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A new experimental approach to detect long‐range conformational changes transmitted between the membrane and cytosolic domains of LmrA, a bacterial multidrug transporter
Author(s) -
Vigano Catherine,
Grimard Vinciane,
Margolles Abelardo,
Goormaghtigh Erik,
van Veen Hendrik W,
Konings Wil N,
Ruysschaert Jean-Marie
Publication year - 2002
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(02)03485-3
Subject(s) - transmembrane domain , cytosol , atp hydrolysis , biophysics , biochemistry , transmembrane protein , atp binding cassette transporter , förster resonance energy transfer , conformational change , lactococcus lactis , quenching (fluorescence) , cyclic nucleotide binding domain , nucleotide , protein structure , biology , atpase , transporter , membrane , enzyme , fluorescence , bacteria , gene , genetics , lactic acid , physics , receptor , quantum mechanics
LmrA confers multidrug resistance to Lactococcus lactis by mediating the extrusion of antibiotics, out of the bacterial membrane, using the energy derived from ATP hydrolysis. Cooperation between the cytosolic and membrane‐embedded domains plays a crucial role in regulating the transport ATPase cycle of this protein. In order to demonstrate the existence of a structural coupling required for the cross‐talk between drug transport and ATP hydrolysis, we studied specifically the dynamic changes occurring in the membrane‐embedded and cytosolic domains of LmrA by combining infrared linear dichroic spectrum measurements in the course of H/D exchange with Trp fluorescence quenching by a water‐soluble attenuator. This new experimental approach, which is of general interest in the study of membrane proteins, detects long‐range conformational changes, transmitted between the membrane‐embedded and cytosolic regions of LmrA. On the one hand, nucleotide binding and hydrolysis in the cytosolic nucleotide binding domain cause a repacking of the transmembrane helices. On the other hand, drug binding to the transmembrane helices affects both the structure of the cytosolic regions and the ATPase activity of the nucleotide binding domain.