The pituitary adenylate cyclase-activating polypeptide (PACAP) receptor is a class II G protein-coupled receptor that contributes to many different cellular functions including neurotransmission, neuronal survival, and synaptic plasticity. The solution structure of the potent antagonist PACAP (residues 6-38) complexed to the N-terminal extracellular (EC) domain of the human splice variant hPAC1-R-short (hPAC1-RS) was determined by NMR. The PACAP peptide adopts a helical conformation when bound to hPAC1-RS with a bend at residue A18 and makes extensive hydrophobic and electrostatic interactions along the exposed -sheet and interconnecting loops of the N-terminal EC domain. Mutagenesis data on both the peptide and the receptor delineate the critical interactions between the C terminus of the peptide and the C terminus of the EC domain that define the high affinity and specificity of hormone binding to hPAC1-RS. These results present a structural basis for hPAC1-RS selectivity for PACAP versus the vasoactive intestinal peptide and also differentiate PACAP residues involved in binding to the N-terminal extracellular domain versus other parts of the full-length hPAC1-RS receptor. The structural, mutational, and binding data are consistent with a model for peptide binding in which the C terminus of the peptide hormone interacts almost exclusively with the N-terminal EC domain, whereas the central region makes contacts to both the N-terminal and other extracellular parts of the receptor, ultimately positioning the N terminus of the peptide to contact the transmembrane region and result in receptor activation.NMR ͉ vasoactive intestinal peptide ͉ G protein-coupled receptor S even transmembrane domain G protein-coupled receptors (GPCRs) are cell surface proteins that transduce signals initiated by hormones or neurotransmitters into the cell (1). Class II GPCRs are a family of receptors that bind structurally related peptide hormones including glucagon, glucagon-like peptides, vasoactive intestinal peptide (VIP), corticotrophinreleasing factor (CRF), parathyroid hormone (PTH), and pituitary adenylate cyclase-activating polypeptide (PACAP) hormone. This family of receptors is capable of regulating intracellular concentrations of cAMP through activation of the adenylate cyclase pathway, and some can also modulate intracellular calcium levels through the phospholipase C pathway. Structurally, they have low homology with other GPCR families but are well conserved within the family. They all contain a relatively large amino-terminal extracellular (EC) domain that plays a critical role in ligand binding. The N-terminal EC hormone-binding domains have common features, typically containing six conserved cysteine residues, two conserved tryptophan residues, and an aspartate residue which has been suggested to be critical for ligand binding (2, 3). Many of the peptide ligands of these receptors have related sequences and can bind to more than one receptor subtype (4).VIP and PACAP are two prototypical neuropeptides that modulate C...
Ion channels play critical roles in signaling processes and are attractive targets for treating various diseases. Here we describe an NMR-based strategy for structural analyses of potassium channel-ligand complexes using KcsA (residues 1-132, with six mutations to impart toxin binding and to mimic the eukaryotic hERG channel). Using this approach, we determined the solution structure of KcsA in complex with the high-affinity peptide antagonist charybdotoxin. The structural data reveal how charybdotoxin binds to the closed form of KcsA and makes specific contacts with the extracellular surface of the ion channel, resulting in pore blockage. This represents the first direct structural information about an ion channel complexed to a peptide antagonist and provides an experimental framework for understanding and interpreting earlier mutational analyses. The strategy presented here overcomes many of the limitations of conventional NMR approaches to helical membrane protein structure determination and can be applied in the study of the binding of druglike molecules to this important class of proteins.
We have recently reported on the development of a La assay to detect reactive molecules by nuclear magnetic resonance (ALARM NMR) to detect reactive false positive hits from high-throughput screening, in which we observed a surprisingly large number of compounds that can oxidize or form covalent adducts with protein thiols groups. In the vast majority of these cases, the covalent interactions are largely nonspecific (e.g., affect many protein targets) and therefore unsuitable for drug development. However, certain thiol-reactive species do appear to inhibit the target of interest in a specific manner. The question then arises as to the potential toxicology risks of developing a drug that can react with protein thiol groups. Here, we report on the evaluation of a large set of ALARM-reactive and -nonreactive compounds against a panel of additional proteins (aldehyde dehydrogenase, superoxide dismutase, and three cytochrome P450 enzymes). It was observed that ALARM-reactive compounds have significantly increased risks of interacting with one or more of these enzymes in vitro. Thus, ALARM NMR seems to be a sensitive tool to rapidly identify compounds with an enhanced risk of producing side effects in humans, including alcohol intolerance, the formation of reactive oxygen species, and drug-drug interactions. In conjunction with other toxicology assays, ALARM NMR should be a valuable tool for prioritizing compounds for lead optimization and animal testing.
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