Insight into the mechanism of serpin-proteinase inhibition from 2D[~1H-~(15)N]NMR studies of the 69 kDa #alpha#_1-Proteinase inhibitor pittsburgh-trypsin covalent

摘要

We have used [1H-’5N]-HSQC NMR to investigate the structural changes that occur in both serpin and proteinase in forming the kinetically trapped covalent protein—protein complex that is the basis for serpin inhibition of serine proteinases. By alternately using ‘5N-alanine specifically-labeled a1-proteinase inhibitor (ct1PI) Pittsburgh (serpin) and bovine trypsin (proteinase). we were able to selectively monitor structural changes in each component of the 69 kDa complex. Residue-specific assignments of four alanines in the reactive center loop and seven other alanines aided interpretation of the spectral changes in the serpin. We found that the majority of the alanine resonances, including those from reactive center loop residues P12, P11, and P9, were at identical positions in covalent complex and in cleaved ct1PI. Five alanines that are close to the contact region with proteinase showed some chemical shift perturbation compared with cleaved a1PI, indicating some degree of structural deformation. With ‘5N label in the proteinase, an HSQC spectrum was obtained that more closely resembled that of a molten globule, suggesting that the structure of the proteinase had been significantly altered as a result of complex formation. Large increases in line width for all ct.1PJ resonances in the covalent complex, with the sole exception of two residues in the flexible N-terminal tail, indicate that, unlike the noncovalent ct1PI-anhydroproteinase complex, the covalent complex is a rigid body of effectively increased molecular weight. We conclude that the mutual perturbations of serpin and proteinase result from steric compression and distortion, rather than simple contact effects. This distortion provides a structural basis for the greatly reduced catalytic efficiency of the proteinase in the complex and hence kinetic trapping of the covalent reaction intermediate.