Structure of a serine protease poised to resynthesize a peptide bond

Abstract

The serine proteases are among the most thoroughly studied enzymes, and numerous crystal structures representing the enzymesubstrate complex and intermediates in the hydrolysis reactions have been reported. Some aspects of the catalytic mechanism remain controversial, however, especially the role of conformational changes in the reaction. We describe here a high-resolution (1.46 Å) crystal structure of a complex formed between a cleaved form of bovine pancreatic trypsin inhibitor (BPTI) and a catalytically inactive trypsin variant with the BPTI cleavage site ideally positioned in the active site for resynthesis of the peptide bond. This structure defines the positions of the newly generated amino and carboxyl groups following the 2 steps in the hydrolytic reaction. Comparison of this structure with those representing other intermediates in the reaction demonstrates that the residues of the catalytic triad are positioned to promote each step of both the forward and reverse reaction with remarkably little motion and with conservation of hydrogen-bonding interactions. The results also provide insights into the mechanism by which inhibitors like BPTI normally resist hydrolysis when bound to their target proteases.trypsin ͉ bovine pancreatic trypsin inhibitor ͉ enzyme mechanisms S erine proteases are found throughout all 3 domains of cellular life and function in a wide range of physiological processes, including digestion, protein maturation and turnover, hemostasis, and immune responses (1). Approximately 0.6% of human protein-encoding genes are predicted to specify serine proteases, and this family is even more prevalent in other organisms, notably the arthropods (2, 3). A large body of biochemical and structural data have established a 2-step mechanism for hydrolysis of peptide bonds by this class of proteases (4), as shown in Scheme 1.The first step of the reaction is a nucleophilic attack by the catalytic serine residue (Ser-195 in trypsin and other members of the chymotrypsin, or S1, family) on the carbonyl carbon atom of the residue labeled P1, generating a covalent acyl-enzyme intermediate and a new peptide amino terminus, on the P1Ј residue. A second nucleophilic attack, by a water molecule, leads to hydrolysis of the acyl-enzyme, releasing the new carboxyl group and restoring the catalytic Ser residue to its initial state.

Sequence and Conformational Specificity in Substrate Recognition

“…This observation is consistent with other evidence that such protease⅐inhibitor complexes represent ideal Michaelis complexes (21). Serine proteases inhibited by canonical inhibitors may undergo rapid acylation, the first step of catalytic cleavage, but progress of the reaction is blocked when the primed-side leaving group residues fail to dissociate from the active site, held in place by a combination of interactions with the enzyme and with the inhibitor scaffold (21,45,46). This leads to a thermodynamic equilibrium in which religation of the peptide bond is strongly favored, explaining why the intact form of the inhibitor is observed crystallographically in enzyme⅐inhibitor complexes.…”

An Acrobatic Substrate Metamorphosis Reveals a Requirement for Substrate Conformational Dynamics in Trypsin Proteolysis

“…Because acyl-enzyme hydrolysis and associated conformational changes have been previously postulated to limit catalytic rates (20,23,28), as starting points for simulations we modeled the acyl-enzymes that would be initially formed upon nucleophilic attack by mesotrypsin Ser-195, prior to any significant conformational changes involving primed side residue movement. After energy minimization, the initial positioning of the P 1 Ј residue in all acyl-enzyme models was consistent with the positioning of the P 1 Ј residue of cleaved BPTI* from the previously reported experimental structure (29). Because of the anticipated slow time scale of large substrate motions such as those evidenced in the APLP2-KD* complex, multiple long simulations (1000ϩ ns/replicate) were conducted for each acyl-enzyme, along with several 100 -200-ns replicates for convergence.…”

“…The motions evidenced by the new crystal structure of cleaved APLP2-KD* appear to facilitate primed side residue dissociation from the enzyme, and thus might help to explain the more rapid proteolysis of this Kunitz domain compared with others. Notably, the only prior reported crystal structure of a postcleavage Kunitz domain, cleaved BPTI* (where BPTI* is BPTI cleaved at the Lys-15-Ala-16 reactive-site bond) bound to rat anionic trypsin, revealed a native-like structure in which the P 1 Ј-P 3 Ј residues were retained in the trypsin active site, poised for peptide bond resynthesis (29). As BPTI is proteolyzed orders of magnitude more slowly than APLP2-KD (Fig.…”

“…DECEMBER 16, 2016 • VOLUME 291 • NUMBER 51 atoms, enabling us to evaluate whether incursion of water into the active site could plausibly limit the catalytic rate. Previous structural studies have revealed the path taken by a hydrolytic water molecule during trypsin catalysis (14), and they have demonstrated through reconstruction of the reaction coordinate that the substrate P 1 Ј leaving group must be displaced prior to acyl-enzyme hydrolysis (29). To identify water molecules in the simulations that might meet geometric criteria to become "hydrolytic" waters, we initially searched for waters that moved to within 3.5 Å of the Lys/Arg-15 carbonyl carbon during the simulations, finding multiple waters in all simulations (shown for the APPI complex in Fig.…”

“…Substrate Primed Side Local Conformational Dynamics Do Not Correlate with Rates of Proteolysis-Because prior work has implicated the spatial retention of primed side leaving group residues by the acyl-enzyme as a likely feature contributing to the slow proteolysis of Kunitz domains and other canonical inhibitors (20,23,28,29), we next examined the movements of P 1 Ј substrate residue Ala-16 throughout the MD simulations. Distance between Ala-16(N) and Arg/Lys-15(C), which is spatially fixed in the active site by the covalent acyl bond, was plotted as a unidimensional reporter of Ala-16 motion (Fig.…”