Analysis of a Naturally Occurring PAR4 Variant By Hydrogen/Deuterium Exchange Reveals Its Structural Rearrangement upon Activation By Thrombin and the Molecular Basis for Altered Reactivity

Protease activated receptors 1 and 4 (PAR1 and PAR4) are GPCRs that mediate thrombin signaling on human platelets. In this dual receptor system, PAR4 is responsible for sustained signaling required for stable platelet aggregation. In addition, common PAR4 polymorphisms affect its reactivity, which contributes to the variability in platelet response among individuals. The threonine residue 120 leads to a variant that is hyper-reactive to agonists and resistant to some antagonist compared to an alanine at this site. The molecular mechanisms by which this substitution influences PAR4 activation are not known. Further, the structural basis for PAR activation in general is also unknown. PARs are activated by enzymatic cleavage of the N-terminus exposing a tethered ligand that interacts with a binding pocket on the receptor's extracellular side. Since the ligand is attached to the receptor, there are likely substantial conformational changes that occur during PAR activation. The goal of this study was to determine the structural rearrangement of PAR4 upon activation by thrombin to test the hypothesis that polymorphisms at position 120 directly contribute to the ligand binding site and the subsequent PAR4 signaling cascades. Here, we used hydrogen-deuterium exchange mass spectrometry to map the conformational rearrangement of the hyper-reactive PAR4 variant (PAR4-120T). Full length human PAR4-120T was expressed in and purified from SF9 insect cells without additional stabilizing modifications. Our initial peptide mapping studies generated 93.5% (344/368) amino acid sequence coverage with multiple overlapping peptides. In the uncleaved (apo) state, the seven transmembrane domains of PAR4 show limited deuterium uptake as a result of being buried in detergent micelles. TM4 and TM5 displayed lower deuterium uptake than the other helices which potentially indicate the formation of dimerization interface, which is in agreement with our previous biochemical studies. Helix 8, a short α-helix between TM7 and C-terminus, showed reduced deuterium uptake. Together with other studies, this may indicate helix 8 plays a role in anchoring the receptor on lipid rafts. In comparison, the N- and C-termini displayed higher solvent accessibility based on the high deuterium uptake. Surprisingly, the tethered ligand sequence of PAR4 (GYPGQV) at the N-terminus has significantly lower solvent accessibility than neighboring regions in the apo state. This suggests it is less flexible and may be localized near the binding pocket even before thrombin cleavage. Comparison of deuterium uptake between apo and thrombin cleaved (active) states of PAR4-120T revealed reduced solvent accessibility at amino acids Cys149-Leu156 at TM3 and Ser329-Ser333 at TM7. This identifies the potential tethered ligand binding region of PAR4, which has not been previously described yet. Interestingly, in our molecular models Thr120 was located near this region spatially, which potentially explains the impact of this residue on PAR4 reactivity. We further observed an increase in deuterium uptake indicating a potential role for extracellular loop III and the connected regions of TM6 and TM7 in the receptor activation mechanism. In summary, our study on PAR4 is the first to elucidate the structural dynamics and molecular rearrangement of a protease activated receptor upon activation. Our results provide the overall molecular architecture of PAR4, uncovers the tethered ligand binding region, and further reveals the molecular basis for the inter-individual platelet reactivity based on the genotype of this key platelet receptor. Altogether, our studies may have significant clinical impact towards explaining the diversity of individual responses to existing and forthcoming therapies.