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The MobM relaxase domain of plasmid pMV158: thermal stability and activity upon Mn2+ and specific DNA binding.

Lorenzo-Díaz F, Dostál L, Coll M, Schildbach JF, Menéndez M, Espinosa M - Nucleic Acids Res. (2011)

Bottom Line: However, whereas Mn(2+) strongly stabilized MobMN199 against thermal denaturation, no protective effect was observed for Mg(2+).The structure of MobMN199 was strongly stabilized by binding to the defined target DNA, indicating the formation of a tight protein-DNA complex.We demonstrate that the oriT recognition by MobMN199 was highly specific and suggest that this protein most probably employs Mn(2+) during pMV158 transfer.

View Article: PubMed Central - PubMed

Affiliation: Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain.

ABSTRACT
Protein MobM, the relaxase involved in conjugative transfer of the streptococcal plasmid pMV158, is the prototype of the MOB(V) superfamily of relaxases. To characterize the DNA-binding and nicking domain of MobM, a truncated version of the protein (MobMN199) encompassing its N-terminal region was designed and the protein was purified. MobMN199 was monomeric in contrast to the dimeric form of the full-length protein, but it kept its nicking activity on pMV158 DNA. The optimal relaxase activity was dependent on Mn(2+) or Mg(2+) cations in a dosage-dependent manner. However, whereas Mn(2+) strongly stabilized MobMN199 against thermal denaturation, no protective effect was observed for Mg(2+). Furthermore, MobMN199 exhibited a high affinity binding for Mn(2+) but not for Mg(2+). We also examined the binding-specificity and affinity of MobMN199 for several substrates of single-stranded DNA encompassing the pMV158 origin of transfer (oriT). The minimal oriT was delimited to a stretch of 26 nt which included an inverted repeat located eight bases upstream of the nick site. The structure of MobMN199 was strongly stabilized by binding to the defined target DNA, indicating the formation of a tight protein-DNA complex. We demonstrate that the oriT recognition by MobMN199 was highly specific and suggest that this protein most probably employs Mn(2+) during pMV158 transfer.

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Sedimentation equilibrium profile of MobMN199 (8 μM of protein in buffer UA) at 35 000 rpm and 20°C (λ=280 nm). The lower part shows the experimental data (circles) and the best fit (solid line) to a single species with Mw=23 101 Da. The upper part shows residuals of the theoretical fit. The inset shows the distribution of sedimentation coefficients of the same MobMN199 protein sample in sedimentation velocity experiments (48 000 rpm, 20°C).
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Figure 2: Sedimentation equilibrium profile of MobMN199 (8 μM of protein in buffer UA) at 35 000 rpm and 20°C (λ=280 nm). The lower part shows the experimental data (circles) and the best fit (solid line) to a single species with Mw=23 101 Da. The upper part shows residuals of the theoretical fit. The inset shows the distribution of sedimentation coefficients of the same MobMN199 protein sample in sedimentation velocity experiments (48 000 rpm, 20°C).

Mentions: Analytical ultracentrifugation analyses showed that the entire MobM protein behaved as an elongated dimer in solution (19). Since the shorter version MobMN199 lacks the last 295 C-terminal amino acids, we wanted to determine its oligomeric state and hydrodynamic properties. The elution volume of the truncated protein in the size-exclusion chromatography used for purification (Figure 1c) suggested that MobMN199 was a monomer. To corroborate this, analytical ultracentrifugation assays were performed at three protein concentrations (2, 8 and 20 µM). The results of sedimentation equilibrium and velocity assays are shown in Figure 2. Sedimentation velocity profiles fit well to a model of single sedimenting species (98.1% of total concentration loaded), with an s20,w value of 2.37 S, and an average molecular mass (Mw,app) of 22 944±2820 Da, which agrees with the monomer mass determined by MALDI-TOF. No improvement in the best-fitting parameters was obtained considering more sedimenting species, an indication of sample homogeneity. At 8 µM, the experimental sedimentation equilibrium data fitted best to a Mw,app of 23 000±700 Da, which agrees with the value obtained from the sedimentation velocity. Similar average molecular masses were determined when MobMN199 concentrations of 2 µM (22 637 Da) and 20 µM (21 985 Da) were used. The frictional ratio ( f/f0) calculated was 1.25, indicating that MobMN199 clearly deviates from the behaviour expected for globular particles ( f / f0 ≅ 1). Moreover, since the results fit to the MobMN199 monomer mass, we can conclude that MobM dimerization requires the C-terminal portion of the protein, which should also comprise the protein dimerization domain. Plasmids bearing mutations that result in deletions of either the 18 or the 30 last codons of gene mobM failed to be transferred between pneumococci (our unpublished observations). In addition, mutations directed to a predicted coiled-coil region in MobM (19) also resulted in plasmids unable to be transferred. Based in all these results, we tentatively propose that the C-terminal moiety of MobM would be involved in other transactions during the transfer process, like dimerization, membrane association and interactions with the coupling protein which would be provided by the auxiliary plasmid.Figure 2.


The MobM relaxase domain of plasmid pMV158: thermal stability and activity upon Mn2+ and specific DNA binding.

Lorenzo-Díaz F, Dostál L, Coll M, Schildbach JF, Menéndez M, Espinosa M - Nucleic Acids Res. (2011)

Sedimentation equilibrium profile of MobMN199 (8 μM of protein in buffer UA) at 35 000 rpm and 20°C (λ=280 nm). The lower part shows the experimental data (circles) and the best fit (solid line) to a single species with Mw=23 101 Da. The upper part shows residuals of the theoretical fit. The inset shows the distribution of sedimentation coefficients of the same MobMN199 protein sample in sedimentation velocity experiments (48 000 rpm, 20°C).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3105389&req=5

Figure 2: Sedimentation equilibrium profile of MobMN199 (8 μM of protein in buffer UA) at 35 000 rpm and 20°C (λ=280 nm). The lower part shows the experimental data (circles) and the best fit (solid line) to a single species with Mw=23 101 Da. The upper part shows residuals of the theoretical fit. The inset shows the distribution of sedimentation coefficients of the same MobMN199 protein sample in sedimentation velocity experiments (48 000 rpm, 20°C).
Mentions: Analytical ultracentrifugation analyses showed that the entire MobM protein behaved as an elongated dimer in solution (19). Since the shorter version MobMN199 lacks the last 295 C-terminal amino acids, we wanted to determine its oligomeric state and hydrodynamic properties. The elution volume of the truncated protein in the size-exclusion chromatography used for purification (Figure 1c) suggested that MobMN199 was a monomer. To corroborate this, analytical ultracentrifugation assays were performed at three protein concentrations (2, 8 and 20 µM). The results of sedimentation equilibrium and velocity assays are shown in Figure 2. Sedimentation velocity profiles fit well to a model of single sedimenting species (98.1% of total concentration loaded), with an s20,w value of 2.37 S, and an average molecular mass (Mw,app) of 22 944±2820 Da, which agrees with the monomer mass determined by MALDI-TOF. No improvement in the best-fitting parameters was obtained considering more sedimenting species, an indication of sample homogeneity. At 8 µM, the experimental sedimentation equilibrium data fitted best to a Mw,app of 23 000±700 Da, which agrees with the value obtained from the sedimentation velocity. Similar average molecular masses were determined when MobMN199 concentrations of 2 µM (22 637 Da) and 20 µM (21 985 Da) were used. The frictional ratio ( f/f0) calculated was 1.25, indicating that MobMN199 clearly deviates from the behaviour expected for globular particles ( f / f0 ≅ 1). Moreover, since the results fit to the MobMN199 monomer mass, we can conclude that MobM dimerization requires the C-terminal portion of the protein, which should also comprise the protein dimerization domain. Plasmids bearing mutations that result in deletions of either the 18 or the 30 last codons of gene mobM failed to be transferred between pneumococci (our unpublished observations). In addition, mutations directed to a predicted coiled-coil region in MobM (19) also resulted in plasmids unable to be transferred. Based in all these results, we tentatively propose that the C-terminal moiety of MobM would be involved in other transactions during the transfer process, like dimerization, membrane association and interactions with the coupling protein which would be provided by the auxiliary plasmid.Figure 2.

Bottom Line: However, whereas Mn(2+) strongly stabilized MobMN199 against thermal denaturation, no protective effect was observed for Mg(2+).The structure of MobMN199 was strongly stabilized by binding to the defined target DNA, indicating the formation of a tight protein-DNA complex.We demonstrate that the oriT recognition by MobMN199 was highly specific and suggest that this protein most probably employs Mn(2+) during pMV158 transfer.

View Article: PubMed Central - PubMed

Affiliation: Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain.

ABSTRACT
Protein MobM, the relaxase involved in conjugative transfer of the streptococcal plasmid pMV158, is the prototype of the MOB(V) superfamily of relaxases. To characterize the DNA-binding and nicking domain of MobM, a truncated version of the protein (MobMN199) encompassing its N-terminal region was designed and the protein was purified. MobMN199 was monomeric in contrast to the dimeric form of the full-length protein, but it kept its nicking activity on pMV158 DNA. The optimal relaxase activity was dependent on Mn(2+) or Mg(2+) cations in a dosage-dependent manner. However, whereas Mn(2+) strongly stabilized MobMN199 against thermal denaturation, no protective effect was observed for Mg(2+). Furthermore, MobMN199 exhibited a high affinity binding for Mn(2+) but not for Mg(2+). We also examined the binding-specificity and affinity of MobMN199 for several substrates of single-stranded DNA encompassing the pMV158 origin of transfer (oriT). The minimal oriT was delimited to a stretch of 26 nt which included an inverted repeat located eight bases upstream of the nick site. The structure of MobMN199 was strongly stabilized by binding to the defined target DNA, indicating the formation of a tight protein-DNA complex. We demonstrate that the oriT recognition by MobMN199 was highly specific and suggest that this protein most probably employs Mn(2+) during pMV158 transfer.

Show MeSH
Related in: MedlinePlus