Atomic structures of two nitroxide spin labels complexed with human thrombin: Comparison with solution studies

Crystal structures of thrombin complexed with two spin labels called pam-V, 4-(2,2,5,5-tetramethyl-pyrrolidine-1 -oxyl)-p-(fluorosulfonyl) benzamidine , and meta-V, 3-(2,2,5,5-tetramethyl-pyrrolidinel-oxyl)-m-(fluorosulfonyl) benzamidine, have been completed at 2.0 and 3.0 Angstrom resolution, respec tively. Previous electron spin resonance studies with these labels gave ris e to a low-resolution "topography map" of thrombin's extended active site. These labels monitor two distinct areas of the thrombin active site: (1) an apolar binding site which manifests itself in an biphasic activation! inhi bition effect on thrombin activity and (2) a region sensitive to alpha-thro mbin autoproteolytic cleavage(s) to gamma-thrombin (Arg75-Tyr76 and/or Arg7 7A-Asn78, and Lys149E-Gly150, chymotrypsin numbering). Para-V was found to bind along the substrate binding cleft, while meta-V was found to bind both at the substrate primary specificity pocket and at a site which interacts with the gamma-cleavage loop. These studies. reaffirm that accurate informa tion may be gained from solution studies and indicates the complementarity of solid-state studies.