title: Atomic structures of two nitroxide spin labels complexed with human thrombin: comparison with solution studies.
abstract: Crystal structures of thrombin complexed with two spin labels called para-V, 4-(2,2,5,5-tetramethylpyrrolidine-1-oxyl)-p-(fluorosulfonyl) benzamidine, and meta-V, 3-(2,2,5,5-tetramethyl-pyrrolidine1-oxyl)-m-(fluorosulfonyl) benzamidine, have been completed at 2.0 and 3.0 A resolution, respectively. Previous electron spin resonance studies with these labels gave rise 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/inhibition effect on thrombin activity and (2) a region sensitive to alpha-thrombin autoproteolytic cleavage(s) to gamma-thrombin (Arg75-Tyr76 and/or Arg77A-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 information may be gained from solution studies and indicates the complementarity of solid-state studies.
authors.affiliation: Department of Chemical and Physical Sciences, The DuPont Merck Pharmaceutical Company, Experimental Station, Wilmington, Delaware 19880, USA.
authors.forename: V L
authors.initials: VL
authors.lastname: Nienaber
authors.forename: L J
authors.initials: LJ
authors.lastname: Berliner
mesh_terms: D001665:Binding Sites; D018360:Crystallography, X-Ray; D003497:Cyclic N-Oxides / Q000737:chemistry* / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D006801:Humans; D008956:Models, Chemical; D008968:Molecular Conformation; D015394:Molecular Structure; D013113:Spin Labels; D013917:Thrombin / Q000737:chemistry* / Q000378:metabolism; D014961:X-Ray Diffraction
chemical_list: C417198:3-(2,2,5,5-tetramethylpyrrolidine-1-oxyl)-3-(fluorosulfonyl)benzamide; D003497:Cyclic N-Oxides; D013113:Spin Labels; C056445:4-(2,2,5,5-tetramethylpyrrolidine-1-oxyl)-4-fluorosulfonylbenzamide; D013917:Thrombin