In Vivo Oligomerization of the F Conjugative Coupling Protein TraD

Haft, Rembrandt J. F.; Gachelet, Eliora; Nguyen, Tran; Toussaint, L. Gerard; Chivian, Dylan; Traxler, Beth

doi:10.1128/jb.00513-07

Cited by 19 publications

(22 citation statements)

References 50 publications

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“…Thus, in addition to the N-terminal region, the C-terminal segment and the NBD region of TrwB were involved in TrwB-TrwB interactions. Similar results were obtained with the T4CPs TraD of the F plasmid and TcpA of plasmid pCW3: the TMD is important for oligomerization, together with another segment of the cytoplasmic domain (23,50). It was suggested that the N-terminal domain of TraD is required for the interaction of the T4CP with the rest of the conjugal machinery, while the region of the soluble domain would only be required for self-interactions.…”

Section: Discussionsupporting

confidence: 70%

“…The cytoplasmic domain of the related TraD protein of plasmid R1 stimulates both transesterase and helicase activities of its cognate relaxase, TraI (41,51). A series of random mutations were shown to affect TraD oligomerization (23). In VirD4, the T4CP of the VirB T4SS of A. tumefaciens, both the periplasmic domain plus key residues of the NBD are required for its location at the cell poles (31); its interaction with the T4SS protein substrate VirE2 does not require the N-terminal TMD (2).…”

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confidence: 99%

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Functional Dissection of the Conjugative Coupling Protein TrwB

et al. 2010

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The conjugative coupling protein TrwB is responsible for connecting the relaxosome to the type IV secretion system during conjugative DNA transfer of plasmid R388. It is directly involved in transport of the relaxase TrwC, and it displays an ATPase activity probably involved in DNA pumping. We designed a conjugation assay in which the frequency of DNA transfer is directly proportional to the amount of TrwB. A collection of point mutants was constructed in the TrwB cytoplasmic domain on the basis of the crystal structure of TrwB⌬N70, targeting the nucleotide triphosphate (NTP)-binding region, the cytoplasmic surface, or the internal channel in the hexamer. An additional set of transfer-deficient mutants was obtained by random mutagenesis. Most mutants were impaired in both DNA and protein transport. We found that the integrity of the nucleotide binding domain is absolutely required for TrwB function, which is also involved in monomer-monomer interactions. Polar residues surrounding the entrance and inside the internal channel were important for TrwB function and may be involved in interactions with the relaxosomal components. Finally, the N-terminal transmembrane domain of TrwB was subjected to random mutagenesis followed by a two-hybrid screen for mutants showing enhanced protein-protein interactions with the related TrwE protein of Bartonella tribocorum. Several point mutants were obtained with mutations in the transmembranal helices: specifically, one proline from each protein may be the key residue involved in the interaction of the coupling protein with the type IV secretion apparatus.

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Section: Discussionsupporting

confidence: 70%

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confidence: 99%

Functional Dissection of the Conjugative Coupling Protein TrwB

et al. 2010

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“…In support of the latter hypothesis, the presence of the membrane-spanning domain altered interactions of native TrwB with some ligands in vitro (31). Moreover, Traxler and colleagues have shown that associations between F plasmid TraD monomers in vivo require N-terminal transmembrane sequences and that formation of stable, higher-order TraD oligomers in the inner membrane also appeared to involve other F proteins (28). Accordingly, the model of Fig.…”

Section: Discussionmentioning

confidence: 58%

Plasmid R1 Conjugative DNA Processing Is Regulated at the Coupling Protein Interface

et al. 2009

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Selective substrate uptake controls initiation of macromolecular secretion by type IV secretion systems in gram-negative bacteria. Type IV coupling proteins (T4CPs) are essential, but the molecular mechanisms governing substrate entry to the translocation pathway remain obscure. We report a biochemical approach to reconstitute a regulatory interface between the plasmid R1 T4CP and the nucleoprotein relaxosome dedicated to the initiation stage of plasmid DNA processing and substrate presentation. The predicted cytosolic domain of T4CP TraD was purified in a predominantly monomeric form, and potential regulatory effects of this protein on catalytic activities exhibited by the relaxosome during transfer initiation were analyzed in vitro. Type IV secretion systems (T4SS) in gram-negative bacteria mediate translocation of macromolecules out of the bacterial cell (14). The transmission of effector proteins and DNA into plant cells or other bacteria via cell-cell contact is one example of their function, and conjugation systems as well as the transferred DNA (T-DNA) delivery system of the phytopathogen Agrobacterium tumefaciens are prototypical of the T4SS family. Macromolecular translocation is achieved by a membranespanning protein machinery comprised of 12 gene products, VirB1 to VirB11 and an associated factor known as the coupling protein (VirD4) (66). The T4SS-associated coupling protein (T4CP) performs a crucial function in recognition of appropriate secretion substrates and governing entry of those molecules to the translocation pathway (7,8,10,30,41). In conjugation systems substrate recognition is applied to the relaxosome, a nucleoprotein complex of DNA transfer initiator proteins assembled specifically at the plasmid origin of transfer (oriT). In current models, initiation of the reactions that provide the single strand of plasmid (T-strand) DNA for secretion to recipient bacteria is expected to resemble the initiation of chromosomal replication (for reviews, see references 18, 54, and 81). Controlled opening of the DNA duplex is required to permit entry of the DNA processing machinery. The task of remodeling the conjugative oriT is generally ascribed to two or three relaxosome auxiliary factors, of host and plasmid origin, which occupy specific DNA binding sites at this locus. Intrinsic to the relaxosome is also a site-and strand-specific DNA transesterase activity that breaks the phosphodiester backbone at nic (5). Upon cleavage, the transesterase enzyme (also called relaxase) forms a reversible phosphotyrosyl linkage to the 5Ј end of the DNA. Duplex unwinding initiating from this site produces the single-stranded T strand to be exported. A wealth of information is available supporting the importance of DNA sequence recognition and binding by relaxosome components at oriT to the transesterase reaction in vitro and for effective conjugative transfer (for reviews, see references 18, 54, and 81). On the other hand, the mechanisms controlling release of the 3Ј-OH generated at nic and the subsequent DNA unwind...

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“…TraD stimulates DNA cleavage at nic in combination with full-length TraI via mechanisms not supported by the isolated transesterase domain (41). TraD requires an additional F plasmid protein(s) to form stable multimers in the membrane (23). We propose that TraD is not yet assembled in the hexameric form required for ATP hydrolysis (58,59).…”

Section: Discussionmentioning

confidence: 99%

Protein and DNA Effectors Control the TraI Conjugative Helicase of Plasmid R1

et al. 2009

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Controlled duplex DNA unwinding is a crucial prerequisite for the expression and maintenance of genomes. Genomemanipulating and -regulating proteins are central to that biological function in recognizing appropriate DNA targets at initiation sequences and unwinding the complementary strands to provide single-stranded DNA (ssDNA) templates for nucleic acid synthesis and other processing reactions. The protein machineries involved include nucleic acid helicases. DNA helicases are powerful enzymes that convert the energy of nucleoside triphosphate hydrolysis to directional DNA strand translocation and separation of the double helix into its constituent single strands (for reviews, see references 13, 14, 16, 38, 55, and 64). By necessity, these enzymes interact with DNA strands via mechanisms independent of sequence recognition. At replication initiation helicases gain controlled access to the doublestranded genome at positions determined by the DNA binding properties of initiator proteins that comprise an origin recognition complex (1,9,17,31,45,66). The mechanisms supporting localized unwinding within the complex include initiatorinduced DNA looping, wrapping, and bending and feature regions of low thermodynamic stability. The exposed ssDNA mediates helicase binding followed by directional translocation along that strand until the enzyme engages the duplex for unwinding.In the MOB F family of conjugation systems, the plasmid DNA strand destined for transfer (T strand) is unwound from its complement by a dedicated conjugative helicase, TraI of F-like plasmids or TrwC of the IncW paradigm. These enzymes are remarkable in that the same polypeptides additionally harbor in a distinct domain a DNA transesterase activity. That function is required to recognize and cleave the precise phosphodiester bond, nic, in the T strand where unwinding of the secretion substrate begins. In current models the conjugative helicases are thus targeted to the transfer origin (oriT) of their cognate plasmid by the high-affinity DNA sequence interactions of their N-terminal DNA transesterase domains. In the bacterial cell, recruitment and activation of the conjugative helicase occur not on naked DNA but within an initiator complex called the relaxosome (67). For the F-like plasmid R1, sequence-specific DNA binding properties of the plasmid proteins TraI, TraY, TraM, and the host integration factor (IHF) direct assembly of the relaxosome at oriT (10,12,29,33,51,52). Integration of protein TraM confers recognition features to the relaxosome, which permit its selective docking to TraD, the coupling protein associated with the conjugative type IV secretion system (T4CP) (2,15,49). In current models, the T4CP forms a hexameric translocation pore at the cytoplasmic membrane that not only governs substrate entry to the envelope spanning type IV secretion machinery but also provides energy for macromolecular transport via ATP hydrolysis (36,50). These models propose that T4CPs provide not only a physical bridge between the plasmid and the type IV ...

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In Vivo Oligomerization of the F Conjugative Coupling Protein TraD

Cited by 19 publications

References 50 publications

Functional Dissection of the Conjugative Coupling Protein TrwB

Functional Dissection of the Conjugative Coupling Protein TrwB

Plasmid R1 Conjugative DNA Processing Is Regulated at the Coupling Protein Interface

Protein and DNA Effectors Control the TraI Conjugative Helicase of Plasmid R1

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