Exploring Transporters within the Small Multidrug Resistance Family Using Solid-State NMR Spectroscopy

Traaseth, Nathaniel J.; Banigan, James R.; Leninger, Maureen

doi:10.1002/9780470034590.emrstm1413

eMagRes 2015

DOI: 10.1002/9780470034590.emrstm1413

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Exploring Transporters within the Small Multidrug Resistance Family Using Solid-State NMR Spectroscopy

Nathaniel J. Traaseth

James R. Banigan

Maureen Leninger

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2021

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Asymmetric protonation of glutamate residues drives a preferred transport pathway in EmrE

Her

Traaseth

2021

Proc. Natl. Acad. Sci. U.S.A.

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EmrE is an Escherichia coli multidrug efflux pump and member of the small multidrug resistance (SMR) family that transports drugs as a homodimer by harnessing energy from the proton motive force. SMR family transporters contain a conserved glutamate residue in transmembrane 1 (Glu14 in EmrE) that is required for binding protons and drugs. Yet the mechanism underlying proton-coupled transport by the two glutamate residues in the dimer remains unresolved. Here, we used NMR spectroscopy to determine acid dissociation constants (pKa) for wild-type EmrE and heterodimers containing one or two Glu14 residues in the dimer. For wild-type EmrE, we measured chemical shifts of the carboxyl side chain of Glu14 using solid-state NMR in lipid bilayers and obtained unambiguous evidence on the existence of asymmetric protonation states. Subsequent measurements of pKa values for heterodimers with a single Glu14 residue showed no significant differences from heterodimers with two Glu14 residues, supporting a model where the two Glu14 residues have independent pKa values and are not electrostatically coupled. These insights support a transport pathway with well-defined protonation states in each monomer of the dimer, including a preferred cytoplasmic-facing state where Glu14 is deprotonated in monomer A and protonated in monomer B under pH conditions in the cytoplasm of E. coli. Our findings also lead to a model, hop-free exchange, which proposes how exchangers with conformation-dependent pKa values reduce proton leakage. This model is relevant to the SMR family and transporters comprised of inverted repeat domains.

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Asymmetric protonation of glutamate residues drives a preferred transport pathway in EmrE

Her

Traaseth

2021

Proc. Natl. Acad. Sci. U.S.A.

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Exploring Transporters within the Small Multidrug Resistance Family Using Solid-State NMR Spectroscopy

Cited by 1 publication

References 70 publications

Asymmetric protonation of glutamate residues drives a preferred transport pathway in EmrE

Asymmetric protonation of glutamate residues drives a preferred transport pathway in EmrE

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