The substrate specificity switch FlhB assembles onto the export gate to regulate type three secretion

Kuhlen, Lucas; Johnson, Steven; Zeitler, Andreas; Bäurle, Sandra; Deme, Justin C.; Caesar, Joseph; Debo, Rebecca; Fisher, Joan M.; Wagner, Samuel; Lea, Susan M.

doi:10.1038/s41467-020-15071-9

Cited by 55 publications

(100 citation statements)

References 48 publications

(69 reference statements)

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“…FliR (10 subunits) and FliP 5 FliQ 4 FliR 1 FlhB 1 (11 subunits) complexes19,30 , and the helical parameter is similar to those of the flagellar axial structures, such as the rod, hook and filament, with about 5.5 subunits per turn of the 1-start helix, which can also be regarded as a tubular structure with 11 protofilaments. However, the 11-fold symmetry feature of the internal part of the M ring is not the one directly associated with the export gate at the center of the M ring.…”

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confidence: 95%

Native structure of flagellar MS ring is formed by 34 subunits with 23-fold and 11-fold subsymmetries

Kawamoto

Miyata

Makino

et al. 2020

Preprint

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The bacterial flagellar MS ring is a transmembrane complex acting as the core of the flagellar motor. It not only acts as the template for rod and C ring assembly but also houses the type III protein export gate for assembly of the rod, hook and filament. The cytoplasmic C ring, involved in torque generation and rotation switch, is directly attached to the MS ring, and a symmetry mismatch between 26-fold MS ring and 34-fold C ring had been a long puzzle as to whether this would play some role in motor function. Although this puzzle seemed to have been resolved by the recent high-resolution structure of the MS ring with 33-fold symmetry with a variation from 32-fold to 35-fold because the C ring also shows a similar symmetry variation, it still remained ambiguous whether their symmetries are matched in the native motor structure. Here we show that the native MS ring structure formed by full-length FliF is 34-fold with no symmetry variation whereas the C ring has a small symmetry variation, indicating a flexibility in C ring assembly to generate small symmetry mismatches. We also show two conformations of FliF in part of its periplasmic region to form the 34-subunit ring with 23-fold and 11-fold subsymmetries in the inner and middle M ring, respectively, to accommodate the export gate at the center of the M ring.

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confidence: 95%

Native structure of flagellar MS ring is formed by 34 subunits with 23-fold and 11-fold subsymmetries

Kawamoto

Miyata

Makino

et al. 2020

Preprint

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“…FliP, FliQ and FliR form a polypeptide channel complex for the translocation of export substrates across the cytoplasmic membrane 6,7 . FlhB associates with the FliP/FliQ/FliR complex and is postulated to coordinate gate opening of the polypeptide channel 8 . FlhA associates not only with the FliP/FliQ/FliR complex but also with the MS ring 9 .…”

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confidence: 99%

The FlgN chaperone activates the Na⁺-driven engine of the flagellar protein export apparatus

Minamino

Kinoshita

Morimoto

et al. 2020

Preprint

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The bacterial flagellar protein export machinery promotes H+-coupled translocation of flagellar proteins to the cell exterior. When the cytoplasmic ATPase complex does not function, the transmembrane export gate complex opens its Na+ channel and continues protein transport. However, it remains unknown how. Here we report that the FlgN chaperone acts as a switch to activate a backup export mechanism for the ATPase complex by activating the Na+-driven engine. Impaired interaction of FlhA with the FliJ subunit of the ATPase complex increased Na+-dependence of flagellar protein export. Deletion of FlgN inhibited protein export in the absence of the ATPase complex but not in its presence. Gain-of-function mutations in FlhA restored not only the FlgN defect but also the FliJ defect. We propose that the interaction of FlgN with FlhA opens the Na+ channel in the export engine, thereby maintaining the protein export activity in the absence of the active ATPase complex.

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“…Our initial results suggested that the length of the amino acid side chain was important for the function of the M-gasket. Thereafter, several structural studies have confirmed that the M-loop is indeed exposed to the channel in both fT3SS and vT3SS (8)(9)(10). In the FliPQR secretion pore, five copies of FliP are present, which results in a local pool of 15 methionine residues that are spread over about 20Å at the base of the channel (Figure 2A).…”

Section: The Methionine Loop In Flip Regulates Membrane Gatingmentioning

confidence: 89%

Controlling membrane barrier during bacterial type-III protein secretion

Hüsing

Look

Guse

et al. 2020

Preprint

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Type-III secretion systems (T3SSs) of the bacterial flagellum and the evolutionarily related injectisome are capable of translocating proteins with a remarkable speed of several thousand amino acids per second. Here, we investigated how T3SSs are able to transport proteins at such a high rate while preventing the leakage of small molecules. Our mutational and evolutionary analyses demonstrate that an ensemble of conserved methionine residues at the cytoplasmic side of the T3SS channel create a deformable gasket (M-gasket) around fast-moving substrates undergoing export. The unique physicochemical features of the M-gasket are crucial to preserve the membrane barrier, to accommodate local conformational changes during active secretion, and to maintain stability of the secretion pore in cooperation with a plug domain (R-plug) and a network of salt-bridges. The conservation of the M-gasket, R-plug, and salt-bridge network suggests a universal mechanism by which the membrane integrity is maintained during high-speed protein translocation in all T3SSs.

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The substrate specificity switch FlhB assembles onto the export gate to regulate type three secretion

Cited by 55 publications

References 48 publications

Native structure of flagellar MS ring is formed by 34 subunits with 23-fold and 11-fold subsymmetries

Native structure of flagellar MS ring is formed by 34 subunits with 23-fold and 11-fold subsymmetries

The FlgN chaperone activates the Na⁺-driven engine of the flagellar protein export apparatus

Controlling membrane barrier during bacterial type-III protein secretion

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The substrate specificity switch FlhB assembles onto the export gate to regulate type three secretion

Cited by 55 publications

References 48 publications

Native structure of flagellar MS ring is formed by 34 subunits with 23-fold and 11-fold subsymmetries

Native structure of flagellar MS ring is formed by 34 subunits with 23-fold and 11-fold subsymmetries

The FlgN chaperone activates the Na+-driven engine of the flagellar protein export apparatus

Controlling membrane barrier during bacterial type-III protein secretion

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The FlgN chaperone activates the Na⁺-driven engine of the flagellar protein export apparatus