A new experimental approach to detect long‐range conformational changes transmitted between the membrane and cytosolic domains of LmrA, a bacterial multidrug transporter

FEBS Letters

Grimard, Vinciane³  Goormaghtigh, Erik³  Margolles, Abelardo¹  Ruysschaert, Jean-Marie³  Konings, Wil N¹  van Veen, Hendrik W²  Vigano, Catherine³ 

[1] Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, 9751 NN Haren, The Netherlands;Department of Pharmacology, University of Cambridge, Cambridge CB2 1QJ, UK;Service de Structure et Fonction des Membranes Biologiques (SFMB), Université Libre de Bruxelles, P.O. Box 206/2, Bd du Triomphe, B1050 Brussels, Belgium

关键词: Multidrug resistance;    LmrA;    Infrared spectroscopy;    Tryptophan fluorescence;    Membrane domain;    Conformational change;    ABC;    ATP binding cassette;    ATPγS;    adenosine-5′-O-(thiotriphosphate);    ATR-FTIR;    attenuated total reflection Fourier transform infrared;    DDM;    dodecyl-β-D-maltoside;    DNR;    daunorubicin;    MDR;    multidrug resistance;    MRP;    multidrug resistance protein;    NBD;    nucleotide binding domain;    Ni-NTA;    Ni2+-nitrilotriacetic acid;    Pi;    inorganic phosphate;    Pgp;    P-glycoprotein;   

DOI  :  10.1016/S0014-5793(02)03485-3

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown