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

TrwC is a DNA strand transferase that catalyzes the initial and final stages of conjugative DNA transfer. We have solved the crystal structure of the N-terminal relaxase domain of TrwC in complex with a 27 base-long DNA oligonucleotide that contains both the recognition hairpin and the scissile phosphate. In addition, a series of ternary structures of protein–DNA complexes with different divalent cations at the active site have been solved. Systematic anomalous difference analysis allowed us to determine unambiguously the nature of the metal bound. Zn²⁺, Ni²⁺ and Cu²⁺ were found to bind the histidine-triad metal binding site. Comparison of the structures of the different complexes suggests two pathways for the DNA to exit the active pocket. They are probably used at different steps of the conjugative DNA-processing reaction. The structural information allows us to propose (i) an enzyme mechanism where the scissile phosphate is polarized by the metal ion facilitating the nucleophilic attack of the catalytic tyrosine, and (ii) a probable sequence of events during conjugative DNA processing that explains the biological function of the relaxase.

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.