Journal of Molecular Biology
Unveiling the Molecular Mechanism of a Conjugative Relaxase: The Structure of TrwC Complexed with a 27-mer DNA Comprising the Recognition Hairpin and the Cleavage Site
Introduction
Bacterial conjugation is a process by which a DNA molecule is transferred from a donor to a recipient bacterium. The mechanism is efficient, and allows bacteria to acquire new adaptive traits such as antibiotic resistance. Thus, it is considered an important mechanism in bacterial evolution.1, 2 The underlying biochemical process can be operationally divided into two steps, DNA processing and DNA transport, each of which is carried out by a specific set of proteins encoded by the tra genes of a given conjugative plasmid. Conjugative DNA processing starts by cleavage of a specific phosphodiester bond (the nic site) in the donor supercoiled DNA by a plasmid-specific relaxase. The resulting nucleoprotein complex, the relaxosome, contacts the transport site, where a multi-protein DNA transport apparatus effects the transfer process of the cleaved DNA strand to the recipient cell. Presumably, the relaxase religates the transferred DNA strand, and finally host proteins replicate both single strands in donor and recipient bacteria to regenerate the double-stranded conjugative plasmid.3, 4
Most conjugative systems contain phylogenetically related relaxases, according to their amino acid sequences.5 Remarkably conspicuous is a histidine triad that has been intimately involved in the catalytic mechanism. A few selected relaxases have been analyzed at a biochemical level. The purified proteins can specifically cleave oligonucleotides containing their respective nic site sequences so that the 5′ phosphoryl end of the cleaved product becomes covalently bound to the hydroxyl group of a specific tyrosyl residue of the protein. Relaxases can then transfer the bound DNA to an appropriate acceptor oligonucleotide by a second DNA strand-transfer reaction, so that a hybrid oligonucleotide is released from the enzyme.6 The P-family relaxase TraI of plasmid RP4 is one of the best analyzed with respect to the biochemical details of these reactions. By using oligonucleotides bound to a solid support, Pansegrau & Lanka7 showed that the TraI oligonucleotide adduct was incapable of carrying out the strand transfer reaction when challenged with a second oligonucleotide containing nic. The interpretation was that a second TraI molecule is required to complete the reaction, presumably by providing a new Tyr to catalyze the second strand transfer reaction.
The F-family relaxase TrwC, from the IncW plasmid R388, is a large protein composed of an N-terminal domain with DNA-relaxase activity and a C-terminal domain with DNA helicase activity.8 It can cleave a supercoiled plasmid DNA containing oriT in vitro in the absence of accessory proteins.9 TrwC contains two active-site tyrosyl residues (instead of the single one in P-family relaxases) that play different functional roles in the DNA processing reactions. Thus, it was proposed that conjugative DNA processing of plasmid R388 occurs by a variant of the flip-flop mechanism used in ϕX-174 replication10 in which the two active tyrosine residues catalyze the initiation and termination steps in conjugative DNA replication.11
The atomic structures of two F-family relaxases, R388 TrwC12 and F plasmid TraI,13 have been reported. The TrwC structure shows a complex of the protein with the nic DNA up to the cleavage site. This complex was shown both as a binary DNA–protein complex and as a ternary complex with a Zn2+ in the three-histidine pocket. Although these structures provided valuable information with respect to the DNA and metal binding sites, they did not reveal the identity of the metal ion and did not provide a complete view of the reaction mechanism catalyzed by relaxases. In particular, both the exit path for the DNA and the position of the scissile phosphate with respect to the catalytic residues were unclear because the DNA used for the DNA–TrwC complex12 did not include any residue downstream of the cleavage site nor the scissile phosphate (A26, according to our naming). Recently, the structure of TraI complexed with a 10 bp oligonucleotide, including the scissile bond but not the full recognition hairpin, has been reported.14 Here we present additional structural information of TrwC with the description of a relaxase ternary complex with a 27-mer DNA oligonucleotide encompassing both the recognition hairpin and the cleavage site. Also, we present a detailed structural analysis of the binding of different metal ions. Finally, we provide a structure where the loop α1-β1 that includes the second catalytic residue Tyr26 is fully traced, in contrast to previous structures where it was disordered. As a result we clarify crucial aspects of the mechanism for the successive DNA-strand transfer reactions catalyzed by relaxases.
Section snippets
Crystal structure of TrwCY18F–DNA27
For this complex a Tyr18Phe TrwC mutant relaxase was used, impairing cleavage of the DNA at the scissile O3′(T25)–P(A26) bond. In this crystal, the space group and crystal packing arrangement is the same as the one observed for the 1OSB structure.12 The overall structures of the protein and the DNA in the DNA27 complex (Figure 1(a)) are similar to those found in the metal-free DNA25 complex structures determined previously.12 Briefly, the DNA folds in a hairpin structure, with a double-helical
Discussion
The atomic structure of TrwC relaxase in complex with the 25-mer nic DNA12 unveiled how the relaxase recognises the extruded inverted repeat hairpin and provided a detailed view of the architecture of the active site. However, since the DNA in the complex did not extend past the cleavage site but ended up before the scissile phosphate, it left some questions unanswered. For example, the precise position of the scissile phosphate was unknown and, thus, the kind of interactions that it could
Protein preparation
The N-terminal relaxase domain of TrwC (residues 1 to 293), and mutant Tyr18Phe (named TrwC and TrwCY18F, respectively, here) were expressed and purified as follows.21 Escherichia coli strain C43-DE322 containing plasmid pSU1588 was grown in a 2 l micro-DCU fermentation system (B. Biotech International). Plasmid pSU1588 carries the trwC fragment corresponding to the protein residues 1 to 293 cloned between the NdeI and BamHI sites of vector pET3a. Overexpression was induced with 0.5 mM IPTG for 2
Acknowledgements
This study was supported by the Ministerio de Educación y Ciencia of Spain (grants BIO2002-03964, GEN2003-20642 and BFU2005-06758/BMC to M.C.; grant BMC2002-00379 to F.C.) and by the Generalitat de Catalunya (grant 2001SGR-346 to M.C.). Synchrotron data collection was supported by the ESRF and the EU. S.R. is the recipient of a predoctoral fellowship from the Generalitat de Catalunya. M.L. acknowledges a fellowship from the Fundación Marqués de Valdecilla IFIMAV.
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2015, VirologyCitation Excerpt :Side chains of Glu119 and Lys214 form a salt bridge. In comparison, metal binding leads to significant conformational changes in various regions in TrwC, including the nickase catalytic residue Tyr18, a loop adjacent to the active site harboring the second catalytic tyrosine residue, and the side chain conformations of the active site histidine residues (Boer et al., 2006), reflecting conformational flexibility in the nickase active site. The pre-configured architecture of the nickase active site observed in MVM NS1 might not exist in TrwC (Boer et al., 2006), thus providing a structural basis for capability of binding multiple types of metal ions in MVM NS1 but failure to bind Mg2+ and Mn2+ in TrwC as well as differences in the metal coordination systems observed in MVM NS1 and TrwC.
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R.B. and S.R. contributed equally to this work.