The transmembrane domain provides nucleotide binding specificity to the bacterial conjugation protein TrwB

Hormaeche, Itsaso; Segura, Rosa L.; Vecino, Ana J.; Goñi, Félix M.; Cruz, Fernando de la; Alkorta, Itziar

doi:10.1016/j.febslet.2006.04.059

Cited by 24 publications

(19 citation statements)

References 39 publications

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“…Instead, transporter components or conditions particular to the membrane environment missing in our analysis may be additionally important to functional assembly of TraD and possibly other T4CPs. 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).…”

Section: Discussionmentioning

confidence: 59%

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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Section: Discussionmentioning

confidence: 59%

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

et al. 2009

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“…This result is consistent with the failure of D⌬2-134, Di⌬7-134, and G-D to exhibit conjugative dominance (Table 2 and data not shown). The importance of the Nterminal domains is further attested by previous studies showing that the N-terminally-truncated CP TrwB⌬N70 has altered stability and nucleotide-binding ability (23,24) and that both TrwB⌬N70 and N-terminally-truncated mutants of RP4 TraG exhibit monomeric behavior in vitro (22,47). The K6 and I59 mutants reveal that the N-terminal region of TraD is also important for assembly beyond the dimer stage: these mutants bound wild-type TraD and formed homodimers by cross-linking but did not form the larger cross-linked structures observed for TraD and TraDiN702 even when the F plasmid was present.…”

Section: Discussionmentioning

confidence: 97%

In Vivo Oligomerization of the F Conjugative Coupling Protein TraD

et al. 2007

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Type IV secretory systems are a group of bacterial transporters responsible for the transport of proteins and nucleic acids directly into recipient cells. Such systems play key roles in the virulence of some pathogenic organisms and in conjugation-mediated horizontal gene transfer. Many type IV systems require conserved "coupling proteins," transmembrane polypeptides that are critical for transporting secreted substrates across the cytoplasmic membrane of the bacterium. In vitro evidence suggests that the functional form of coupling proteins is a homohexameric, ring-shaped complex. Using a library of tagged mutants, we investigated the structural and functional organization of the F plasmid conjugative coupling protein TraD by coimmunoprecipitation, cross-linking, and genetic means. We present direct evidence that coupling proteins form stable oligomeric complexes in the membranes of bacteria and that the formation of some of these complexes requires other F-encoded functions. Our data also show that different regions of TraD play distinct roles in the oligomerization process. We postulate a model for in vivo oligomerization and discuss the probable participation of individual domains of TraD in each step.

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“…The biological relevance of these findings remains to be evaluated, but it should be noted that several studies have identified functions of the various T4CP domains that could contribute-directly or indirectly-to substrate binding. The NTDs, for example, are unlikely to directly bind substrates, but studies have identified several NTD functions, including binding interactions with other channel subunits, stimulatory effects on T4CP oligomerization and catalytic activity, and contributions to T4CP spatial positioning in the cell (17,(64)(65)(66)(67), that could indirectly affect substrate docking. By virtue of their possible accessibility to secretion substrates in the cytoplasm, the NBDs and CTDs might coordinate with the AAD to recruit substrates through direct substrate engagement.…”

Section: Discussionmentioning

confidence: 99%

The All-Alpha Domains of Coupling Proteins from the Agrobacterium tumefaciens VirB/VirD4 and Enterococcus faecalis pCF10-Encoded Type IV Secretion Systems Confer Specificity to Binding of Cognate DNA Substrates

et al. 2015

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Bacterial type IV coupling proteins (T4CPs) bind and mediate the delivery of DNA substrates through associated type IV secretion systems (T4SSs). T4CPs consist of a transmembrane domain, a conserved nucleotide-binding domain (NBD), and a sequence-variable helical bundle called the all-alpha domain (AAD). In the T4CP structural prototype, plasmid R388-encoded TrwB, the NBD assembles as a homohexamer resembling RecA and DNA ring helicases, and the AAD, which sits at the channel entrance of the homohexamer, is structurally similar to N-terminal domain 1 of recombinase XerD. Here, we defined the contributions of AADs from the Agrobacterium tumefaciens VirD4 and Enterococcus faecalis PcfC T4CPs to DNA substrate binding. AAD deletions abolished DNA transfer, whereas production of the AAD in otherwise wild-type donor strains diminished the transfer of cognate but not heterologous substrates. Reciprocal swaps of AADs between PcfC and VirD4 abolished the transfer of cognate DNA substrates, although strikingly, the VirD4-AAD PcfC chimera (VirD4 with the PcfC AAD) supported the transfer of a mobilizable plasmid. Purified AADs from both T4CPs bound DNA substrates without sequence preference but specifically bound cognate processing proteins required for cleavage at origin-of-transfer sequences. The soluble domains of VirD4 and PcfC lacking their AADs neither exerted negative dominance in vivo nor specifically bound cognate processing proteins in vitro. Our findings support a model in which the T4CP AADs contribute to DNA substrate selection through binding of associated processing proteins. Furthermore, MOBQ plasmids have evolved a docking mechanism that bypasses the AAD substrate discrimination checkpoint, which might account for their capacity to promiscuously transfer through many different T4SSs. IMPORTANCEFor conjugative transfer of mobile DNA elements, members of the VirD4/TraG/TrwB receptor superfamily bind cognate DNA substrates through mechanisms that are largely undefined. Here, we supply genetic and biochemical evidence that a helical bundle, designated the all-alpha domain (AAD), of T4SS receptors functions as a substrate specificity determinant. We show that AADs from two substrate receptors, Agrobacterium tumefaciens VirD4 and Enterococcus faecalis PcfC, bind DNA without sequence or strand preference but specifically bind the cognate relaxases responsible for nicking and piloting the transferred strand through the T4SS. We propose that interactions of receptor AADs with DNA-processing factors constitute a basis for selective coupling of mobile DNA elements with type IV secretion channels. Bacterial type IV secretion systems (T4SSs) translocate DNA and protein substrates to target cells, generally by a mechanism requiring direct cell-to-cell contact (1). These systems are phylogenetically widely distributed among many Gram-negative and -positive bacterial species (2, 3). In both cell types, the T4SSs function as conjugation machines by delivering DNA substrates to bacterial recipient cells (3-5). Many G...

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The transmembrane domain provides nucleotide binding specificity to the bacterial conjugation protein TrwB

Cited by 24 publications

References 39 publications

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

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

In Vivo Oligomerization of the F Conjugative Coupling Protein TraD

The All-Alpha Domains of Coupling Proteins from the Agrobacterium tumefaciens VirB/VirD4 and Enterococcus faecalis pCF10-Encoded Type IV Secretion Systems Confer Specificity to Binding of Cognate DNA Substrates

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