THE CRYSTAL STRUCTURE OF [[alpha]]-THROMBIN WITH A NEW TYPE OF INHIBITOR: AERUGINOSIN 298-A. J. L. Rios Steiner, A. Tulinsky, Dept. of Chemistry, Michigan State University, E. Lansing MI 48824

Thrombin is a serine protease that plays different and important roles in the blood coagulation process. One of its most important functions is to convert fibrinogen to fibrin, the major component of a blood clot. It is one of the most studied targets towards the development and design for new thrombotic and antithrombotic drugs.

Aeruginosin 298-A (C₃₀H₄₈O₇N₆) is a unique tetrapeptide isolated from the fresh water blue-green alga Microscytis aeruginosa. Aeruginosin exhibits inhibition selectivity for thrombin and trypsin (IC₅₀ of 0.3 mg/mL and 1.0 mg/mL, respectively) but displays no inhibition towards papain, chymotrypsin, elastase or plasmin. Crystallographic data for its complex with hirugen-thrombin were collected to 2.1Å resolution. The ternary complex crystallizes in the monoclinic system C2, where a=71.97, b=72.48, c=72.24Å, [[beta]]=100.9deg.. The structure refinement near completion has R=15.6% and wR=17.3%.

This inhibitor binds to the catalytic site of thrombin in a D-PheProArg chloromethylketone-like fashion, where the argininol group is fixed into the S1 site by the Aspl89 and the carboxy-6-hydroxy octahydroindole group occupies the S2 site flanked by His57, Tyr60A and Trp60D. The P3 and the P4 sites are occupied by an L-Leu and a 4-hydroxyphenyllactic acid group, respectively. These sites are well positioned mainly due to the hydrogen bonding network present between the carbonyl and NH groups of Gly216 with the amide and carbonyl groups of the P3 and P2 residues coupled with a H-bond between the terminal hydroxyl of the P4 residue and the amide group of Gly219.

Crystals were prepared using diffusion techniques. The structure was solved using molecular replacement techniques (XPLOR) and refined using the PROLSQ program.

We would like to thank Dr. M. Murakami and Professor N. Fusetani, University of Tokyo, for providing a sample of the Aeruginosin 298-A.