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.