The X-Ray Crystal Structure of Thrombin in Complex with Nα-2-Naphthylsulfonyl-L-3-Amidino-Phenylalanyl-4-Methylpiperidide: The Beneficial Effect of Filling Out an Empty Cavity

Abstract

The 2.5 Å structure of bovine ε-thrombin in complex with Nα-2-naphthyl-sulfonyl-L-3-amidinophenylalanyl-4-methylpiperidide (L-NAF'AMP) was solved and crystallographically refined to an R-value of 0.19. The L-NAPAMP moiety is completely and unambiguosly defined in the electron density. NAPAMP binds almost identical to the related 4-methyl deficient 3-amidino-phenylalanyl derivative TAPAP. The overall binding geometry appears dominated by the fixation of the 3-amidinophenyl ring in thrombin's S1-pocket and the hydrogen bonds to Gly 216, irrespective of the presence or absence of a substituent in the 4-position of the piperidine ring. The additional 4-methyl group gives rise to a 17-fold better binding. The more complete spatial occupancy of the hydrophobic S2-cavity therefore accounts for a decrease in free energy of binding of 15 kcal/mol, a value comparable with that anticipated for filling up a stable empty cavity of similar size by a methyl group.

Key Words: :

• Thrombin
• Antithrombotics
• Inhibitors
• X-ray crystal structure
• inhibitor complex