Packing of the Extracellular Domain Hydrophobic Core Has Evolved to Facilitate Pentameric Ligand-gated Ion Channel Function

Abstract

Protein function depends on conformational flexibility and folding stability. Loose packing of hydrophobic cores is not infrequent in proteins, as the enhanced flexibility likely contributes to their biological function. Here, using experimental and computational approaches, we show that eukaryotic pentameric ligand-gated ion channels are characterized by loose packing of their extracellular domain ␤-sandwich cores, and that loose packing contributes to their ability to rapidly switch from closed to open channel states in the presence of ligand. Functional analyses of GABA A receptors show that increasing the ␤-core packing disrupted GABA-mediated currents, with impaired GABA efficacy and slowed GABA current activation and desensitization. We propose that loose packing of the hydrophobic ␤-core developed as an evolutionary strategy aimed to facilitate the allosteric mechanisms of eukaryotic pentameric ligand-gated ion channels.Proteins are conformationally dynamic and require thermodynamic stability and structural flexibility to function (1-5). Stability and flexibility are interdependent, with higher stability increasing structural rigidity, which in many cases reduces protein activity (6). Proteins are only marginally stable, with ⌬G folding values mostly between Ϫ3 and Ϫ15 kcal/ mol (7). Trade-offs between thermodynamic stability and conformational flexibility are observed during protein evolution, with novel or specialized functions often acquired at the expense of stability (1,2,8,9). New-function mutations, however, rarely involve active site residues (10, 11) but occur with high frequencies in hydrophobic cores (12). Core mutations, although, are highly demanding in terms of stability, because a well packed hydrophobic core is the major factor that drives protein folding and maintains a stably folded structure through entropic ("hydrophobic effect") and enthalpic (van der Waals interactions) forces (13-16).Protein cores are normally packed with amino acid residues with bulky hydrophobic side chains that efficiently fill the space (17, 18). Core mutations tend to loosen the packing and create "defects" (empty or water-filled cavities) that decrease thermodynamic stability up to several kcal/moles (3, 19 -21). Numerous proteins (e.g. p53 (22), serpins (23), and hemagglutinin (24, 25)) have evolved localized core packing defects that enhance their biological activity at the cost of their stability.Recently, the crystal structure of the extracellular domain of the nicotinic acetylcholine receptor (nAChR) 3 ␣1 subunit revealed a hydrophilic water-filled cavity buried in the hydrophobic core of the protein and it was hypothesized that loose core packing is important for nAChR activity (26). nAChRs are members of the pentameric ligand-gated ion channel (pLGIC) superfamily, which includes ␥-aminobutyric acid (GABA A Rs), serotonin (5-HT 3 Rs), and glycine (GlyRs) receptors. For these receptors, binding of neurotransmitter to the extracellular ligand-binding domain triggers rapid structural rearrangements (in the ...

Characterization of the dynamic resting state of a pentameric ligand-gated ion channel by cryo-electron microscopy and simulations

“…Interestingly, relatively loose packing of the ECD core has been proposed as a gating strategy specific to eukaryotic members of this channel family [46] ; substantiation in eukaryotic receptors of the the dynamic patterns we report here for GLIC may prove a valuable extension of the present work.…”

Structure-Based Approaches to Antigen-Specific Therapy of Myasthenia Gravis

“…This role of the hydration pocket on the conformation flexibility/dynamics of the nAChR ECD is supported by recent molecular dynamics studies [34]. This model also suggests that the specific location of the hydration cavity is important for a particular class of pentameric LGICs [35]. A practical implication of these observations is that one can design stabilization mutants of LGICs, including nAChR ECD, by structure-guided modifications of such packing defects, which are evolved for intrinsic ion channel functions but may be detrimental to recombinant production of proteins as therapeutic antigen.…”

String method solution of the gating pathways for a pentameric ligand-gated ion channel

“…S1), this bridge is part of a largely conserved triplet, D32-R192-D122, of which R192-D122 is highly conserved and essential for function (27,28), and mutation of D32 in GLIC causes loss of function (28). Breaking of D32-R192 and β-sandwich expansion likely cause changes that influence interdomain communication via deflection of the pre-M1 R192 residue (29)(30)(31), or through noncovalent interactions, as indicated in Fig. 1B (observed in string simulations and described below).…”