ß-SHEET REARRANGEMENT BURIES ARGININE SIDE CHAINS IN THE HYDROPHOBIC CORE OF CLEAVED ANTICHYMOTRYPSIN HINGE REGION VARIANTS. Lukacs, C.M., University of Pennsylvania, Departments of Chemistry and Medicine, Philadelphia, PA 19104

The hallmark of serpin (serine protease inhibitor) function is a massive ß-sheet rearrangement involving the insertion of the P1-side of the cleaved reactive loop as strand s4A in ß-sheet A. This structural transition results in greatly enhanced stability and is required for inhibitory activity. Current dogma suggests that small hydrophobic residues at the P-even positions of the reactive loop (e.g., P14, P12, P10) facilitate this structural transition since these residues must pack in the hydrophobic core of the cleaved serpin. We have undertaken the X-ray crystal structures of cleaved P14, P12, and P10 arginine variants of antichymotrypsin (ACT), and our results challenge this dogma. All three variants show greatly enhanced thermostability upon cleavage, yet the P14 (T345R) and P12 (A347R) variants are substrates of chymotrypsin and elastase. Upon reactive loop cleavage as a substrate of chymotrypsin, A347R-ACT (P12) undergoes full strand s4A insertion despite the resultant burial of the bulky P12 arginine side chain in the hydrophobic core, and similar results are expected from the P14 variant. This feature has profound implications for structure-function and structure-stability relationships in ACT and as other members of the serpin superfamily.