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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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ITC binding analysis of Mn2+ by MobMN199. Raw data (top) for the injection of 1.5 mM MnCl2 into a solution of MobMN199 (75 µM) and integrated heats of injection (bottom) are shown. The solid line shows the best data fit assuming a Kb of 2.3 (±0.4) × 106 M−1 and a ΔHb, of −27.3 ± 0.3 kcal mol−1.
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Figure 6: ITC binding analysis of Mn2+ by MobMN199. Raw data (top) for the injection of 1.5 mM MnCl2 into a solution of MobMN199 (75 µM) and integrated heats of injection (bottom) are shown. The solid line shows the best data fit assuming a Kb of 2.3 (±0.4) × 106 M−1 and a ΔHb, of −27.3 ± 0.3 kcal mol−1.

Mentions: The affinity of MobMN199 for Mn2+ and Mg2+ was examined by ITC. Titrations were performed at 17°C to preserve the native state of MobMN199 during the experiment. The analysis of the binding isotherms (Figure 6) showed that MobMN199 binds Mn2+ with high affinity and one-to-one stoichiometry. The equilibrium constant (Kb) for the complex formation was 2.3 (±0.4) × 106 M−1 and the enthalpy change (ΔHb) −27.3±0.3 kcal mol−1 (average of three experiments). These results evidenced that binding of Mn2+ to MobMN199 was enthalpically driven since the entropy change (ΔSb = −65.2±0.7 cal mol−1 K−1) was largely unfavourable. The high affinity of Mn2+ for MobMN199 is consistent with the strong stabilization observed in the CD profiles at high cation concentrations (Figure 4). Further, the high values calculated for the enthalpic and entropic contributions indicate the existence of significant conformational rearrangements in the structure of MobMN199 upon Mn2+ binding. In contrast, no evidence of Mg2+ binding to MobMN199 was found neither from direct binding assays nor from the protein titration with Mn2+ in the presence of 10 mM Mg2+ (metal competition for the same site should have decreased the apparent affinity of MobMN199 for Mn2+).Figure 6.


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)

ITC binding analysis of Mn2+ by MobMN199. Raw data (top) for the injection of 1.5 mM MnCl2 into a solution of MobMN199 (75 µM) and integrated heats of injection (bottom) are shown. The solid line shows the best data fit assuming a Kb of 2.3 (±0.4) × 106 M−1 and a ΔHb, of −27.3 ± 0.3 kcal mol−1.
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Related In: Results  -  Collection

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Figure 6: ITC binding analysis of Mn2+ by MobMN199. Raw data (top) for the injection of 1.5 mM MnCl2 into a solution of MobMN199 (75 µM) and integrated heats of injection (bottom) are shown. The solid line shows the best data fit assuming a Kb of 2.3 (±0.4) × 106 M−1 and a ΔHb, of −27.3 ± 0.3 kcal mol−1.
Mentions: The affinity of MobMN199 for Mn2+ and Mg2+ was examined by ITC. Titrations were performed at 17°C to preserve the native state of MobMN199 during the experiment. The analysis of the binding isotherms (Figure 6) showed that MobMN199 binds Mn2+ with high affinity and one-to-one stoichiometry. The equilibrium constant (Kb) for the complex formation was 2.3 (±0.4) × 106 M−1 and the enthalpy change (ΔHb) −27.3±0.3 kcal mol−1 (average of three experiments). These results evidenced that binding of Mn2+ to MobMN199 was enthalpically driven since the entropy change (ΔSb = −65.2±0.7 cal mol−1 K−1) was largely unfavourable. The high affinity of Mn2+ for MobMN199 is consistent with the strong stabilization observed in the CD profiles at high cation concentrations (Figure 4). Further, the high values calculated for the enthalpic and entropic contributions indicate the existence of significant conformational rearrangements in the structure of MobMN199 upon Mn2+ binding. In contrast, no evidence of Mg2+ binding to MobMN199 was found neither from direct binding assays nor from the protein titration with Mn2+ in the presence of 10 mM Mg2+ (metal competition for the same site should have decreased the apparent affinity of MobMN199 for Mn2+).Figure 6.

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