their crystallographically determined conformations to engage in productive, "canonical" interactions (i.e. similar to those observed between trypsin and bovine pancreatic trypsin inhibitor) with proteases. The need to deform a sequence prior to binding, and/or cleavage may become rate-limiting for hydrolysis of protein substrates, suggesting that the efficiency of proteolysis of a target sequence within a protein surface loop may be sensitive to small changes in the size of the loop, and that this dependence may differ among related proteases. To test this hypothesis we synthesized 5 substrates with sequences of increasing length replacing residues 44-51. Each substrate contained a 6-residue P4 to P2' sequence previously shown to be highly labile for cleavage by t-PA (12) and flanking regions that varied from no amino acids, SNase (I), to 4 amino acids, SNase (V) (Table II). For both trypsin and t-PA, SNase (I) was the least efficiently cleaved substrate. Trypsin and t-PA differed, however, in that catalytic efficiency of trypsin-mediated cleavage increased 7.5-fold as the length of the introduced sequences increased from 6 to 10 residues, while efficiency of t-PA-mediated cleavage increased 38-fold (Table II) as the length increased from 6 to 8 residues. Increased efficiency for t-PA-mediated cleavage was largely due to a greater than 15-fold decrease in Km, from >= 140 µM for SNase (I) to 9.3 µM for SNase (V). By contrast, Km for trypsin-mediated cleavage of the same variants decreased less than 3-fold.

                              
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Table II
Kinetic constants for hydrolysis by trypsin and t-PA of a series of SNase variants (I-V) that contain an optimal P4-P2' t-PA target sequence (PFGRSA) and that vary insert length from 6 to 10 residues by addition of Gly or Pro residues at subsites flanking the P4-P2' target sequence

Selective Proteolysis of SNase Variants by t-PA-- SNase variants (VI) and (VII) were obtained by replacing the wild-type sequence of residues 44-51 with sequences HYGRSG and QRGRSA, which had been identified as t-PA selective within a peptide context (12, 14). Proteolysis of the variant nucleases was highly selective with hydrolysis observable only at the inserted sequences (Fig. 2). Both variants (VI) and (VII) were hydrolyzed by t-PA with a kcat/Km value of 670 M-1 s-1 (Table III), similar to kcat/Km values of 322 and 850 M-1 s-1 for hydrolysis of the respective analogous peptide substrates (12, 14). There are striking differences for the individual constants kcat and Km for protein versus peptide cleavage, and these will be discussed below. u-PA proteolyzed the variants less efficiently than t-PA with kcat/Km values 230-fold less than t-PA for (VI) and 92-fold less than t-PA for (VII). For (VI) this decrease was largely due to a decreased kcat (136-fold), while the decreased value for (VII) was more evenly influenced by both kcat and Km. The 230- and 92-fold t-PA/u-PA selectivities for cleavage of HYGRSG and QRGRSA in a protein context compare favorably with the 21- and 19-fold selectivities observed in the peptide context. To further the comparison between t-PA and u-PA we examined cleavage of variants (I) and (V) by u-PA. These were characterized by kcat/Km values of 1.5 and 25 M-1 s-1 respectively, affording t-PA/u-PA selectivities of 19-fold for (I) and 44-fold for (V).

                              
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Table III
Kinetic parameters for hydrolysis by t-PA and u-PA of SNase variants containing t-PA or u-PA specific target sequences identified in peptides

Selective Proteolysis of SNase Variants by u-PA-- We also assayed SNase variants (VIII) and (IX) containing the u-PA specific target sequence GSGRSA in loops of varying size. u-PA cleaved both substrates more efficiently than did t-PA (Table III, Fig. 2). The variant containing GSGRSA within an 8-residue insert (VIII) displayed selectivity for u-PA cleavage of 12-fold, whereas the variant containing a 14-residue insert was cleaved by u-PA with a higher efficiency (kcat/Km = 900 M-1 s-1) and with a u-PA/t-PA selectivity of 47-fold. The analogous sequence in a peptide context was cleaved with a u-PA/t-PA specificity of 63 so that, by contrast to cleavage of t-PA selective variants (VI) and (VII), cleavage of the u-PA selective sequence was slightly less selective in a protein relative to a peptide context. The differing reactivities of SNase variants (VIII) and (IX) suggest that u-PA requires substrate mobility to fully exploit selectivity inherent in a primary sequence. kcat/Km values for both (VIII) and (IX) were similar to or less than the kcat/Km values of 740-4700 M-1 s-1 for hydrolysis of the analogous peptide substrates containing the sequence GSGRSA surrounded by different flanking sequences.

Reduction of kcat and Km for Sequences in a Protein Context-- As noted above, kcat/Km values were within 5-fold for analogous protein and peptide substrates, whereas partitioning between kcat and Km were much different. Km values for hydrolysis by t-PA or u-PA of SNase variants (I-IX) are reduced from 10- to 200-fold relative to those for analogous peptide substrates. Variants (III-VIII) have Km values of 8.9-18 µM, whereas analogous peptides possess Km values of 600 µM to 4 mM (Table III). For example, the Km value for hydrolysis of SNase variant (VI) containing the sequence HYGRSG by t-PA of 18 µM (Table III) is over 200-fold lower than Km for the analogous peptide of 4 mM (13), while the Km value for hydrolysis of SNase variant (VII) containing the sequence QRGRSA of 8.9 µM is 120-fold lower than Km for the analogous peptide of 1.5 mM (5). kcat values were also decreased for the cleavage of protein substrates. The largest kcat values for cleavage of SNase variants by t-PA or u-PA were 0.01-0.018 s-1 (Tables II and III), whereas kcat values for analogous peptide substrates were 1.3-3.7 s-1 (12-14).

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Kinetic Analysis of Protein Substrates Enhances Understanding of Proteases-- Protease activity and specificity are usually studied using 1-4 amino acid peptides containing chromogenic or fluorogenic leaving groups. These small molecules provide convenient and mechanistically informative substrates, but the absence of a peptide amide bond between P1 and P1' and the lack of amino acids on the prime side of the scissile bond limit the conclusions that can be drawn from their use. Synthetic peptides containing amino acids on both sides of the scissile bond overcome these limitations by providing useful information about preferences for prime site occupancy and activity toward amide bonds that are chemically identical to linkages in natural substrates. Proteases, however, have evolved to hydrolyze proteins. Understanding this hydrolysis is especially important for highly selective proteases like t-PA and u-PA, which exist in a milieu of folded proteins and must discriminate between their physiologic target, plasminogen, and all other proteins they encounter. To understand how t-PA and u-PA accomplish this task, it is necessary to examine how the activity of these two enzymes is enhanced toward plasminogen and diminished toward other proteins, and our study was designed to begin to explore positive and negative determinants for this proteolysis.

SNase Is an Informative Substrate for Kinetic Analysis of Proteolysis-- One disadvantage for the use of protein substrates is that in contrast to peptide substrates, which can usually be assumed to lack ordered structure (an assumption readily testable by NMR or circular dichroism), the structure of protein substrates may vary depending on the sequence at the target site. Such structural variation would not prevent comparison of proteolysis of a given variant by u-PA versus t-PA or t-PA versus trypsin but might obscure comparison of different variants by the same protease. To mitigate this problem we chose SNase as our substrate scaffold. SNase is one of the most mutagenized and structurally characterized proteins known, and previous studies had revealed that the 43-52 loop was accessible to trypsin and could be altered without changing the structure of the rest of the protein. In fact, early work by Anfinsen and co-workers (36, 37) had noted that this site could be cleaved to generate two fragments, which could then be separated and reconstituted to restore catalysis.

Our alterations within the 43-52 loop do reduce catalysis by the SNase variants, but this reduction is modest compared with retention by the variants of 10-12 orders of magnitude catalytic rate enhancements relative to the nonenzyme-catalyzed rate. Indeed, choosing a target site for mutation near the active site is an ideal strategy for obtaining and comparing protein substrates since mutating a loop distant from the active site might disrupt nearby structures without providing any means for assessing this disruption by monitoring catalysis. An example of the usefulness of possessing a target site whose mutation will perturb but not abolish catalysis is the finding that kcat/Km for hydrolysis of DNA by the SNase variants varies by only 10-fold, a similarity that supports the belief that informative conclusions can be drawn from conservative comparison of kinetic constants for their proteolysis. Finally, an additional advantage of using SNase is that its extensive characterization by x-ray crystallography and NMR spectroscopy facilitates molecular modeling of the protease-substrate complex to gain insights into the structural basis for the kinetic results we obtain and structural investigations of any particularly interesting recombinant substrates.

Sequence Preferences for Peptide Cleavage by t-PA and u-PA Are Preserved in Protein Substrates-- Prior to these experiments we did not know whether the sequence requirements that mediate t-PA/u-PA specificity for small peptide substrates would also apply to protein substrates. If interactions stabilizing substrate-like conformations, such as those at S1 and surrounding subsites, are similar for both protein and peptide substrates, then the selectivity exhibited for the cleavage of peptide substrates would be maintained in corresponding protein substrates. If, conversely, protein substrates contribute other critical interactions that do not form with peptide substrates, specificity conferred by optimal subsite interactions might be less important for protein substrates. Our finding that t-PA/u-PA selectivity is similar for catalysis of the SNase variants and peptide substrates suggests that the molecular determinants defining optimal sequence selectivity for peptide substrates also influence cleavage of protein substrates.

The selective proteolysis of the SNase variants in our study is in contrast to our previous observation that t-PA was unable to cleave a t-PA labile primary sequence placed into an internal, trypsin accessible site within ornithine decarboxylase (15). Apparently the lack of cleavage of the engineered ornithine decarboxylase was due to unfavorable structure or restricted mobility at that particular target site and not to a general inability of t-PA to hydrolyze sequences within nonphysiological protein targets. Highly selective cleavage by t-PA and u-PA at introduced sites suggests that these and similar proteases can be used for structural mapping, cleavage of fusion proteins, and separation of protein domains.

t-PA Requires a Longer Target Loop for Optimal Cleavage than Does Trypsin-- Highly selective proteases possess many mechanisms to enhance cleavage of physiologic target proteins while discouraging cleavage of nontarget proteins. The 5-fold greater dependence on insert size for t-PA relative to trypsin suggests that t-PA is more sensitive to substrate mobility and may require that more amino acids be properly oriented by the enzyme before the sequence can bind in a catalytically productive alignment. Such sensitivity would have obvious advantages for a protease, like t-PA, that must avoid systemic cleavage of nontarget proteins since cleavage of nonphysiologic protein substrates require that two prerequisites be fulfilled (i) that the substrates contain an amino acid sequence that is optimal for cleavage and (ii) that the sequence be displayed within a flexible loop. The requirement for more interactions with substrate is similar to our earlier finding that t-PA has a greater need for direct interactions with substrate residues P3 and P4 to achieve maximal catalysis of peptide substrates than trypsin (15).

Reduction of kcat and Km for Sequences in a Protein Context-- Our observation that Km is reduced for cleavage of labile sequences placed within a protein context agrees with our previous finding that Km decreases 950-fold upon incorporation of a t-PA labile sequence into an amino-terminal sequence extension of ornithine decarboxylase (15). The large reduction in Km may be a key distinction between stringently selective proteases and their close relatives that exhibit broad specificity. Consistent with this hypothesis, Km values for the cleavage of SNase variants (I-V) by trypsin, 11-31 µM, are only 3-9-fold lower than the Km value for trypsin-mediated hydrolysis of a peptide containing the same PFGRSA sequence from P4 to P2', 103 µM (12). SPase I, a signal peptidase from E. coli that is not a member of the chymotrypsin family, exhibits a 50-100-fold decrease in Km for cleavage of a protein relative to analogous peptide substrates (38), suggesting that enhanced binding of protein substrates may also characterize other classes of protease.

Whereas Km decreased for the variants studied, kcat also decreased relative to cleavage of analogous peptide substrates. At least two explanations could account for the reduction in kcat. First, as discussed by Thornton and co-workers (16, 17), there appears to be a requirement for deformation of a protein's native structure to obtain a scissile bond in a substrate-like conformation. Inducing or stabilizing this nonnative conformation represents an enthalpic cost, which may manifest itself as a reduction in kcat. A second explanation is that protein substrates provide determinants for diverse interactions with proteases and that many of these interactions are nonproductive, yielding reductions in both kcat and Km.

Conclusion-- To achieve selective proteolysis of protein substrates, proteases must possess one or more mechanisms to enhance hydrolysis of a specific peptide bond within a selected substrate and separate mechanisms to discourage hydrolysis at other potential substrate sequences. Understanding these mechanisms will require kinetic analysis of the interactions of proteases with protein substrates. We find that t-PA and u-PA hydrolyze nonphysiological protein substrates and that cleavage remains dependent upon the same determinants of specificity noted for hydrolysis of small peptide substrates. Our finding that loop size and target sequence play critical roles in defining the susceptibility of protein substrates to cleavage by t-PA and u-PA suggests mechanisms for the development of specificity in the fibrinolytic system and may reflect general strategies for control of selective proteolysis. Finally, the ability to cleave engineered protein substrates suggests that it may be possible to generate a wide range of new proteolytic specificities and hydrolyze selected native proteins by engineering subsite specificity of highly selective proteases.

    ACKNOWLEDGEMENT

We are grateful to Dr. Margaret Phillips and Dr. Donald Doyle for critical evaluation of this manuscript.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grants RO1 HL52475 and P01 HL31950 (to E. L. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported by National Institutes of Health predoctoral training grant T32GMO8203.

** Assistant investigator with the Howard Hughes Medical Institute. To whom correspondence may be addressed: Dept. of Pharmacology, Howard Hughes Medical Institute, University of Texas Southwestern Medical Ctr., Dallas, TX 75325-9050. Fax: 214-648-5095; E-mail: Corey{at}howie.swmed.edu.

par To whom correspondence may be addressed: Dept. of Vascular Biology (VB-1), The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037. Fax: 619-784-7323.

1 The abbreviations used are: t-PA, tissue-type plasminogen activator; SNase, staphylococcal nuclease; u-PA, urokinase-type plasminogen activator; Plg, plasminogen.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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