Topology characterization of a benzodiazepine‐binding β‐rich domain of the GABAA receptor α1 subunit

• Published in: Protein science Vol. 14; no. 10; pp. 2622 - 2637
• Main Authors: Xu, Zhiwen, Fang, Shisong, Shi, Haifeng, Li, Hoiming, Deng, Yiqun, Liao, Yinglei, Wu, Jiun‐Ming, Zheng, Hui, Zhu, Huaimin, Chen, Hueih‐Min, Tsang, Shui Ying, Xue, Hong
• Format: Journal Article
• Language: English
• Published: Bristol Cold Spring Harbor Laboratory Press 01.10.2005
• Subjects: AChBP, acetylcholine‐binding protein; Ala‐scanning; BFR, Bod‐ipy‐FL Ro‐1986; BZ, benzodiazepine; CD, circular dichroism; circular dichroism; epitope mapping; FA, fluorescence anisotropy; fluorescence spectroscopy; fold recognition; FRET, fluorescence resonance energy transfer; GABAA, type A γ‐aminobutyric acid; GdCl, guanidine hydrochloride; Ig, immunoglobulin; LGIC, ligand‐gated ion channel; mAb, monoclonal antibody; MPB, membrane‐proximal β‐rich; secondary structure; stability; thermodynamic analysis
• ISSN: 0961-8368; 1469-896X
• DOI: 10.1110/ps.051555205

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 β‐rich (MPB) domain in the C139–L269 segment of the receptor α1 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 β‐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.