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Altering CLC stoichiometry by reducing non-polar side-chains at the dimerization interface

Altering CLC stoichiometry by reducing non-polar side-chains at the dimerization interface
Mersch, KaceyOzturk, Tugba N.Park, KunwoongLim, Hyun-HoRobertson, Janice L.
DGIST Authors
Mersch, Kacey; Ozturk, Tugba N.; Park, Kunwoong; Lim, Hyun-Ho; Robertson, Janice L.
Issue Date
Journal of Molecular Biology, 433(8), 166886
Author Keywords
membrane proteinoligomerizationCLC-ec1van der Waalslipid bilayer
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. © 2021 Elsevier Ltd
Academic Press
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