التفاصيل البيبلوغرافية
| العنوان: |
The Multidrug Transporter MdfA Deviates from the Canonical Model of Alternating Access of MFS Transporters. |
| المؤلفون: |
Yardeni, Eliane H.1 (AUTHOR), Mishra, Smriti1,2 (AUTHOR), Stein, Richard A.2 (AUTHOR), Bibi, Eitan1 (AUTHOR) e.bibi@weizmann.ac.il, Mchaourab, Hassane S.1,2 (AUTHOR) hassane.mchaourab@vanderbilt.edu |
| المصدر: |
Journal of Molecular Biology. Sep2020, Vol. 432 Issue 20, p5665-5680. 16p. |
| مصطلحات موضوعية: |
*SPIN labels, *X-ray crystallography, *DRUG accessibility, *ESCHERICHIA coli, *DEER, *BILAYER lipid membranes |
| مستخلص: |
The prototypic multidrug (Mdr) transporter MdfA from Escherichia coli efflux chemically‐ dissimilar substrates in exchange for protons. Similar to other transporters, MdfA purportedly functions by alternating access of a central substrate binding pocket to either side of the membrane. Accordingly, MdfA should open at the cytoplasmic side and/or laterally toward the membrane to enable access of drugs into its pocket. At the end of the cycle, the periplasmic side is expected to open to release drugs. Two distinct conformations of MdfA have been captured by X-ray crystallography: An outward open (O o) conformation, stabilized by a Fab fragment, and a ligand-bound inward-facing (I f) conformation, possibly stabilized by a mutation (Q131R). Here, we investigated how these structures relate to ligand-dependent conformational dynamics of MdfA in lipid bilayers. For this purpose, we combined distances measured by double electron-electron resonance (DEER) between pairs of spin labels in MdfA, reconstituted in nanodiscs, with cysteine cross-linking of natively expressed membrane-embedded MdfA variants. Our results suggest that in a membrane environment, MdfA assumes a relatively flexible, outward-closed/inward-closed (O c /I c) conformation. Unexpectedly, our data show that neither the substrate TPP nor protonation induces large-scale conformational changes. Rather, we identified a substrate-responsive lateral gate, which is open toward the inner leaflet of the membrane but closes upon drug binding. Together, our results suggest a modified model for the functional conformational cycle of MdfA that does not invoke canonical elements of alternating access. Unlabelled Image • We investigated the conformational states of the multidrug transporter MdfA, embedded in a lipid bilayer. • We found that the resting state of MdfA is a flexible outward-closed/inward-closed conformation. • We identified substrate-induced movements between TMs that are uncoupled from the conceptual rigid-body movements in MFS transporters. • We describe a putative lateral gate that allows the recruitment of lipophilic drugs from the inner leaflet of the membrane. • We conclude that MdfA does not follow the canonical rocker-switch models of MFS transporters. [ABSTRACT FROM AUTHOR] |
| قاعدة البيانات: |
Academic Search Index |
