Insight into structural remodeling of the FlhA ring responsible for bacterial flagellar type III protein export

Terahara, Naoya; Inoue, Yumi; Kodera, Noriyuki; Morimoto, Yusuke V.; Uchihashi, Takayuki; Imada, Katsumi; Ando, Toshio; Namba, Keiichi; Minamino, Tetsuo

doi:10.1126/sciadv.aao7054

Cited by 53 publications

(105 citation statements)

References 45 publications

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“…FlhA acts as an energy transducer along with FliH, FliI and FliJ (Minamino, Morimoto, Hara, Aldridge, & Namba, ; Minamino, Morimoto, Hara, & Namba, ; Morimoto et al, ). The FlhA C ring provides binding sites for FliH, FliI, FliJ, flagellar export chaperones and export substrates along with the C‐terminal cytoplasmic domain of FlhB (FlhB C ) and coordinates flagellar protein export with assembly (Bange et al, ; Evans, Poulter, Terentjev, Hughes, & Fraser, ; Inoue, Morimoto, Namba, & Minamino, ; Kinoshita, Hara, Imada, Namba, & Minamino, ; Minamino, González‐Pedrajo, Kihara, Namba, & Macnab, ; Minamino et al, ; Minamino & Macnab, ; Terahara et al, ).…”

Section: Introductionmentioning

confidence: 99%

Mutational analysis of the C‐terminal cytoplasmic domain of FlhB, a transmembrane component of the flagellar type III protein export apparatus in Salmonella

et al. 2019

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The flagellar protein export apparatus switches its substrate specificity when hook length has reached approximately 55 nm in Salmonella. The C-terminal cytoplasmic domain of FlhB (FlhB C ) is involved in this switching process. FlhB C consists of FlhB CN and FlhB CC polypeptides. FlhB CC has a flexible C-terminal tail (FlhB CCT ).FlhB CC is involved in substrate recognition, and conformational rearrangements of FlhB CN -FlhB CC boundary are postulated to be required for the export switching. However, it remains unknown how it occurs. To clarify this question, we carried out mutational analysis of highly conserved residues in FlhB C . The flhB(E230A) mutation reduced the FlhB function. The flhB(E11S) mutation restored the protein transport activity of the flhB(E230A) mutant to the wild-type level, suggesting that the interaction of FlhB CN with the extreme N-terminal region of FlhB is required for flagellar protein export. The flhB(R320A) mutation affected hydrophobic interaction networks in FlhB CC , thereby increasing insolubility of FlhB C . The R320A mutation also affected the export switching, thereby producing longer hooks with the filament attached. C-terminal truncations of FlhB CCT induced a conformational change of FlhB CN -FlhB CC boundary, resulting in a loose hook length control. We propose that FlhB CCT may control conformational arrangements of FlhB CN -FlhB CC boundary through the hydrophobic interaction networks of FlhB CC . K E Y W O R D S bacterial flagella, FlhB, hook, substrate specificity switching, type III protein export

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Section: Introductionmentioning

confidence: 99%

Mutational analysis of the C‐terminal cytoplasmic domain of FlhB, a transmembrane component of the flagellar type III protein export apparatus in Salmonella

et al. 2019

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“…At the core of the secretion system is the highly conserved export apparatus (EA) 4,5 , which is made up of five predicted transmembrane (TM) proteins (SctR, SctS, SctT, SctU and SctV in the vT3SS; FliP, FliQ, FliR, FlhB and FlhA in the fT3SS). FlhA/SctV has been shown to form a nonameric ring [6][7][8] , consisting of a large cytoplasmic domain situated below a hydrophobic domain predicted to contain 72 helices. This structure was proposed to surround an "export gate" through which substrates would enter the secretion pathway.…”

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confidence: 99%

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

et al. 2020

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Protein secretion through type-three secretion systems (T3SS) is critical for motility and virulence of many bacteria. Proteins are transported through an export gate containing three proteins (FliPQR in flagella, SctRST in virulence systems). A fourth essential T3SS protein (FlhB/SctU) functions to "switch" secretion substrate specificity once the growing hook/ needle reach their determined length. Here, we present the cryo-electron microscopy structure of an export gate containing the switch protein from a Vibrio flagellar system at 3.2 Å resolution. The structure reveals that FlhB/SctU extends the helical export gate with its four predicted transmembrane helices wrapped around FliPQR/SctRST. The unusual topology of the FlhB/SctU helices creates a loop wrapped around the bottom of the closed export gate. Structure-informed mutagenesis suggests that this loop is critical in gating secretion and we propose that a series of conformational changes in the T3SS trigger opening of the gate through interactions between FlhB/SctU and FliPQR/SctRST.

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“…For instance, in the Epsilonproteobacterium H. pylori, which has a three-tiered regulatory hierarchy of flagellar expression and assembly, including three sigma factors and a FlgM feedback loop (Niehus et al, 2004), researchers have found that several flagellar structural proteins are involved in flagellar regulation. These proteins include the basal body component FlhA, an essential part of the basal body and the assembly platform (Schmitz et al, 1997;Rust et al, 2009;Minamino et al, 2016a,b;Terahara et al, 2018) as well as FlhF, considered a signal recognition particle (SRP)like protein important for flagellar localization (Bange et al, 2007;Kazmierczak and Hendrixson, 2013;Kondo et al, 2017); additionally the TCSs FlgS-FlgR and ArsS-ArsR are involved (Pflock et al, 2006;Loh et al, 2010;Marcus et al, 2016;Xiong et al, 2019).…”

Section: Protein-protein Interaction Sensing In Flagellar Assembly Rementioning

confidence: 99%

Protein Activity Sensing in Bacteria in Regulating Metabolism and Motility

2020

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Bacteria have evolved complex sensing and signaling systems to react to their changing environments, most of which are present in all domains of life. Canonical bacterial sensing and signaling modules, such as membrane-bound ligand-binding receptors and kinases, are very well described. However, there are distinct sensing mechanisms in bacteria that are less studied. For instance, the sensing of internal or external cues can also be mediated by changes in protein conformation, which can either be implicated in enzymatic reactions, transport channel formation or other important cellular functions. These activities can then feed into pathways of characterized kinases, which translocate the information to the DNA or other response units. This type of bacterial sensory activity has previously been termed protein activity sensing. In this review, we highlight the recent findings about this non-canonical sensory mechanism, as well as its involvement in metabolic functions and bacterial motility. Additionally, we explore some of the specific proteins and protein-protein interactions that mediate protein activity sensing and their downstream effects. The complex sensory activities covered in this review are important for bacterial navigation and gene regulation in their dynamic environment, be it host-associated, in microbial communities or free-living.

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Insight into structural remodeling of the FlhA ring responsible for bacterial flagellar type III protein export

Abstract: Cooperative remodeling of the FlhA ring terminates hook assembly and initiates filament assembly at the hook tip.

Cited by 53 publications

References 45 publications

Mutational analysis of the C‐terminal cytoplasmic domain of FlhB, a transmembrane component of the flagellar type III protein export apparatus in Salmonella

Mutational analysis of the C‐terminal cytoplasmic domain of FlhB, a transmembrane component of the flagellar type III protein export apparatus in Salmonella

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

Protein Activity Sensing in Bacteria in Regulating Metabolism and Motility

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