Institute for Biomolecular Science, Gakushuin University, Mejiro,Tokyo 171-8588, Japan
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Abstract |
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Keywords: amino acid replacement/inhibitory activity/ovomucoid domain 3/protease inhibitor/proteinprotein interaction
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Introduction |
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We have been studying the structurefunction relationship of protease inhibitors using genetically engineered site-specific mutants of the Streptomyces subtilisin inhibitor (SSI) (Kojima et al., 1990a,b
, 1991
, 1993
, 1994a
; Takeuchi et al., 1992
), ovomucoid domain 3 (Kojima et al., 1994b
), squash-family inhibitor (Kojima et al., 1996
) and natural mutants of SSI (SIL proteins) (Kojima et al., 1994c
; Taguchi et al., 1994
; Terabe et al., 1994
, 1995
). We previously succeeded in inducing inhibitory activity toward trypsin or chymotrypsin by replacing the P1-site residue of SSI with Lys or Tyr (Kojima et al., 1990a
), respectively. This indicates that the P1-site residue of protease inhibitors is the main determinant of the inhibitor's specificity of action. Similarly, we showed that chicken ovomucoid domain 3 (OMCHI3) acquires inhibitory activity toward chymotrypsin or trypsin when its P1-site residue (Ala) is replaced by Met or Lys, respectively (Kojima et al., 1994b
). However, the inhibitory activities of these OMCHI3 mutants (Ki = 4.12x107 M for the P1-Met mutant acting on chymotrypsin and Ki = 1.34x105 M for the P1-Lys mutant acting on trypsin) are not as strong as those of naturally-occurring inhibitors (Ki = 10101011 M).
Therefore, we attempted to convert OMCHI3(P1Met) and OMCHI3(P1Lys) into stronger inhibitors of chymotrypsin or trypsin by additional amino acid replacements around the reactive site. We also hoped to clarify the contribution of residues in specific positions to the inhibitors' interactions with these proteases. In the case of SSI, we have shown that MetGly replacement at the P4 site of the P1-Lys mutant resulted in an increase of inhibitory activity toward trypsin (Kojima et al., 1990b
). Laskowski and co-workers have been studying the sequenceactivity relationship of ovomucoid domain 3 extensively using natural mutants isolated from more than 100 species of bird (Empie and Laskowski, 1982
; Laskowski et al., 1987
). However, natural mutants do not necessarily possess the amino acid replacements that are of the greatest interest to investigators. Therefore, we have used site-directed mutagenesis to produce desirable mutants.
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Materials and methods |
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Restriction enzymes and DNA-modifying enzymes were purchased from Takara Shuzo (Kyoto, Japan), Toyobo (Osaka, Japan) and BioRad (Hercules, CA). Two proteases, bovine chymotrypsin (TLCK-treated) and trypsin (TPCK-treated), and their synthetic substrates, N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe p-nitroanilide and N-benzoyl-L-Arg p-nitroanilide, were obtained from Sigma (St Louis, MO). Other chemicals were of reagent grade for biochemical research.
Site-directed mutagenesis
The PstIEcoRI fragment of the OMCHI3 gene (Kojima et al., 1994b) was inserted into the PstIEcoRI site of pTZ18U and the constructed plasmid was then introduced into Escherichia coli CJ236. Single-stranded DNA containing uracil bases was prepared by infection of the helper phage M13KO7, and site-directed mutagenesis was carried out using mutation primers, according to the method of Kunkel (1985). Double and triple mutants were prepared from single and double mutants, respectively, and so on. The amino acid sequences of OMCHI3 mutants used as templates, the nucleotide sequences of the mutation primers and the obtained mutants are summarized in Table I
.
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Expression and purification of OMCHI3 mutants
The mutated OMCHI3 was expressed in E.coli and purified essentially as described previously (Kojima et al., 1994b). Large-scale cultivation of E.coli JM105 transformed by the expression plasmid was started by inoculation of a small-scale overnight culture. Gene expression was induced by adding isopropylthio-ß-D-galactopyranoside to a final concentration of 1 mM when A550 = 1.0, and the cultivation was continued overnight. Proteins in the culture supernatant were precipitated with ammonium sulfate, and subjected to ion-exchange chromatography on DE-52 with a gradient of NaCl. The ovomucoid-containing fractions were then applied to gel-filtration on Sephadex G-50 or Superdex 75. The concentrations of the purified OMCHI3 mutants were determined by the modified method of Lowry (Bensadoun and Weinstein, 1976
).
Measurement of inhibitor constants (Ki) of OMCHI3 mutants
Chymotrypsin or trypsin was incubated with OMCHI3 mutants at various molar ratios in 200 µl of 0.1 M TrisHCl (pH 7.8) at 25°C to allow complexes to form, and then added to 800 µl of 0.0806 mM N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe p-nitroanilide (for chymotrypsin) or 0.231 mM N-benzoyl-L-Arg p-nitroanilide (for trypsin) in the buffer. The absorbance at 410 nm at 25°C was monitored to measure the residual activity of the protease. Inhibitor constants were estimated from these inhibition profiles according to a procedure that has been described previously (Kojima et al., 1994b).
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Results |
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Protease inhibitors of chymotrypsin have been isolated from various natural sources. The amino acid sequences around the reactive site and the Ki values of several ovomucoid domain 3 and chymotrypsin inhibitors from other families of protease inhibitors are listed in Table II. All of the natural variants of ovomucoid domain 3 that strongly inhibit chymotrypsin possess a Tyr residue at the P2' site (Laskowski et al., 1987
), whereas OMCHI3 has an Asp residue at this site. Therefore, we replaced the Asp residue at the P2' site of OMCHI3(P1Met) with a Tyr. OMCHI3(P1Met, P2'Ala) was also prepared to examine the effect on binding with chymotrypsin of the removal of the negatively charged Asp residue and the introduction of an aromatic ring. The Asp
Tyr replacement at the P2' site was also introduced into wild-type OMCHI3.
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Inhibition of chymotrypsin by OMCHI3(P1Met)-derived mutants
The OMCHI3 mutants secreted from E.coli were purified to homogeneity by chromatography, and some amino acid replacements introduced into the mutants were confirmed by sequence analysis of the purified mutant proteins.
The Ki value of OMCHI3 (P1Met, P2'Tyr) was estimated to be 1.17x1011 M, showing that this mutant is a strong inhibitor of chymotrypsin. Since the Ki value of OMCHI3(P1Met) was 4.12x107 M (Kojima et al., 1994b), the Asp
Tyr replacement at the P2' site made OMCHI3(P1Met) about 35 000-fold more effective as an inhibitor of chymotrypsin. Similarly 35 000-fold enhancement of the interaction between chymotrypsin and the wild-type OMCHI3 was observed after Asp
Tyr replacement at the P2' site; the Ki value of OMCHI3(P2'Tyr) was estimated to be 1.45x108 M and that of wild-type OMCHI3 was reported by Laskowski et al. (1989) to be 5.1x105 M. These Ki values show that the increase in the chymotrypsin-binding ability of wild-type OMCHI3 or OMCHI3(P2'Tyr) induced by Ala
Met replacement at the P1 site is about 1200-fold, which is weaker than the effect of Asp
Tyr replacement at the P2' site. Hence, OMCHI3(P2'Tyr) is a more effective inhibitor of chymotrypsin than OMCHI3(P1Met).
We produced OMCHI3(P1Met,P2'Ala) to examine the effects of removing a negative charge and of introducing an aromatic ring by AspTyr replacement at the P2' site. OMCHI3(P1Met,P2'Ala) had a Ki value of 3.19x1010 M, which showed that the negative charge at the P2' site weakened the interaction of OMCHI3(P1Met,P2'Ala) with chymotrypsin by a factor of 1300. In contrast, the introduction of an aromatic ring into the P2' site resulted in a 27-fold enhancement of binding. These results indicate that the increased chymotrypsin-binding ability of ovomucoid domain 3 with Asp
Tyr replacement at the P2' site is mainly due to the removal of the negative charge.
On the other hand, removal of a positive charge from the P3' site of a strong chymotrypsin inhibitor, OMCHI3(P1 Met,P2'Tyr), by ArgAla replacement resulted in conversion to a 18 400-fold less effective inhibitor with a Ki value of 2.15x107 M. This finding indicates that a positive charge at the P3' site is essential for strong binding of an inhibitor with chymotrypsin.
The amino acid sequence of OMCHI3(P1Met,P2'Tyr), a strong inhibitor of chymotrypsin, differs from that of ovomucoid domain 3 of silver pheasant (OMSVP3) by only one amino acid at the P4-site, which is Asp in OMCHI3(P1Met,P2'Tyr) and Ala in OMSVP3. Empie and Laskowski (1982) reported that the Ki value of OMSVP3 is 5.6x1012 M. Thus the contribution of the P4 site to the interaction with chymotrypsin is small compared with those of the P1, P2' or P3' sites.
The Ki values and amino acid sequences around the reactive site of the genetically engineered variants of ovomucoid domain 3 are summarized in Figure 1.
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The amino acid sequences and Ki values of trypsin inhibitors isolated from natural sources are listed in Table III. This data shows that an aromatic amino acid is conserved at the P2' site in OMCHI2 and pancreatic secretory trypsin inhibitors (PSTI) that are homologous to OMCHI3. Therefore, we replaced an Asp residue at the P2' site of OMCHI3(P1Lys) with a Tyr or an Ala to examine the effect on trypsin inhibition of removing a negative charge and of introducing an aromatic ring at the P2' site.
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Then, we prepared three derivatives of OMCHI3(P1Lys), each of which possessed two of the three replacements described above, to examine the combined effects of these mutations on trypsin inhibition. Finally, a mutant with P4, P2' and P3' site replacements was prepared, because it was expected to be a strong inhibitor of trypsin.
Inhibition of trypsin by OMCHI3(P1Lys)-derived mutants
The Ki values of the three OMCHI3(P1Lys)-derived mutants possessing single amino acid replacement were 1.27x107 M for OMCHI3(P1Lys,P2'Tyr), 2.45x105 M for OMCHI3 (P1Lys, P3'Ala) and 2.59x106 M for OMCHI3(P1Lys, P4Ala). Comparing these results with the Ki value for OMCHI3(P1Lys), 1.34x105 M (Kojima et al., 1994b), shows that Asp
Tyr replacement at the P2' site increased the binding of OMCHI3(P1Lys) with trypsin most effectively, by a factor of 106; the Asp
Ala replacement at the P4 site resulted in only a 5-fold increase in trypsin binding and the Arg
Ala replacement at the P3' site resulted in conversion to a less effective inhibitor.
As was the case for chymotrypsin inhibition, the increase in inhibitory activity toward trypsin induced by AspTyr replacement at the P2' site of OMCHI3(P1Lys) was mainly due to removal of the negative charge from that site, since OMCHI3(P1Lys,P2'Ala) with a Ki value of 3.52x107 M was about 30-fold more effective than OMCHI3(P1Lys) and was only 2.8-fold less effective than OMCHI3(P1Lys,P2'Tyr) as a trypsin inhibitor.
Combining two effective mutations, AspTyr at the P2' site and Asp
Ala at the P4 site, converted OMCHI3(P1Lys) to a more effective trypsin inhibitor with a Ki value of 3.08x108 M. The Ki values also show that the ratio of binding enhancement induced by introducing both of these mutations into OMCHI3(P1Lys) was similar to the sum of the enhancements induced by the same mutations when they were introduced into OMCHI3(P1Lys) separately.
When the ArgAla replacement at the P3' site was introduced into OMCHI3(P1Lys) the interaction with trypsin was weakened; however, introducing this substitution into OMCHI3(P1Lys, P2'Tyr) and OMCHI3(P1Lys, P4Ala) enhanced their binding abilities by a factor of 1.8 and 2.7, respectively: the Ki value of OMCHI3(P1Lys, P2'Tyr, P3'Ala) was 7.11x108 M and that of OMCHI3(P1Lys, P4Ala, P3'Ala) was 9.65x107 M. These results also show that OMCHI3 (P1Lys, P2'Tyr, P3'Ala) and OMCHI3(P1Lys, P4Ala, P3'Ala) are 345- and 25-fold more effective as inhibitors of trypsin, respectively, compared with OMCHI3(P1Lys, P3'Ala).
When all three mutations were introduced into OMCHI3 (P1Lys) the resultant mutant had a Ki value of 1.44x109 M and was about 10 000-fold more effective as a trypsin inhibitor than OMCHI3(P1Lys). Based on the Ki values, the binding enhancement induced by introducing a fourth mutation into a triple mutant was 670-fold for the AspTyr replacement at the P2' site, 49-fold for the Asp
Ala replacement at the P4 site and 21-fold for the Arg
Ala replacement at the P3' site.
These results are summarized in Figure 2.
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Discussion |
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Why is the Tyr residue at the P2' site of ovomucoid domain 3 conserved? Ardelt and Laskowski (1991) suggested that rigidification of the main chain, achieved through stacking of the aromatic ring of Tyr and the pyrrolidine ring of Pro at the P4' site, may facilitate the binding of the inhibitor to the protease. The Pro at the P4' site is also conserved in ovomucoid domain 3. We observed increased inhibition of chymotrypsin or trypsin by AlaTyr replacement at the P2' site in the P1-site mutants of OMCHI3, although this effect was smaller than that induced by Asp
Ala replacement at the same site. Therefore, the enhanced affinity of wild-type OMCHI3 for chymotrypsin induced by Asp
Tyr replacement at the P2' site seems to be due to the removal of the negative charge and to the stacking effect of an aromatic ring, rather than being due to the recognition by chymotrypsin of the Tyr residue as a P1-site residue of an inhibitor.
In contrast to the increased inhibition of chymotrypsin and trypsin induced by the AspTyr replacement at the P2' site, Arg
Ala replacement at the P3' site of OMCHI3(P1Met, P2'Tyr) dramatically weakened the interaction with chymotrypsin. The presence of an Arg residue at the P3' site of both ovomucoid domain 3 and barley chymotrypsin inhibitor 2 (Svendsen et al., 1980
), an inhibitor belonging to the other family of serine protease inhibitors, suggests the presence of an electrostatic interaction or salt bridge between the Arg residue of the inhibitor and a negative charge(s) in the substrate-binding pocket of chymotrypsin. The chymotrypsin structure, shown in Figure 3
, demonstrates that Asp35 and Asp64 are candidates for such an interaction. Attempts by Collins et al. (1990) to convert PSTI to an effective inhibitor of chymotrypsin showed that introducing an Arg into the P3' site in addition to Lys
Leu replacement at the P1 site was required for strong inhibition of chymotrypsin; subsequent X-ray crystallographic analysis of the inhibitorenzyme complex indicated the presence of a salt bridge between the Arg residue at the P3' site of the inhibitor and Asp residues in the substrate-binding pocket of chymotrypsin (Hecht et al., 1991
). The importance of an Arg residue at the P3' site for interaction with a substrate was also suggested. Schellenberger et al. (1994) investigated the subsite structures of several serine proteases using acyl-transfer to the peptide nucleophile, and found that a peptide having an Arg at the P3' site was a good substrate for the reaction catalyzed by chymotrypsin. These results strongly indicate that the Arg residue at the P3' site is an essential requirement for strong interaction with chymotrypsin.
Why is a salt bridge required for the interaction of chymotrypsin with its cognate inhibitor or substrate? The P1-site residue of ovomucoid domain 3, which strongly inhibits chymotrypsin, is Met or Leu (Empie and Laskowski, 1982) in spite of the presence of a Tyr residue at the P2' site, which is a more favorable amino acid as a substrate for chymotrypsin. If the Arg residue at the P3' site of ovomucoid domain 3 is replaced, will the Tyr residue at the P2' site be recognized as a P1-site residue? The Tyr residue at the P2' site is required for rigidification of the main chain around the reactive site through its interaction with a Pro residue at the P4' site (Ardelt and Laskowski, 1991
). Therefore, it seems likely that the Arg residue at the P3' site of a chymotrypsin inhibitor is necessary so that the Met or Leu residue in its neighborhood is recognized as a P1-site residue.
When we used trypsin as the target protease, the ArgAla replacement at the P3' site resulted in increased inhibitory activity of OMCHI3(P1Lys)-derived mutants, although the interaction with OMCHI3(P1Lys) was weakened by this replacement. This suggests the presence of a positive charge in the substrate-binding pocket of trypsin. The trypsin structure, shown in Figure 3
, shows that Lys61 is a candidate for electrostatic repulsion with an Arg residue at the P3' site of an inhibitor. This residue does not correspond to, but is near, the Asp64 of chymotrypsin, which is responsible for the strong interaction with the Arg residue at the P3' site of chymotrypsin inhibitors. These structural differences in the substrate-binding pockets of chymotrypsin and trypsin seem to result in the Arg
Ala replacement at the P3' site having opposite effects on the interactions with these proteases.
We postulate that the decrease in the interaction with trypsin induced by the ArgAla replacement at the P3' site of OMCHI3(P1Lys) is due to the less positively charged atmosphere around the reactive site resulting from the combination of negative charges at the P4 and P2' sites and the Arg
Ala replacement at the P3' site. Therefore, when the negative charge of Asp residue at the P4 and/or P2' sites was removed by replacement, the Arg
Ala replacement at the P3' site strengthened the interaction with trypsin.
Similarly, increased binding to trypsin after AspAla replacement at the P4 site of OMCHI3(P1Lys) derivatives seemed to be due to the atmosphere around the reactive site becoming more positively charged rather than to the removal of electrostatic repulsion between trypsin and the Asp residue of OMCHI3, because the substrate-binding pocket of trypsin has no obvious negative charges that contact the P4-site residue of the inhibitor, as shown in Figure 3
. By contrast, comparison of the Ki values of OMSVP3 and OMCHI3(P1Met, P2'Tyr) showed that the effect on the interaction with chymotrypsin of Asp
Ala replacement at the P4 site was small, suggesting that the substrate-binding region of chymotrypsin lacks charged residues that contact the P4 site of the inhibitor.
These arguments clearly indicate that the strength of inhibition of chymotrypsin and trypsin by ovomucoid domain 3 is determined by not only the P1 site but also the electrostatic properties of the amino acids around the reactive site. Although the P2' site has to be a Tyr to reduce the mobility of the main chain, the P4- and P3'-site residues, in addition to the P1 site, have to possess different properties to achieve strong inhibition of either chymotrypsin or trypsin. The Arg residue at the P3' site of chymotrypsin inhibitors seems to be required so that the Leu or Met in its neighborhood is recognized as a P1 site, and the non-charged residues around the reactive site of trypsin inhibitors seem to be needed to highlight the positive charge of the P1 site. These requirements result in different amino acid requirements around the reactive site for strong inhibition of two proteases, chymotrypsin and trypsin. In the present study, we replaced the P4- and P3'-site residues of OMCHI3 (P1Lys) with Ala. Replacements with other polar, not charged, amino acids at these sites may produce more effective inhibitors of trypsin in the future.
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Acknowledgments |
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Notes |
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References |
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Received May 26, 1999; accepted June 30, 1999.