Autoinhibitory Regulation of TrwK, an Essential VirB4 ATPase in Type IV Secretion Systems

Peña, Alejandro; Ripoll‐Rozada, Jorge; Zunzunegui, Sandra; Cabezón, Elena; Cruz, Fernando de la; Aréchaga, Ignacio

doi:10.1074/jbc.m110.208942

Cited by 18 publications

(20 citation statements)

References 55 publications

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“…First, we explored the putative interactions between TrwD and TrwK, as there is evidence for a partnership between these proteins during pilus morphogenesis (15). In this work, we demonstrate that the formation of TrwK-TrwD complexes is not dependent on ATP hydrolysis and that the regulatory C-terminal domain of TrwK (27) is not involved in these protein-protein interactions. However, truncated variants of TrwD, including either the NTD or the CTD, were unable to interact with TrwK, suggesting that the linker region between both domains plays an essential role in these interactions.…”

Section: Discussionmentioning

confidence: 74%

“…Protein samples eluted from the HisTrap column were run in an SDS-polyacrylamide gel, transferred to a nitrocellulose membrane, and incubated with anti-TrwK (27) or anti-TrwD rabbit antiserum (8). Images were obtained after incubation with an IRDye anti-rabbit IgG (goat) antibody conjugate (Thermo Scientific), using an Odyssey scanner (LI-COR Biosciences).…”

Section: Methodsmentioning

confidence: 99%

“…Recently, we have shown that the C-terminal end of TrwK plays a regulatory role on the ATPase activity of the protein, since removal of the last ␣ helix of TrwK induced a large increase in ATP turnover relative to that for wild-type TrwK (27). This autoinhibitory region prevents futile ATP hydrolysis, suggesting that interaction with specific partners of the transport machinery could stimulate TrwK ATPase activity.…”

Section: Interactions Between Trwd and Trwkmentioning

confidence: 99%

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Functional Interactions of VirB11 Traffic ATPases with VirB4 and VirD4 Molecular Motors in Type IV Secretion Systems

Ripoll‐Rozada

Zunzunegui

Cruz

et al. 2013

Journal of Bacteriology

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Pilus biogenesis and substrate transport by type IV secretion systems require energy, which is provided by three molecular motors localized at the base of the secretion channel. One of these motors, VirB11, belongs to the superfamily of traffic ATPases, which includes members of the type II secretion system and the type IV pilus and archaeal flagellar assembly apparatus. Here, we report the functional interactions between TrwD, the VirB11 homolog of the conjugative plasmid R388, and TrwK and TrwB, the motors involved in pilus biogenesis and DNA transport, respectively. Although these interactions remained standing upon replacement of the traffic ATPase by a homolog from a phylogenetically related conjugative system, namely, TraG of plasmid pKM101, this homolog could not replace the TrwD function for DNA transfer. This result suggests that VirB11 works as a switch between pilus biogenesis and DNA transport and reinforces a mechanistic model in which VirB11 proteins act as traffic ATPases by regulating both events in type IV secretion systems. Type IV secretion systems (T4SSs) export proteins and virulence effectors to other bacteria and eukaryotic cells (1-3). They are also responsible for genetic exchange between bacteria during conjugation (4, 5). T4SSs are large macromolecular assemblies formed by 12 different protein subunits, named VirB1 to VirB11 and VirD4, following the Agrobacterium tumefaciens T4SS nomenclature. Three of these proteins (VirD4, VirB4, and VirB11) are hexameric ATPases (6-8) that provide the energy for substrate transport and T4SS biogenesis.VirB11 proteins are traffic ATPases that belong to a large AAA ϩ secretion protein superfamily, which also includes proteins of type II secretion systems and the type IV pilus biogenesis and archaeal flagellar assembly machineries (9). All of them are soluble, hexameric proteins located at the cytoplasmic side of the inner membrane. The monomer is characterized by the presence of an N-terminal domain (NTD) and a C-terminal domain (CTD), which are connected by a flexible linker that plays a key role in the catalytic cycle (10-12). Comparison of the crystal structures of VirB11 from Brucella suis (13) and its homolog in Helicobacter pylori, HP0525 (10,14), revealed that this linker is responsible for a large domain swap of the NTD over the CTD without affecting the hexameric assembly (13).VirB11 was reported to assist VirB4 during pilus biogenesis by dislocating pilin subunits from the inner membrane to the periplasmic space, thus promoting pilus polymerization (15). VirB4 proteins are the largest and most conserved components of T4SSs. TrwK, the VirB4 homolog of plasmid R388, consists of a hexameric double ring with a barrel-shaped structure (16). The atomic structure of the C-terminal domain of the VirB4 homolog in Thermoanaerobacter pseudethanolicus was recently obtained (17), revealing a striking structural similarity with TrwB, the VirD4 homolog of the R388 plasmid. TrwB was extensively characterized as a DNA-dependent ATPase (6, 18), playing an ess...

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Section: Discussionmentioning

confidence: 74%

Section: Methodsmentioning

confidence: 99%

Section: Interactions Between Trwd and Trwkmentioning

confidence: 99%

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Functional Interactions of VirB11 Traffic ATPases with VirB4 and VirD4 Molecular Motors in Type IV Secretion Systems

Ripoll‐Rozada

Zunzunegui

Cruz

et al. 2013

Journal of Bacteriology

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“…The structure is very similar to the computer-generated models on the basis of TrwB (14,15). A negative stain model of a complex formed by a VirB4 monomer bound to the core subunits VirB7/ VirB9/VirB10 of plasmid pKM101 has also been reported (21).…”

mentioning

confidence: 60%

“…On the basis of computer predictions, molecular models of the CTD have been proposed (14,15). These models have been built using the structure of TrwB, the coupling protein of conjugative R388, as a template.…”

mentioning

confidence: 99%

The Hexameric Structure of a Conjugative VirB4 Protein ATPase Provides New Insights for a Functional and Phylogenetic Relationship with DNA Translocases

Peña

Matilla

Martín‐Benito

et al. 2012

Journal of Biological Chemistry

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Background: VirB4 ATPases are involved in protein transport in T4SS. Results: The structure of the conjugative VirB4 homologue TrwK has been determined by single-particle electron microscopy. Conclusion: TrwK forms hexamers and binds preferentially G4-quadruplex DNA as the coupling protein TrwB. Significance: The results provide structural and biochemical evidence for a common evolutionary scenario between DNA and protein translocases.

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Spectrophotometric Assays to Quantify the Activity of T4SS ATPases

Cabezón

Aréchaga

2019

Horizontal Gene Transfer

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Autoinhibitory Regulation of TrwK, an Essential VirB4 ATPase in Type IV Secretion Systems

Cited by 18 publications

References 55 publications

Functional Interactions of VirB11 Traffic ATPases with VirB4 and VirD4 Molecular Motors in Type IV Secretion Systems

Functional Interactions of VirB11 Traffic ATPases with VirB4 and VirD4 Molecular Motors in Type IV Secretion Systems

The Hexameric Structure of a Conjugative VirB4 Protein ATPase Provides New Insights for a Functional and Phylogenetic Relationship with DNA Translocases

Spectrophotometric Assays to Quantify the Activity of T4SS ATPases

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