Journal of Molecular Biology
Volume 266, Issue 2, 21 February 1997, Pages 441-461
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Regular article
Binding of amino acid side-chains to S1 cavities of serine proteinases1

https://doi.org/10.1006/jmbi.1996.0781Get rights and content

Abstract

The P1 or primary specificity residue of standard mechanism canonical protein inhibitors of serine proteinases, inserts into the S1 primary specificity cavity of the cognate enzyme upon enzyme-inhibitor complex formation. Both natural evolution and protein engineering often change the P1 residue to greatly alter the specificity and the binding strength. To systematize such results we have obtained all 20 coded P1 variants of one such inhibitor, turkey ovomucoid third domain, by recombinant DNA technology. The variants were extensively characterized. The association equilibrium constants were measured at pH 8.30, 21 (±2)°C, for interaction of these variants with six well characterized serine proteinases with hydrophobic S1 cavities. The enzyme names are followed by the best, worst and most specific coded residue for each. Bovine chymotrypsin Aα (Tyr, Pro, Trp), porcine pancreatic elastase (Leu/Ala, Arg, Ala), subtilisin Carlsberg (Cys, Pro, Glu), Streptomyces griseus proteinase A (Cys, Pro, Leu) and B (Cys, Pro, Lys) and human leukocyte elastase (Ile, Asp, Ile). The data set was merged with Ka values for five non-coded variants at P1 of turkey ovomucoid third domain obtained in our laboratory by enzymatic semisynthesis. The ratios of the highest to the lowest Ka for each of the six enzymes range from 106 to 108. The dominant force for binding to these pockets is the hydrophobic interaction. Excess steric bulk (too large for the pocket), awkward shape (Pro, Val and Ile), polarity (Ser) oppose interaction. Ionic charges, especially negative charges on Glu and Asp are strongly unfavorable. The Pearson product moment correlations for all the 15 enzyme pairs were calculated. We suggest that these may serve as a quantitative description of the specificity of the enzymes at P1. The sets of Streptomyces griseus proteinases A and B and of the two elastases are strongly positively correlated. Strikingly, chymotrypsin and pancreatic elastase are negatively correlated (−0.10). Such correlations can be usefully extended to many other enzymes and to many other binding pockets to provide a general measure of pocket binding specificity.

Introduction

Substrate and inhibitor recognition by most serine proteinases involves inter alia the binding of the P1 residue (see Figure 1 for explanation of Schechter & Berger, 1967 numbering) to the S1 cavity of the enzyme. This is probably the most famous of protein-protein recognition motifs. Millions of students learn that trypsin is specific for Arg and Lys P1 side-chains because of the presence of Asp189(chymotrypsinogen numbering) at the bottom of its S1 pocket. Similarly, chymotrypsin prefers Tyr, Trp, Phe, Leu and Met P1 residues because of its commodious S1 pocket with neutral Ser189 at the bottom of it.

Ovomucoid is a major glycoprotein in avian eggwhites whose single polypeptide chain consists of three tandem Kazal family domains, each with a single, actual or putative, reactive site for inhibition of serine proteinases Kato et al 1976, Kato et al 1978, Kato et al 1987. Rhodes et al. (1960) showed that ovomucoids from various, closely related species of birds exhibit striking variation in inhibitory activity and specificity. We have found that such variation persists when third domains (Figure 1) are examined alone (Empie & Laskowski, 1982; S. J. Park et al., unpublished results) and that it is due to much more rapid fixation of mutations in the enzyme-inhibitor contact region compared to the rest of the inhibitor molecule Laskowski et al 1987, Laskowski et al 1990, Apostol et al 1993. We have attributed this hypervariability to positive Darwinian selection (Laskowski et al., 1988).

The reasons for the choice of turkey ovomucoid third domain as the wild type are the approximately equal inhibition of all six enzymes we study and the ready availability. Ovomucoid third domains are among the best studied of all small proteins and of protein inhibitors of serine proteinases. The three-dimensional structures of two free ovomucoid third domains, Japanese quail Weber et al 1981, Papamokos et al 1982 and silver pheasant (Bode et al., 1985) were determined both in virgin (reactive site intact) and in modified (reactive site hydrolyzed) forms (Musil et al., 1991). The three-dimensional structures of complexes of turkey ovomucoid third domain with Streptomyces griseus proteinase B Fujinaga et al 1982, Read et al 1983, human leukocyte elastase (Bode et al., 1986) and bovine αchymotrypsin (Fujinaga et al., 1987) were also determined. There are several more structures that are either unpublished or now in progress, both in Martinsried and in Edmonton. The most relevant to this paper is that Huang, Bateman and James intend to obtain the structures of all 20 coded X18 OMTKY3 in complex with SGPB. Some of these structures e.g. Gly18, Ala18 and Leu18 (short form) are already completed (Huang et al., 1995), many others await publication Huang 1995, Bateman et al 1996, others are in refinement. These studies as well as the interpretation of the huge volume of thermodynamic data available at Purdue lead to the following conclusions (1) residue 18 always serves as the P1 residue even when its binding to the S1 cavity is locally deleterious, (2) the changes in the inhibitor and enzyme conformation on complex formation are very small, nearly lock and key, (3) with the exception of Pro18, the nature of X18 has little effect on the interaction between the enzyme and the remaining residues of the inhibitor. Thus, for our purposes, the inhibitor-enzyme system serves as a molecular vise. It positions X18 into the S1 cavity and by subtraction of Gly18 data (see below) it allows us to measure just the X18…S1 interaction.

We have already reported Ka values for 14 variants of ovomucoid third domain at X18 (P1 residue) interacting with six serine proteinases (Bigler et al., 1993). Of these 14 variants, nine involved coded residues, while five were non-coded. Of the nine coded variants, six were obtained directly, while three were calculated from other data by making (reasonable) additivity assumptions. Most of the variants employed were generated by semisynthesis (Wieczorek & Laskowski, 1983).

Here, we describe a set of techniques announced by us earlier (Lu et al., 1992) for the production of coded ovomucoid third domain variants. We apply these techniques to the production of all 20 coded X18 ovomucoid third domain variants. We directly measure the Ka values for all these, interacting with the same six serine proteinases as employed by Bigler et al. (1993). We combine our complete set of 20 with the set of five non-coded residues (Abu, Ape, Ahx, Ahp and Hse) provided by Bigler et al. (1993). The non-coded set Ka values were also all obtained by direct measurements. The combined set consists of 25×6=150 Ka values. It allows for a great number of comparisons both internally and to literature values. Only some of these are provided below.

Section snippets

The Ka values

Table 1 lists the Ka values for all 20 coded variants of X18OMTKY3. We were fortunate that in spite of the huge range of these data (see below) all the values fit into our dynamic range of measurements (roughly 103 M−1 to 1013 M−1, the upper bound is lower for some enzymes e.g. HLE). As was already pointed out by Bigler et al. (1993) the set of all coded amino acids allows for only a few structurally simple pairwise comparisons. The homologous series e.g. Gly, Ala; Asp, Glu; Asn, Gln; Val, Ile

Construction of the OM3D expression vector

The steps in the construction of the expression vector for the turkey ovomucoid third domain as a downstream fusion to secreted IgG-binding domains of staphylococcal protein A are shown schematically in Figure 9. Briefly, a 0.18 kbp HincII/BamH I fragment from a plasmid containing a molecular clone of the chicken ovomucoid cDNA, pOM100 Stein et al 1978, Lai et al 1979, was purifed by gel electrophoresis and inserted into SmaI/BamHI-digested pEZZ318 (Nilsson & Abrahmsén, 1990). This HincII/BamHI

Acknowledgements

B. O’Malley’s gift of pOM100 made the molecular biology much easier. We thank B. Nilsson for the gift of pEZZ318 and for the suggestion of RV308 as an expression host. Discussions with K. Huang, K. S. Bateman and M. N. G. James about their X-ray structures had a big impact on the interpretation. We are grateful to L. Smillie, J. Travis and the late M. Laskowski Sr for gifts of enzymes that are used as standards for validating the commercial ones. L. Smillie additionally reviewed his old data on

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    Present addresses: W. Lu, Scripps Research Institute, 10666 N. Torrey Pines Road, La Jolla, CA 92037, USA; I. Apostol, Somatogen, Inc., 5797 Central Avenue, Boulder, CO 80301, USA; N. Warne, Genetics Institute, Drug Product Development, 1 Burtt Road, Andover, MA 01810, USA; R. Wynn, The DuPont Merck Pharmaceutical Company, Experimental Station, Rte. 141, Wilmington, DE 19880, USA; S. Anderson, Dept of Molecular Biology and Biochemistry, Rutgers University, New Brunswick, NJ 08903, USA; E. Ogin, Interferon Sciences, Inc., 783 Jersey Ave New Brunswick, NJ 08901, USA; I. Rothberg, Dept of Chemistry, Olson Laboratories, Rutgers University-Newark, 73 Warren St., Newark, NJ 07102, USA.

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