Zooming in on a small multidrug transporter reveals details of asymmetric protonation

Shen, Jana

doi:10.1073/pnas.1810814115

Cited by 2 publications

(2 citation statements)

References 22 publications

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“…Alternative proton-binding site and coupling between key aspartates highlight the intricacy of the compensatory transport mechanism of NhaA. Coupled protonation by two adjacent aspartates is common (26) and has been demonstrated in the one-proton exchange mechanism of a multidrug transporter AcrB by a simulation study (17); however, the coupling between two distant aspartates enabled through the protoncoupled hydrogen-bonding network at the flexible crossing of two disrupted helices (as in K300A and K300Q/D163N NhaA) has not been demonstrated before. We speculate that alternative proton-binding site and long-distance coupling may represent important general mechanisms of proton-coupled transport in secondary active transporters.…”

Section: Concluding Discussionmentioning

confidence: 99%

Alternative proton-binding site and long-distance coupling in Escherichia coli sodium–proton antiporter NhaA

Henderson

Huang

Beckstein

et al. 2020

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

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Escherichia coli NhaA is a prototypical sodium–proton antiporter responsible for maintaining cellular ion and volume homeostasis by exchanging two protons for one sodium ion; despite two decades of research, the transport mechanism of NhaA remains poorly understood. Recent crystal structure and computational studies suggested Lys300 as a second proton-binding site; however, functional measurements of several K300 mutants demonstrated electrogenic transport, thereby casting doubt on the role of Lys300. To address the controversy, we carried out state-of-the-art continuous constant pH molecular dynamics simulations of NhaA mutants K300A, K300R, K300Q/D163N, and K300Q/D163N/D133A. Simulations suggested that K300 mutants maintain the electrogenic transport by utilizing an alternative proton-binding residue Asp133. Surprisingly, while Asp133 is solely responsible for binding the second proton in K300R, Asp133 and Asp163 jointly bind the second proton in K300A, and Asp133 and Asp164 jointly bind two protons in K300Q/D163N. Intriguingly, the coupling between Asp133 and Asp163 or Asp164 is enabled through the proton-coupled hydrogen-bonding network at the flexible intersection of two disrupted helices. These data resolve the controversy and highlight the intricacy of the compensatory transport mechanism of NhaA mutants. Alternative proton-binding site and proton sharing between distant aspartates may represent important general mechanisms of proton-coupled transport in secondary active transporters.

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Section: Concluding Discussionmentioning

confidence: 99%

Alternative proton-binding site and long-distance coupling in Escherichia coli sodium–proton antiporter NhaA

Henderson

Huang

Beckstein

et al. 2020

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

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“…A similar monomer-dimer equilibrium was also observed for EmrE, a well-studied SMR family member, on MS analysis ( 38 ). Structural information obtained from cryo-electron microscopy, X-ray crystallography, and nuclear magnetic resonance (NMR) spectroscopy, along with other experimental data, have established that EmrE functions as an asymmetric antiparallel dimer ( 39 – 48 ). For EmrE, an essential Glu14 residue (sharing conservation with Glu15 in AceI), is involved in drug-proton binding during efflux.…”

Section: Discussionmentioning

confidence: 99%

Assembly and regulation of the chlorhexidine-specific efflux pump AceI

Bolla

Howes

Fiorentino

et al. 2020

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

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Few antibiotics are effective against Acinetobacter baumannii, one of the most successful pathogens responsible for hospital-acquired infections. Resistance to chlorhexidine, an antiseptic widely used to combat A. baumannii, is effected through the proteobacterial antimicrobial compound efflux (PACE) family. The prototype membrane protein of this family, AceI (Acinetobacter chlorhexidine efflux protein I), is encoded for by the aceI gene and is under the transcriptional control of AceR (Acinetobacter chlorhexidine efflux protein regulator), a LysR-type transcriptional regulator (LTTR) protein. Here we use native mass spectrometry to probe the response of AceI and AceR to chlorhexidine assault. Specifically, we show that AceI forms dimers at high pH, and that binding to chlorhexidine facilitates the functional form of the protein. Changes in the oligomerization of AceR to enable interaction between RNA polymerase and promoter DNA were also observed following chlorhexidine assault. Taken together, these results provide insight into the assembly of PACE family transporters and their regulation via LTTR proteins on drug recognition and suggest potential routes for intervention.

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Zooming in on a small multidrug transporter reveals details of asymmetric protonation

Cited by 2 publications

References 22 publications

Alternative proton-binding site and long-distance coupling in Escherichia coli sodium–proton antiporter NhaA

Alternative proton-binding site and long-distance coupling in Escherichia coli sodium–proton antiporter NhaA

Assembly and regulation of the chlorhexidine-specific efflux pump AceI

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