title: Molecular modeling and mutagenesis reveals a tetradentate binding site for Zn2+ in GABA(A) alphabeta receptors and provides a structural basis for the modulating effect of the gamma subunit.
abstract: Gamma-aminobutyric acid type A receptors (GABA(A)-R) containing alpha1beta2gamma2 subunits are weakly inhibited by Zn2+, whereas receptors containing only the alpha1beta2 subunits are strongly inhibited. We built homology models of the ion pores of alpha1beta2 and alpha1beta2gamma2 GABA(A)-R using coordinates of the nicotinic acetylcholine receptor as a template. Threading the GABA(A)-R beta2 sequence onto this template placed the 17' histidine and the 20' glutamate residues at adjacent locations in the mouth of the pore, such that a nearly ideal tetradentate site for Zn2+ was formed from two histidine and two glutamate residues between adjacent beta subunits in the alpha1beta2 GABA(A)-R. Following optimization with CHARMM, the distance between the alpha-carbons of the adjacent histidine residues was approximately 9.2 A, close to the ideal distance for a Zn2+ binding site. Loss of inhibition by Zn2+ in alpha1beta2gamma2 GABA(A)-R can be explained by the geometry of these residues in the arrangement alpha1beta2gamma2alpha1beta2, in which the nearest C-alpha-C-alpha distance between the histidine residues is 15.5 A, too far apart for an energetically optimal Zn2+ binding site. We then mutated the gamma subunit at the 17' and/or 20' positions. Zn2+ inhibition was not restored in alpha1beta2gamma2 (I282H) receptors. A novel finding is that the modeling shows the native 20' lysine in gamma2 can compete with Zn2+ for binding to the inserted 17' histidine. Sensitivity to Zn2+ was restored in the double mutant receptor, alpha1beta2gamma2 (I282H; K285E), in which the competition with lysine was removed and a more favorable Zn2+ binding site was formed.
authors.affiliation: Department of Anesthesia, Stanford University School of Medicine, Stanford, California 94305-5117, USA. trudell@stanford.edu
authors.forename: James R
authors.initials: JR
authors.lastname: Trudell
authors.forename: Minerva E
authors.initials: ME
authors.lastname: Yue
authors.forename: Edward J
authors.initials: EJ
authors.lastname: Bertaccini
authors.forename: Andrew
authors.initials: A
authors.lastname: Jenkins
authors.forename: Neil L
authors.initials: NL
authors.lastname: Harrison
mesh_terms: D001665:Binding Sites; D008958:Models, Molecular; D016296:Mutagenesis; D021122:Protein Subunits / Q000737:chemistry / Q000502:physiology*; D011963:Receptors, GABA-A / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D011978:Receptors, Nicotinic; D017386:Sequence Homology, Amino Acid; D015032:Zinc / Q000737:chemistry*
chemical_list: D021122:Protein Subunits; D011963:Receptors, GABA-A; D011978:Receptors, Nicotinic; D015032:Zinc