title: Topology characterization of a benzodiazepine-binding beta-rich domain of the GABAA receptor alpha1 subunit.
abstract: Structural investigation of GABAA receptors has been limited by difficulties imposed by its trans-membrane-complex nature. In the present study, the topology of a membrane-proximal beta-rich (MPB) domain in the C139-L269 segment of the receptor alpha1 subunit was probed by mapping the benzodiazepine (BZ)-binding and epitopic sites, as well as fluorescence resonance energy transfer (FRET) analysis. Ala-scanning and semiconservative substitutions within this segment revealed the contribution of the phenyl rings of Y160 and Y210, the hydroxy group of S186 and the positive charge on R187 to BZ-binding. FRET with the bound BZ ligand indicated the proximity of Y160, S186, R187, and S206 to the BZ-binding site. On the other hand, epitope-mapping using the monoclonal antibodies (mAbs) against the MPB domain established a clustering of T172, R173, E174, Q196, and T197. Based on the lack of FRET between Trp substitutionally placed at R173 or V198 and bound BZ, this epitope-mapped cluster is located on a separate end of the folded protein from the BZ-binding site. Mutations of the five conserved Cys and Trp residues in the MPB domain gave rise to synergistic and rescuing effects on protein secondary structures and unfolding stability that point to a CCWCW-pentad, reminiscent to the CWC-triad "pin" of immunoglobulin (Ig)-like domains, important for the structural maintenance. These findings, together with secondary structure and fold predictions suggest an anti-parallel beta-strand topology with resemblance to Ig-like fold, having the BZ-binding and the epitopic residues being clustered at two different ends of the fold.
authors.affiliation: Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China.
authors.forename: Zhiwen
authors.initials: Z
authors.lastname: Xu
authors.forename: Shisong
authors.initials: S
authors.lastname: Fang
authors.forename: Haifeng
authors.initials: H
authors.lastname: Shi
authors.forename: Hoiming
authors.initials: H
authors.lastname: Li
authors.forename: Yiqun
authors.initials: Y
authors.lastname: Deng
authors.forename: Yinglei
authors.initials: Y
authors.lastname: Liao
authors.forename: Jiun-Ming
authors.initials: JM
authors.lastname: Wu
authors.forename: Hui
authors.initials: H
authors.lastname: Zheng
authors.forename: Huaimin
authors.initials: H
authors.lastname: Zhu
authors.forename: Hueih-Min
authors.initials: HM
authors.lastname: Chen
authors.forename: Shui Ying
authors.initials: SY
authors.lastname: Tsang
authors.forename: Hong
authors.initials: H
authors.lastname: Xue
mesh_terms: D000596:Amino Acids / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D001569:Benzodiazepines / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D018604:Epitope Mapping / Q000379:methods; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D006801:Humans; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011963:Receptors, GABA-A / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D000596:Amino Acids; C546240:GABRA1 protein, human; D011963:Receptors, GABA-A; D001569:Benzodiazepines