Clinical studies indicate that partial agonists of the G-protein-coupled, free fatty acid receptor 1 GPR40 enhance glucose-dependent insulin secretion and represent a potential mechanism for the treatment of type 2 diabetes mellitus. Full allosteric agonists (AgoPAMs) of GPR40 bind to a site distinct from partial agonists and can provide additional efficacy. We report the 3.2-Å crystal structure of human GPR40 (hGPR40) in complex with both the partial agonist MK-8666 and an AgoPAM, which exposes a novel lipid-facing AgoPAM-binding pocket outside the transmembrane helical bundle. Comparison with an additional 2.2-Å structure of the hGPR40-MK-8666 binary complex reveals an induced-fit conformational coupling between the partial agonist and AgoPAM binding sites, involving rearrangements of the transmembrane helices 4 and 5 (TM4 and TM5) and transition of the intracellular loop 2 (ICL2) into a short helix. These conformational changes likely prime GPR40 to a more active-like state and explain the binding cooperativity between these ligands.

Narcolepsy type 1 (NT1) is a chronic neurological disorder that impairs the brain’s ability to control sleep-wake cycles. Current therapies are limited to the management of symptoms with modest effectiveness and substantial adverse effects. Agonists of the orexin receptor 2 (OX2R) have shown promise as novel therapeutics that directly target the pathophysiology of the disease. However, identification of drug-like OX2R agonists has proven difficult. Here we report cryo-electron microscopy structures of active-state OX2R bound to an endogenous peptide agonist and a small-molecule agonist. The extended carboxy-terminal segment of the peptide reaches into the core of OX2R to stabilize an active conformation, while the small-molecule agonist binds deep inside the orthosteric pocket, making similar key interactions. Comparison with antagonist-bound OX2R suggests a molecular mechanism that rationalizes both receptor activation and inhibition. Our results enable structure-based discovery of therapeutic orexin agonists for the treatment of NT1 and other hypersomnia disorders.

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p70 ribosomal S6 kinase (p70S6K) is a downstream effector of the mTOR signaling pathway involved in cell proliferation, cell growth, cell-cycle progression, and glucose homeostasis. Multiple phosphorylation events within the catalytic, autoinhibitory, and hydrophobic motif domains contribute to the regulation of p70S6K. We report the crystal structures of the kinase domain of p70S6K1 bound to staurosporine in both the unphosphorylated state and in the 3-phosphoinositide-dependent kinase-1-phosphorylated state in which Thr-252 of the activation loop is phosphorylated. Unphosphorylated p70S6K1 exists in two crystal forms, one in which the p70S6K1 kinase domain exists as a monomer and the other as a domain-swapped dimer. The crystal structure of the partially activated kinase domain that is phosphorylated within the activation loop reveals conformational ordering of the activation loop that is consistent with a role in activation. The structures offer insights into the structural basis of the 3-phosphoinositide-dependent kinase-1-induced activation of p70S6K and provide a platform for the rational structure-guided design of specific p70S6K inhibitors.The ribosomal S6 kinase family belongs to the AGC 3 subfamily of serine-threonine protein kinases. In humans two forms of p70 ribosomal S6 kinases (S6K1 and S6K2) have been reported that are encoded by two different genes (RPS6KB1 and RPS6KB2), respectively (1, 2). RPS6KB1 encodes two isoforms that differ only at the N termini by 23 amino acid residues (2). The longer form of S6K1 contains an N-terminal nuclear localization signal, whereas the shorter isoform of S6K1 predominantly localizes in the cytosol.Several substrates of p70S6K have been identified including 40 S ribosomal protein S6, insulin substrate (IRS1), preapoptotic protein BAD, eukaryotic initiation factor (elF4B), eukaryotic elongation factor (eEF2K) and cAMP-response element modulator (CREMt) (3). The most studied substrate is the 40 S ribosomal protein S6, a major component of the machinery involved in protein synthesis in mammalian cells, suggesting that p70S6K plays a role in regulating translation.Several observations suggest a role for p70S6K in cancer (4, 5). For example, upstream regulators of p70S6K are deregulated in multiple types of cancer, and gene and protein overexpression is observed in various cancers (4, 5). In addition, p70S6K is also a downstream kinase of insulin receptor-mediated signaling and is a potential therapeutic target for the management of obesity and diabetes as shown by enhanced metabolic rate and insulin sensitivity in p70S6K knock-out mice (4, 5).The activation of p70S6K requires multiple phosphorylation events in both the kinase and autoinhibitory domains (Fig. 1). The C-terminal autoinhibitory domain, which is believed to block phosphorylation within the hydrophobic motif and the activation loop, is phosphorylated by upstream kinases such as ERK (6, 7). Other activating phosphorylation events occur at Thr-412 in the hydrophobic motif by mTOR (mammalian target of rapam...

BackgroundThe unique S28 family of proteases is comprised of the carboxypeptidase PRCP and the aminopeptidase DPP7. The structural basis of the different substrate specificities of the two enzymes is not understood nor has the structure of the S28 fold been described.ResultsThe experimentally phased 2.8 Å crystal structure is presented for human PRCP. PRCP contains an α/β hydrolase domain harboring the catalytic Asp-His-Ser triad and a novel helical structural domain that caps the active site. Structural comparisons with prolylendopeptidase and DPP4 identify the S1 proline binding site of PRCP. A structure-based alignment with the previously undescribed structure of DPP7 illuminates the mechanism of orthogonal substrate specificity of PRCP and DPP7. PRCP has an extended active-site cleft that can accommodate proline substrates with multiple N-terminal residues. In contrast, the substrate binding groove of DPP7 is occluded by a short amino-acid insertion unique to DPP7 that creates a truncated active site selective for dipeptidyl proteolysis of N-terminal substrates.ConclusionThe results define the structure of the S28 family of proteases, provide the structural basis of PRCP and DPP7 substrate specificity and enable the rational design of selective PRCP modulators.