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

We have used [H-1-N-15]-HSQC NMR to investigate the structural changes that occur in both serpin and proteinase in forming the kinetically trapped cov alent protein-protein complex that is the basis for serpin inhibition of se rine proteinases. By alternately using N-15-alanine specifically-labeled al pha (1)-proteinase inhibitor (alpha 1PI) Pittsburgh (serpin) and bovine try psin (proteinase), we were able to selectively monitor structural changes i n each component of the 69 kDa complex. Residue-specific assignments of fou r alanines in the reactive center loop and seven other alanines aided inter pretation of the spectral changes in the serpin. We found that the majority of the alanine resonances, including those from reactive center loop resid ues P12, P11, and P9, were at identical positions in covalent complex and i n cleaved alpha 1PI. Five alanines that are close to the contact region wit h proteinase showed some chemical shift perturbation compared with cleaved alpha 1PI, indicating some degree of structural deformation. With N-15 labe l in the proteinase, an HSQC spectrum was obtained that more closely resemb led that of a molten globule, suggesting that the structure of the proteina se had been significantly altered as a result of complex formation. Large i ncreases in line width for all alpha 1PI resonances in the covalent complex , with the sole exception of two residues in the flexible N-terminal tail, indicate that, unlike the noncovalent alpha 1PI-anhydroproteinase complex, the covalent complex is a rigid body of effectively increased molecular wei ght. We conclude that the mutual perturbations of serpin and proteinase res ult from steric compression and distortion, rather than simple contact effe cts. This distortion provides a structural basis for the greatly reduced ca talytic efficiency of the proteinase in the complex and hence kinetic trapp ing oil the covalent reaction intermediate.