The ATPase activity of the DNA transporter TrwB is modulated by protein TrwA: implications for a common assembly mechanism of DNA translocating motors

Conjugative systems contain an essential integral membrane protein involved in DNA transport called the Type IV coupling protein (T4CP). The T4CP of conjugative plasmid R388 is TrwB, a DNA-dependent ATPase. Biochemical and structural data suggest that TrwB uses energy released from ATP hydrolysis to pump DNA through its central channel by a mechanism similar to that used by F1-ATPase or ring helicases. For DNA transport, TrwB couples the relaxosome (a DNA-protein complex) to the secretion channel. In this work we show that TrwA, a tetrameric oriT DNA-binding protein and a component of the R388 relaxosome, stimulates TrwBDeltaN70 ATPase activity, revealing a specific interaction between the two proteins. This interaction occurs via the TrwA C-terminal domain. A 68-kDa complex between TrwBDeltaN70 and TrwA C-terminal domain was observed by gel filtration chromatography, consistent with a 1:1 stoichiometry. Additionally, electron microscopy revealed the formation of oligomeric TrwB complexes in the presence, but not in the absence, of TrwA protein. TrwBDeltaN70 ATPase activity in the presence of TrwA was further enhanced by DNA. Interestingly, maximal ATPase rates were achieved with TrwA and different types of dsDNA substrates. This is consistent with a role of TrwA in facilitating the interaction between TrwB and DNA. Our findings provide a new insight into the mechanism by which TrwB recruits the relaxosome for DNA transport. The process resembles the mechanism used by other DNA-dependent molecular motors, such as the RuvA/RuvB system, to be targeted to the DNA followed by hexamer assembly

The C-terminal domain of TrwK, the VirB4 protein of the conjugative plasmid R388, displays a critical regulatory function

Trabajo presentado al "35th FEBS Congress: Molecules of Life" celebrado en Gotemburgo (Suecia) del 26 de junio al 1 de julio de 2010.Type IV secretion systems (T4SS) translocate DNA and protein substrates across the cell envelope of bacteria to a widely distributed number of eukaryotic and prokaryotic target cells. Conjugative bacteria use T4SS to mediate the transfer of DNA and proteins to recipient cells. T4SS are macromolecular assemblies composed of 11 mating pair subunits (VirB1 to VirB11) and a coupling protein (VirD4) that span inner and outer bacterial membranes. Three of these subunits are ATPases that energize DNA and protein substrate transfer as well as pilus assembly. VirB4 proteins are the largest and most evolutionarily conserved proteins in T4SS and are essential for virulence and plasmid transfer.VirB4 proteins have been suggested to energize substrate translocation across the T4SS. Recently, we demonstrated that TrwK, the VirB4 homologue in the R388 conjugative system, is able to hydrolyze ATP in the absence of potential substrates. The atomic structure of VirB4 proteins is unknown. Based on computing predictions, a model of the C-terminal (residues 413 to 772) of Escherichia coli TrwK has been suggested using the atomic coordinates of the coupling protein TrwB of plasmid R388 as a template. Secondary structure predictions of TrwK and TrwB revealed the presence of three α-helices in the C-terminus that are conserved in all VirB4 proteins but are absent in TrwB. Therefore, we decided to generate truncated variants of TrwK where these α-helical structures were sequentially removed and we have analyzed their in vitro and in vivo properties. The results suggest that the C-terminal end of VirB4 proteins could play a key functional regulatory role in the biological activity of T4SS.Peer Reviewe

Functional interactions of VirB11 traffic ATPases with VirB4 and VirD4 molecular motors in type IV secretion systems

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. © 2013, American Society for Microbiology.This work was supported by Ministerio de Economía y Competitividad (MINECO, Spain) grants BFU2011-22874 (to E.C. and I.A.) and BFU2011-26608 (to F.D.L.C.) and by European VII Framework Program grants 248919/FP7-ICT-2009-4 and 282004/FP7-HEALTH.2011.2.3.1-2 (to F.D.L.C.). J.R.-R. was supported by a fellowship from the University of Cantabria.Peer Reviewe

The ATPase activity of the DNA transporter TrwB is modulated by protein TrwA. Implications for a common assembly mechanism of DNA translocating motors

Conjugative systems contain an essential integral membrane protein involved in DNA transport called the Type IV coupling protein (T4CP). The T4CP of conjugative plasmid R388 is TrwB, a DNA-dependent ATPase. Biochemical and structural data suggest that TrwB uses energy released from ATP hydrolysis to pump DNA through its central channel by a mechanism similar to that used by F1-ATPase or ring helicases. For DNA transport, TrwB couples the relaxosome (a DNA-protein complex) to the secretion channel. In this work we show that TrwA, a tetrameric oriT DNA-binding protein and a component of the R388 relaxosome, stimulates TrwBΔN70 ATPase activity, revealing a specific interaction between the two proteins. This interaction occurs via the TrwA C-terminal domain. A 68-kDa complex between TrwBΔN70 and TrwA C-terminal domain was observed by gel filtration chromatography, consistent with a 1:1 stoichiometry. Additionally, electron microscopy revealed the formation of oligomeric TrwB complexes in the presence, but not in the absence, of TrwA protein. TrwBΔN70 ATPase activity in the presence of TrwA was further enhanced by DNA. Interestingly, maximal ATPase rates were achieved with TrwA and different types of dsDNA substrates. This is consistent with a role of TrwA in facilitating the interaction between TrwB and DNA. Our findings provide a new insight into the mechanism by which TrwB recruits the relaxosome for DNA transport. The process resembles the mechanism used by other DNA-dependent molecular motors, such as the RuvA/RuvB system, to be targeted to the DNA followed by hexamer assembly. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.This work was supported in part by Grant BFU2005-02718 (Spanish Ministry of Education) (to E. C.) and Grants BFU2005-03477/BMC (Spanish Ministry of Education) and LSHM-CT-2005_019023 (European VI Framework Program) (to F. C.).Peer Reviewe

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

Type IV secretion systems (T4SS) mediate the transfer of DNA and protein substrates to target cells. TrwK, encoded by the conjugative plasmid R388, is a member of the VirB4 family, comprising the largest and most conserved proteins of T4SS. In a previous work we demonstrated that TrwK is able to hydrolyze ATP. Here, based on the structural homology of VirB4 proteins with the DNA-pumping ATPase TrwB coupling protein, we generated a series of variants of TrwK where fragments of the C-terminal domain were sequentially truncated. Surprisingly, the in vitro ATPase activity of these TrwK variants was much higher than that of the wild-type enzyme. Moreover, addition of a synthetic peptide containing the amino acid residues comprising this C-terminal region resulted in the specific inhibition of the TrwK variants lacking such domain. These results indicate that the C-terminal end of TrwK plays an important regulatory role in the functioning of the T4SS

Analysis of the function of Sp6 and Sp8 transcription factors in the limb ectoderm

Resumen del póster presentado al XXXIX Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Salamanca del 5 al 8 de septiembre de 2016.We have recently identified the crucial role played by Sp6 and Sp8, two transcription factors members of the Sp family, during limb development. In their absence or substantial reduction no limbs form and their progressive dose reduction associates a spectrum of limb malformations ranking from syndactyly through Split-hand/split-foot malformation, to olygodactyly. When digits form, they exhibit bidorsal tips. We have postulated that Sp6 and Sp8 are necessary, downstream of Wnt/B-catenin, to activate Fgf8 and downstream of Bmp signaling, possible cooperating with Smads, to activate En1, the marker of the ventral ectoderm. Currently we are exploring these suspected protein-protein interactions by co-immunoprecipitation (CoIP) and by bimolecular fluorescence complementation (BiFC). To this end, Sp6, Sp8 and Smads were tagged with Myc or FLAG epitopes to their N-terminal end and with YFP full length or YFP moieties to their C-terminal end. We show by CoIP and BiFC that Sp8 homodimerizes. To determine the region involved in Sp8 interaction, we have generated different truncated versions of Sp8. Our results show that Sp8 homodimerizes at its C-terminal domain. Our study underscores the relevance of Sp6/8 in mediating B-catenin and Bmp signaling pathways in the limb ectoderm.Peer Reviewe

Analysis of the mechanism underlying the function of Sp6 and Sp8 in the limb ectoderm

Resumen del póster presentado al 11th Meeting Spanish Society for Developmental Biology, celebrado en Girona del 19 al 21 de octubre de 2016.We have recently shown that the transcription factors Sp6 and Sp8 are conjointly necessary for limb development as no limbs form in their absence or substantial reduction. The study of the allelic series of double Sp6;Sp8 mutant also revealed that mice with only a functional copy of Sp8 exhibit the Split-hand/split-foot malformation. Based on the mo- lecular study of mutant limbs, we have postulated that Sp6 and Sp8 are necessary in the limb ectoderm, downstream of Wnt/ß-catenin, to activate Fgf8 and downstream of Bmp signaling, possibly cooperating with Smads for dorso-ven- tral patterning. Here we have used co-immunoprecipitation (CoIP) and bimolecular fluorescence complementation (BiFC) to explore these suspected protein-protein interactions. To this end, Sp6, Sp8 and Smads were tagged with Myc or Flag epitopes to their N-terminal end and with YFP full length or YFP moieties to their C-terminal end. By CoIP and BiFC we show that Sp6 and Sp8 can homo and heterodimerize and that they can also interact with Smads. Finally, by generating different truncated versions of Sp8 we resolve the protein regions responsible of these interaction. Our study underscores the mechanisms by which Sp6/8 meditare Wnt/ß -catenin and Bmp signaling in the limb ectoderm.Peer Reviewe

Functional Dissection of the Conjugative Coupling Protein TrwB▿

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 TrwBΔN70, 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

Analysis of Sp6 and Sp8 protein-protein interactions and DNA binding during limb development

Resumen del póster presentado al Experimental Biology Meeting, celebrado en Chicago (US) del 22 al 26 de abril de 2017.Sp6 and Sp8, two members of the specificity protein (Sp) family of zinc-finger transcription factors, are expressed in the limb ectoderm. Their importance in limb development was demonstrated by the analysis of double null mutants that exhibited a range of limb malformations that correlated with the dose of Sp6/Sp8 gene products remained (Haro et al., 2014). Thus, the progressive reduction led to predictable morphologies that transit from syndactyly, to split-hand/foot malformation phenotype, oligodactyly, truncation and finally amelia. In addition, when digits form these show a double dorsal tip. The molecular study of these mutants revealed that Sp6 and Sp8 work together downstream to Wnt/bcatenin to activate Fgf8 and downstream of Bmp signaling, possible cooperating with Smads, to activate En1, the ventral determinant in limb patterning. Here, to further the understanding of Sp6/8 function, we have investigated Sp6/8 suspected protein-protein interactions by co-immunoprecipitation (CoIP) and by bimolecular fluorescence complementation (BiFC). To this end, Sp6, Sp8 and Smads were tagged with Myc or FLAG epitopes to their N-terminal end and with YFP full length or YFP moieties to their C-terminal end. Our results show that Sp6/8 form homo and heterodimers and that they form complexes with BMP R-Smads. All these interactions require the C-terminal domain of Sps. We are currently determining Sp6/8 DNA binding activity by luciferase reporter and EMSA assays.Grant BFU2014-57216-P to MAR from the Spanish Government.Peer Reviewe

ATPase Activity and Oligomeric State of TrwK, the VirB4 Homologue of the Plasmid R388 Type IV Secretion System▿ †

Type IV secretion systems (T4SS) mediate the transfer of DNA and protein substrates to target cells. TrwK, encoded by the conjugative plasmid R388, is a member of the VirB4 family, comprising the largest and most conserved proteins of T4SS. VirB4 was suggested to be an ATPase involved in energizing pilus assembly and substrate transport. However, conflicting experimental evidence concerning VirB4 ATP hydrolase activity was reported. Here, we demonstrate that TrwK is able to hydrolyze ATP in vitro in the absence of its potential macromolecular substrates and other T4SS components. The kinetic parameters of its ATPase activity have been characterized. The TrwK oligomerization state was investigated by analytical ultracentrifugation and electron microscopy, and its effects on ATPase activity were analyzed. The results suggest that the hexameric form of TrwK is the catalytically active state, much like the structurally related protein TrwB, the conjugative coupling protein