Inmaculada Matilla

Universidad de Cantabria, Santander, Cantabria, Spain

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Publications (5)13.95 Total impact

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    ABSTRACT: VirB4 proteins are ATPases essential for pilus biogenesis and protein transport in Type IV secretion systems. These proteins contain a motor domain which shares structural similarities with the motor domains of DNA translocases, such as the VirD4/TrwB conjugative coupling proteins and the chromosome segregation pump FtsK. Here, we report the three dimensional structure of full length TrwK, the VirB4 homologue in the conjugative plasmid R388, determined by single particle electron microscopy. The structure consists of a hexameric double ring with a barrel-shaped structure. Docking the atomic coordinates of the crystal structures of TrwB and FtsK into the EM map revealed a better fit for FtsK. Interestingly, we have found that, likewise TrwB, TrwK is able to bind DNA, with higher affinity for G4 quadruplex structures than for single stranded DNA. Furthermore, TrwK exerts a dominant negative effect on the ATPase activity of TrwB, which reflects an interaction between the two proteins. Our studies provide new insights into the structure-function relationship and the evolution of these DNA and protein translocases.
    Journal of Biological Chemistry 10/2012; · 4.65 Impact Factor
  • Conference Paper: Congress
    International Plasmid Biology Conference, Santander (Spain); 09/2012
  • Conference Paper: Congress
    Biophysical Aspects of Type IV Secretion System., Bilbao (Spain); 09/2012
  • [Show abstract] [Hide abstract]
    ABSTRACT: TrwB is a DNA-dependent ATPase involved in DNA transport during bacterial conjugation. The protein presents structural similarity to hexameric molecular motors such as F(1)-ATPase, FtsK, or ring helicases, suggesting that TrwB also operates as a motor, using energy released from ATP hydrolysis to pump single-stranded DNA through its central channel. In this work, we have carried out an extensive analysis with various DNA substrates to determine the preferred substrate for TrwB. Oligonucleotides with G-rich sequences forming G4 DNA structures were the optimal substrates for TrwB ATPase activity. The protein bound with 100-fold higher affinity to G4 DNA than to single-stranded DNA of the same sequence. Moreover, TrwB formed oligomeric protein complexes only with oligonucleotides presenting such a G-quadruplex DNA structure, consistent with stoichiometry of six TrwB monomers to G4 DNA, as demonstrated by gel filtration chromatography and analytical ultracentrifugation experiments. A protein-DNA complex was also formed with unstructured oligonucleotides, but the molecular mass corresponded to one monomer protein bound to one oligonucleotide molecule. Sequences capable of forming G-quadruplex structures are widespread through genomes and are thought to play a biological function in transcriptional regulation. They form stable structures that can obstruct DNA replication, requiring the action of specific helicases to resolve them. Nevertheless, TrwB displayed no G4 DNA unwinding activity. These observations are discussed in terms of a possible role for TrwB in recognizing G-quadruplex structures as loading sites on the DNA.
    Journal of Biological Chemistry 04/2010; 285(23):17537-44. · 4.65 Impact Factor
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    ABSTRACT: 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.
    Journal of Biological Chemistry 09/2007; 282(35):25569-76. · 4.65 Impact Factor