Structure of FlgK reveals the divergence of the bacterial Hook-Filament Junction of Campylobacter

Bulieris, Paula V.; Shaikh, Nausad H.; Freddolino, Peter L.; Samatey, Fadel A.

doi:10.1038/s41598-017-15837-0

Cited by 15 publications

(11 citation statements)

References 39 publications

(54 reference statements)

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“…Our method resolves the interaction interface between flagellar proteins FlgL and FlgK, which form the junction between the flagellar filament and flagellar hook 23 . Extrapolating from our docked model of the monomers and a previous model of the FlgK ring from Campylobacter jejuni 24 , we infer the configuration of an 11-mer ring of FlgL inside an 11-mer ring of FlgK 24 (Fig. 4 , Supplementary Fig.…”

Section: Resultsmentioning

confidence: 95%

Large-scale discovery of protein interactions at residue resolution using co-evolution calculated from genomic sequences

et al. 2021

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Increasing numbers of protein interactions have been identified in high-throughput experiments, but only a small proportion have solved structures. Recently, sequence coevolution-based approaches have led to a breakthrough in predicting monomer protein structures and protein interaction interfaces. Here, we address the challenges of large-scale interaction prediction at residue resolution with a fast alignment concatenation method and a probabilistic score for the interaction of residues. Importantly, this method (EVcomplex2) is able to assess the likelihood of a protein interaction, as we show here applied to large-scale experimental datasets where the pairwise interactions are unknown. We predict 504 interactions de novo in the E. coli membrane proteome, including 243 that are newly discovered. While EVcomplex2 does not require available structures, coevolving residue pairs can be used to produce structural models of protein interactions, as done here for membrane complexes including the Flagellar Hook-Filament Junction and the Tol/Pal complex.

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Section: Resultsmentioning

confidence: 95%

Large-scale discovery of protein interactions at residue resolution using co-evolution calculated from genomic sequences

et al. 2021

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“…The β-sandwich structure of xcFlgL D2, which consists of a three-β-strand sheet and a five-β-strand sheet, is observed in the functionally related FlgK protein (RMSD values of 1.8–1.9 Å with the D2 domain of bpFlgK for ~80 Cα atoms) (Fig. 4b,c ) 18 , 19 . Moreover, the D2 domains of FlgL and FlgK both form accessory structures that are appended to the primarily α-helical D1 domain.…”

Section: Resultsmentioning

confidence: 99%

“…2e,f ). Notably, the four-segment arrangement of FlgL D1 is recapitulated in the D1 domains of other flagellar junction and filament proteins, such as FlgK, flagellin, and FliD, although their sequences and functions are highly diverse (Figs 1 and 3 ) 10 , 18 , 19 , 25 . In structure overlays, each of the S1, S2, and S4 segments is represented by α-helices that are identical in terms of direction and position.…”

Section: Resultsmentioning

confidence: 99%

“…Thus, bpFlgL is expected to interact with bpFlgK in the flagellum through a quasi-homotypic interaction between the D2 domains of the two proteins. Similarly, the D2 domain of FlgK was proposed to interact with the variable D3 and D4 domains of FlgE 19 . To reveal the functional role of the D1 or D2 domains that we have proposed here, future structural studies of the FlgL-flagellin interaction are required.…”

Section: Discussionmentioning

confidence: 99%

“…Moreover, the FlgL and FlgK proteins use the same export chaperone, FlgN, for their delivery to an export gate protein and for the prevention of premature aggregation and proteolysis in the cytosol 15 – 17 . Recent structural studies of the truncated Burkholderia pseudomallei FlgK (bpFlgK) and Campylobacter jejuni FlgK proteins revealed that FlgK contains a four-helix bundle structure 18 , 19 . However, FlgL has not been structurally characterized, and it is thus unknown how FlgL assembles into the upper layer of the junction and links the hook to the filament at the top of FlgK.…”

Section: Introductionmentioning

confidence: 99%

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Crystal structure of FlgL and its implications for flagellar assembly

Hong

Kim

Song

et al. 2018

Sci Rep

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Bacteria move toward attractants and away from repellants by rotating their flagellum. The bacterial flagellum assembles through the ordered organization of more than 30 different proteins. Among the diverse flagellar proteins, FlgL forms the junction between the hook and the filament in the flagellum together with FlgK and provides a structural base where flagellin, a filament-forming protein, is inserted for the initiation of filament elongation. However, the functional and structural information available for FlgL is highly limited. To provide structural insights into the cross-linkage between the FlgL junction and the flagellin filament, we determined the crystal structures of FlgL from gram-positive Bacillus cereus (bcFlgL) and gram-negative Xanthomonas campestris (xcFlgL). bcFlgL contains one domain (D1), whereas xcFlgL adopts a two-domain structure that consists of the D1 and D2 domains. The constant D1 domain of FlgL adopts a rod structure that is generated by four longitudinal segments. This four-segment structure is recapitulated in filament and junction proteins but not in hook and rod proteins, allowing us to propose a junction-filament assembly mechanism based on a quasi-homotypic interaction. The D2 domain of xcFlgL resembles that of another junction protein, FlgK, suggesting the structural and functional relatedness of FlgL and FlgK.

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Helicobacter pylori FlgN binds its substrate FlgK and the flagellum ATPase FliI in a similar manner observed for the FliT chaperone

Dhindwal,

Boniecki,

Moore

2024

Protein Science

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In bacterial flagellum biogenesis, secretion of the hook‐filament junction proteins FlgK and FlgL and completion of the flagellum requires the FlgN chaperone. Similarly, the related FliT chaperone is necessary for the secretion of the filament cap protein FliD and binds the flagellar export gate protein FlhA and the flagellum ATPase FliI. FlgN and FliT require FliJ for effective substrate secretion. In Helicobacter pylori, neither FlgN, FliT nor FliJ have been annotated. We demonstrate that the genome location of HP1120 is identical to that of flgN in other flagellated bacteria and that HP1120 is the homologue of Campylobacter jejuni FlgN. A modeled HP1120 structure contains three α‐helices and resembles the FliT chaperone, sharing a similar substrate‐binding pocket. Using pulldowns and thermophoresis, we show that both HP1120 and a HP1120Δ126‐144 deletion mutant bind to FlgK with nanomolar affinity, but not to the filament cap protein FliD, confirming that HP1120 is FlgN. Based on size‐exclusion chromatography and multi‐angle light scattering, H. pylori FlgN binds to FlgK with 1:1 stoichiometry. Overall structural similarities between FlgN and FliT suggest that substrate recognition on FlgN primarily involves an antiparallel coiled‐coil interface between the third helix of FlgN and the C‐terminal helix of the substrate. A FlgNΔ126‐144 N100A, Y103A, S111I triple mutant targeting this interface significantly impairs the binding of FlgK. Finally, we demonstrate that FlgNΔ126‐144, like FliT, binds with sub‐micromolar affinity to the flagellum ATPase FliI or its N‐terminal domain. Hence FlgN and FliT likely couple delivery of low‐abundance export substrates to the flagellum ATPase FliI.This article is protected by copyright. All rights reserved.

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Structure of FlgK reveals the divergence of the bacterial Hook-Filament Junction of Campylobacter

Cited by 15 publications

References 39 publications

Large-scale discovery of protein interactions at residue resolution using co-evolution calculated from genomic sequences

Large-scale discovery of protein interactions at residue resolution using co-evolution calculated from genomic sequences

Crystal structure of FlgL and its implications for flagellar assembly

Helicobacter pylori FlgN binds its substrate FlgK and the flagellum ATPase FliI in a similar manner observed for the FliT chaperone

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