Helix Dipole Movement and Conformational Variability Contribute to Allosteric GDP Release in Gα i Subunits † , ‡

Heterotrimeric G proteins (Gαβγ) transmit signals from activated G protein coupled receptors (GPCRs) to downstream effectors through a guanine nucleotide signaling cycle. Numerous studies indicate that the carboxy-terminal α5 helix of Gα subunits participate in Gα-receptor binding, and previous EPR studies suggest this receptor-mediated interaction induces a rotation and translation of the α5 helix of the Gα subunit [Oldham et al., Nat. Struct. Mol. Biol., 13: 772-7 (2006)]. Based on this result, an engineered disulfide bond was designed to constrain the α5 helix of Gαi1 into its EPR-measured receptor-associated conformation through the introduction of cysteines at positions 56 in the α1 helix and 333 in the α5 helix (I56C/Q333C Gαi1). A functional mimetic of the EPR-measured α5 helix dipole movement upon receptor association was additionally created by introduction of a positive charge at the amino-terminus of this helix, D328R Gαi1. Both proteins exhibit dramatically elevated basal nucleotide exchange. The 2.9 Å resolution crystal structure of the I56C/Q333C Gαi1 in complex with GDP-AlF4− reveals the shift of the α5 helix toward the guanine nucleotide-binding site that is anticipated by EPR measurements. The structure of the I56C/Q333C Gαi1 subunit further revealed altered positions for the switch regions and throughout the Gαi1 subunit, accompanied by significantly elevated crystallographic temperature factors. Combined with previous evidence in the literature, the structural analysis supports the critical role of electrostatics of the α5 helix dipole and overall conformational variability during nucleotide release

Unifying view of mechanical and functional hotspots across class A GPCRs

G protein-coupled receptors (GPCRs) are the largest superfamily of signaling proteins. Their activation process is accompanied by conformational changes that have not yet been fully uncovered. Here, we carry out a novel comparative analysis of internal structural fluctuations across a variety of receptors from class A GPCRs, which currently has the richest structural coverage. We infer the local mechanical couplings underpinning the receptors' functional dynamics and finally identify those amino acids whose virtual deletion causes a significant softening of the mechanical network. The relevance of these amino acids is demonstrated by their overlap with those known to be crucial for GPCR function, based on static structural criteria. The differences with the latter set allow us to identify those sites whose functional role is more clearly detected by considering dynamical and mechanical properties. Of these sites with a genuine mechanical/dynamical character, the top ranking is amino acid 7x52, a previously unexplored, and experimentally verifiable key site for GPCR conformational response to ligand binding. \ua9 2017 Ponzoni et al

Irreversible Platelet Activation Requires Protease-Activated Receptor 1-Mediated Signaling to Phosphatidylinositol Phosphates

Thrombin induces platelet activation through an early, reversible stage of platelet aggregation, which is followed by a later, irreversible stage of platelet aggregation. Without intervention, events leading to pathological platelet activation can result in vessel occlusion, acute coronary syndrome, and stroke. Therefore, a better understanding of events leading to platelet-mediated clot formation may provide insight into new therapeutic targets. Once activated, protease activated receptors (PARs) are essential in regulating events leading to platelet aggregation. We have determined a signaling cascade through PAR1, which involves phosphatidylinositol (PI) kinases, phosphatidylinositol bisphosphate (PIP2), and Rap1 activation (independent of P2Y12) in the formation of a stable platelet aggregate. The putative phosphatidylinositol-3 kinase (PI3K) inhibitor LY294002 was found to reduce basal and PAR-stimulated PIP2 levels by mass spectrometry and to inhibit PAR1-mediated stable platelet aggregation. Rap1 activation in platelets (during time points corresponding to the late, irreversible phase of aggregation) was found to require the PI signaling pathway. Perturbation of PI3K signaling by isoform-selective inhibitors had differential effects on Rap1 activation through PAR1 and PAR4. Hence, it is possible to disrupt lipid signaling pathways involved in stable clot formation without inhibiting early clot formation, offering a new potential target for antiplatelet therapy

Myristoylation Exerts Direct and Allosteric Effects on Gα Conformation and Dynamics in Solution

Coupling of heterotrimeric G proteins to activated G protein-coupled receptors results in nucleotide exchange on the Gα subunit, which in turn decreases its affinity for both Gβγ and activated receptors. N-Terminal myristoylation of Gα subunits aids in membrane localization of inactive G proteins. Despite the presence of the covalently attached myristoyl group, Gα proteins are highly soluble after GTP binding. This study investigated factors facilitating the solubility of the activated, myristoylated protein. In doing so, we also identified myristoylation-dependent differences in regions of Gα known to play important roles in interactions with receptors, effectors, and nucleotide binding. Amide hydrogen–deuterium exchange and site-directed fluorescence of activated proteins revealed a solvent-protected amino terminus that was enhanced by myristoylation. Furthermore, fluorescence quenching confirmed that the myristoylated amino terminus is in proximity to the Switch II region in the activated protein. Myristoylation also stabilized the interaction between the guanine ring and the base of the α5 helix that contacts the bound nucleotide. The allosteric effects of myristoylation on protein structure, function, and localization indicate that the myristoylated amino terminus of Gα_i functions as a myristoyl switch, with implications for myristoylation in the stabilization of nucleotide binding and in the spatial regulation of G protein signaling