title: Structural model of rho1 GABAC receptor based on evolutionary analysis: Testing of predicted protein-protein interactions involved in receptor assembly and function.
abstract: The homopentameric rho1 GABA(C) receptor is a ligand-gated ion channel with a binding pocket for gamma-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 omega-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) rho1 receptors and demonstrate how the integration of computational and experimental approaches can aid discovery of functionally important interactions.
authors.affiliation: Department of Bioengineering, University of Illinois at Chicago, Chicago, Illinois 60612, USA.
authors.forename: Larisa
authors.initials: L
authors.lastname: Adamian
authors.forename: Hélène A
authors.initials: HA
authors.lastname: Gussin
authors.forename: Yan Yuan
authors.initials: YY
authors.lastname: Tseng
authors.forename: Niraj J
authors.initials: NJ
authors.lastname: Muni
authors.forename: Feng
authors.initials: F
authors.lastname: Feng
authors.forename: Haohua
authors.initials: H
authors.lastname: Qian
authors.forename: David R
authors.initials: DR
authors.lastname: Pepperberg
authors.forename: Jie
authors.initials: J
authors.lastname: Liang
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001499:Bayes Theorem; D001665:Binding Sites / Q000235:genetics; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics; D019143:Evolution, Molecular; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000502:physiology; D010802:Phylogeny; D054730:Protein Interaction Domains and Motifs / Q000235:genetics; D025941:Protein Interaction Mapping / Q000379:methods*; D055503:Protein Multimerization; D018079:Receptors, GABA / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D029867:Xenopus Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: C428944:AChBP protein, Lymnaea; D002352:Carrier Proteins; C087200:GABA-C receptor; D018079:Receptors, GABA; D029867:Xenopus Proteins; C523978:rho1 GABA(C) receptor, human