Orphan receptor ligand discovery by pickpocketing pharmacological neighbors.

Understanding the pharmacological similarity of G protein-coupled receptors (GPCRs) is paramount for predicting ligand off-target effects, drug repurposing, and ligand discovery for orphan receptors. Phylogenetic relationships do not always correctly capture pharmacological similarity. Previous family-wide attempts to define pharmacological relationships were based on three-dimensional structures and/or known receptor-ligand pairings, both unavailable for orphan GPCRs. Here, we present GPCR-CoINPocket, a novel contact-informed neighboring pocket metric of GPCR binding-site similarity that is informed by patterns of ligand-residue interactions observed in crystallographically characterized GPCRs. GPCR-CoINPocket is applicable to receptors with unknown structure or ligands and accurately captures known pharmacological relationships between GPCRs, even those undetected by phylogeny. When applied to orphan receptor GPR37L1, GPCR-CoINPocket identified its pharmacological neighbors, and transfer of their pharmacology aided in discovery of the first surrogate ligands for this orphan with a 30% success rate. Although primarily designed for GPCRs, the method is easily transferable to other protein families.

The elusive π-helix.

Central to protein architecture is the local arrangement or secondary structure of the polypeptide backbone. Thirty to forty percent of protein domains are α-helices with 3.6 residues per turn. π-Helices, in which the peptide chain is more loosely coiled (4.4 residues per turn), have also been proposed. However, such structures necessitate an energetically unfavorable ∼1Å central helical hole. We show that rather than being composed of idealized π-helices, helical regions formed from putative π-helices actually consist of a series of concatenated wide turns with unique elliptical configurations. These structures have a larger helical radius akin to that of a π-helix, but without the loss of favorable cross-core van der Waals interactions. This not only obviates the helical void, but also endows proteins with important functionalities, including metal ion coordination, enhanced flexibility and specific enzyme-substrate binding interactions.

The pore domain outer helix contributes to both activation and inactivation of the HERG K+ channel.

Ion flow in many voltage-gated K(+) channels (VGK), including the (human ether-a-go-go-related gene) hERG channel, is regulated by reversible collapse of the selectivity filter. hERG channels, however, exhibit low sequence homology to other VGKs, particularly in the outer pore helix (S5) domain, and we hypothesize that this contributes to the unique activation and inactivation kinetics in hERG K(+) channels that are so important for cardiac electrical activity. The S5 domain in hERG identified by NMR spectroscopy closely corresponded to the segment predicted by bioinformatics analysis of 676 members of the VGK superfamily. Mutations to approximately every third residue, from Phe(551) to Trp(563), affected steady state activation, whereas mutations to approximately every third residue on an adjacent face and spanning the entire S5 segment perturbed inactivation, suggesting that the whole span of S5 experiences a rearrangement associated with inactivation. We refined a homology model of the hERG pore domain using constraints from the mutagenesis data with residues affecting inactivation pointing in toward S6. In this model the three residues with maximum impact on activation (W563A, F559A, and F551A) face out toward the voltage sensor. In addition, the residues that when mutated to alanine, or from alanine to valine, that did not express (Ala(561), His(562), Ala(565), Trp(568), and Ile(571)), all point toward the pore helix and contribute to close hydrophobic packing in this region of the channel.

Wide turn diversity in protein transmembrane helices implications for G-protein-coupled receptor and other polytopic membrane protein structure and function.

Previously, we showed that perturbations of protein transmembrane helices are manifested as one of three types of noncanonical structures (wide turns, tight turns, and kinks), which, compared with alpha-helices, are evident by distinctive Calpha(i)-->Calpha(x) distances. In this study, we report the analysis of more than 3000 transmembrane helices in 244 crystal structures from which we identified 70 wide turns (29 proline- and 41 nonproline-induced). Based on differences in the Calpha(i)-->Calpha(i)(-4) and Calpha(i)-->Calpha(i)(-5) profiles, we show that wide turns can be subclassified into three distinct subclasses (W(1), W(2), and W(3)) that differ with regard to the number and position of backbone i --> i-5 H-bonds formed N-terminal to the perturbing or signature proline or nonproline residue. Although wide turns generally produce changes in helical direction of 20 degrees to 30 degrees and a lateral shift in the helical axis, some of the W(3) subclass are associated with changes of <5 degrees . We also show that the distinct architectural features of wide turns allow the carbonyl bond of the i-4th residue, which is located on the widened loop of a wide turn, to be directed away from the helical axis. This provides regions of flexibility within helical regions allowing, for example, unique opportunities for interhelical H-bonding, including interactions with glycine zipper motifs, and for ion and cofactor binding. Furthermore, differences in wide-turn subtype usage by related protein family members, such as the G-protein-coupled receptors rhodopsin and the beta2-adrenergic receptor, can significantly affect the orientation and position of residues critical for ligand binding and receptor activation.

Mutation of a single TMVI residue, Phe(282), in the beta(2)-adrenergic receptor results in structurally distinct activated receptor conformations.

We showed previously that Phe(303) in transmembrane segment (TM) VI of the alpha(1B)-adrenergic receptor (alpha(1B)-AR), a residue conserved in many G protein-coupled receptors (GPCRs), is critically involved in coupling agonist binding with TM helical movement and G protein activation. Here the equivalent residue, Phe(282), in the beta(2)-AR was evaluated by mutation to glycine, asparagine, alanine, or leucine. Except for F282N, which exhibits attenuated basal and maximal isoproterenol stimulation, the Phe(282) mutants display varying degrees of constitutive activity (F282L > F282A > F282G), and as shown by the results of substituted cysteine accessibility method (SCAM) studies, induce movement of endogenous cysteine(s) into the water-accessible ligand-binding pocket. For F282A, movement is confined to Cys(285) in TMVI, whereas F282L induces movement of both Cys(285) in TMVI and Cys(327) in TMVII. Further, engineered cysteine-sensor studies indicate that F282L causes movement of TMVI, both above and below an apparent kink-inducing TMVI proline (Pro(288)), whereas that due to F282A is confined to the domain below Pro(288). A plausible interpretation of these data is that receptor activation involves rigid body movement of TMVI which, because of its Pro(288)-induced kink, acts as a pivot to transduce and amplify the agonist-induced conformational change in the upper domain, to a change in the lower domain required for productive receptor-G protein coupling.