Protein Structure and Folding
Negative Stain Single-particle EM of the Maltose Transporter in Nanodiscs Reveals Asymmetric Closure of MalK2 and Catalytic Roles of ATP, MalE, and Maltose*
Conformational Dynamics of the Maltose Transporter

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The Escherichia coli MalE-MalFGK2 complex is one of the best characterized members of the large and ubiquitous family of ATP-binding cassette (ABC) transporters. It is composed of a membrane-spanning heterodimer, MalF-MalG; a homodimeric ATPase, MalK2; and a periplasmic maltose receptor, MalE. Opening and closure of MalK2 is coupled to conformational changes in MalF-MalG and the alternate exposition of the substrate-binding site to either side of the membrane. To further define this alternate access mechanism and the impact of ATP, MalE, and maltose on the conformation of the transporter during the transport cycle, we have reconstituted MalFGK2 in nanodiscs and analyzed its conformations under 10 different biochemical conditions using negative stain single-particle EM. EM map results (at 15–25 Å resolution) indicate that binding of ATP to MalK2 promotes an asymmetric, semi-closed conformation in accordance with the low ATPase activity of MalFGK2. In the presence of MalE, the MalK dimer becomes fully closed, gaining the ability to hydrolyze ATP. In the presence of ADP or maltose, MalE·MalFGK2 remains essentially in a semi-closed symmetric conformation, indicating that release of these ligands is required for the return to the initial state. Taken together, this structural information provides a rationale for the stimulation of MalK ATPase activity by MalE as well as by maltose.

ABC transporter
electron microscopy (EM)
membrane protein
membrane transporter reconstitution
single-particle analysis
translocation
transport

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*

This work was supported by Canadian Institutes of Health Research Grants 86693MOP (to I. R.) and 74525MOP (to F. D.), the Natural Sciences and Engineering Research Council of Canada Grant RGPIN-2014-04798 (to I. R.), a Bourse de Prestige from the Groupe d'Étude des Protéines Membranaires (which is supported by the Fonds de la Recherche en Santé du Québec) (to L. F.), and a summer fellowship from the Natural Sciences and Engineering Research Council of Canada Create Training Program in Bionanomachines (to J. I.). The authors declare that they have no conflicts of interest with the contents of this article.

This article contains supplemental Table S1 and Figs. S1–S7.

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The abbreviations used are:

    ABC

    ATP-binding cassette

    NBD

    nucleotide-binding domain

    TMD

    transmembrane domain

    ATPγS

    adenosine 5′-O-(thiotriphosphate)

    AMP-PNP

    adenosine 5′-(β,γ-iminotriphosphate)

    PDB

    Protein Data Bank

    BMOE

    bismaleimidoethane.