Outward open conformation of a Major Facilitator Superfamily multidrug/H+ antiporter provides insights into switching mechanism

Nagarathinam, Kumar; Nakada-Nakura, Yoshiko; Parthier, C.; Terada, Tohru; Juge, Narinobu; Jaenecke, F.; Liu, Kehong; Hotta, Yunhon; Miyaji, Takaaki; Omote, Hiroshi; Iwata, So; Nomura, Norimichi; Stubbs, Milton T.; Tanabe, Mikio

doi:10.1038/s41467-018-06306-x

Cited by 53 publications

(67 citation statements)

References 53 publications

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“…Of note, the alanine substitution of Y30, which interacts with Cm but not DXC, exerted deleterious effect on the ability of E26T/D34M/A150E to confer cellular resistance against DXC. This finding may be explained by the involvement of Y30 in stabilizing the outward-facing conformation 25 . Additionally, we found that DXC reduced the ability of substrate-binding-site mutants to confer cellular resistance against Cm, regardless of whether the mutated protein residues bind DXC or Cm, thereby implying that DXC inhibits the export of Cm by E26T/D34M/ A150E, and vice versa.…”

Section: Dxc/h + Coupling In E26t/d34m/a150ementioning

confidence: 91%

“…1d) may occur during the substrate-induced deprotonation of A150E. The deprotonation of side-chain carboxylate of A150E, which is likely favored by the alkaline pH in the cytoplasm, may subsequently promote E26T/D34M/A150E to switch from its inward-facing state to the outward-facing conformation 25 .…”

Section: Capture Of Two Cm-binding Modes In the Crystals At The Intementioning

confidence: 99%

“…By contrast, MdfA from Escherichia coli, a polyspecific MFS multidrug transporter in the ubiquitous drug/H + antiporter-1 (DHA1) subfamily, can export physicochemically disparate compounds that lack a common chemical structure 12,13 . Previous studies suggested that MdfA is mechanistically distinct from LacY [14][15][16][17][18][19][20][21] , although they share the same protein fold [22][23][24][25][26] . Notably, the interactions between MdfA and its substrates are often hydrophobic and ionic in nature, direct H-bonds are scanty 22,26 .…”

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confidence: 99%

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Structure and mechanism of a redesigned multidrug transporter from the Major Facilitator Superfamily

Symerský

2020

Sci Rep

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The rapid increase of multidrug resistance poses urgent threats to human health. Multidrug transporters prompt multidrug resistance by exporting different therapeutics across cell membranes, often by utilizing the H+ electrochemical gradient. MdfA from Escherichia coli is a prototypical H+ -dependent multidrug transporter belonging to the Major Facilitator Superfamily. Prior studies revealed unusual flexibility in the coupling between multidrug binding and deprotonation in MdfA, but the mechanistic basis for this flexibility was obscure. Here we report the X-ray structures of a MdfA mutant E26T/D34M/A150E, wherein the multidrug-binding and protonation sites were revamped, separately bound to three different substrates at resolutions up to 2.0 Å. To validate the functional relevance of these structures, we conducted mutational and biochemical studies. Our data elucidated intermediate states during antibiotic recognition and suggested structural changes that accompany the substrate-evoked deprotonation of E26T/D34M/A150E. These findings help to explain the mechanistic flexibility in drug/H+ coupling observed in MdfA and may inspire therapeutic development to preempt efflux-mediated antimicrobial resistance.

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Section: Dxc/h + Coupling In E26t/d34m/a150ementioning

confidence: 91%

Section: Capture Of Two Cm-binding Modes In the Crystals At The Intementioning

confidence: 99%

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confidence: 99%

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Structure and mechanism of a redesigned multidrug transporter from the Major Facilitator Superfamily

Symerský

2020

Sci Rep

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“…The I f crystal structure was determined from the nonfunctional mutant Q131R with bound deoxycholate and other ligands (5), while for the more recent O o structure, crystallization was facilitated by a Fab antibody fragment attached to the cytoplasmic side of the protein. (6) The solution structure of MdfA solubilized in detergent at neutral pH, was explored by double electronelectron resonance (DEER) measurements on a series of double cysteine mutants of MdfA labeled with two different spin labels -a pair of nitroxide labels (NO) or a pair of C2-Gd labels (see Fig. 1a).…”

Section: Introductionmentioning

confidence: 99%

Drug binding sites in the multidrug transporter MdfA in detergent solution and in lipid nanodiscs

Goldfarb

Bahrenberg

Yardeni

et al. 2020

Preprint

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MdfA, a member of the major facilitator superfamily (MFS), is a multidrug/proton antiporter from E. coli that has been considered a model for secondary multidrug (Mdr) transporters. Its transport mechanism, driven by a proton gradient, is associated with conformational changes, which accompany the recruitment of drugs and their release. In this work, we applied double-electron electron resonance (DEER) spectroscopy to locate the binding site of one of its substrates, tetraphenylphosphonium (TPP) within available crystal structures. We carried out Gd(III)-nitroxide distance measurements between MdfA labeled with a Gd(III) tag and the TPP analog mito-TEMPO (bearing the nitroxide moiety). Data were obtained both for MdfA solubilized in detergent micelles (n-dodecyl-β-D-maltopyranoside (DDM)), and reconstituted into lipid nanodiscs (ND). For both DDM and ND, the average position of the substrate at a neutral pH was found to be close to the ligand position in the If (inward facing) crystal structure, with the DDM environment exhibiting a somewhat better agreement than the ND environment. We therefore conclude that the If structure provides a good description for substrate-bound MdfA in DDM solution, while in ND the structure is slightly modified. A second binding site was found for the ND sample situated at the cytoplasmic side, towards the end of transmembrane helix 7 (TM7). In addition, we used DEER distance measurements on Gd(III) doubly labeled MdfA to track conformational changes within the periplasmic and cytoplasmic sides associated with substrate binding. We detected significant differences in the periplasmic side of MdfA, with the ND featuring a more closed conformation than in DDM, in agreement with earlier reports. The addition of TPP led to a noticeable conformational change in the periplasmic face in ND, attributed to a movement of TM10. This change was not observed in DDM.Statement of SignificanceMdfA is multidrug transporter from E. coli, which exhibits multidrug efflux activities with an unusually broad spectrum of drug specificities. While it has been established that solute transport by similar transporters is coupled to significant conformational changes, previous studies raised the possibility that this is not the case for MdfA. Moreover, it is not clear how MdfA functionally accommodates chemically dissimilar substrates. Towards resolving these open questions, we used double-electron electron resonance distance measurements to determine the binding site of a spin labeled drug analog within available crystal structures of MdfA and to examine how MdfA responds conformationally to drug binding. Moreover, we explored how these two are affected by the media, detergent micelles vs lipid nanodiscs.

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“…Several entries have already been submitted to BSM-Arc, in various formats, sizes, and annotation styles. BSM-00001, BSM-00002, BSM-00003, BSM-00004, BSM-00006, BSM-00007, and BSM-00009 pertain to MD simulations (Bekker et al , 2019aInaba et al 2018;Oda et al 2018;Numoto et al 2018;Nagarathinam et al 2018), while BSM-00005 pertains to molecular docking (Kawabata et al 2017) and BSM-00011 and BSM-00012 to homology models (Ishizuka et al 2017;Kimura et al 2017). All the projects concerning MD simulations include representative structures, but BSM-00001 also includes all the raw trajectory data including topologies and preparation files.…”

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confidence: 99%

The Biological Structure Model Archive (BSM-Arc): an archive for in silico models and simulations

2020

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We present the Biological Structure Model Archive (BSM-Arc, https://bsma.pdbj.org), which aims to collect raw data obtained via in silico methods related to structural biology, such as computationally modeled 3D structures and molecular dynamics trajectories. Since BSM-Arc does not enforce a specific data format for the raw data, depositors are free to upload their data without any prior conversion. Besides uploading raw data, BSM-Arc enables depositors to annotate their data with additional explanations and figures. Furthermore, via our WebGL-based molecular viewer Molmil, it is possible to recreate 3D scenes as shown in the corresponding scientific article in an interactive manner. To submit a new entry, depositors require an ORCID ID to login, and to finally publish the data, an accompanying peer-reviewed paper describing the work must be associated with the entry. Submitting their data enables researchers to not only have an external backup but also provide an opportunity to promote their work via an interactive platform and to provide third-party researchers access to their raw data.

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Outward open conformation of a Major Facilitator Superfamily multidrug/H+ antiporter provides insights into switching mechanism

Cited by 53 publications

References 53 publications

Structure and mechanism of a redesigned multidrug transporter from the Major Facilitator Superfamily

Structure and mechanism of a redesigned multidrug transporter from the Major Facilitator Superfamily

Drug binding sites in the multidrug transporter MdfA in detergent solution and in lipid nanodiscs

The Biological Structure Model Archive (BSM-Arc): an archive for in silico models and simulations

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