31VH

Alcalase/CI-2A complex at 277K

Summary for 31VH

• Entry DOI: 10.2210/pdb31vh/pdb
• Descriptor: KerA, Subtilisin-chymotrypsin inhibitor-2A, CALCIUM ION, ... (6 entities in total)
• Functional Keywords: alcalase, chymotrypsin inhibitor 2a (ci-2a), atomic-resolution crystallography, hydrolase
• Biological source: Hordeum vulgare; More
• Total number of polymer chains: 2
• Total formula weight: 34800.76

Authors

Rypniewski, W. (deposition date: 2026-06-24, release date: 2026-10-07, Last modification date: 2026-10-14)

Primary citation

Raczynska, J.E.,Jedrzejczak, R.,Dauter, M.,Ostergaard, P.R.,Wilson, K.S.,Rypniewski, W.
Catalytic hydrogen bonds in alcalase-CI-2A complexes revealed by ultra-high-resolution crystallography.
J.Struct.Biol., 218:108377-108377, 2026

PubMed Abstract: 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 Å resolution, while an additional 4 °C (277 K) structure of the native complex was determined at 1.05 Å 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 °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 °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.

PubMed: 42790760
DOI: 10.1016/j.jsb.2026.108377

Experimental method

X-RAY DIFFRACTION (1.05 Å)