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 Å 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.Thrombin is a trypsin-like protease endowed with important physiological functions that are mediated and regulated by interaction with numerous macromolecular substrates, receptors, and inhibitors (1). Fenton (2) was the first to recognize that thrombin selectivity toward macromolecular substrates depends on interactions with exosites that extend beyond the active site. Exosite I is positioned some 15 Å away from the active site (3) and occupies a domain analogous to the Ca 2ϩ -binding loop of trypsin and chymotrypsin (4). Recognition of macromolecular substrates or receptors responsible for the procoagulant, prothrombotic, signaling, and anticoagulant functions of thrombin depends on the structural integrity of this exosite (1). In general, exosite-dependent binding is kinetically limiting as a recognition strategy of macromolecular targets by enzymes involved in blood coagulation (5). Structural and site-directed mutagenesis data document the important role of exosite I in the interaction of thrombin with fibrinogen (6 -9) and the protease-activated receptors (PARs) 2 PAR1 (7, 10 -13) and PAR3 (7,14,15). Likewise, abundant structural and functional data document how thrombin recognizes substrates at the active site (3, 16, 17), including fibrinogen (18), PAR4 (15,19), and factor XIII (20, 21). On the other hand, structural elucidation of how substrates bridge the active site and exosite I in their binding to thrombin has been challenging. PAR1 is a premiere prothrombotic and signaling factor (22), the most specific physiological substrate of thrombin in terms of k cat /K m values (7), and a most relevant target for crystallization studies. PARs are members of the G-protein-coupled receptor superfamily and play important roles in blood coagulation, inflammation, cancer, and embryogenesis (23-28). Four PARs have been cloned, and they all share the same mechanism of ...

Ligand Binding to Anion-binding Exosites Regulates Conformational Properties of Thrombin

“…The PAR3(44 -56) and PAR1(49 -62) sequences were shown previously by x-ray crystallography to interact with thrombin ABE I (35,36). In our HDX control studies, PAR3(44 -56) has not been observed to bypass ABE I and move on to bind ABE II.…”

Autoactivation of Thrombin Precursors

“…The conceptual framework of the E*-E equilibrium extends to other classes of enzymes such as caspases and explains how small molecules can induce activation of the pro-enzyme (43) by stabilizing the E form. Prethrombin-2 exists in the E* and E forms (23,26) and carries a P1-P4 sequence in the activation domain that closely resembles that of PAR1, the most specific substrate of thrombin (44). What prevents autoactivation in prethrombin-2 is burial of Arg-15 in the activation domain, an unanticipated feature uncovered when the structure of this zymogen was solved for the first time in the free form (23).…”

Design, synthesis and biological evaluation of new peptide-based ureas and thioureas as potential antagonists of the thrombin receptor PAR1

“…Up to now, there is not structural information on the binding sites of these PAR1 antagonists to be used for structure-based design of new antagonists. However, recent mutagenesis studies on thrombin and/or PAR1 [34][35][36][37][38][39][40][41][42], X-ray of thrombin crystallized with diverse N-terminal fragments of PAR1 [43,44], and NMR studies on the (Ala 26 -Hse 103 ) N-terminal sequence of PAR1 [45] have shown that the first thrombin/PAR1 interaction is produced between the exosite I of thrombin and the hirudin-like sequence of PAR1 (K 51 YEPF 55 ), and that this first interaction is essential and determinant for high affinity. It seems that the hydrophobic residues F34, I82, L65 and Y76, and the basic residues R67 and R73, of the exosite I of thrombin are important for high affinity.…”