Structural basis for thrombin activation of a protease-activated receptor: inhibition of intramolecular liganding.
Protease-activated G protein-coupled receptors (PAR1-4) are tethered-ligand receptors that are activated by proteolytic cleavage of the extracellular domain (exodomain) of the receptor. PAR1, the prototypic member of the PAR family, is the high-affinity thrombin receptor of platelets and vascular endothelium and plays a critical role in blood coagulation, thrombosis, and inflammation. Here, we describe the solution structure of the thrombin-cleaved exodomain of PAR1. The side chains of a hydrophobic hirudin-like (Hir) sequence and adjacent anionic motif project into solution. Docking of the exodomain Hir sequence to exosite I of thrombin reveals that the tethered ligand in the cleaved exodomain bends away from thrombin, leaving its active site available to another large macromolecular substrate. The N-terminal ligand is longer than anticipated and forms an intramolecular complex with a region located in the C terminus of the exodomain. Mutational analysis confirmed that this C-terminal region is a ligand binding site for both intra- and intermolecular ligands. A lipidated-ligand binding site peptide was found to be an effective inhibitor of thrombin-induced platelet aggregation.
Seeley
Stacy
S
Division of Hematology/Oncology and Department of Medicine, New England Medical Center, Boston, MA 02111, USA.
Covic
Lidija
L
Jacques
Suzanne L
SL
Sudmeier
James
J
Baleja
James D
JD
Kuliopulos
Athan
A
D000595:Amino Acid Sequence; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D044462:Receptors, Proteinase-Activated / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013917:Thrombin / Q000737:chemistry / Q000378:metabolism*
D008024:Ligands; D044462:Receptors, Proteinase-Activated; D013917:Thrombin