Activation Related Conformational Changes in GPCRs

In lieu of a high-resolution crystal structure for a fully activated GPCR, activation-related changes in rhodopsin and other receptors have been extensively probed using indirect biochemical and biophysical methods [94, 96, 97]. These data provide a set of empirical restraints for modeling receptor activation. However, comprehensive modeling of the GPCR activation mechanism has yet to overcome multiple hurdles. GPCR activation is a dynamic, multistage, and multicomponent process, involving not only large-scale rearrangements of loops and local flexibility of TM helices, but also the binding of G proteins, as well as structured water, ions, and lipid moieties. Further, while experimentally derived constraints are frequently employed to facilitate accurate model creation, these data are often inconsistent due to variations of GPCR behavior in different assays, cell types, and culture conditions.

The TM domains exhibit a high degree of structural similarity and include several universally conserved features, such as the D(E)RY motif, suggesting the possibility of a common activation mechanism conserved among GPCR types .43, 96] . Further experimental and theoretical evaluation in different receptors is required to establish those conformational changes that are receptor specific and those which are part of a universal activation mechanism. Indeed, the outward dislocation of the TM6 cytoplasmic end has been indicated by numerous indirect biochemical and spectroscopic studies, as reviewed in Park et al. [43]. The recently solved crystal structures of opsin also confirm a significant 6-7-A outward displacement of the TM6 cytoplasmic end [44, 98]. This conformational change in TM6 forms a binding site for a G-a C-terminal peptide, which forms additional contacts with the D(E)RY motif .98] . The general requirement for G protein interaction suggests that this motion of the TM6 cytoplasmic portion is a common feature of GPCR activation. However, other conformational rearrangements such as the "rotamer toggle switch" may be less universal. The "toggle switch" mechanism was initially suggested based on an observed correlation between rotamer changes in the conserved amino acid W6.48 and enhancement of the adjacent proline kink in TM6 [96, 99-101]. While the TM6 proline kink angle is dramatically increased in both ligand-free opsin structures, no rotamer switch in W6.48 or other aromatic residues of the binding site was observed [44, 98]. One must therefore be judicious in incorporating particular experimental restraints or structural features during model development.

Despite the complexity of structural rearrangements in GPCRs and the paucity of their structural data availability, several modeling approaches have attempted to reproduce and/or predict activation-related conformational change in different GPCR families. The majority of these studies explicitly use experimental data as structural constraints to guide model optimization or select "best" models from a large set generated by molecular dynamic (MD)

or molecular mechanic (MC) calculations. In work by the Fanelli group, comparative MD simulations of several wild-type, inactivated, and constitutively active receptor mutants were performed to reproduce functional features of the receptor active state [102] . This study focused on the D(E)RY ionic lock of the a1b adrenergic receptor and found that a weakening of interactions with the D(E)RY arginine and an increase in solvent accessibility at the cyto-plasmic surface correlated with receptor activation. However, additional biochemical and computational evidence suggests that the D(E)RY motif should be considered in the context of supramolecular complexes with Ga and/or other GPCR homodimers/heterodimers, rather than in calculations considering only a single GPCR monomer [103-105] . New horizons for analysis of activation- related conformational changes in GPCRs have been opened by the recently solved high-resolution structure of ligand-free opsin and the Ops*-Ga-CT peptide complex [44, 98]. The Ops*-Ga-CT complex may also prove useful for deciphering the structural basis of Ga selectivity and building more reliable models of GPCR-G-aPy activation complexes.

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