How nanoscale protein interactions determine the mesoscale dynamic organisation of bacterial outer membrane proteins

Chavent, Matthieu; Duncan, Anna L.; Rassam, Patrice; Birkholz, Oliver; Hélie, Jean; Reddy, Tyler; Beliaev, Dmitry; Hambly, Ben; Piehler, Jacob; Kleanthous, Colin; Sansom, Mark S.P.

doi:10.1038/s41467-018-05255-9

Cited by 54 publications

(55 citation statements)

References 68 publications

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“…In WT and Δ tolC cells that did feature puncta, colE1-TR formed clusters of 12–20 molecules per punctum, where each cluster had a diameter of less than the microscope’s 0.5 μm resolution. The quantity of molecules and the size of the puncta were in agreement with previous studies of BtuB clusters (25, 26), supporting the assumption that the puncta comprised clusters of colE1-TR bound to BtuB. We further verified that punctum formation and localization was not an artifact of Cy3 conjugation: a fusion construct of GFP to the C-terminus of colE1-TR also displayed the same cluster formation characteristics (Fig.…”

Section: Resultssupporting

confidence: 91%

Colicin E1 opens its hinge to plug TolC

Budiardjo

Stevens

Calkins

et al. 2019

Preprint

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17The double membrane architecture of Gram-negative bacteria forms a barrier 18 that is effectively impermeable to extracellular threats. Accordingly, researchers have 19shown increasing interest in developing antibiotics that target the accessible, surface-20 exposed proteins embedded in the outer membrane. TolC forms the outer membrane 21 channel of an antibiotic efflux pump in Escherichia coli. Drawing from prior observations 22 that colicin E1, a toxin produced by and lethal to E. coli, can bind to the TolC channel, 23we investigate the capacity of colicin E1 fragments to 'plug' TolC and inhibit its efflux 24 function. First, using single-molecule fluorescence, we show that colicin E1 fragments 25 that do not include the cytotoxic domain localize at the cell surface. Next, using real-time 26 efflux measurements and minimum inhibitory concentration assays, we show that 27 exposure of wild-type E. coli to fragments of colicin E1 indeed disrupts TolC efflux and 28 heightens bacterial susceptibility to four common classes of antibiotics. This work 29 demonstrates that extracellular plugging of outer membrane transporters can serve as a 30 novel method to increase antibiotic susceptibility. In addition to the utility of these protein 31 fragments as starting points for the development of novel antibiotic potentiators, the 32 variety of outer membrane protein colicin binding partners provides an array of options 33 that would allow our method to be used to inhibit other outer membrane protein 34 functions. 35 36Significance 37 We find that fragments of a protein natively involved in intraspecies bacterial 38 warfare can be exploited to plug the E. coli outer membrane antibiotic efflux machinery. 39This plugging disables a primary form of antibiotic resistance. Given the diversity of 40 bacterial species of similar bacterial warfare protein targets, we anticipate that this 41 3 method of plugging is generalizable to disabling the antibiotic efflux of other 42 proteobacteria. Moreover, given the diversity of the targets of bacterial warfare proteins, 43 this method could be used for disabling the function of a wide variety of other bacterial 44 outer membrane proteins. 45 46

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Section: Resultssupporting

confidence: 91%

Colicin E1 opens its hinge to plug TolC

Budiardjo

Stevens

Calkins

et al. 2019

Preprint

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“…The design of mesoscale synthetic protein assemblies is becoming increasingly powerful to create new materials [40][41][42] and functions 43,44 . Moreover, as we are only beginning to grasp the complexity of proteome self-organization, new approaches are needed for characterizing and understanding mesoscale properties of protein self-assembly in cells 19,20,32,[45][46][47][48][49][50] . In this context, our synthetic system constitutes a powerful tool to interrogate biological mechanisms of protein assembly.…”

Section: Discussionmentioning

confidence: 99%

Designer protein assemblies with tunable phase diagrams in living cells

et al. 2020

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Proteins self-organization is a hallmark of biological systems. Physico-chemical principles governing protein-protein interactions have long been known. However, the principles by which such nanoscale interactions generate diverse phenotypes of mesoscale assemblies, including phase-separated compartments, remain challenging to characterize. To illuminate such principles, we create a system of two proteins designed to interact and form mesh-like assemblies. We devise a novel strategy to map high-resolution phase diagrams in living cells, which provide self-assembly signatures of this system. The structural modularity of the two protein components allows straightforward modification of their molecular properties, enabling us to characterize how interaction affinity impacts the phase diagram and material state of the assemblies in vivo. The phase diagrams and their dependence on interaction affinity were captured by theory and simulations, including out-of-equilibrium effects seen in growing cells. Finally, we find that cotranslational protein binding suffices to recruit an mRNA to the designed micron-scale structures.

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“…The top panel shows a schematic diagram of an E. coli cell, with the areas of outer membrane studied via CG simulation (yellow square), by mesoscale simulation (blue square), and by experimental single molecule tracking (green circle) shown to scale. The lower two panels are snapshots from CG (left) and meso (right) simulations of OMP clustering (see main text and ref (688) for details).…”

Section: Increasing Complexitymentioning

confidence: 99%

Computational Modeling of Realistic Cell Membranes

Marrink¹,

Corradi

Souza³

et al. 2019

Chem. Rev.

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585

488

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Cell membranes contain a large variety of lipid types and are crowded with proteins, endowing them with the plasticity needed to fulfill their key roles in cell functioning. The compositional complexity of cellular membranes gives rise to a heterogeneous lateral organization, which is still poorly understood. Computational models, in particular molecular dynamics simulations and related techniques, have provided important insight into the organizational principles of cell membranes over the past decades. Now, we are witnessing a transition from simulations of simpler membrane models to multicomponent systems, culminating in realistic models of an increasing variety of cell types and organelles. Here, we review the state of the art in the field of realistic membrane simulations and discuss the current limitations and challenges ahead.

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How nanoscale protein interactions determine the mesoscale dynamic organisation of bacterial outer membrane proteins

Cited by 54 publications

References 68 publications

Colicin E1 opens its hinge to plug TolC

Colicin E1 opens its hinge to plug TolC

Designer protein assemblies with tunable phase diagrams in living cells

Computational Modeling of Realistic Cell Membranes

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