l-Isoaspartate 115 of porcine β-trypsin promotes crystallization of its complex with bdellastasin

Bdellastasin is a 59‐amino‐acid, cysteine‐rich, antistasin‐type inhibitor of sperm acrosin, plasmin and trypsin, isolated from the medicinal leech Hirudo medicinalis. The complex formed between bdellastasin and porcine β‐trypsin has previously been crystallized in the presence of PEG in a tetragonal crystal form of space group P43212 and has now been found to crystallize under high‐salt conditions in the enantiomorphic space group P41212. These structures have been solved and refined to 2.8 and 2.7 Å resolution, respectively. Bdellastasin turns out to have an antistasin‐like fold exhibiting a bis‐domainal structure. In the second new crystal form, the flexible N‐terminal subdomain is rotated with respect to the C‐­terminal subdomain by about 90°, fitting into a cavity formed by symmetry‐related trypsin molecules. The canonical inhibitor–proteinase interaction is restricted to the primary binding loop comprising residues Leu31–Lys36 of bdellastasin. During the refinement, a bound sodium ion occupying the calcium‐binding site of the porcine β‐trypsin component was discovered. This sodium ion is coordinated in a tetragonal–pyramidal manner, with the geometry of the enclosing loop slightly changed compared with the loop in the presence of calcium. In the crystal form of space group P43212, the electron density for residue 115 of porcine β‐trypsin clearly indicates the presence of a β‐isomerized l‐aspartic acid, which is placed in spatial proximity to segment Thr144–­Gly148 of a symmetry‐related trypsin molecule. This is the first structurally observed example of an l‐isoaspartate in β‐­trypsin originating from Asn. A comparison with other known crystal structures of porcine β‐trypsin–macromolecular inhibitor complexes suggests that the deamidation, isomerization and racemization of Asn115 is the key step in crystallization.