The Role of SurA PPIase Domains in Preventing Aggregation of the Outer-Membrane Proteins tOmpA and OmpT

Humes, Julia R.; Schiffrin, Bob; Calabrese, Antonio N.; Higgins, A.J.; Westhead, David R.; Brockwell, David J.; Radford, Sheena E.

doi:10.1016/j.jmb.2019.01.032

Cited by 27 publications

(56 citation statements)

References 91 publications

(131 reference statements)

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“…In the presence of OmpX, regions in SurA that are protected from deuterium uptake upon substrate binding cluster to the core domain. No change in protection in P2 was detected in the presence of OmpX, consistent with the tag transfer XL-MS results and with previous results which have shown that P2 is not required to prevent the aggregation of the small (8-stranded) OmpA 28 . Intriguingly, two regions of SurA (residues 46-72 in the N-terminal region and 212-239 in P1) ( Fig.…”

Section: Conformational Changes In Sura Upon Omp Bindingsupporting

confidence: 92%

“…SurA binds unfolded OmpX with low affinity (K d,app of ~800 nM), as measured by microscale thermophoresis (MST) ( Supplementary Fig. 8), similar to the affinity of SurA for other OMPs 28,43,44 . SurA-OmpX complexes were assembled by rapid dilution of urea-denatured OmpX into a solution of SurA (final concentrations: 5 µM OmpX, 5 µM SurA, 0.24 M urea) (see Methods) immediately prior to crosslinking with DSBU.…”

Section: Sura Binds Its Omp Substrates At Multiple Interaction Sitesmentioning

confidence: 59%

“…Despite its key role in OMP biogenesis and bacterial virulence 51,52 , how SurA binds its OMP substrates both specifically, but weakly 28,44,49 , and how it is able to protect its clients from aggregation and deliver them to BAM for folding into the OM, remain poorly understood in molecular detail. Previous NMR studies have shown that OmpX, tOmpA and FhuA are dynamically disordered when bound to SurA 40 -42 .…”

Section: Discussionmentioning

confidence: 99%

“…By contrast, the results presented here show that SurA binds its OMP clients at multiple sites 64 , predominantly involving the core domain as indicated by tag-transfer XL and HDX. The additional binding capacity in the P1 and P2 domains may be employed in a substrate-specific manner, as suggested by the finding that P2 is required to suppress the aggregation of the 10-stranded OmpT, but not the 8-strand tOmpA 28 . Previous results have also shown that deletion of P2 can perturb SurA function in vivo, as measured by a decrease in the amount of assembled LamB in a BAMcompromised strain 29 .…”

Section: Dynamic Interplay Between Sura and Its Substratesmentioning

confidence: 99%

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

Calabrese

Schiffrin

Watson

et al. 2019

Preprint

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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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Section: Conformational Changes In Sura Upon Omp Bindingsupporting

confidence: 92%

Section: Sura Binds Its Omp Substrates At Multiple Interaction Sitesmentioning

confidence: 59%

Section: Discussionmentioning

confidence: 99%

Section: Dynamic Interplay Between Sura and Its Substratesmentioning

confidence: 99%

See 2 more Smart Citations

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

Calabrese

Schiffrin

Watson

et al. 2019

Preprint

Self Cite

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“…BAM and BAM(∆BamB) was over-produced in E. coli C43 according to established protocols (Iadanza et al, 2016;Roman-Hernandez et al, 2014). For preparation of SurA and SurA-OmpA complexes, both proteins were over-produced separately in 1 L of cultures as described previously (Humes et al, 2019). Both were harvested by centrifugation, lysed in a cell disruptor…”

Section: Protein Production and Purificationmentioning

confidence: 99%

Inter-membrane association of the Sec and BAM translocons for bacterial outer-membrane biogenesis

Alvira

Watkins

Troman

et al. 2019

Preprint

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SUMMARYThe outer-membrane of Gram-negative bacteria is critical for surface adhesion, pathogenicity, antibiotic resistance and survival. The major constituent – hydrophobic β-barrel Outer-Membrane Proteins (OMPs) – are secreted across the inner-membrane through the Sec-translocon for delivery to periplasmic chaperones e.g. SurA, which prevent aggregation. OMPs are then offloaded to the β-Barrel Assembly Machinery (BAM) in the outer-membrane for insertion and folding. We show the Holo-TransLocon (HTL: an assembly of the protein-channel core-complex SecYEG, the ancillary sub-complex SecDF, and the membrane ‘insertase’ YidC) contacts SurA and BAM through periplasmic domains of SecDF and YidC, ensuring efficient OMP maturation. Our results show the trans-membrane proton-motive-force (PMF) acts at distinct stages of protein secretion: for SecA-driven translocation across the inner-membrane through SecYEG; and to communicate conformational changes via SecDF to the BAM machinery. The latter presumably ensures efficient passage of OMPs. These interactions provide insights of inter-membrane organisation, the importance of which is becoming increasingly apparent.

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Single‐molecule approaches reveal outer membrane protein biogenesis dynamics

2022

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Outer membrane proteins (OMPs) maintain the viability of Gram-negative bacteria by functioning as receptors, transporters, ion channels, lipases, and porins. Folding and assembly of OMPs involves synchronized action of chaperones and multi-protein machineries which escort the highly hydrophobic polypeptides to their target outer membrane in a folding competent state. Previous studies have identified proteins and their involvement along the OMP biogenesis pathway. Yet, the mechanisms of action and the intriguing ability of all these molecular machines to work without the typical cellular energy source of ATP, but solely based on thermodynamic principles, are still not well understood. Here, we highlight how different single-molecule studies can shed additional light on the mechanisms and kinetics of OMP biogenesis.

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The Role of SurA PPIase Domains in Preventing Aggregation of the Outer-Membrane Proteins tOmpA and OmpT

Cited by 27 publications

References 91 publications

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

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

Inter-membrane association of the Sec and BAM translocons for bacterial outer-membrane biogenesis

Single‐molecule approaches reveal outer membrane protein biogenesis dynamics

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