7CVS

Crystal structure of the C85A/L194A mutant CLC-ec1 with Fab fragment

7CVS の概要

• エントリーDOI: 10.2210/pdb7cvs/pdb
• 分子名称: H(+)/Cl(-) exchange transporter ClcA, antibody Fab fragment heavy chain, antibody Fab fragment light chain, ... (4 entities in total)
• 機能のキーワード: clc, chloride, dimer interface, transport protein
• 由来する生物種: Escherichia coli MS 198-1; 詳細
• タンパク質・核酸の鎖数: 6
• 化学式量合計: 194597.28

構造登録者

Park, K.,Mersch, K.,Robertson, J.,Lim, H.-H. (登録日: 2020-08-27, 公開日: 2021-09-01, 最終更新日: 2024-10-23)

主引用文献

Mersch, K.,Ozturk, T.N.,Park, K.,Lim, H.H.,Robertson, J.L.
Altering CLC stoichiometry by reducing non-polar side-chains at the dimerization interface.
J.Mol.Biol., 433:166886-166886, 2021

PubMed Abstract: CLC-ec1 is a Cl/H antiporter that forms stable homodimers in lipid bilayers, with a free energy of -10.9 kcal/mol in 2:1 POPE/POPG lipid bilayers. The dimerization interface is formed by four transmembrane helices: H, I, P and Q, that are lined by non-polar side-chains that come in close contact, yet it is unclear as to whether their interactions drive dimerization. To investigate whether non-polar side-chains are required for dimer assembly, we designed a series of constructs where side-chain packing in the dimer state is significantly reduced by making 4-5 alanine substitutions along each helix (H-ala, I-ala, P-ala, Q-ala). All constructs are functional and three purify as stable dimers in detergent micelles despite the removal of significant side-chain interactions. On the other hand, H-ala shows the unique behavior of purifying as a mixture of monomers and dimers, followed by a rapid and complete conversion to monomers. In lipid bilayers, all four constructs are monomeric as examined by single-molecule photobleaching analysis. Further study of the H-helix shows that the single mutation L194A is sufficient to yield monomeric CLC-ec1 in detergent micelles and lipid bilayers. X-ray crystal structures of L194A reveal the protein re-assembles to form dimers, with a structure that is identical to wild-type. Altogether, these results demonstrate that non-polar membrane embedded side-chains play an important role in defining dimer stability, but the stoichiometry is highly contextual to the solvent environment. Furthermore, we discovered that L194 is a molecular hot-spot for defining dimerization of CLC-ec1.

PubMed: 33617898
DOI: 10.1016/j.jmb.2021.166886

実験手法

X-RAY DIFFRACTION (3.01 Å)