X-ray analysis of complexes of carbonic anhydrase II with 1,3-oxazole-containing sulfonamide derivatives elucidates the structural basis for their exceptionally high inhibitory potency

Carbonic anhydrase II (CA2) is a zinc metalloenzyme that catalyzes the reversible hydration of CO₂ and is widely used as a model for structure-guided inhibitor development. Previously reported crystal structures of human CA II (α-hCA2) in complex with the picomolar sulfonamide inhibitors 8V5 and 8V8 (PDB entries 5nee and 5nea) did not allow the complete ligand geometry of 8V5 to be resolved. To clarify the structural basis of binding, we determined high-resolution (1.5-1.7 Å) crystal structures of bovine CA II (α-bCA2) in complex with both inhibitors. α-bCA2 was selected as a crystallographically robust homolog, and structural alignment with α-hCA2 showed an r.m.s.d. of <0.44 Å across C^α atoms, confirming a near-identical active-site architecture. The α-bCA2-8V8 complex reproduced the canonical sulfonamide-zinc coordination pattern. In contrast, the α-bCA2-8V5 structure revealed continuous electron density for the entire ligand, including the morpholine group, which was not observed previously. Re-refinement of the α-hCA2-8V5 model based on these data resulted in an improved fit to density, indicating that the absence of the morpholine density in the original model is consistent with partial ligand modification or variability in the ligand state during crystallization. These results demonstrate that cross-isoform structural comparison can resolve ambiguities in ligand modeling and provide a reliable framework for the rational design of selective CA2 inhibitors.