Crystal structure of thrombin bound to the uncleaved extracellular fragment of PAR1

Abstract

Abundant structural information exists on how thrombin recognizes ligands at the active site or at exosites separate from the active site region, but remarkably little is known about how thrombin recognizes substrates that bridge both the active site and exosite I. The case of the protease-activated receptor PAR1 is particularly relevant in view of the plethora of biological effects associated with its activation by thrombin. Here, we present the 1.8 A resolution structure of thrombin S195A in complex with a 30-residue long uncleaved extracellular fragment of PAR1 that documents for the first time a productive binding mode bridging the active site and exosite I. The structure reveals two unexpected features of the thrombin-PAR1 interaction. The acidic P3 residue of PAR1, Asp(39), does not hinder binding to the active site and actually makes favorable interactions with Gly(219) of thrombin. The tethered ligand domain shows a considerable degree of disorder even when bound to thrombin. The results fill a significant gap in our understanding of the molecular mechanisms of recognition by thrombin in ways that are relevant to other physiological substrates.

FIGURE 1.

X-ray crystal structure of thrombin in complex with the uncleaved extracellular fragment of PAR1. Thrombin is shown in the standard Bode orientation (3), with the active site in the center and exosite I on the right. Thrombin is rendered in surface representation (wheat), and PAR1 is rendered in stick representation (yellow). The 2F_o − F_c electron density map (light green mesh) for the uncleaved PAR1 fragment is contoured at 0.9σ. Residues of thrombin interacting with PAR1 through molecular contacts within 4 Å are colored in orange (hydrophobic contacts) and marine (polar contacts). The uncleaved extracellular fragment of PAR1 engages both the active site and exosite I in productive binding modes.

FIGURE 2.

Details of the cleavage site and tethered ligand of PAR1. Fragment ⁴⁰PRSFLL⁴⁵, spanning the P2–P4′ positions, is shown with its 2F_o − F_c electron density map (light green mesh) contoured at 0.9σ. The peptide bond between Arg⁴¹ and Ser⁴² can be assigned with confidence, vouching for an intact PAR1 fragment. Downstream of Phe⁴³, the electron density becomes weaker, underscoring disorder in most of the tethered ligand.

FIGURE 3.

Molecular contacts at the active site for the thrombin-PAR1 complex. Thrombin is rendered in surface representation color-coded according to atom type (carbon, white; nitrogen, blue; oxygen, red; and sulfur, yellow), and PAR1 is rendered in stick representation (yellow). Residues of thrombin within 4 Å of PAR1 are labeled in black. PAR1 residues are labeled in red. Arg⁴¹ penetrates the primary specificity pocket and is partially covered in this view by the side chain of Glu¹⁹². Pro⁴⁰ at the P2 position makes strong hydrophobic interactions with Pro^60b, Pro^60c, and Trp^60d, and Leu³⁸ at the P4 position occupies the aryl-binding site formed by Trp²¹⁵, Ile¹⁷⁴, and Leu⁹⁹. Asp³⁹ at the P3 position makes a polar interaction with the backbone N of Gly²¹⁹, which is reinforced by a water-mediated contact contributed by w271. H-bonds between thrombin and PAR1 are listed in Table 2.