The traffic ATPase PilF interacts with the inner membrane platform of the DNA translocator and type IV pili from <i>Thermus thermophilus</i>

Kruse, Kerstin; Salzer, Ralf; Averhoff, Beate

doi:10.1002/2211-5463.12548

Cited by 13 publications

(6 citation statements)

References 64 publications

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“…Furthermore, the addition of ATPγS results in the appearance of a minor higher molecular weight peak with a large molecular weight distribution (between 350 and 600 kDa), indicative of larger aggregates (Fig 5D). These observations are consistent with previous reports on isolated PilB homologs from other systems that were observed as monomers, dimers and hexamers [12,[39][40][41][42][43][44][45].…”

Section: Full-length Pilb-pilz-fimx Complexessupporting

confidence: 93%

“…Since the ND0/ND1 and ND2 domains are connected by what is expected to be a negatively charged flexible linker (see below), the ND0/ND1 domain is expected to be free to make contacts with interaction partners in the inner membrane platform. One strong candidate to mediate such interactions is PilM since interactions between PilB and PilM homologs have been observed for T4P of M. xanthus [54], T. thermophilus [41], P. aeruginosa [55], the bundle-forming pilus machinery of enteropathogenic E. coli [56] and the T2SS of X. campestris [35]. Furthermore, complexes between PilB and PilM homologs in T2SS have been crystallized: full-length ATPase GspE with the cytosolic domain of GspL from V. vulnificus [52] and EpsE ND1 domain with the cytosolic domain of EpsL from V. cholerae [57].…”

Section: Plos Pathogensmentioning

confidence: 99%

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The PilB-PilZ-FimX regulatory complex of the Type IV pilus from Xanthomonas citri

et al. 2021

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Type IV pili (T4P) are thin and flexible filaments found on the surface of a wide range of Gram-negative bacteria that undergo cycles of extension and retraction and participate in a variety of important functions related to lifestyle, defense and pathogenesis. During pilus extensions, the PilB ATPase energizes the polymerization of pilin monomers from the inner membrane. In Xanthomonas citri, two cytosolic proteins, PilZ and the c-di-GMP receptor FimX, are involved in the regulation of T4P biogenesis through interactions with PilB. In vivo fluorescence microscopy studies show that PilB, PilZ and FimX all colocalize to the leading poles of X. citri cells during twitching motility and that this colocalization is dependent on the presence of all three proteins. We demonstrate that full-length PilB, PilZ and FimX can interact to form a stable complex as can PilB N-terminal, PilZ and FimX C-terminal fragments. We present the crystal structures of two binary complexes: i) that of the PilB N-terminal domain, encompassing sub-domains ND0 and ND1, bound to PilZ and ii) PilZ bound to the FimX EAL domain within a larger fragment containing both GGDEF and EAL domains. Evaluation of PilZ interactions with PilB and the FimX EAL domain in these and previously published structures, in conjunction with mutagenesis studies and functional assays, allow us to propose an internally consistent model for the PilB-PilZ-FimX complex and its interactions with the PilM-PilN complex in the context of the inner membrane platform of the X. citri Type IV pilus.

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Section: Full-length Pilb-pilz-fimx Complexessupporting

confidence: 93%

Section: Plos Pathogensmentioning

confidence: 99%

The PilB-PilZ-FimX regulatory complex of the Type IV pilus from Xanthomonas citri

et al. 2021

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“…Filament assembly is ATP-dependent and occurs at an inner membrane platform which contains PilC, PilM, PilN and PilO 6 . In Thermus thermophilus, assembly of pilins into a T4P filament depends on the assembly ATPase PilF, which interacts with the inner membrane platform via PilM 7 . Two retraction ATPases, PilT1 and PilT2, are essential for T4P depolymerisation 8,9 .…”

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confidence: 99%

Cryo-electron microscopy reveals two distinct type IV pili assembled by the same bacterium

et al. 2020

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Type IV pili are flexible filaments on the surface of bacteria, consisting of a helical assembly of pilin proteins. They are involved in bacterial motility (twitching), surface adhesion, biofilm formation and DNA uptake (natural transformation). Here, we use cryo-electron microscopy and mass spectrometry to show that the bacterium Thermus thermophilus produces two forms of type IV pilus ('wide' and 'narrow'), differing in structure and protein composition. Wide pili are composed of the major pilin PilA4, while narrow pili are composed of a so-far uncharacterized pilin which we name PilA5. Functional experiments indicate that PilA4 is required for natural transformation, while PilA5 is important for twitching motility.

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“…Most of these proteins form a stable supramolecular complex that spans the bacterial envelope from the cytoplasm to the outer membrane [17]. PilB is proposed to dock with this stable T4PM complex on the cytoplasmic side and hydrolyze ATP to power the assembly of pilin monomers into the base of a growing T4P filament [17,20,21]. PilB and all proteins in this stable complex are required for T4P assembly and deletions of their genes result in a non-piliated and non-motile phenotype [10,18].…”

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confidence: 99%

Cyclic-di-GMP and ADP bind to separate domains of PilB as mutual allosteric effectors

Dye

Yang

2020

Biochemical Journal

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PilB is the assembly ATPase for the bacterial type IV pilus (T4P), and as a consequence, it is essential for T4P-mediated bacterial motility. In some cases, PilB has been demonstrated to regulate the production of exopolysaccharide (EPS) during bacterial biofilm development independently of or in addition to its function in pilus assembly. While the ATPase activity of PilB resides at its C-terminal region, the N terminus of a subset of PilBs forms a novel cyclic-di-GMP (cdG)-binding domain. This multi-domain structure suggests that PilB binds cdG and adenine nucleotides through separate domains which may influence the functionality of PilB in both motility and biofilm development. Here, Chloracidobacterium thermophilum PilB is used to investigate ligand binding by its separate domains and by the full-length protein. Our results confirm the specificity of these individual domains for their respective ligands and demonstrate communications between these domains in the full-length protein. It is clear that when the N-and the C-terminal domains of PilB bind to cdG and ADP, respectively, they mutually influence each other in conformation and in their binding to ligands. We propose that the interactions between these domains in response to their ligands play critical roles in modulating or controlling the functions of PilB as a regulator of EPS production and as the T4P assembly ATPase.The type IV pilus machinery (T4PM) in Gram-negative bacteria is made of a dozen T4P or Pil proteins including the T4P assembly ATPase PilB (or PilF in T. thermophilus and a few others) [10,[17][18][19]. Most of these proteins form a stable supramolecular complex that spans the bacterial envelope from the cytoplasm to the outer membrane [17]. PilB is proposed to dock with this stable T4PM complex on the cytoplasmic side and hydrolyze ATP to power the assembly of pilin monomers into the base of a growing T4P filament [17,20,21]. PilB and all proteins in this stable complex are required for T4P assembly and deletions of their genes result in a non-piliated and non-motile phenotype [10,18]. The C-terminus of PilB contains the structure of an AAA+ ATPase which crystallizes as a hexamer [22][23][24][25]. Based on the hexameric structures of these ATPase domains, it is proposed that PilB utilizes a symmetric rotary mechanism to hydrolyze ATP [23][24][25][26]. That is, two ATP molecules are hydrolyzed to ADP simultaneously by two protomers at the opposite corners of the hexameric ring. Another pair adjacent to these two would hydrolyze ATP next. It is proposed that the hydrolysis of ATP by the C-terminal ATPase domain of PilB results in the movement of the N-terminal domain which is mechanically coupled to the insertion of pilins into the pilus through other components in the T4PM [22]. More recently, the full-length PilB of Chloracidobacterium thermophilum (CtPilB) was shown to be a hexamer in solution with robust ATPase activity in vitro [27]. All available evidence supports the model that the ATPase activity of PilB endowed by its C terminus ...

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The traffic ATPase PilF interacts with the inner membrane platform of the DNA translocator and type IV pili from Thermus thermophilus

Cited by 13 publications

References 64 publications

The PilB-PilZ-FimX regulatory complex of the Type IV pilus from Xanthomonas citri

The PilB-PilZ-FimX regulatory complex of the Type IV pilus from Xanthomonas citri

Cryo-electron microscopy reveals two distinct type IV pili assembled by the same bacterium

Cyclic-di-GMP and ADP bind to separate domains of PilB as mutual allosteric effectors

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