Atomic structures of two nitroxide spin labels complexed with human thrombin: comparison with solution studies |
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Authors: | Nienaber V L Berliner L J |
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Institution: | (1) Department of Chemical and Physical Sciences, The DuPont Merck Pharmaceutical Company, Experimental Station, Wilmington, Delaware, 19880;(2) Department of Chemistry, The Ohio State University, Columbus, Ohio, 43210;(3) Present address: Department of Structural Biology, Abbott Laboratories, Abbott Park, Illinois, 60064-3500;(4) Department of Chemistry, The Ohio State University, Columbus, Ohio, 43210 |
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Abstract: | Crystal structures of thrombin complexed with two spin labels called para-V, 4-(2,2,5,5-tetramethyl-pyrrolidine-1-oxyl)-p-(fluorosulfonyl) benzamidine, and meta-V, 3-(2,2,5,5-tetramethyl-pyrrolidine-1-oxyl)-m-(fluorosulfonyl) benzamidine, have been completed at 2.0 and 3.0 Å 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 -thrombin autoproteolytic cleavage(s) to -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 -cleavage loop. These studies reaffirm that accurate information may be gained from solution studies and indicates the complementarity of solid-state studies. |
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Keywords: | Thrombin spin labels X-ray crystallography electron spin resonance serine protease |
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