Calcium-associated Pro367 ring puckering supports conformational selection in a thermophilic lipase
Year: 2026
Type: preprint
Abstract: Thermophilic bacterial lipases must combine structural stability with the conformational flexibility required for interfacial activation, yet the local organization of their metal-binding sites remains incompletely understood. Here, we determined two X-ray crystal structures of the family I.5 lipase from Geobacillus kaustophilus ( Gk Lip ). The 2.47-Å orthorhombic model contains a Ca²⁺ ion at the conserved metal-binding site, whereas the 2.30-Å anisotropic triclinic model contains four non-equivalent protein chains in which no Ca²⁺ ion was modeled. Gk Lip retains the canonical α/β-hydrolase fold, the Ser114-Asp318-His359 catalytic triad, the Phe17/Gln115 oxyanion-hole region, an extended lid, and a conserved Zn²⁺-binding site. In the orthorhombic structure, Gly287, Glu361, Asp366, and Pro367 contribute to the Ca²⁺ coordination environment. Pro367 adopts a Cγ-endo pucker in the Ca²⁺-containing structure; three Ca²⁺-free chains also adopt the endo pucker, whereas one adopts a Cγ-exo pucker. The presence of both Pro367 pucker states in the Ca²⁺-free structure is consistent with calcium preferentially stabilizing a pre-existing, coordination-compatible conformation. This local organization is further supported by an Arg35-centered network of direct and water-mediated contacts. Comparison with the 94%-identical open G. thermocatenulatus lipase reveals substantial lid rearrangement despite strong conservation of the protein core. Together, these structures highlight localized conformational variability at the calcium-binding region within an otherwise highly conserved thermophilic lipase scaffold.
Language: English
Subfield: Structural Biology
Domain: Life Sciences
Open Access status: green
Funder: Deutsches Elektronen-Synchrotron
OpenAlex ID: W7221047988