LOW TEMPERATURE STRUCTURE OF THE THROMBIN-HIRUGEN COMPLEX AT 1.7Å RESOLUTION. S. P. Bajaj*#, I. I. Mathews*, J. H. Matthews*, A. Tulinsky*, *Department of Chemistry, Michigan State University, East Lansing, MI-48824; and #Departments of Medicine and Biochemistry, St. Louis University Medical School, St. Louis, MO - 63110
Thrombin is a serine proteinase that converts fibrinogen to fibrin. Hirugen is a 12-residue hirudin-like peptide that binds to thrombin in the fibrinogen exosite. The room temperature structure of active site D-PhePro Arg chloromethyl ketone inhibited thrombin at 1.9Å resolution and the thrombin-hirugen complex at 2.2Å resolution have been reported. Since thrombin is an important target of anticoagulation therapy, we determined its structure at 150K to further refine localization of the protein and solvent atoms. Such information could be useful in improving the design of potent thrombin inhibitors.
The thrombin-hirugen complex at 150K has been refined to a current R-value of 0.176 at 1.7Å resolution. Although the folding is strikingly the same as that determined at room temperature (RMS [[Delta]] for C[[alpha]] atoms ~0.28Å), the average B-factor of the protein and solvent atoms is about 8Å2 lower and some residues show alternate conformations. Both the active site serine and the Sl aspartate are held in optimal configurations poised for catalysis by a chain of highly ordered wafer molecules that link up to the protein. A water molecule is hydrogen bonded to the OG of Serl95 and Glyl93N in the oxyanion hole that must be displaced on substrate binding. Glu192 is pointing away from the active site and at low temperature is held in position by a hydrogen bonding network of water molecules which extends to the exosite. Additional water molecules stabilize several [[beta]]-strands near turns or in regions apparently missing hydrogen bonds. The octahedrally coordinated Na+ -binding site of the fast form of thrombin has four well-defined water molecules, while an octahedrally coordinated intermolecular Na+ -site has three. A tetrahedral cluster of five peaks around crystallographic 2-fold rotation axis could be a phosphate ion. In addition, many other solvent molecules have been located that were not noted in the room temperature structure. Thus, the low temperature structure reveals biologically important features that were not apparent in the room temperature structure.