Crystal structures of murine thrombin in complex with the extracellular fragments of murine protease-activated receptors PAR3 and PAR4

摘要

It has been proposed that the cleaved form of protease-activated receptor 3 (PAR3) acts as a cofactor for thrombin cleavage and activation of PAR4 on murine platelets, but the molecular basis of this physiologically important effect remains elusive. X-ray crystal structures of murine thrombin bound to extracellular fragments of the murine receptors PAR3 (~(38)SFNGGPQNTFEEFPLSDIE~(56)) and PAR4 (~(51)KSSDKPNPR↓GYPGKFCANDSDTLELPASSQA~(81), ↓ = site of cleavage) have been solved at 2.0 and 3.5 A resolution, respectively. The cleaved form of PAR3, traced in the electron density maps from Gln-44 to Glu-56, makes extensive hydrophobic and electrostatic contacts with exosite Ⅰ of thrombin through the fragment ~(47)FEEFPLSDIE~(56). Occupancy of exosite Ⅰ by PAR3 allosterically changes the conformation of the 60-loop and shifts the position of Trp-60d ≈ 10 A with a resulting widening of the access to the active site. The PAR4 fragment, traced entirely in the electron density maps except for five C-terminal residues, clamps Trp-60d, Tyr-60a, and the aryl-binding site of thrombin with Pro-56 and Pro-58 at the P2 and P4 positions and engages the primary specificity pocket with Arg-59. The fragment then leaves the active site with Gly-60 and folds into a short helical turn that directs the backbone away from exosite Ⅰ and over the autolysis loop. The structures demonstrate that thrombin activation of PAR4 may occur with exosite Ⅰ available to bind cofactor molecules, like the cleaved form of PAR3, whose function is to promote substrate diffusion into the active site by allosterically changing the conformation of the 60-loop.