Packing of the Extracellular Domain Hydrophobic Core is Evolutionarily Optimized to Facilitate Pentameric Ligand-Gated Ion Channel Activation

Protein function depends on protein dynamics as well as structure. Local loose packing of hydrophobic cores is not infrequent in proteins, as the enhanced flexibility likely contributes to their biological function. The crystal structure of the extracellular domain of the nicotinic acetylcholine receptor (nAChR) α1 subunit revealed a hydrophilic water-filled cavity formed by Thr-52 and Ser-126 buried in the hydrophobic core of the protein. Mutation of these residues to bulky hydrophobic amino acids substantially reduced acetylcholine activated channel current suggesting loose-packing of the β-sandwich core is important for nAChR function. Intriguingly, structure-based sequence alignment suggests the presence of loose packing of the hydrophobic β-sandwich core in other pentameric ligand-gated ion channels (pLGICs), whereas tight packing is observed in the crystal structures of the nonchannel homolog, acetylcholine binding protein (AChBP). Here, we examined the generality and importance of this loose packing for pLGIC function using experimental and computational approaches. Mutating aligned residues in the related heteropentameric GABA-A receptor disrupted GABA-mediated currents. Mutations in the β2 subunit had the largest effects suggesting distinct requirements for subunit flexibility in receptor activation. Using FoldX, we examined the energetic cost of mutating residues in the hydrophobic core on protein folding in AChBP, prokaryotic and eukaryotic pLGICs as a measure of protein stability. Interestingly, a loss in protein stability appears correlated to the ability of pLGICs to rapidly switch from closed to open channel states in the presence of ligand. Overall, we suggest that loose packing of the hydrophobic core likely developed as an evolutionary strategy aimed to optimize the specialized allosteric mechanisms of pLGICs.