Structural Superimposition

Abstact

Serine proteases catalyze peptide-bond hydrolysis through a conserved catalytic triad and an extensive network of hydrogen bonds that facilitate proton transfer and stabilization of reaction intermediates. Despite decades of study, the nature of the hydrogen-bonding interactions within the catalytic machinery remains incompletely understood. Here, we report ultra-high-resolution crystal structures of alcalase, a subtilisin-family serine protease, in complex with native chymotrypsin inhibitor 2A (CI-2 A) and with a low-affinity M59P inhibitor variant. The cryogenic structures were determined at 0.74 A resolution, while an additional 4 degrees C (277 K) structure of the native complex was determined at 1.05 A resolution. The data allowed visualization and refinement of numerous hydrogen atoms within the active site and at the enzyme-inhibitor interface. Comparison of the 4 degrees C and cryogenic structures revealed a systematic shortening of hydrogen-bond donor-acceptor distances upon cooling, consistent with the global lattice contraction. A notable exception was the hydrogen bond between the catalytic residues Ser221 and His64, which remained unusually short at both temperatures. In the 4 degrees C structure, the corresponding hydrogen atom is associated with weak and diffuse electron density, suggesting increased proton mobility within this interaction. The structures further reveal temperature-dependent differences in interactions involving the scissile peptide bond of the inhibitor and the conserved backbone carbonyl oxygen of Ser125. These observations support a model in which Ser125 participates in a concerted hydrogen-bond network coupled to proton transfer within the catalytic triad. Together, the results provide atomic-level insights into hydrogen bonding, protonation equilibria, and catalytic mechanism in subtilisin-like serine proteases.