Functional properties and structural requirements of the plasmid pMV158-encoded MobM relaxase domain

Abstract

A crucial element in the horizontal transfer of mobilizable and conjugative plasmids is the relaxase, a single-stranded endonuclease that nicks the origin of transfer (oriT) of the plasmid DNA. The relaxase of the pMV158 mobilizable plasmid is MobM (494 residues). In solution, MobM forms a dimer through its C-terminal domain, which is proposed to anchor the protein to the cell membrane and to participate in type 4 secretion system (T4SS) protein-protein interactions. In order to gain a deeper insight into the structural MobM requirements for efficient DNA catalysis, we studied two endonuclease domain variants that include the first 199 or 243 amino acid residues (MobMN199 and MobMN243, respectively). Our results confirmed that the two proteins behaved as monomers in solution. Interestingly, MobMN243 relaxed supercoiled DNA and cleaved single-stranded oligonucleotides harboring oriTpMV158, whereas MobMN199 was active only on supercoiled DNA. Protein stability studies using gel electrophoresis and mass spectrometry showed increased susceptibility to degradation at the domain boundary between the N- and C-terminal domains, suggesting that the domains change their relative orientation upon DNA binding. Overall, these results demonstrate that MobMN243 is capable of nicking the DNA substrate independently of its topology and that the amino acids 200 to 243 modulate substrate specificity but not the nicking activity per se. These findings suggest that these amino acids are involved in positioning the DNA for the nuclease reaction rather than in the nicking mechanism itself.

Fig 1

Genetic map and origin of transfer (oriT) of plasmid pMV158. (A) Scheme of pMV158. Arrowheads indicate the direction of transcription of the four protein-coding genes (mobM, copG, repB, and tetL). The positions of the oriT and the origins of the leading-strand (dso) and lagging-strand (ssoU and ssoA) syntheses are shown. (B) DNA sequence of the oriT of pMV158 (coordinates 3564 to 3605). The three identified inverted repeats (IR1, IR2, and IR3) are indicated by arrows, and the nick site is indicated by a vertical arrowhead. The oligonucleotides used in this study are also depicted (Table 1).

Fig 2

Overexpression and purification of the MobMN243 protein. (A) Fractions from the different purification steps of MobMN243 were analyzed by electrophoresis on 12% SDS-PAGE gels. Samples were loaded in the following order: noninduced cultures (lane 1), cultures after induction with IPTG (lane 2) and rifampin (lane 3), supernatant of a total cell lysate (lane 4), supernatant after polyethyleneimine precipitation (lane 5), supernatant of the ammonium sulfate precipitation step dialysis against buffer A plus 300 mM NaCl (lane 6), and protein soluble fraction after dialysis (lane 7). The sample was loaded onto a heparin-agarose column and then subjected to a gel filtration column (lane 8). M indicates the molecular mass standards (in kDa). (B) Comparative analysis of the gel filtration elution profiles of MobM (gray dashed line), MobMN243 (continuous black line), and MobMN199 (continuous gray line) subjected to 1 ml/min isocratic flow (buffer A plus 300 mM NaCl) in a HiLoad Superdex 200 column.

Fig 3

MobMN243 oligomerization state studied by analytical ultracentrifugation profile. The sedimentation equilibrium profile (35,000 rpm and 20°C) of 20 μM MobMN243 in buffer UA is shown. The lower part shows the experimental data (circles) and the best fit (solid line) to a single species with a molecular size of 31,000 ± 1,000 Da. The upper part shows residuals of the theoretical fit. The inset shows the distribution of sedimentation coefficients of the same MobMN243 protein sample in sedimentation velocity experiments (48,000 rpm at 20°C).

Fig 4

Nicking activity of MobMN199 and MobMN243. (A) Relaxation assays of pMV158 by MobMN199 and MobMN243. Supercoiled pMV158 DNA samples (8 nM) were incubated with (8, 16, 32, 64, 128, or 256 nM) or without (−) MobMN199 or MobMN243 and supplemented with 8 mM MnCl₂ at 30°C for 20 min. Generation of relaxed forms (FII) from scDNA forms (FI) was analyzed by electrophoresis on 1% agarose gels stained with ethidium bromide (1 μg/ml). The weak band above relaxed forms (FII) has been observed before (20) and might correspond to relaxed DNA dimers. (B) Nicking activity of MobMN199 and MobMN243 on single-strand DNA harboring regions of the pMV158 oriT (19nic4). The nicking reaction yields 19- and 4-mer (not shown) oligonucleotide products. 19nic4 (60 nM) was incubated with a range of protein concentrations (60, 120, 240, 480, and 960 nM) at 30°C for 20 min in the presence of 8 mM MnCl₂. The reaction was stopped with 0.1% SDS and proteinase K (1 mg/ml) for 30 min at 37°C, and samples were electrophoretically analyzed in a denaturing 20% PAA–7 M urea gel and visualized by a phosphorimager. Band quantification was done using the QuantityOne software (Bio-Rad) for both panels.

Fig 5

Isolation of the ssDNA-MobMN243 stable complexes after IR1+12pA oligonucleotide cleavage. (A) Scheme of the substrate and reaction products expected after IR1+12pA nicking by MobMN243. (B) DNA analysis. IR1+12pA 5′-labeled (Cy5) oligonucleotide (60 nM) was incubated with different concentrations of MobMN243 (128, 256, and 512 μM) for 20 min at 30°C in the presence of 8 mM MnCl₂. The reaction was stopped by incubation with 0.1% SDS and proteinase K (100 μg/ml) for 30 min at 37°C. Samples were loaded in a denaturing 20% PAA gel (7 M urea), and DNA was visualized in a phosphorimager platform. (C) Protein analysis. MobMN243 (10 μM) was incubated with DNA (10 or 20 μM) in the same conditions used for the DNA analysis. Samples were visualized by SDS-PAGE (12%) and SYPRO Ruby staining. A stable adduct corresponding to the MobMN243 protein attached to a 21-mer ssDNA fragment was identified (MobMN243-DNA21).

Fig 6

MobMN243 ligand-dependent proteolysis as analyzed by 12% SDS-PAGE. Lane 1, full-length MobM (57.9 kDa), incubated for 2 months at 4°C. Lane 2, MobMN243 (28.4 kDa), fresh sample. Lane 3, MobMN243 incubated for 2 months at 4°C. Lane 4, MobMN243, incubated for 2 months at 4°C in the presence of MnCl₂ (15 mM final concentration). Lane 5, MobMN243 incubated for 2 months at 4°C in the presence of IR1+8 (180 μM). Lane 6, MobMN243 incubated for 2 months at 4°C in the presence of MnCl₂ plus IR1+8. Lane 7, MobMN199 (23.1 kDa), nondegraded fresh sample, included for comparison to the degraded samples in lanes 1 to 6. The marker lane (M) was loaded with SeeBlue prestained standards (Invitrogen).