Functional Rotation Induced by Alternating Protonation States in the Multidrug Transporter AcrB: All-Atom Molecular Dynamics Simulations

Yamane, Tsutomu; Murakami, Satoshi; Ikeguchi, Mitsunori

doi:10.1021/bi400119v

Cited by 39 publications

(89 citation statements)

References 51 publications

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“…R2 helices collectively rotate about R1 in the anti-clockwise direction on the membrane plane (Fig. 8a), resembling the O-to-L transition revealed by the crystal structures and discussed by Eicher et al 19 Such backbone movements were not reported by the previous all-atom simulations; 19,25 however, similar motion has been observed in the most recent hybrid coarse-grained simulations. 42 Along with the backbone changes, the sidechains of essential residues, Asp407, Asp408, Lys940, Thr978 and Arg941 rearrange, which leads to, among others, the movement of Lys940 from Thr978 to Asp408.…”

Section: Concluding Discussionsupporting

confidence: 75%

“…The two proton model however contradicts another recent MD study which showed that protonation of both Asp407 and Asp408 destabilizes the O state. 25 By testing a combination of protonation states of Asp407/Asp408 for the O protomer, the latter study supported a one proton model, in which protonation and deprotonation of Asp408 alone drives the conformational cycling of AcrB. 25 …”

Section: Introductionmentioning

confidence: 88%

“…23,24 Notwithstanding, it remains unclear which specific residue(s) is(are) responsible for proton binding/release. Further, although models have been proposed, 19,25 the detailed mechanism by which proton translocation is coupled to the conformational conversion from T to O and O to L states remains unclear. The objective of this work is to address these two important questions using the state-of-the-art continuous constant pH molecular dynamics (CpHMD) simulations.…”

Section: Introductionmentioning

confidence: 99%

“…26,27 Conventional MD, which has been used in all previous MD studies of AcrB, 18,19,25,28 assumes fixed protonation states. By contrast, CpHMD allows ionizable residues to titrate during dynamics in response to changes in the conformational environment and solution pH.…”

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Constant pH Molecular Dynamics Reveals How Proton Release Drives the Conformational Transition of a Transmembrane Efflux Pump

Yue

Chen²,

Zgurskaya

et al. 2017

J. Chem. Theory Comput.

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AcrB is the inner-membrane transporter of E. coli AcrAB-TolC tripartite efflux complex, which plays a major role in the intrinsic resistance to clinically important antibiotics. AcrB pumps a wide range of toxic substrates by utilizing the proton gradient between periplasm and cytoplasm. Crystal structures of AcrB revealed three distinct conformational states of the transport cycle, substrate access, binding and extrusion, or loose (L), tight (T) and open (O) states. However, the specific residue(s) responsible for proton binding/release and the mechanism of proton-coupled conformational cycling remain controversial. Here we use the newly developed membrane hybridsolvent continuous constant pH molecular dynamics technique to explore the protonation states and conformational dynamics of the transmembrane domain of AcrB. Simulations show that both Asp407 and Asp408 are deprotonated in the L/T states, while only Asp408 is protonated in the O state. Remarkably, release of a proton from Asp408 in the O state results in large conformational changes, such as the lateral and vertical movement of transmembrane helices as well as the salt-bridge formation between Asp408 and Lys940 and other sidechain rearrangements among essential residues. Consistent with the crystallographic differences between the O and L protomers, simulations offer dynamic details of how proton release drives the O-to-L transition in AcrB and address the controversy regarding the proton/drug stoichiometry. This work offers a significant step towards characterizing the complete cycle of proton-coupled drug transport in AcrB and further validates the membrane hybrid-solvent CpHMD technique for studies of proton-coupled transmembrane proteins which are currently poorly understood.

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Section: Concluding Discussionsupporting

confidence: 75%

Section: Introductionmentioning

confidence: 88%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Constant pH Molecular Dynamics Reveals How Proton Release Drives the Conformational Transition of a Transmembrane Efflux Pump

Yue

Chen²,

Zgurskaya

et al. 2017

J. Chem. Theory Comput.

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“…The carboxyl group of Asp408 was indeed shown to have an unusually high pK a of 7.4, which would help in the facile binding and release of the proton under physiological conditions (107). A recent MD simulation study (108) suggests that in the extrusion protomer, Asp408 becomes protonated, but Asp407 remains deprotonated. In this scheme, the translocation of one proton across the IM would be sufficient to cause the conformational changes in AcrB, resulting in the extrusion of the drug molecule(s).…”

Section: Figmentioning

confidence: 98%

The Challenge of Efflux-Mediated Antibiotic Resistance in Gram-Negative Bacteria

2015

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SUMMARY The global emergence of multidrug-resistant Gram-negative bacteria is a growing threat to antibiotic therapy. The chromosomally encoded drug efflux mechanisms that are ubiquitous in these bacteria greatly contribute to antibiotic resistance and present a major challenge for antibiotic development. Multidrug pumps, particularly those represented by the clinically relevant AcrAB-TolC and Mex pumps of the resistance-nodulation-division (RND) superfamily, not only mediate intrinsic and acquired multidrug resistance (MDR) but also are involved in other functions, including the bacterial stress response and pathogenicity. Additionally, efflux pumps interact synergistically with other resistance mechanisms (e.g., with the outer membrane permeability barrier) to increase resistance levels. Since the discovery of RND pumps in the early 1990s, remarkable scientific and technological advances have allowed for an in-depth understanding of the structural and biochemical basis, substrate profiles, molecular regulation, and inhibition of MDR pumps. However, the development of clinically useful efflux pump inhibitors and/or new antibiotics that can bypass pump effects continues to be a challenge. Plasmid-borne efflux pump genes (including those for RND pumps) have increasingly been identified. This article highlights the recent progress obtained for organisms of clinical significance, together with methodological considerations for the characterization of MDR pumps.

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Studying dynamics without explicit dynamics: A structure‐based study of the export mechanism by AcrB

Simsir¹,

Broutin

Mus‐Veteau³

et al. 2020

Proteins

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RND family proteins are transmembrane proteins identified as large spectrum drug transporters involved in multi-drug resistance. A prototypical case in this superfamily, responsible for antibiotic resistance in selected gram negative bacteria, is AcrB. AcrB forms a trimer using the proton motive force to efflux drugs, implementing a functional rotation mechanism. Unfortunately, the size of the system (1049 amino-acid per monomer and membrane) has prevented a systematic dynamical exploration, so that the mild understanding of this coupled transport jeopardizes our ability to counter it. The large number of crystal structures of AcrB prompts studies to further our understanding of the mechanism. To this end, we present a novel strategy based on two key ingredients which are to study dynamics by exploiting information embodied in the numerous crystal structures obtained to date, and to systematically consider subdomains, their dynamics, and their interactions. Along the way, we identify the subdomains responsible for dynamic events, refine the states (A,B,E) of the functional rotation mechanism, and analyze the evolution of intramonomer and intermonomer interfaces along the functional cycle. Our analysis shows the relevance of AcrB's efflux mechanism as a template within the HAE1 family but not beyond. It also paves the way to targeted simulations exploiting the most relevant degrees of freedom at certain steps, and also to a targeting of specific interfaces to block the drug efflux. Our work shows that complex dynamics can be unveiled from static snapshots, a strategy that may be used on a variety of molecular machines of large size.

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Functional Rotation Induced by Alternating Protonation States in the Multidrug Transporter AcrB: All-Atom Molecular Dynamics Simulations

Cited by 39 publications

References 51 publications

Constant pH Molecular Dynamics Reveals How Proton Release Drives the Conformational Transition of a Transmembrane Efflux Pump

Constant pH Molecular Dynamics Reveals How Proton Release Drives the Conformational Transition of a Transmembrane Efflux Pump

The Challenge of Efflux-Mediated Antibiotic Resistance in Gram-Negative Bacteria

Studying dynamics without explicit dynamics: A structure‐based study of the export mechanism by AcrB

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