L-Isoaspartate 115 of porcine beta-trypsin promotes crystallization of itscomplex with bdellastasin

Bdellastasin is a 59-amino-acid, cysteine-rich, antistasin-type inhibitor o f sperm acrosin, plasmin and trypsin, isolated from the medicinal leech Hir udo medicinalis. The complex formed between bdellastasin and porcine beta-t rypsin has previously been crystallized in the presence of PEG in a tetrago nal crystal form of space group P4(3)2(1)2 and has now been found to crysta llize under high-salt conditions in the enantiomorphic space group P4(1)2(1 )2. These structures have been solved and refined to 2.8 and 2.7 Angstrom r esolution, respectively. Bdellastasin turns out to have an antistasin-like fold exhibiting a bis-domainal structure. In the second new crystal form, t he flexible N-terminal subdomain is rotated with respect to the C-terminal subdomain by about 90 degrees, fitting into a cavity formed by symmetry-rel ated trypsin molecules. The canonical inhibitor-proteinase interaction is r estricted to the primary binding loop comprising residues Leu31-Lys36 of bd ellastasin. During the refinement, a bound sodium ion occupying the calcium -binding site of the porcine beta-trypsin component was discovered. This so dium ion is coordinated in a tetragonal- pyramidal manner, with the geometr y of the enclosing loop slightly changed compared with the loop in the pres ence of calcium. In the crystal form of space group P4(3)2(1)2, the electro n density for residue 115 of porcine beta-trypsin clearly indicates the pre sence of a beta-isomerized L-aspartic acid, which is placed in spatial prox imity to segment Thr144-Gly148 of a symmetry-related trypsin molecule. This is the first structurally observed example of an L-isoaspartate in beta-tr ypsin originating from Asn. A comparison with other known crystal structure s of porcine beta-trypsin-macromolecular inhibitor complexes suggests that the deamidation, isomerization and racemization of Asn115 is the key step i n crystallization.