The structure of the minimal relaxase domain of MobA at 2.1 A resolution

Abstract

The plasmid R1162 encodes proteins that enable its conjugative mobilization between bacterial cells. It can transfer between many different species and is one of the most promiscuous of the mobilizable plasmids. The plasmid-encoded protein MobA, which has both nicking and priming activities on single-stranded DNA, is essential for mobilization. The nicking, or relaxase, activity has been localized to the 186 residue N-terminal domain, called minMobA. We present here the 2.1 A X-ray structure of minMobA. The fold is similar to that seen for two other relaxases, TraI and TrwC. The similarity in fold, and action, suggests these enzymes are evolutionary homologs, despite the lack of any significant amino acid similarity. MinMobA has a well- defined target DNA called oriT. The active site metal is observed near Tyr25, which is known to form a phosphotyrosine adduct with the substrate. A model of the oriT substrate complexed with minMobA has been made, based on observed substrate binding to TrwC and TraI. The model is consistent with observations of substrate base specificity, and provides a rationalization for elements of the likely enzyme mechanism.

Figure 1

Electron density for minMobA protein. These stereo pictures display several active site residues with (a) a 3.0 Å MAD-phased F_o electron density map and (b) a 2.1 Å SIGMAA-weighted 2F_o–F_c electron density map; these maps are contoured at 0.8 σ. The active site Mn atom is shown, bound by three histidine residues, along with the catalytic Tyr 25 and other nearby residues.

Figure 1

Electron density for minMobA protein. These stereo pictures display several active site residues with (a) a 3.0 Å MAD-phased F_o electron density map and (b) a 2.1 Å SIGMAA-weighted 2F_o–F_c electron density map; these maps are contoured at 0.8 σ. The active site Mn atom is shown, bound by three histidine residues, along with the catalytic Tyr 25 and other nearby residues.

Figure 2

Ribbon drawing of minMobA. This stereo picture illustrates the fold of the protein and displays its secondary structural elements. Residues along the backbone are labeled to aid in following the polypeptide path. The active site Mn atom and Tyr side chain are also shown.

Figure 3

(a) Superposition of minMobA and TrwC. This stereo picture shows the least squares superposition of minMobA and TrwC complexed with DNA. The Cα trace of minMobA is shown in black bonds. The N and C termini are labeled. The active site metal atom and Tyr 25 side chain are also shown. The N-terminal region of TrwC, which has a similar fold to minMobA, is shown in thin, gray bonds. The N-terminus is labeled Nc. The C-terminal region of TrwC, for which there is no counterpart in minMobA, is shown in thick, gray bonds and helps enclose the single-stranded stretch of DNA. The phosphate backbone of the 25mer oligonucleotide complexed with TrwC is shown with thick, dark gray bonds. (b) Superposition of active site residues and metal. This stereo picture shows the superposition of the bound metal atom and several active site residues of minMobA with their counterparts in TrwC and TraI. Residues of minMobA are shown with thick, black bonds and are labeled whereas TraI residues are shown with thin, black bonds and TrwC with gray. Shown are the metal atom and its three histidine ligands and the catalytic tyrosine (Y18 for TrwC and Y16 for TraI). Also shown is a conserved acidic residue (D85 for TrwC and D81 for TraI) that has been hypothesized to act as the catalytic base.

Figure 3

(a) Superposition of minMobA and TrwC. This stereo picture shows the least squares superposition of minMobA and TrwC complexed with DNA. The Cα trace of minMobA is shown in black bonds. The N and C termini are labeled. The active site metal atom and Tyr 25 side chain are also shown. The N-terminal region of TrwC, which has a similar fold to minMobA, is shown in thin, gray bonds. The N-terminus is labeled Nc. The C-terminal region of TrwC, for which there is no counterpart in minMobA, is shown in thick, gray bonds and helps enclose the single-stranded stretch of DNA. The phosphate backbone of the 25mer oligonucleotide complexed with TrwC is shown with thick, dark gray bonds. (b) Superposition of active site residues and metal. This stereo picture shows the superposition of the bound metal atom and several active site residues of minMobA with their counterparts in TrwC and TraI. Residues of minMobA are shown with thick, black bonds and are labeled whereas TraI residues are shown with thin, black bonds and TrwC with gray. Shown are the metal atom and its three histidine ligands and the catalytic tyrosine (Y18 for TrwC and Y16 for TraI). Also shown is a conserved acidic residue (D85 for TrwC and D81 for TraI) that has been hypothesized to act as the catalytic base.

Figure 4

The structure-based sequence alignment of minMobA with TraI and TrwC. The sequence numbering and secondary structural elements of minMobA are shown on top. The secondary structural elements of TrwC and TraI are shown at bottom. The conserved catalytic tyrosine and metal-binding histidines are highlighted with black. Conserved hydrophobic core residues are highlighted in dark gray. A conserved arginine, involved in DNA binding, and a putative catalytic acidic residue are highlighted in light gray.

Figure 5

(a) R1162 oriT (b) Drawing of minMobA-33mer model complex. This stereo picture shows the Cα trace of minMobA in a model complex with a 33mer oligonucleotide. The bound Mn atom and catalytic Tyr 25 residue are shown and labeled along with several protein side chains that interact with the DNA in the model complex. Nucleotides mentioned in the narrative are also labeled. (c) Surface electrostatic potential of minMobA. Electronegative charges are shown in red, and positive charges are shown in blue. The bound DNA of the model complex is represented as a trace of the phosphate backbone.

Figure 5

(a) R1162 oriT (b) Drawing of minMobA-33mer model complex. This stereo picture shows the Cα trace of minMobA in a model complex with a 33mer oligonucleotide. The bound Mn atom and catalytic Tyr 25 residue are shown and labeled along with several protein side chains that interact with the DNA in the model complex. Nucleotides mentioned in the narrative are also labeled. (c) Surface electrostatic potential of minMobA. Electronegative charges are shown in red, and positive charges are shown in blue. The bound DNA of the model complex is represented as a trace of the phosphate backbone.

Figure 5

(a) R1162 oriT (b) Drawing of minMobA-33mer model complex. This stereo picture shows the Cα trace of minMobA in a model complex with a 33mer oligonucleotide. The bound Mn atom and catalytic Tyr 25 residue are shown and labeled along with several protein side chains that interact with the DNA in the model complex. Nucleotides mentioned in the narrative are also labeled. (c) Surface electrostatic potential of minMobA. Electronegative charges are shown in red, and positive charges are shown in blue. The bound DNA of the model complex is represented as a trace of the phosphate backbone.

Figure 6

In vitro cleavage of 35mer oligonucleotide by minMobA variants. Cleavage reactions were begun by mixing protein with radiolabeled 35mer oligonucleotide containing the R1162 oriT sequence. Reactions were stopped at various time points by addition of EDTA, and the reaction mixtures were separated on 12% SDS-PAGE gel. In each gel, each lane contains the separated components from the reaction stopped after a time ranging from 0 (left) to 60 (right) minutes. The bottom bands correspond to the unreacted oligonucleotide, and the top bands represent the protein-oligonucleotide adduct. Each gel shows the reaction time course with a particular minMobA variant: (a) wild-type, (b) E74A, (c) E74Q, (d) E74AE76A.