Dissecting the Functions of Conserved Prolines within Transmembrane Helices of the D2 Dopamine Receptor

G protein-coupled receptors (GPCRs) contain a number of conserved proline residues in their transmembrane helices, and it is generally assumed these play important functional and/or structural roles. Here we use unnatural amino acid mutagenesis, employing α-hydroxy acids and proline analogues, to examine the functional roles of five proline residues in the transmembrane helices of the D2 dopamine receptor. The well-known tendency of proline to disrupt helical structure is important at all sites, while we find no evidence for a functional role for backbone amide cis–trans isomerization, another feature associated with proline. At most proline sites, the loss of the backbone NH is sufficient to explain the role of the proline. However, at one site, P210^(5.50), a substituent on the backbone N appears to be essential for proper function. Interestingly, the pattern in functional consequences that we see is mirrored in the pattern of structural distortions seen in recent GPCR crystal structures

Functional Probes of Drug−Receptor Interactions Implicated by Structural Studies: Cys-Loop Receptors Provide a Fertile Testing Ground

Structures of integral membrane receptors provide valuable models for drug–receptor interactions across many important classes of drug targets and have become much more widely available in recent years. However, it remains to be determined to what extent these images are relevant to human receptors in their biological context and how subtle issues such as subtype selectivity can be informed by them. The high precision structural modifications enabled by unnatural amino acid mutagenesis on mammalian receptors expressed in vertebrate cells allow detailed tests of predictions from structural studies. Using the Cys-loop superfamily of ligand-gated ion channels, we show that functional studies lead to detailed binding models that, at times, are significantly at odds with the structural studies on related invertebrate proteins. Importantly, broad variations in binding interactions are seen for very closely related receptor subtypes and for varying drugs at a given binding site. These studies highlight the essential interplay between structural studies and functional studies that can guide efforts to develop new pharmaceuticals

Binding Interactions with the Complementary Subunit of Nicotinic Receptors

The agonist-binding site of nicotinic acetylcholine receptors (nAChRs) spans an interface between two subunits of the pentameric receptor. The principal component of this binding site is contributed by an α subunit, and it binds the cationic moiety of the nicotinic pharmacophore. The other part of the pharmacophore, a hydrogen bond acceptor, has recently been shown to bind to the complementary non-α subunit via the backbone NH of a conserved Leu. This interaction was predicted by studies of ACh-binding proteins and confirmed by functional studies of the neuronal (CNS) nAChR, α4β2. The ACh-binding protein structures further suggested that the hydrogen bond to the backbone NH is mediated by a water molecule and that a second hydrogen bonding interaction occurs between the water molecule and the backbone CO of a conserved Asn, also on the non-α subunit. Here, we provide new insights into the nature of the interactions between the hydrogen bond acceptor of nicotinic agonists and the complementary subunit backbone. We studied both the nAChR of the neuromuscular junction (muscle-type) and a neuronal subtype, (α4)2(β4)3. In the muscle-type receptor, both ACh and nicotine showed a strong interaction with the Leu NH, but the potent nicotine analog epibatidine did not. This interaction was much attenuated in the α4β4 receptor. Surprisingly, we found no evidence for a functionally significant interaction with the backbone carbonyl of the relevant Asn in either receptor with an array of agonists

An Unusual Pattern of Ligand-Receptor Interactions for the α7 Nicotinic Acetylcholine Receptor, with Implications for the Binding of Varenicline

The α7 nicotinic acetylcholine receptor shows broad pharmacology, complicating the development of subtype-specific nicotinic receptor agonists. Here we use unnatural amino acid mutagenesis to characterize binding to α7 by the smoking cessation drug varenicline (Chantix; Pfizer, Groton, CT), an α4β2-targeted agonist that shows full efficacy and modest potency at the α7 receptor. We find that unlike binding to its target receptor, varenicline does not form a cation-π interaction with TrpB, further supporting a unique binding mode for the cationic amine of nicotinic agonists at the α7 receptor. We also evaluate binding to the complementary face of the receptor’s binding site by varenicline, the endogenous agonist acetylcholine, and the potent nicotine analog epibatidine. Interestingly, we find no evidence for functionally important interactions involving backbone NH and CO groups thought to bind the canonical agonist hydrogen bond acceptor of the nicotinic pharmacophore, perhaps reflecting a lesser importance of this pharmacophore element for α7 binding. We also show that the Trp55 and Leu119 side chains of the binding site’s complementary face are important for the binding of the larger agonists epibatidine and varenicline, but dispensable for binding of the smaller, endogenous agonist acetylcholine

Selvamicin, an atypical antifungal polyene from two alternative genomic contexts

The bacteria harbored by fungus-growing ants produce a variety of small molecules that help maintain a complex multilateral symbiosis. In a survey of antifungal compounds from these bacteria, we discovered selvamicin, an unusual antifungal polyene macrolide, in bacterial isolates from two neighboring ant nests. Selvamicin resembles the clinically important antifungals nystatin A1 and amphotericin B, but it has several distinctive structural features: a noncationic 6-deoxymannose sugar at the canonical glycosylation site and a second sugar, an unusual 4-O-methyldigitoxose, at the opposite end of selvamicin’s shortened polyene macrolide. It also lacks some of the pharmacokinetic liabilities of the clinical agents and appears to have a different target. Whole genome sequencing revealed the putative type I polyketide gene cluster responsible for selvamicin’s biosynthesis including a subcluster of genes consistent with selvamicin’s 4-O-methyldigitoxose sugar. Although the selvamicin biosynthetic cluster is virtually identical in both bacterial producers, in one it is on the chromosome, in the other it is on a plasmid. These alternative genomic contexts illustrate the biosynthetic gene cluster mobility that underlies the diversity and distribution of chemical defenses by the specialized bacteria in this multilateral symbiosis.National Institutes of Health/[R01 GM086258]/NIH/Estados UnidosNational Institutes of Health/[U19 AI09673]/NIH/Estados UnidosUniversidad de Costa Rica//UCR/Costa RicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigación en Biología Celular y Molecular (CIBCM

In Vivo Incorporation of Non-canonical Amino Acids by Using the Chemical Aminoacylation Strategy: A Broadly Applicable Mechanistic Tool

We describe a strategy for incorporating non-canonical amino acids site-specifically into proteins expressed in living cells, involving organic synthesis to chemically aminoacylate a suppressor tRNA, protein expression in Xenopus oocytes, and monitoring protein function, primarily by electrophysiology. With this protocol, a very wide range of non-canonical amino acids can be employed, allowing both systematic structure–function studies and the incorporation of reactive functionalities. Here, we present an overview of the methodology and examples meant to illustrate the versatility and power of the method as a tool for investigating protein structure and function