Intramolecular allosteric communication in dopamine D2 receptor revealed by evolutionary amino acid covariation

Abstract

The structural basis of allosteric signaling in G protein-coupled receptors (GPCRs) is important in guiding design of therapeutics and understanding phenotypic consequences of genetic variation. The Evolutionary Trace (ET) algorithm previously proved effective in redesigning receptors to mimic the ligand specificities of functionally distinct homologs. We now expand ET to consider mutual information, with validation in GPCR structure and dopamine D2 receptor (D2R) function. The new algorithm, called ET-MIp, identifies evolutionarily relevant patterns of amino acid covariations. The improved predictions of structural proximity and D2R mutagenesis demonstrate that ET-MIp predicts functional interactions between residue pairs, particularly potency and efficacy of activation by dopamine. Remarkably, although most of the residue pairs chosen for mutagenesis are neither in the binding pocket nor in contact with each other, many exhibited functional interactions, implying at-a-distance coupling. The functional interaction between the coupled pairs correlated best with the evolutionary coupling potential derived from dopamine receptor sequences rather than with broader sets of GPCR sequences. These data suggest that the allosteric communication responsible for dopamine responses is resolved by ET-MIp and best discerned within a short evolutionary distance. Most double mutants restored dopamine response to wild-type levels, also suggesting that tight regulation of the response to dopamine drove the coevolution and intramolecular communications between coupled residues. Our approach provides a general tool to identify evolutionary covariation patterns in small sets of close sequence homologs and to translate them into functional linkages between residues.allostery | G protein-coupled receptors | residue covariation | Evolutionary Trace I dentifying residues that coevolved to maintain or acquire fitness properties is critical for understanding protein structure, function, and evolution (1). Previous studies have shown that covarying residue pairs, those that exhibit correlated amino acid changes in large multiple sequence alignments, tend to form structural contacts (2-7), enhancing predictions of protein 3D structures (8-11). Covariation can also involve distal residues, but the function of these at-a-distance couplings is elusive and has been attributed to background noise, alternative protein conformations, or subunit interactions of protein homooligomers (5,7,12). Alternately, distal covarying residue pairs could indicate allosteric couplings (6,(13)(14)(15)(16)(17)(18).The possibility of capturing intramolecular allosteric communication by amino acid covariation analysis of protein family sequences has not been extensively explored. Nonproximal thermodynamic coupling between correlated residue pairs was noted in 274 PDZ domains (14), but the relationship to allostery is still debated (19,20). It may be that distinctive allosteric mechanisms, even among close homologs, limit the extraction of allosteric couplings from ...

Applications of sequence coevolution in membrane protein biochemistry

“…When combined with MI, coevolving residue pair predictions are closer in structural space. This combined method was used to identify, and confirm through mutagenesis, allosteric networks and proximity of coevolving pairs in the dopamine D2 receptor [59]. These results corroborate studies showing that reweighting of phylogeny and conservation in MI can improve predictions [52,60,61].…”

“…Bias in the phylogeny of the alignment can result in poor prediction of evolutionary couplings, and thus deeper alignments, which can have less phylogenetic bias, generally improve predictive power [7,163,164]. In contrast, explicitly modeling phylogeny in an alignment, such as with evolutionary trace methods [56,58,59] and other phylogeny-based covariation methods [57], can help identify gene-wide covariation. Although in sequence coevolution methods phylogeny of the alignment is typically not modeled directly, correcting for the similarity of sequences in an alignment has been used to improve SCA, MI and direct methods [52,60,163], which accounts for experimental sampling bias.…”

Allosteric communication pipelines in G-protein-coupled receptors

“…The ET analysis (ETA) has been applied to redesign receptor mutations that could mimic ligand specificities and also mutations that switch the signaling bias [49••, 50•]. More recently Sung et al incorporated mutual information (MI) of the co-variation of distal residue pairs in the ETA, to identify residue pairs in dopamine D2 receptor that are involved in allosteric communication [51]. Sung et al used ETA to predict a network of residues involved in allosteric communication in the dopamine receptor D2, shown as yellow spheres in Figure 2A.…”

Functional Diversification of Tripeptidyl Peptidase and Endopeptidase Sedolisins in Fungi

“…Diverge [47] should theoretically contemplate this by using likelihood models. A recent version of Evolutionary Trace 405 [48] includes mutual information in order to substantiate its predictions. We combined SDPfox [35], to identify CDPs, with MISTIC [36], to confirm CDPs as SDPs.…”