Mapping of the Docking of SecA onto the Chaperone SecB by Site-directed Spin Labeling: Insight into the Mechanism of Ligand Transfer During Protein Export

Crane, Jennine M.; Mao, Chunfeng; Lilly, Angela A.; Smith, Virginia F.; Suo, Yang; Hubbell, Wayne L.; Randall, Linda L.

doi:10.1016/j.jmb.2005.08.022

Cited by 46 publications

(42 citation statements)

References 21 publications

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“…1A). This alignment shows that Rv1957 has several clusters of key residues critical for SecB oligomerization and interaction with substrates or with its specific partner SecA at the Sec translocon (19)(20)(21)(22)(23)(24)(25). Previous work Author contributions: P.G.…”

Section: Resultsmentioning

confidence: 98%

“…has shown that there are several conserved interaction sites for SecB on SecA, i.e., at the N-terminal and at the C-terminal zincbinding region of SecA (20,24,26). Remarkably, the housekeeping SecA1 from M. tuberculosis, which does not have the cysteine residues involved in zinc binding at the extreme C-terminal region, retains all of the conserved neighboring residues involved in contact with SecB (19).…”

Section: Resultsmentioning

confidence: 99%

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SecB-like chaperone controls a toxin–antitoxin stress-responsive system in Mycobacterium tuberculosis

Bordes

Cirinesi

Ummels

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

106

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A major step in the biogenesis of newly synthesized precursor proteins in bacteria is their targeting to the Sec translocon at the inner membrane. In Gram-negative bacteria, the chaperone SecB binds nonnative forms of precursors and specifically transfers them to the SecA motor component of the translocase, thus facilitating their export. The major human pathogen Mycobacterium tuberculosis is an unusual Gram-positive bacterium with a well-defined outer membrane and outer membrane proteins. Assistance to precursor proteins by chaperones in this bacterium remains largely unexplored. Here we show that the product of the previously uncharacterized Rv1957 gene of M. tuberculosis can substitute for SecB functions in Escherichia coli and prevent preprotein aggregation in vitro. Interestingly, in M. tuberculosis, Rv1957 is clustered with a functional stress-responsive higB-higA toxin-antitoxin (TA) locus of unknown function. Further in vivo experiments in E. coli and in Mycobacterium marinum strains that do not possess the TA-chaperone locus show that the severe toxicity of the toxin was entirely inhibited when the antitoxin and the chaperone were jointly expressed. We found that Rv1957 acts directly on the antitoxin by preventing its aggregation and protecting it from degradation. Taken together, our results show that the SecB-like chaperone Rv1957 specifically controls a stress-responsive TA system relevant for M. tuberculosis adaptive response.DnaK | molecular chaperones | trigger factor | protease

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Section: Resultsmentioning

confidence: 98%

Section: Resultsmentioning

confidence: 99%

SecB-like chaperone controls a toxin–antitoxin stress-responsive system in Mycobacterium tuberculosis

Bordes

Cirinesi

Ummels

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

106

View full text Add to dashboard Cite

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“…4 The reader is referred to that publication for a detailed description of whether the line shapes of those spectra reflect isotropic motion and whether resolved spectral components are due to multiple dynamic states of the nitroxide in slow exchange or are due to anisotropic motion of a nitroxide in one dynamic state.…”

Section: Resultsmentioning

confidence: 99%

“…Our application of site-directed spin labeling and EPR to the study of SecB has previously allowed us to map the docking interface between SecB and its specific binding partner SecA, 4 and now we have examined the promiscuous interactions of SecB as a chaperone. The large area of the chaperone that is involved in contacts with ligand indicates that numerous routes are available on the surface.…”

Section: Discussionmentioning

confidence: 99%

“…We have previously determined the orientation of the docking of SecA onto SecB 4 and now we investigate the binding of ligand. When physiologic ligands, precursors of polypeptides of 30 -50 kDa in a non-native state, that are destined for export to the periplasmic space bind to SecB, the portion of the polypeptide that makes contact with the chaperone spans a minimal stretch of 150 amino acids.…”

Section: Introductionmentioning

confidence: 99%

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Sites of Interaction of a Precursor Polypeptide on the Export Chaperone SecB Mapped by Site-directed Spin Labeling

Crane

Suo

Lilly

et al. 2006

Journal of Molecular Biology

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SummaryExport of protein into the periplasm of Escherichia coli via the general secretory system requires that the transported polypeptides be devoid of stably folded tertiary structure. Capture of the precursor polypeptides before they fold is achieved by the promiscuous binding to the chaperone SecB. SecB delivers its ligand to export sites through its specific binding to SecA, a peripheral component of the membrane translocon. At the translocon the ligand is passed from SecB to SecA and subsequently through the SecYEG channel. We have previously used site-directed spin labeling and electron paramagnetic resonance spectroscopy to establish a docking model between SecB and SecA. Here we report use of the same strategy to map the pathway of a physiologic ligand, the unfolded form of precursor galactose-binding protein, on SecB. Our set of SecB variants each containing a single cysteine, which was used in the previous study, has been expanded to forty-eight residues which cover 49% of the surface of SecB. The residues on SecB involved in contacts were identified as those that, upon addition of the unfolded polypeptide ligand, showed changes in spectral line shape consistent with restricted motion of the nitroxide. We conclude that the bound precursor makes contact with a large portion of the surface of the small chaperone. The sites on SecB that interact with the ligand are compared with the previously identified sites that interact with SecA and a model for transfer of the ligand is discussed.

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Efficient Building Blocks for Solid‐Phase Peptide Synthesis of Spin Labeled Peptides for Electron Paramagnetic Resonance and Dynamic Nuclear Polarization Applications

et al. 2019

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Specific spin labeling allows the site‐selective investigation of biomolecules by EPR and DNP enhanced NMR spectroscopy. A novel spin labeling strategy for commercially available Fmoc‐amino acids is developed. In this approach, the PROXYL spin label is covalently attached to the hydroxyl side chain of three amino acids hydroxyproline (Hyp), serine (Ser) and tyrosine (Tyr) by a simple three‐step synthesis route. The obtained PROXYL containing building‐blocks are N‐terminally protected by the Fmoc‐protection group, which makes them applicable for the use in solid‐phase peptide synthesis (SPPS). This approach allows the insertion of the spin label at any desired position during SPPS, which makes it more versatile than the widely used post synthetic spin labeling strategies. For the final building‐blocks, the radical activity is proven by EPR. DNP enhanced solid‐state NMR experiments employing these building‐blocks in a TCE solution show enhancement factors of up to 26 for 1H and 13C (1H→13C cross‐polarization). To proof the viability of the presented building‐blocks for insertion of the spin label during SPPS the penta‐peptide Acetyl‐Gly‐Ser(PROXYL)‐Gly‐Gly‐Gly was synthesized employing the spin labeled Ser building‐block. This peptide could successfully be isolated and the spin label activity proved by EPR and DNP NMR measurements, showing enhancement factors of 12.1±0.1 for 1H and 13.9±0.5 for 13C (direct polarization).

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Mapping of the Docking of SecA onto the Chaperone SecB by Site-directed Spin Labeling: Insight into the Mechanism of Ligand Transfer During Protein Export

Cited by 46 publications

References 21 publications

SecB-like chaperone controls a toxin–antitoxin stress-responsive system in Mycobacterium tuberculosis

SecB-like chaperone controls a toxin–antitoxin stress-responsive system in Mycobacterium tuberculosis

Sites of Interaction of a Precursor Polypeptide on the Export Chaperone SecB Mapped by Site-directed Spin Labeling

Efficient Building Blocks for Solid‐Phase Peptide Synthesis of Spin Labeled Peptides for Electron Paramagnetic Resonance and Dynamic Nuclear Polarization Applications

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