Crystal structure of cancer chemopreventive Bowman-Birk inhibitor in ternary complex with bovine trypsin at 2.3 A resolution. Structural basis of Janus-faced serine protease inhibitor specificity.

Understanding molecular recognition on a structural basis is an objective with broad academic and applied significance. In the complexes of serine proteases and their proteinaceous inhibitors, recognition is governed mainly by residue P1 in accord with primary serine protease specificity. The bifunctional soybean Bowman-Birk inhibitor (sBBI) should, therefore, interact at LysI16 (subdomain 1) with trypsin and at LeuI43 (subdomain 2) with chymotrypsin. In contrast with this prediction, a 2:1 assembly with trypsin was observed in solution and in the crystal structure of sBBI in complex with trypsin, determined at 2.3 A resolution by molecular replacement. Strikingly, P1LeuI43 of sBBI was fully embedded into the S(1) pocket of trypsin in contrast to primary specificity. The triple-stranded beta-hairpin unique to the BBI-family and the surface loops surrounding the active site of the enzyme formed a protein-protein-interface far extended beyond the primary contact region. Polar residues, hydrophilic bridges and weak hydrophobic contacts were predominant in subdomain 1, interacting specifically with trypsin. However, close hydrophobic contacts across the interface were characteristic of subdomain 2 reacting with both trypsin and chymotrypsin. A Met27Ile replacement shifted the ratio with trypsin to the predicted 1:1 ratio. Thus, the buried salt-bridge responsible for trypsin specificity was stabilised in a polar, and destabilized in a hydrophobic, environment. This may be used for adjusting the specificity of protease inhibitors for applications such as insecticides and cancer chemopreventive agents.

Dimeric crystal structure of a Bowman-Birk protease inhibitor from pea seeds.

The trypsin/chymotrypsin inhibitors from winter pea seeds (PsTI) are members of the Bowman-Birk protease inhibitor (BBPI) family. The crystal structure of the isoform PsTI-IVb was determined by molecular replacement at 2.7 A resolution using the X-ray co-ordinates of the soybean inhibitor as a search model. The inhibitor crystallized with a nearly perfect 2-fold symmetric dimer in the asymmetric unit. Although the overall structure is very similar to that seen in other BBPIs, there are notable new structural features. Unlike the previously reported X-ray structures of BBPIs, the structure of PsTI-IVb includes the C-terminal segment of the molecule. The C-terminal tail of each subunit is partly beta-stranded and interacts with the 2-fold symmetry-related subunit, forming a beta-sheet with strands A and B of this subunit. The dimer is mainly stabilized by a large internal hydrogen-bonded network surrounded by two hydrophobic links. Fluorescence anisotropy decay measurements show that residues Tyr59 and Tyr43 are mobile in the picosecond time scale with a large amplitude. The fluorescence study and a molecular model of the simultaneous binding of PsTI-IVb to porcine trypsin and bovine chymotrypsin are compatible only with a monomeric state of the functional molecule in solution.

Mutational analysis of disulfide bonds in the trypsin-reactive subdomain of a Bowman-Birk-type inhibitor of trypsin and chymotrypsin--cooperative versus autonomous refolding of subdomains.

It is widely believed that protein folding is a hierarchical process proceeding from secondary structure via subdomains and domains towards the complete tertiary structure. Accordingly, protein subdomains should behave as independent folding units. However, this prediction would underestimate the well-established structural significance of tertiary context and domain interfaces in proteins. The principal objective of this work was to distinguish between autonomous and cooperative refolding of protein subdomains by means of mutational analysis. The double-headed Bowman-Birk inhibitor of trypsin and chymotrypsin of known crystal structure was selected for study. The relative orientation of the two subdomains is stabilized by intramolecular and water-mediated hydrogen bonds and close ion pairs across a polar domain interface. The binary arrangement of a trypsin-reactive and a chymotrypsin-reactive subdomain facilitates the distinction of local and global irregularities in the mutants of this protein by means of functional assays. The functional consequences of five replacements in the S-S bond framework of the trypsin-reactive subdomain are analyzed in the present report. The mutants were subjected to refolding experiments in a refolding buffer and on trypsin-Sepharose as a template with complementary structure leading into a fully active state. The stability of the variants was assessed by means of subsequent equilibration experiments in solution. The mutants may be grouped into the following two classes: the class-I mutations located within beta-strand A are characterized by a breakdown of the trypsin- and the chymotrypsin-reactive subdomain upon refolding in solution and a complicated behavior in the equilibration experiments; by contrast, the Class-II mutations (beta-strand B) display rather local perturbations and a reversible return to the initial ratio of the two subdomains. This points to a significance of polar interactions connecting the beta-strand A of the trypsin-reactive with the chymotrypsin-reactive subdomain. In conclusion, the polar domain interface appears as a major refolding unit of the Bowman-Birk inhibitor.

Proteolytic cleavage of soybean Bowman-Birk inhibitor monitored by means of high-performance capillary electrophoresis. Implications for the mechanism of proteinase inhibitors.

The hydrolysis of the soybean Bowman-Birk inhibitor in the presence of catalytic amounts of bovine trypsin and the formation of the non-covalent enzyme-inhibitor complex with an equimolar amount of enzyme are monitored by means of high-performance capillary electrophoresis (HPCE). The inhibitor is cleaved in the trypsin-reactive and more slowly in the chymotrypsin-reactive subdomain. HPCE proves itself as the only reliable analytical tool to monitor these reactions in clear contrast to classical electrophoretic, chromatographic and enzymatic methods. The most efficient separation of the intact and the two active site cleaved forms of the inhibitor was achieved in borate buffer at pH 10.0. The pH dependence of the rate constant and the final extent of hydrolysis reveal the stability of the enzyme inhibitor complex as a central aspect of the mechanism of proteinase inhibitors.

Crystal structure of the bifunctional soybean Bowman-Birk inhibitor at 0.28-nm resolution. Structural peculiarities in a folded protein conformation.

The Bowman-Birk inhibitor from soybean is a small protein that contains a binary arrangement of trypsin-reactive and chymotrypsin-reactive subdomains. In this report, the crystal structure of this anticarcinogenic protein has been determined to 0.28-nm resolution by molecular replacement from crystals grown at neutral pH. The crystal structure differs from a previously determined NMR structure [Werner, M. H. & Wemmer, D. E. (1992) Biochemistry 31, 999-1010] in the relative orientation of the two enzyme-insertion loops, in some details of the main chain trace, in the presence of favourable contacts in the trypsin-insertion loop, and in the orientation of several amino acid side chains. The proximity of Met27 and Gln48 in the X-ray structure contradicts the solution structure, in which these two side chains point away from each other. The significant effect of a Met27-->Ile replacement on the inhibitory activity of the chymotrypsin-reactive subdomain agrees with the X-ray structure. Exposed hydrophobic patches, the presence of charged amino acid residues, and the presence of water molecules in the protein interior are in contrast to standard proteins that comprise a hydrophobic core and exposed polar amino acids.

Template-directed protein folding into a metastable state of increased activity.

The principal objective of this work was to distinguish between kinetic and thermodynamic reaction control in protein folding. The deleterious effects of a specific mutation on spontaneous refolding competence were analyzed for this purpose. A Bowman-Birk-type proteinase inhibitor of trypsin and chymotrypsin was selected as a double-headed model protein to facilitate the detection of functional irregularities by the use of functional assays. The parent protein spontaneously folds into a single, fully active and thermodynamically stable state in a redox buffer after reduction/denaturation. By contrast, the properties of a P'1Ser-->Pro variant in the trypsin-reactive subdomain differ before and after refolding on trypsin-Sepharose. A heterogenous and thermodynamically dominant population of conformers was attained in solution. However, the enzyme-inhibitory activity of the variant was dramatically increased in the presence of trypsin-Sepharose and a stoichiometric ratio of the two subdomains was obtained as expected for a single conformation. The subsequent return for the initial mixture of conformers in solution reveals a high kinetic barrier late in the folding process. The template facilitates folding kinetically, as shown by a rate acceleration of more than four orders of magnitude. The final state was also the thermodynamically favoured one on the template, due to its increased affinity for the enzyme. The long-range effects on folding kinetics and the partial activity, and the absence of free sulfhydryl groups after refolding in solution indicate rearrangements between closely related conformers late in folding. The importance of minor structural distortions in immobilized trypsin suggests a close structural analogy between the final and the transition state of protein folding.

Chemical synthesis, molecular cloning and expression of gene coding for a Bowman-Birk-type proteinase inhibitor.

A gene coding for a Bowman-Birk-type proteinase inhibitor was synthesized chemically, cloned and expressed in Escherichia coli as a fusion protein with a beta-galactosidase fragment. The corresponding mutant inhibitor, carrying a P1 = Arg16 instead of Lys and an Ile27 instead of Met was obtained after cyanogen bromide cleavage, refolding and affinity chromatography on trypsin-Sepharose. Dissociation constants of complexes with trypsin of this mutant and wild-type Bowman-Birk inhibitor are identical within experimental error. This is explained by differential patterns of hydrogen bonds between side-chains of Arg or Lys in proteinase inhibitors and the primary specificity pocket of trypsin.