Reconstitution of the activity of RND efflux pumps: a “bottom-up” approach

Puvanendran, Dhenesh; Cece, Quentin; Picard, Martin

doi:10.1016/j.resmic.2017.11.004

Cited by 6 publications

(2 citation statements)

References 59 publications

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“…The path toward the successful reconstitution of a tripartite pump is interspersed with many pitfalls, and several laboratories have contributed to reach a sensible framework to reach this goal. The milestones, major results along this path, and possible improvements are reviewed in refs and . For a long time, protocols were based on the reconstitution of the RND pump in the presence or absence of its periplasmic adaptor.…”

Section: Assembly and Function Of The Rnd-based Tripartite Complexesmentioning

confidence: 99%

Structure, Assembly, and Function of Tripartite Efflux and Type 1 Secretion Systems in Gram-Negative Bacteria

et al. 2021

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Tripartite efflux pumps and the related type 1 secretion systems (T1SSs) in Gram-negative organisms are diverse in function, energization, and structural organization. They form continuous conduits spanning both the inner and the outer membrane and are composed of three principal components—the energized inner membrane transporters (belonging to ABC, RND, and MFS families), the outer membrane factor channel-like proteins, and linking the two, the periplasmic adaptor proteins (PAPs), also known as the membrane fusion proteins (MFPs). In this review we summarize the recent advances in understanding of structural biology, function, and regulation of these systems, highlighting the previously undescribed role of PAPs in providing a common architectural scaffold across diverse families of transporters. Despite being built from a limited number of basic structural domains, these complexes present a staggering variety of architectures. While key insights have been derived from the RND transporter systems, a closer inspection of the operation and structural organization of different tripartite systems reveals unexpected analogies between them, including those formed around MFS- and ATP-driven transporters, suggesting that they operate around basic common principles. Based on that we are proposing a new integrated model of PAP-mediated communication within the conformational cycling of tripartite systems, which could be expanded to other types of assemblies.

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Section: Assembly and Function Of The Rnd-based Tripartite Complexesmentioning

confidence: 99%

Structure, Assembly, and Function of Tripartite Efflux and Type 1 Secretion Systems in Gram-Negative Bacteria

et al. 2021

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“…The connection between both protein units is supported by several AcrA bridging both into a functional unit. For all three subunits high-resolution structures were obtained and even more recently a high-resolution structure of the entire tripartite unit was obtained [10][11][12][13]. The TolC-AcrA-AcrB complex and his homologues have a multitude of functions and in particular are involved in antibiotic resistance as the complex can shuffle actively toxic compounds outside the bacteria.…”

Section: Introductionmentioning

confidence: 99%

Single TolC-AcrA complex formation monitored by time dependent single-channel electrophysiology

Winterhalter

Bodrenko

Zewdie

et al. 2022

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Characterizing protein-protein interaction on a single molecular level is a challenge, experimentally as well as the interpretation of the data. For example, Gram-negative bacteria contain protein complexes spanning the outer and inner cell wall being able to efflux effectively cell toxic substances. Within the search for antibiotics with novel modes of action, inhibition of the efflux pump assembly is an interesting possible target. Recent seminal work revealed the high-resolution structure of the tripartite composition TolC-AcrA-AcrB. Here, we reconstitute a single TolC homotrimer into a planar lipid membrane and follow the assembly of AcrA using the modulation of the ion current through TolC during binding. In particular, the presence of AcrA increases the average ionic current through TolC and, moreover, reduces the ion-current fluctuations caused by flickering of TolC. Here, we demonstrate that statistical properties of ion-current fluctuations (the power spectral density) provide a complementary measure of the interaction of the TolC-AcrA complex with potential inhibitors. Both characteristics, the average current and the current noise, taken into consideration together, provide more robust information.

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TolC-AcrA complex formation monitored by time dependent single-channel electrophysiology

et al. 2023

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Reconstitution of the activity of RND efflux pumps: a “bottom-up” approach

Cited by 6 publications

References 59 publications

Structure, Assembly, and Function of Tripartite Efflux and Type 1 Secretion Systems in Gram-Negative Bacteria

Structure, Assembly, and Function of Tripartite Efflux and Type 1 Secretion Systems in Gram-Negative Bacteria

Single TolC-AcrA complex formation monitored by time dependent single-channel electrophysiology

TolC-AcrA complex formation monitored by time dependent single-channel electrophysiology

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