Dihydrostreptomycin Directly Binds to, Modulates, and Passes through the MscL Channel Pore

Wray, Robin; Iscla, Irene; Gao, Ya; Li, Hua; Wang, Junmei; Blount, Paul

doi:10.1371/journal.pbio.1002473

Cited by 41 publications

(96 citation statements)

References 70 publications

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“…Our model schematically depicted in Fig. 8, is also in good agreement with a recent FRET spectroscopic study suggesting that the top of the C-terminal bundle separates to create a larger entrance to the pore, allowing molecules such as streptomycin to pass through the channel 45 . However, the rest of the helical bundle end remains associated during the gating cycle and likely functions both as a molecular sieve and as a stabilizer of the MscL oligomeric structure [15][16][17]46 .…”

Section: Discussionsupporting

confidence: 89%

Structural Dynamics of the MSCL C-Terminal Domain

Martinac

Bavi

Cortes

et al. 2017

Biophysical Journal

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The large conductance mechanosensitive channel (MscL), acts as an osmoprotective emergency valve in bacteria by opening a large, water-filled pore in response to changes in membrane tension. In its closed configuration, the last 36 residues at the C-terminus form a bundle of five α-helices co-linear with the five-fold axis of symmetry. Here, we examined the structural dynamics of the C-terminus of EcMscL using site-directed spin labelling electron paramagnetic resonance (SDSL EPR) spectroscopy. These experiments were complemented with computational modelling including molecular dynamics (MD) simulations and finite element (FE) modelling. Our results show that under physiological conditions, the C-terminus is indeed an α-helical bundle, located near the five-fold symmetry axis of the molecule. Both experiments and computational modelling demonstrate that only the top part of the C-terminal domain (from the residue A110 to E118) dissociates during the channel gating, while the rest of the C-terminus stays assembled. This result is consistent with the view that the C-terminus functions as a molecular sieve and stabilizer of the oligomeric MscL structure as previously suggested.The bacterial mechanosensitive channel of large conductance (MscL), has become a prototype molecular system in the study of mechanotransduction and its evolutionary origins 1,2 . The three-dimensional (3D) structure of MscL from Mycobacterium tuberculosis (MtMscL) in its closed conformation was resolved at 3.5 Å 3 . The channel is a homopentamer consisting of N-and C-terminal domains facing the cytoplasm and TM1 and TM2 transmembrane helices connected by a periplasmic loop [3][4][5][6] . The closed channel structure was also determined in the lipid bilayer by site-directed spin labelling electron paramagnetic resonance (SDSL EPR) spectroscopy 7 . Moreover, the open channel structure was also determined by SDSL EPR spectroscopy 8 allowing for estimation of the size of the open channel pore in a very good agreement with electrophysiological sieving studies 9 . Later on, an improved open E. coli MscL (EcMscL) 3D structure was determined using both ensemble and single molecule site-directed fluorofore labelling (SDFL) FRET spectroscopy in combination with MD simulations 6,10,11 in agreement with the X-ray structure of an expanded form of an archaeal MscL homolog from Methanosarcina acetivorans 12 . While the overall gating-related structural changes in MscL have largely been established 8,11,13,14 the structural dynamics and physiological role of the C-terminal domain has thus far been controversial. Several studies have suggested that the C-terminus should remain intact during gating and could function as a molecular sieve 15,16 , while others suggested that this helical bundle should actively participate in gating and that opening of the MscL channel was accompanied by complete dissociation of the bundle 13,17 . More recently, the X-ray structure of the EcMscL C-terminus domain was obtained at 1. 45 Å 18 , suggesting that even in the absence ...

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Section: Discussionsupporting

confidence: 89%

Structural Dynamics of the MSCL C-Terminal Domain

Martinac

Bavi

Cortes

et al. 2017

Biophysical Journal

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“…The model of aminoglycoside induced death proposed from this work is consistent with evidence from other groups previous work, requires the presence of membrane pores and membrane potential to drive aminoglycoside bactericidal activity. Aminoglycosides enter the cell through an unknown mechanism, possibly through channels such as mscL 57 , which occurs long before a loss of membrane integrity. Once aminoglycosides enter the cell they bind ribosomes, disrupt a majority of translating 70S particles and cause mistranslation of protein 13,30 .…”

Section: Discussionmentioning

confidence: 99%

Membrane voltage dysregulation driven by metabolic dysfunction underlies bactericidal activity of aminoglycosides

Bruni

Kralj

2020

Preprint

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Aminoglycosides are broad-spectrum antibiotics whose mechanism of bactericidal activity has been under debate. It is widely accepted, however, that membrane voltage potentiates aminoglycoside activity, which is ascribed to voltage dependent drug uptake. In this paper, we measured the single cell response of Escherichia coli treated with aminoglycosides and discovered that the bactericidal action arises not from the downstream effects of voltage dependent drug uptake, but rather directly from dysregulated membrane potential. In the absence of voltage, aminoglycosides are taken into cells and exert bacteriostatic effects by inhibiting translation. However, cell killing was immediate upon re-polarization. The hyperpolarization arose from altered ATP flux, which induced a reversal of the F1Fo-ATPase to hydrolyze ATP and generated the deleterious voltage. Heterologous expression of an ATPase inhibitor from Salmonella completely eliminated bactericidal activity, while loss of the F-ATPase significantly reduced the electrophysiological response to aminoglycosides. Our data support a model of voltage induced death, which could be resolved in real-time at the single cell level, and separates the mechanisms of aminoglycoside bacteriostasis and bactericide in E. coli.

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“…Molecular dynamic (MD) simulations for the passage of DHS through the MscL pore: "passing through" competition. Molecular docking was conducted with a representative structure of a 150-ns molecular dynamics trajectory of Eco-MscL with a DHS molecule within the pocket formed by the periplasmic loops, as previously described (Wray et al, 2016). The binding pocket for 011A was identified by the SiteID module of the Sybyl-X2.11 software package and flexible-ligand docking was performed with the Glide module of the Schrodinger software package; the sites were as previous (Wray et al, 2016;Wray et al, 2019).…”

Section: In Vivo Assaysmentioning

confidence: 99%

An agonist of the MscL channel affects multiple bacterial species and increases membrane permeability and potency of common antibiotics

Wray

Herrera

Iscla

et al. 2019

Molecular Microbiology

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Summary The bacterial MscL channel normally functions as an emergency release valve discharging cytoplasmic solutes upon osmotic stress. The channel opens and passes molecules up to 30 Å and its pore is the largest of any gated channel. Opening the MscL pore inappropriately is detrimental to the bacterial cell, suggesting MscL as a potential novel drug target. A small‐molecule compound, 011A, has been shown to increase sensitivity of the Escherichia coli MscL channel, slow growth, and even decrease viability of quiescent cultures. The mscL gene is highly conserved and found in the vast majority of bacterial species, including pathogens. Here, we test the hypothesis that 011A can influence the growth and viability of other bacterial species, specifically Staphylococcus aureus and Mycobacterium smegmatis, in a MscL‐dependent manner. Furthermore, we demonstrate that the 011A compound can increase potency of other antibiotics, presumably by permeabilizing the membrane and allowing easier access of the antibiotic into the cytoplasm. Thus, MscL activators have potential as novel broad‐spectrum antibiotics or adjuvants that work with antibiotics to selectively allow passage across bacterial membranes.

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Dihydrostreptomycin Directly Binds to, Modulates, and Passes through the MscL Channel Pore

Cited by 41 publications

References 70 publications

Structural Dynamics of the MSCL C-Terminal Domain

Structural Dynamics of the MSCL C-Terminal Domain

Membrane voltage dysregulation driven by metabolic dysfunction underlies bactericidal activity of aminoglycosides

An agonist of the MscL channel affects multiple bacterial species and increases membrane permeability and potency of common antibiotics

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