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Structural model of ρ1 GABA C receptor based on evolutionary analysis: Testing of predicted protein–protein interactions involved in receptor assembly and function
Author(s) -
Adamian Larisa,
Gussin Hélène A.,
Tseng Yan Yuan,
Muni Niraj J.,
Feng Feng,
Qian Haohua,
Pepperberg David R.,
Liang Jie
Publication year - 2009
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.247
Subject(s) - salt bridge , mutant , biophysics , receptor , protein subunit , gating , chemistry , cys loop receptors , ion channel , protein structure , biochemistry , biology , acetylcholine receptor , gene , nicotinic acetylcholine receptor
The homopentameric ρ1 GABA C receptor is a ligand‐gated ion channel with a binding pocket for γ‐aminobutyric acid (GABA) at the interfaces of N ‐terminal extracellular domains. We combined evolutionary analysis, structural modeling, and experimental testing to study determinants of GABA C receptor assembly and channel gating. We estimated the posterior probability of selection pressure at amino acid residue sites measured as ω‐values and built a comparative structural model, which identified several polar residues under strong selection pressure at the subunit interfaces that may form intersubunit hydrogen bonds or salt bridges. At three selected sites (R111, T151, and E55), mutations disrupting intersubunit interactions had strong effects on receptor folding, assembly, and function. We next examined the role of a predicted intersubunit salt bridge for residue pair R158–D204. The mutant R158D, where the positively charged residue is replaced by a negatively charged aspartate, yielded a partially degraded receptor and lacked membrane surface expression. The membrane surface expression was rescued by the double mutant R158D–D204R, where positive and negative charges are switched, although the mutant receptor was inactive. The single mutants R158A, D204R, and D204A exhibited diminished activities and altered kinetic profiles with fast recovery kinetics, suggesting that R158–D204 salt bridge perhaps stabilizes the open state of the GABA C receptor. Our results emphasize the functional importance of highly conserved polar residues at the protein–protein interfaces in GABA C ρ1 receptors and demonstrate how the integration of computational and experimental approaches can aid discovery of functionally important interactions.