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Topology characterization of a benzodiazepine-binding -rich domain of the GABA_A receptor ₁ subunit

¹ Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China
² Institute of BioAgricultural Sciences, Academia Sinica, Taipei 115, Taiwan

(RECEIVED April 29, 2005; FINAL REVISION June 28, 2005; ACCEPTED July 4, 2005)

Structural investigation of GABA_A receptors has been limited by difficulties imposed by its trans-membrane-complex nature. In the present study, the topology of a membrane-proximal -rich (MPB) domain in the C139–L269 segment of the receptor ₁ 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 -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.

Keywords: Ala-scanning; circular dichroism; fluorescence spectroscopy; fold recognition; epitope mapping; secondary structure; stability; thermodynamic analysis

Abbreviations: AChBP, acetylcholine-binding protein • BFR, Bod-ipy-FL Ro-1986 • BZ, benzodiazepine • CD, circular dichroism • FA, fluorescence anisotropy • FRET, fluorescence resonance energy transfer • GABA_A, type A -aminobutyric acid • GdCl, guanidine hydrochloride • Ig, immunoglobulin • LGIC, ligand-gated ion channel • mAb, monoclonal antibody • MPB, membrane-proximal -rich

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051555205.

Reprint requests to: Hong Xue, Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China; e-mail: hxue{at}ust.hk; fax: +(852) 23581552.

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