Chaperones Skp and SurA dynamically expand unfolded outer membrane protein X and synergistically disassemble oligomeric aggregates

AbstractPeriplasmic chaperones Skp and SurA are essential players in outer membrane protein (OMP) biogenesis. They prevent unfolded OMPs from misfolding during their passage through the periplasmic space and aid in the disassembly of OMP aggregates under cellular stress conditions. However, functionally important links between interaction mechanisms, structural dynamics, and energetics that underpin both Skp and SurA association with OMPs have remained largely unresolved. Here, using single-molecule fluorescence spectroscopy, we dissect the conformational dynamics and thermodynamics of Skp and SurA binding to unfolded OmpX, and explore their disaggregase activities. We show that both chaperones expand unfolded OmpX distinctly and induce microsecond chain reconfigurations in the client OMP structure. We further reveal that Skp and SurA bind their substrate in a fine-tuned thermodynamic process via enthalpy–entropy compensation. Finally, we observed synergistic activity of both chaperones in the disaggregation of oligomeric OmpX aggregates. Our findings provide an intimate view into the multi-faceted functionalities of Skp and SurA and the fine-tuned balance between conformational flexibility and underlying energetics in aiding chaperone action during OMP biogenesis.

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Inter-domain dynamics in the chaperone SurA and multi-site binding to its unfolded outer membrane protein clients

10.1101/2019.12.19.882696 ◽

2019 ◽

Author(s):

Antonio N. Calabrese ◽

Bob Schiffrin ◽

Matthew Watson ◽

Theodoros K. Karamanos ◽

Martin Walko ◽

...

Keyword(s):

Membrane Protein ◽

Outer Membrane ◽

Single Molecule ◽

Conformational Changes ◽

Outer Membrane Protein ◽

Conformational Dynamics ◽

Core Domain ◽

The Core ◽

Single Molecule Fret ◽

Dynamics Simulations

AbstractThe periplasmic chaperone SurA plays a key role in outer membrane protein (OMP) biogenesis. E. coli SurA comprises a core domain and two peptidylprolyl isomerase domains (P1 and P2), but how it binds its OMP clients and the mechanism(s) of its chaperone action remain unclear. Here, we have used chemical cross-linking, hydrogen-deuterium exchange, single-molecule FRET and molecular dynamics simulations to map the client binding site(s) on SurA and to interrogate the role of conformational dynamics of the chaperone’s domains in OMP recognition. We demonstrate that SurA samples a broad array of conformations in solution in which P2 primarily lies closer to the core/P1 domains than suggested by its crystal structure. Multiple binding sites for OMPs are located primarily in the core domain, with binding of the unfolded OMP resulting in conformational changes between the core/P1 domains. Together, the results portray a model in which unfolded OMP substrates bind in a cradle formed between the SurA domains, with structural flexibility between its domains assisting OMP recognition, binding and release.

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Conformational dynamics and membrane interactions of the E. coli outer membrane protein FecA: A molecular dynamics simulation study

Biochimica et Biophysica Acta (BBA) - Biomembranes ◽

10.1016/j.bbamem.2012.08.021 ◽

2013 ◽

Vol 1828 (2) ◽

pp. 284-293 ◽

Cited By ~ 41

Author(s):

Thomas J. Piggot ◽

Daniel A. Holdbrook ◽

Syma Khalid

Keyword(s):

Molecular Dynamics ◽

Molecular Dynamics Simulation ◽

Membrane Protein ◽

Outer Membrane ◽

Simulation Study ◽

Outer Membrane Protein ◽

Conformational Dynamics ◽

Dynamics Simulation ◽

Membrane Interactions ◽

E Coli

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Developing a Single-Molecule Platform to Understand Outer Membrane Protein Biogenesis

Biophysical Journal ◽

10.1016/j.bpj.2018.11.2682 ◽

2019 ◽

Vol 116 (3) ◽

pp. 497a

Author(s):

Megan Mitchell ◽

Marcelo Sousa

Keyword(s):

Membrane Protein ◽

Outer Membrane ◽

Single Molecule ◽

Outer Membrane Protein ◽

Protein Biogenesis

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A growing toolbox of techniques for studying β-barrel outer membrane protein folding and biogenesis

Biochemical Society Transactions ◽

10.1042/bst20160020 ◽

2016 ◽

Vol 44 (3) ◽

pp. 802-809 ◽

Cited By ~ 9

Author(s):

Jim E. Horne ◽

Sheena E. Radford

Keyword(s):

Protein Folding ◽

Membrane Protein ◽

Outer Membrane ◽

Single Molecule ◽

Outer Membrane Protein ◽

Lipid Membrane ◽

Water Soluble ◽

Membrane Protein Folding ◽

Over 40 Years

Great strides into understanding protein folding have been made since the seminal work of Anfinsen over 40 years ago, but progress in the study of membrane protein folding has lagged behind that of their water soluble counterparts. Researchers in these fields continue to turn to more advanced techniques such as NMR, mass spectrometry, molecular dynamics (MD) and single molecule methods to interrogate how proteins fold. Our understanding of β-barrel outer membrane protein (OMP) folding has benefited from these advances in the last decade. This class of proteins must traverse the periplasm and then insert into an asymmetric lipid membrane in the absence of a chemical energy source. In this review we discuss old, new and emerging techniques used to examine the process of OMP folding and biogenesis in vitro and describe some of the insights and new questions these techniques have revealed.

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Membrane protein dynamics in different environments: simulation study of the outer membrane protein X in a lipid bilayer and in a micelle

European Biophysics Journal ◽

10.1007/s00249-010-0626-7 ◽

2010 ◽

Vol 40 (1) ◽

pp. 39-58 ◽

Cited By ~ 12

Author(s):

Alexandra Choutko ◽

Alice Glättli ◽

César Fernández ◽

Christian Hilty ◽

Kurt Wüthrich ◽

...

Keyword(s):

Membrane Protein ◽

Outer Membrane ◽

Protein Dynamics ◽

Lipid Bilayer ◽

Simulation Study ◽

Outer Membrane Protein ◽

Protein X ◽

Membrane Protein Dynamics

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Colicin E1 Fragments Potentiate Antibiotics by Plugging TolC

10.1101/692251 ◽

2019 ◽

Author(s):

S. Jimmy Budiardjo ◽

Jacqueline J. Deay ◽

Anna L. Calkins ◽

Virangika K. Wimalasena ◽

Daniel Montezano ◽

...

Keyword(s):

Membrane Protein ◽

Outer Membrane ◽

Single Molecule ◽

Outer Membrane Protein ◽

Efflux Pump ◽

Bacterial Species ◽

Outer Membrane Proteins ◽

E Coli ◽

Colicin E1 ◽

Antibiotic Efflux

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

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