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Allosteric modulation of G protein-coupled receptors (GPCRs) is a major paradigm in drug discovery. Despite decades of research, a molecular-level understanding of the general principles that govern the myriad pharmacological effects exerted by GPCR allosteric modulators remains limited. The M4 muscarinic acetylcholine receptor (M4 mAChR) is a validated and clinically relevant allosteric drug target for several major psychiatric and cognitive disorders. In this study, we rigorously quantified the affinity, efficacy, and magnitude of modulation of two different positive allosteric modulators, LY2033298 (LY298) and VU0467154 (VU154), combined with the endogenous agonist acetylcholine (ACh) or the high-affinity agonist iperoxo (Ipx), at the human M4 mAChR. By determining the cryo-electron microscopy structures of the M4 mAChR, bound to a cognate Gi1 protein and in complex with ACh, Ipx, LY298-Ipx, and VU154-Ipx, and applying molecular dynamics simulations, we determine key molecular mechanisms underlying allosteric pharmacology. In addition to delineating the contribution of spatially distinct binding sites on observed pharmacology, our findings also revealed a vital role for orthosteric and allosteric ligand–receptor–transducer complex stability, mediated by conformational dynamics between these sites, in the ultimate determination of affinity, efficacy, cooperativity, probe dependence, and species variability. There results provide a holistic framework for further GPCR mechanistic studies and can aid in the discovery and design of future allosteric drugs.

Human 12-lipoxygenase (12-LOX) is an enzyme involved in platelet activation and is a promising target for antiplatelet therapies. Despite the clinical importance of 12-LOX, the exact mechanisms of how it affects platelet activation are unclear, and the lack of structural information has limited drug discovery efforts. In this study, we used single-particle cryoelectron microscopy to determine the high-resolution structures (1.7 Å - 2.8 Å) of human 12-LOX for the first time. Our results showed that 12-LOX can exist in multiple oligomeric states, from monomer to hexamer, which may impact its catalytic activity and membrane association. We also identified different conformations within a 12-LOX dimer, likely representing different time points in its catalytic cycle. Furthermore, we were able to identify small molecules bound to the 12-LOX structures. The active site of the 12-LOX tetramer is occupied by an endogenous 12-LOX inhibitor, a long-chain acyl-Coenzyme A. Additionally, we found that the 12-LOX hexamer can simultaneously bind to arachidonic acid and ML355, a selective 12-LOX inhibitor that has passed a phase I clinical trial for treating heparin-induced thrombocytopenia and has received fast-track designation by the FDA. Overall, our findings provide novel insights into the assembly of 12-LOX oligomers, its catalytic mechanism, and small molecule binding, paving the way for further drug development targeting the 12-LOX enzyme.Key PointsThe first full-length structures of human arachidonate 12S-Lipoxygenase (12-LOX)Reveals mechanisms of oligomeric and conformational statesUncovers natural inhibitor of 12S-Lipoxygenase (12-lox)Reveals a binding site of inhibitor ML355

Allosteric modulation of G protein-coupled receptors (GPCRs) is a major paradigm in drug discovery. Despite decades of research, a molecular level understanding of the general principals that govern the myriad pharmacological effects exerted by GPCR allosteric modulators remains limited. The M4 muscarinic acetylcholine receptor (M4 mAChR) is a well-validated and clinically relevant allosteric drug target for several major psychiatric and cognitive disorders. Here, we present high-resolution cryo-electron microscopy structures of the M4 mAChR bound to a cognate Gi1 protein and the high affinity agonist, iperoxo, in the absence and presence of two different positive allosteric modulators, LY2033298 or VU0467154. We have also determined the structure of the M4 mAChR-Gi1 complex bound to its endogenous agonist, acetylcholine (ACh). Structural comparisons, together with molecular dynamics, mutagenesis, and pharmacological validations, have provided in-depth insights into the role of structure and dynamics in orthosteric and allosteric ligand binding, global mechanisms of receptor activation, cooperativity, probe-dependence, and species variability; all key hallmarks underpinning contemporary GPCR drug discovery.

Within the family of purinergic receptors, the P2X1 receptor is a ligand-gated ion channel that plays a role in urogenital, immune and cardiovascular function. Specifically, the P2X1 receptor has been implicated in controlling smooth muscle contractions of the vas deferens and therefore has emerged as an exciting drug target for male contraception. In addition, the P2X1 receptor contributes to smooth muscle contractions of the bladder and is a target to treat bladder dysfunction. Finally, platelets and neutrophils have populations of P2X1 receptors that could be targeted for thrombosis and inflammatory conditions. Drugs that specifically target the P2X1 receptor have been challenging to develop, and only recently have small molecule antagonists of the P2X1 receptor been available. However, these ligands need further biological validation for appropriate selectivity and drug-like properties before they will be suitable for use in preclinical models of disease. Although the atomic structure of the P2X1 receptor has yet to be determined, the recent discovery of several other P2X receptor structures and improvements in the field of structural biology suggests that this is now a distinct possibility. Such efforts may significantly improve drug discovery efforts at the P2X1 receptor.

G protein–coupled receptors (GPCRs) are critical regulators of cellular function acting via heterotrimeric G proteins as their primary transducers with individual GPCRs capable of pleiotropic coupling to multiple G proteins. Structural features governing G protein selectivity and promiscuity are currently unclear. Here, we used cryo-electron microscopy (cryo-EM) to determine structures of the cholecystokinin (CCK) type 1 receptor (CCK1R) bound to the CCK peptide agonist, CCK-8 and 2 distinct transducer proteins, its primary transducer Gq, and the more weakly coupled Gs. As seen with other Gq/11–GPCR complexes, the Gq–α5 helix (αH5) bound to a relatively narrow pocket in the CCK1R core. Surprisingly, the backbone of the CCK1R and volume of the G protein binding pocket were essentially equivalent when Gs was bound, with the Gs αH5 displaying a conformation that arises from “unwinding” of the far carboxyl-terminal residues, compared to canonically Gs coupled receptors. Thus, integrated changes in the conformations of both the receptor and G protein are likely to play critical roles in the promiscuous coupling of individual GPCRs., Cryo-EM structures of the G protein-coupled receptor CCK1R bound to the CCK peptide agonist CCK-8 and two distinct transducer proteins – its primary transducer Gq, and the more weakly coupled Gs – reveal unexpected modes of G protein interaction.

G protein-coupled receptors (GPCRs) are critical regulators of cellular function acting via heterotrimeric G proteins as their primary transducers with individual GPCRs capable of pleiotropic coupling to multiple G proteins. Structural features governing G protein selectivity and promiscuity are currently unclear. Here we used cryo-electron microscopy to determine structures of the CCK1R bound to the CCK peptide agonist, CCK-8 and two distinct transducer proteins, its primary transducer Gq, and the more weakly coupled Gs. As seen with other Gq/11-GPCR complexes, the Gq-α5 helix bound to a relatively narrow pocket in the CCK1R core. Surprisingly, the backbone of the CCK1R and volume of the G protein binding pocket was essentially equivalent when Gs was bound, with the Gs α5 helix displaying a conformation that arises from “unwinding” of the far C-terminal residues, compared to canonically Gs coupled receptors. Thus, integrated changes in the conformations of both the receptor and G protein play critical roles in the promiscuous coupling of individual GPCRs.One-Sentence SummaryCryo-EM structures of the CCK-1R reveal key mechanisms for promiscuous G protein coupling.

The β subunit of adenosine monophosphate (AMP)-activated protein kinase (AMPK), which exists as two isoforms (β1 and β2) in humans, has a carbohydrate-binding module (CBM) that interacts with glycogen. Although the β1- and β2-CBMs are structurally similar, with strictly conserved ligand-contact residues, they show different carbohydrate affinities. β2-CBM shows the strongest affinity for both branched and unbranched oligosaccharides and it has recently been shown that a Thr insertion into β2-CBM (Thr101) forms a pocket to accommodate branches. This insertion does not explain why β2-CBM binds all carbohydrates with stronger affinity. Herein, it is shown that residue 134 (Val for β2 and Thr for β1), which does not come into contact with a carbohydrate, appears to account for the affinity difference. Characterisation by NMR spectroscopy, however, suggests that mutant β2-Thr101Δ/Val134Thr differs from that of β1-CBM, and mutant β1-Thr101ins/Thr134Val differs from that of β2-CBM. Furthermore, these mutants are less stable to chemical denaturation, relative to that of wild-type β-CBMs, which confounds the affinity analyses. To support the importance of Thr101 and Val134, the ancestral CBM has been constructed. This CBM retains Thr101 and Val134, which suggests that the extant β1-CBM has a modest loss of function in carbohydrate binding. Because the ancestor bound carbohydrate with equal affinity to that of β2-CBM, it is concluded that residue 134 plays an indirect role in carbohydrate binding.

Human leukocyte antigen (HLA)-C*06:02 is identified as the allele associated with the highest risk for the development of the autoimmune skin disease psoriasis. However, the diversity and mode of peptide presentation by the HLA-C*06:02 molecule remains unclear. Here, we describe the endogenous peptide repertoire of ∼3,000 sequences for HLA-C*06:02 that defines the peptide-binding motif for this HLA allomorph. We found that HLA-C*06:02 predominantly presents nonamer peptides with dominant arginine anchors at the P2 and P7 positions and a preference for small hydrophobic residues at the C terminus (PΩ). To determine the structural basis of this selectivity, we determined crystal structures of HLA-C*06:02 in complex with two self-peptides (ARTELYRSL and ARFNDLRFV) and an analogue of a melanocyte autoantigen (ADAMTSL5, VRSRR-abu-LRL) implicated in psoriasis. These structures revealed that HLA-C*06:02 possesses a deep peptide-binding groove comprising two electronegative B- and E-pockets that coincide with the preference for P2 and P7 arginine anchors. The ADAMTSL5 autoantigen possessed a P7-Leu instead of the P7-Arg residue, but nevertheless was accommodated within the HLA-C*06:02 antigen-binding cleft. Collectively, our results provide the structural basis for understanding peptide repertoire selection in HLA-C*06:02.

Understanding the kinetics of proteins interacting with their ligands is important for characterizing molecular mechanism. However, it can be difficult to determine the extent and nature of these interactions for weakly formed protein-ligand complexes that have lifetimes of micro- to milliseconds. Nuclear magnetic resonance (NMR) spectroscopy is a powerful solution-based method for the atomic-level analysis of molecular interactions on a wide range of timescales, including micro- to milliseconds. Recently the combination of thermodynamic experiments using isothermal titration calorimetry (ITC) with kinetic measurements using ZZ-exchange and CPMG relaxation dispersion NMR spectroscopy have been used to determine the kinetics of weakly interacting protein systems. This chapter describes the application of ITC and NMR to understand the differences in the kinetics of carbohydrate binding by the β1- and β2-carbohydrate-binding modules of AMP-activated protein kinase.

Understanding the kinetics of proteins interacting with their ligands is important for characterizing molecular mechanism. However, it can be difficult to determine the extent and nature of these interactions for weakly formed protein-ligand complexes that have lifetimes of micro- to milliseconds. Nuclear magnetic resonance (NMR) spectroscopy is a powerful solution-based method for the atomic-level analysis of molecular interactions on a wide range of timescales, including micro- to milliseconds. Recently the combination of thermodynamic experiments using isothermal titration calorimetry (ITC) with kinetic measurements using ZZ-exchange and CPMG relaxation dispersion NMR spectroscopy have been used to determine the kinetics of weakly interacting protein systems. This chapter describes the application of ITC and NMR to understand the differences in the kinetics of carbohydrate binding by the β1- and β2-carbohydrate-binding modules of AMP-activated protein kinase.

Expression of HLA-C varies widely across individuals in an allele-specific manner. This variation in expression can influence efficacy of the immune response, as shown for infectious and autoimmune diseases. MicroRNA binding partially influences differential HLA-C expression, but the additional contributing factors have remained undetermined. Here we use functional and structural analyses to demonstrate that HLA-C expression is modulated not just at the RNA level, but also at the protein level. Specifically, we show that variation in exons 2 and 3, which encode the α1/α2 domains, drives differential expression of HLA-C allomorphs at the cell surface by influencing the structure of the peptide-binding cleft and the diversity of peptides bound by the HLA-C molecules. Together with a phylogenetic analysis, these results highlight the diversity and long-term balancing selection of regulatory factors that modulate HLA-C expression., HLA-C expression levels correlate with immune responses to pathogens and autoimmunity, and vary in an allele-specific manner across individuals. Here the authors identify factors that drive differential expression of HLA-C allomorphs at the cell surface, and influence the structure of the peptide-binding cleft and diversity of peptides bound by HLA-C molecules.

AMP-activated protein kinase interacts with oligosaccharides and glycogen through the carbohydrate-binding module (CBM) containing the beta-subunit, for which there are two isoforms (beta(1) and beta(2)). Muscle-specific beta(2)-CBM, either as an isolated domain or in the intact enzyme, binds carbohydrates more tightly than the ubiquitous beta(1)-CBM. Although residues that contact carbohydrate are strictly conserved, an additional threonine in a loop of beta(2)-CBM is concurrent with an increase in flexibility in beta(2)-CBM, which may account for the affinity differences between the two isoforms. In contrast to beta(1)-CBM, unbound beta(2)-CBM showed microsecond-to-millisecond motion at the base of a beta-hairpin that contains residues that make critical contacts with carbohydrate. Upon binding to carbohydrate, similar microsecond-to-millisecond motion was observed in this beta-hairpin and the loop that contains the threonine insertion. Deletion of the threonine from beta(2)-CBM resulted in reduced carbohydrate affinity. Although motion was retained in the unbound state, a significant loss of motion was observed in the bound state of the beta(2)-CBM mutant. Insertion of a threonine into the background of beta(1)-CBM resulted in increased ligand affinity and flexibility in these loops when bound to carbohydrate. However, these mutations indicate that the additional threonine is not solely responsible for the differences in carbohydrate affinity and protein dynamics. Nevertheless, these results suggest that altered protein dynamics may contribute to differences in the ligand affinity of the two naturally occurring CBM isoforms.

AMP-activated protein kinase (AMPK) is an αβγ heterotrimer that is important in regulating energy metabolism in all eukaryotes. The β-subunit exists in two isoforms (β1 and β2) and contains a carbohydrate-binding module (CBM) that interacts with glycogen. The two CBM isoforms (β1- and β2-CBM) are near identical in sequence and structure, yet show differences in carbohydrate-binding affinity. β2-CBM binds linear carbohydrates with 4-fold greater affinity than β1-CBM and binds single α1,6-branched carbohydrates up to 30-fold tighter. To understand these affinity differences, especially for branched carbohydrates, we determined the NMR solution structure of β2-CBM in complex with the single α1,6-branched carbohydrate glucosyl-β-cyclodextrin (gBCD) which supported the dynamic nature of the binding site, but resonance broadening prevented defining where the α1,6 branch bound. We therefore solved the X-ray crystal structures of β1- and β2-CBM, in complex with gBCD, to 1.7 and 2.0 Å (1 Å=0.1 nm) respectively. The additional threonine (Thr101) of β2-CBM expands the size of the surrounding loop, creating a pocket that accommodates the α1,6 branch. Hydrogen bonds are formed between the α1,6 branch and the backbone of Trp99 and Lys102 side chain of β2-CBM. In contrast, the α1,6 branch could not be observed in the β1-CBM structure, suggesting that it does not form a specific interaction. The orientation of gBCD bound to β1- and β2-CBM is supported by thermodynamic and kinetic data obtained through isothermal titration calorimetry (ITC) and NMR. These results suggest that AMPK containing the muscle-specific β2-isoform may have greater affinity for partially degraded glycogen.