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The goal of designing safer, more effective drugs has led to tremendous interest in molecular mechanisms through which ligands can precisely manipulate the signaling of G-protein-coupled receptors (GPCRs), the largest class of drug targets. Decades of research have led to the widely accepted view that all agonists—ligands that trigger GPCR activation—function by causing rearrangement of the GPCR’s transmembrane helices, opening an intracellular pocket for binding of transducer proteins. Here we demonstrate that certain agonists instead trigger activation of free fatty acid receptor 1 by directly rearranging an intracellular loop that interacts with transducers. We validate the predictions of our atomic-level simulations by targeted mutagenesis; specific mutations that disrupt interactions with the intracellular loop convert these agonists into inverse agonists. Further analysis suggests that allosteric ligands could regulate the signaling of many other GPCRs via a similar mechanism, offering rich possibilities for precise control of pharmaceutically important targets. Ligands that activate GPCRs generally do so by stabilizing a particular conformation of the transmembrane helices. Here, the authors reveal a distinct activation mechanism where a ligand instead stabilizes a particular intracellular loop conformation.

GPR40 (FFA1) is a fatty acid receptor whose activation results in potent glucose lowering and insulinotropic effects in vivo. Several reports illustrate that GPR40 agonists exert glucose lowering in diabetic humans. To assess the mechanisms by which GPR40 partial agonists improve glucose homeostasis, we evaluated the effects of MK-2305, a potent and selective partial GPR40 agonist, in diabetic Goto Kakizaki rats. MK-2305 decreased fasting glucose after acute and chronic treatment. MK-2305-mediated changes in glucose were coupled with increases in plasma insulin during hyperglycemia and glucose challenges but not during fasting, when glucose was normalized. To determine the mechanism(s) mediating these changes in glucose metabolism, we measured the absolute contribution of precursors to glucose production in the presence or absence of MK-2305. MK-2305 treatment resulted in decreased endogenous glucose production (EGP) driven primarily through changes in gluconeogenesis from substrates entering at the TCA cycle. The decrease in EGP was not likely due to a direct effect on the liver, as isolated perfused liver studies showed no effect of MK-2305 ex vivo and GPR40 is not expressed in the liver. Taken together, our results suggest MK-2305 treatment increases glucose stimulated insulin secretion (GSIS), resulting in changes to hepatic substrate handling that improve glucose homeostasis in the diabetic state. Importantly, these data extend our understanding of the underlying mechanisms by which GPR40 partial agonists reduce hyperglycemia.

Niemann-Pick C1-like protein (NPC1L1) mediates the absorption of dietary cholesterol in the proximal region of the intestine, a process that is blocked by cholesterol absorption inhibitors (CAIs), including ezetimibe (EZE). Using a proteomic approach, we demonstrate that NPC1L1 is the protein to which EZE and its analogs bind. Next, we determined the site of interaction of EZE analogs with NPC1L1 by exploiting the different binding affinities of mouse and dog NPC1L1 for the radioligand analog of EZE, [³H]AS. Chimeric and mutational studies indicate that high-affinity binding of [³H]AS to dog NPC1L1 depends on molecular determinants present in a 61-aa region of a large extracellular domain (loop C), where Phe-532 and Met-543 appear to be key contributors. These data suggest that the [³H]AS-binding site resides in the intestinal lumen and are consistent with preclinical data demonstrating in vivo efficacy of a minimally bioavailable CAI. Furthermore, these determinants of [³H]AS binding lie immediately adjacent to a hotspot of human NPC1L1 polymorphisms correlated with hypoabsorption of cholesterol. These observations, taken together with the recently described binding of cholesterol to the N terminus (loop A) of the close NPC1L1 homologue, NPC1, may provide a molecular basis for understanding EZE inhibition of NPC1L1-mediated cholesterol absorption. Specifically, EZE binding to an extracellular site distinct from where cholesterol binds prevents conformational changes in NPC1L1 that are necessary for the translocation of cholesterol across the membrane.

Key Points Membrane transport proteins mediate the movement of molecules into, or out of, cells, intracellular organelles and across epithelia. Despite their abundance in the genome and evident importance for the cell, we know little about their structure and function, largely because their hydrophobic and metastable nature makes them difficult to study. Recent developments have allowed initial insight into the structure and mechanism of membrane transport proteins. One example is the lactose permease from Escherichia coli , a member of the major facilitator superfamily. This membrane protein uses free energy released from the energetically downhill translocation of H + in response to an electrochemical H + gradient to drive the accumulation of specific sugars against a concentration gradient. Extensive use of site-directed mutagenesis demonstrates that only six amino acid residues are irreplaceable with respect to active lactose transport. Furthermore, mutants engineered for various biochemical and biophysical approaches provide structural information about how the helices are packed and how the irreplaceable residues interact to catalyse transport. Through this work, charge pairs have been identified that mediate substrate binding and H + translocation. The residues that are irreplaceable for activity are conserved in other members of the oligosaccharide/H + symport subfamily, but are not found in other members of the major facilitator superfamily. Despite this, it is thought that relatively few residues will be critical for transport in these other families of membrane transport proteins and that the conformational changes involved will be largely rigid body movements of the transmembrane helices. Membrane transport proteins catalyse the movement of molecules into and out of cells and organelles, but their hydrophobic and metastable nature often makes them difficult to study by traditional means. Novel approaches that have been developed and applied to one membrane transport protein, the lactose permease from Escherichia coli , are now being used to study various other membrane proteins.

Deletion of 5 residues (Δ 5) from the central cytoplasmic loop of the lactose permease of Escherichia coli has no significant effect on expression or activity, whereas Δ 12 leads to increased rates of permease turnover after membrane insertion and decreased transport activity, and Δ 20 abolishes insertion and activity. By expressing Δ 12 or Δ 20 in two halves, both expression and activity are restored to levels approximating wild type. Replacing deleted residues with random hydrophilic amino acids also leads to full recovery. However, introduction of hydrophobic residues decreases expression and activity in a context-dependent manner. Thus, a minimum length of the central cytoplasmic loop is vital for proper insertion, stability, and efficient transport activity, because of constraints at the cytoplasmic ends of helices VI and VII. Furthermore, the results are consistent with the idea that the middle cytoplasmic loop provides a temporal delay between insertion of the first six helices into the membrane before insertion of the second six helices.

Integration of biochemical and biophysical data on the lactose permease of Escherichia coli has culminated in a molecular model that predicts substrate–protein proximities which include interaction of a hydroxyl group in the galactopyranosyl ring with Glu269. In order to test this hypothesis, we studied covalent modification of carboxyl groups with carbodiimides using electrospray ionization mass spectrometry (ESI‐MS) and demonstrate that substrate protects the permease against carbodiimide reactivity. Further more, a significant proportion of the decrease in carbodiimide reactivity occurs specifically in a nanopeptide containing Glu269. In contrast, carbodiimide reactivity of mutant Glu269→Asp that exhibits lower affinity is unaffected by substrate. By monitoring the ability of different substrate analogs to protect against carbodiimide modification of Glu269, it is suggested that the C‐3 OH group of the galactopyranosyl ring may play an important role in specificity, possibly by H‐bonding with Glu269. The approach demonstrates that mass spectrometry can provide a powerful means of analyzing ligand interactions with integral membrane proteins.

Luminescence resonance energy transfer with a lanthanide like Tb3+as donor is a useful technique for estimating intra- and intermolecular distances in macromolecules. However, the technique usually requires the use of a bulky chelator with a flexible linker attached to a Cys residue to bind Tb3+and, for intramolecular studies, an acceptor fluorophor attached to another Cys residue in the same protein. Here, an engineered EF- hand motif is incorporated into the central cytoplasmic loop of the lactose permease of Escherichia coli generating a high-affinity site for Tb3+(KTb 3+≈ 4.5 µM) or Gd3+(KGd 3+≈ 2.3 µM). By exciting a Trp residue in the coordination sequence, Tb3+bound to the EF-hand motif is sensitized specifically, and the efficiency of energy transfer to strategically placed Cys residues labeled with fluorophors is measured. In this study, we use the technique to measure distance from the EF-hand in the central cytoplasmic loop of lactose permease to positions 179 or 169 at the center or periplasmic end of helix VI, respectively. The average calculated distances of ≈23 Å (position 179) and ≈33 Å (position 169) observed with three different fluorophors as acceptors agree well with the geometry of a slightly tilted α-helix. The approach should be of general use for studying static and dynamic aspects of polytopic membrane protein structure and function.

GPR40 agonists are effective antidiabetic agents believed to lower glucose through direct effects on the beta cell to increase glucose stimulated insulin secretion. However, not all GPR40 agonists are the same. Partial agonists lower glucose through direct effects on the pancreas, whereas GPR40 AgoPAMs may incorporate additional therapeutic effects through increases in insulinotrophic incretins secreted by the gut. Here we describe how GPR40 AgoPAMs stimulate both insulin and incretin secretion in vivo over time in diabetic GK rats. We also describe effects of AgoPAMs in vivo to lower glucose and body weight beyond what is seen with partial GPR40 agonists in both the acute and chronic setting. Further comparisons of the glucose lowering profile of AgoPAMs suggest these compounds may possess greater glucose control even in the presence of elevated glucagon secretion, an unexpected feature observed with both acute and chronic treatment with AgoPAMs. Together these studies highlight the complexity of GPR40 pharmacology and the potential additional benefits AgoPAMs may possess above partial agonists for the diabetic patient.

The goal of designing safer, more effective drugs has led to tremendous interest in molecular mechanisms through which ligands can precisely manipulate signaling of G-protein-coupled receptors (GPCRs), the largest class of drug targets. Decades of research have led to the widely accepted view that all agonists-ligands that trigger GPCR activation-function by causing rearrangement of the GPCR's transmembrane helices, opening an intracellular pocket for binding of transducer proteins. Here we demonstrate that certain agonists instead trigger activation of free fatty acid receptor 1 by directly rearranging an intracellular loop that interacts with transducers. We validate the predictions of our atomic-level simulations by targeted mutagenesis; specific mutations which disrupt interactions with the intracellular loop convert these agonists into inverse agonists. Further analysis suggests that allosteric ligands could regulate signaling of many other GPCRs via a similar mechanism, offering rich possibilities for precise control of pharmaceutically important targets.

Glu-126 (helix IV) and Arg-144 (helix V) are charge paired and play a critical role in substrate binding in the lactose permease of Escherichia coli. When Glu-126 is replaced with Asp, the permease has relatively high activity, implying that helix V has sufficient flexibility to allow Arg-144 to accommodate the decreased length of the carboxylate-containing side chain of Asp-126. Helices IV and V contain five Gly residues at positions 115, 121, 141, 147, and 150, all of which are conserved in the oligosaccharide / H+symport family of the major facilitator superfamily. To test the notion that these residues may contribute to conformational flexibility, each residue was replaced with Ala in either the wild type or the Glu-126→ Asp mutant. Although the replacements are well tolerated in the wild type, the mutations severely inactivate substrate binding and transport in the Glu-126→ Asp background, with the exception of Gly-121→ Ala, which retains significant activity. Strikingly, moreover, in two instances Gly-150→ Ala and Gly-141→ Ala, significant activity is recovered when Ala residues at approximately parallel positions in helix IV (Ala-122 or Ala-127, respectively) are replaced with Gly. In addition to providing further evidence that the major determinants for substrate binding in the permease are at the interface between helices IV and V, the findings indicate that the region is conformationally flexible.