Atomistic insights into human Cys-loop receptors by solution NMR

Abstract

Cys-loop receptors are pentameric ligand-gated ion channels (pLGICs) mediating fast neurotransmission in the central and peripheral nervous systems. They are important targets for many currently used clinical drugs, such as general anesthetics, and for allosteric modulators with potential therapeutic applications. Here, we provide an overview of advances in the use of solution NMR in structural and dynamic characterization of ion channels, particularly human Cys-loop receptors. We present challenges to overcome and realistic solutions for achieving high-resolution structural information for this family of receptors. We discuss how subtle structural differences among homologous channels define unique channel pharmacological properties and advocate the necessity to determine high-resolution structures for individual receptor subtypes. Finally, we describe drug binding to the Cys-loop receptors’ TMD identified by solution NMR and the associated dynamics changes relevant to channel functions.

Conformational Changes Underlying Desensitization of the Pentameric Ligand-Gated Ion Channel ELIC

“…In recent years, X-ray structures of several pLGICs, both from prokaryotic (Bocquet et al, 2009; Hilf and Dutzler, 2008, 2009) and eukaryotic origins (Hassaine et al, 2014; Hibbs and Gouaux, 2011; Miller and Aricescu, 2014), have been determined. These crystal structures and the cryo-electron microscopy structure of the hetero-pentameric nicotinic acetylcholine receptor (nAChR) from Torpedo (Unwin, 2005) along with other structural data (Bondarenko et al, 2012; Bondarenko et al, 2014; Mowrey et al, 2013b; Mowrey et al, 2014) have greatly enriched our knowledge of the structure-function relationship of pLGICs.…”

Solution NMR Studies of Anesthetic Interactions with Ion Channels

“…Recent advances in solution NMR spectroscopy have made it possible to determine structures of various ion channels with molecular weights less than 80 kDa (Mowrey et al, 2015; B. OuYang et al, 2013; Oxenoid & Chou, 2016; Schnell & Chou, 2008).…”

“…Nuclear magnetic resonance (NMR) spectroscopy has a wide range of applications, including the determination of protein structures, characterization of protein-ligand interactions, and detection of changes in protein conformations and dynamics upon ligand binding (Kay, 2016; Kim, Howell, Van Horn, Jeon, & Sanders, 2009; Kitevski-LeBlanc & Prosser, 2012; Liang & Tamm, 2016; Liu, Horst, Katritch, Stevens, & Wuthrich, 2012; Oxenoid & Chou, 2013, 2016; Rosenzweig & Kay, 2016; Ye, Van Eps, Zimmer, Ernst, & Prosser, 2016; Zhuang et al, 2013). For the specific purpose of understanding anesthetic interactions with proteins, NMR has been used to determine the structures of anesthetic targets (Bondarenko et al, 2012; Bondarenko et al, 2014; Bondarenko, Tillman, Xu, & Tang, 2010; Cui et al, 2012; Ma, Brandon, et al, 2008; Ma, Liu, Li, Tang, & Xu, 2005; Ma, Tillman, et al, 2008; Mowrey, Cui, et al, 2013; Mowrey, Kinde, Xu, & Tang, 2015; Tang, Mandal, & Xu, 2002), identify anesthetic binding sites in proteins (Bondarenko, Mowrey, Liu, Xu, & Tang, 2013; Bondarenko et al, 2014; Bondarenko, Yushmanov, Xu, & Tang, 2008; Kinde, Bondarenko, et al, 2016; Kinde, Bu, et al, 2016; Mowrey, Liu, et al, 2013; Tang, Eckenhoff, & Xu, 2000), characterize the direct interactions between proteins and anesthetics over a broad range of binding affinities (Bondarenko et al, 2013; Bondarenko et al, 2008; Canlas, Cui, Li, Xu, & Tang, 2008; Cui et al, 2008; Ma, Brandon, et al, 2008; Tang et al, 2000; Tang, Hu, Liachenko, & Xu, 1999; Xu, Seto, Tang, & Firestone, 2000; Xu, Tang, Firestone, & Zhang, 1996), and determine how anesthetic binding affects protein structures and dynamics (Canlas et al, 2008; Cui et al, 2008; Cui, Canlas, Xu, & Tang, 2010; Mowrey, Liu, et al, 2013).…”

Escherichia coli Protein Expression System for Acetylcholine Binding Proteins (AChBPs)

“…Furthermore, attempts have been made to use NMR techniques to monitor ligand induced conformational changes of the nAChR in solution. Isotopically labelled ( 15 N and 13 C) ligand binding domains of bacterial homologs have been expressed for this purpose, but these failed to oligomerize as a pentamer in solution and therefore lacked an intact ligand binding pocket [ 35 , 36 ]. AChBP naturally assembles as a pentamer in solution and therefore is a suitable candidate for such studies.…”