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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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Temperature-associated changes in the secondary-structure of MobMN199. (a) Thermal denaturation profiles of the protein (15 µM) measured at 218 nm in presence or absence of divalent metals (free protein, black; 15 mM MgCl2, orange; 0.015 mM MnCl2, dark blue; 0.2 mM MnCl2, red; 1.5 mM MnCl2, green; 8 mM MnCl2, purple; and 15 mM MnCl2, blue). Data fit (continuous lines) assumed the superimposition of two apparently independent transitions whose thermodynamic parameters are shown in Table 2. The inset compares the thermal denaturation profiles of MobMN199 in CD2 (solid symbols) and ITC (open symbols) buffers in the absence (black) and in the presence (purple) of 8 mM MnCl2. (b) Far-UV CD spectra registered before denaturation (10°C, solid lines) or under renaturing conditions (heated samples cooled to 10°C, dashed lines) without metal (black), and in the presence of 15 mM Mg2+ (orange) or 15 mM Mn2+ (blue). A spectrum of MobMN199 at 85°C (solid grey line) is also depicted.
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Figure 4: Temperature-associated changes in the secondary-structure of MobMN199. (a) Thermal denaturation profiles of the protein (15 µM) measured at 218 nm in presence or absence of divalent metals (free protein, black; 15 mM MgCl2, orange; 0.015 mM MnCl2, dark blue; 0.2 mM MnCl2, red; 1.5 mM MnCl2, green; 8 mM MnCl2, purple; and 15 mM MnCl2, blue). Data fit (continuous lines) assumed the superimposition of two apparently independent transitions whose thermodynamic parameters are shown in Table 2. The inset compares the thermal denaturation profiles of MobMN199 in CD2 (solid symbols) and ITC (open symbols) buffers in the absence (black) and in the presence (purple) of 8 mM MnCl2. (b) Far-UV CD spectra registered before denaturation (10°C, solid lines) or under renaturing conditions (heated samples cooled to 10°C, dashed lines) without metal (black), and in the presence of 15 mM Mg2+ (orange) or 15 mM Mn2+ (blue). A spectrum of MobMN199 at 85°C (solid grey line) is also depicted.

Mentions: To determine the influence of Mn2+ and Mg2+ on MobMN199 structural stability, CD spectroscopic studies were performed in the absence or presence of either cation (Figure 4a). Changes in the ellipticity associated with protein denaturation were monitored at 218 nm while the temperature was increased from 10 to 90°C at the rate of 50°C/h or 20°C/h with similar results. Denaturation of the unbound protein started around 15°C (Figure 4a; black triangles). The ellipticity strongly decreased as the temperature was increased, showing a denaturation profile that slightly deviated from the behaviour expected for a two-state transition (Figure 4). Addition of Mn2+ at one-to-one stoichiometry strongly modified MobMN199 denaturation, shifting the apparent transition temperature by ∼5°C. A further increase in Mn2+ concentration clearly showed the presence of at least two different processes, particularly at the higher Mn2+ concentrations tested. A very different picture was observed when the cation used was Mg2+: even at 15 mM, the metal concentration typically used in DNA-nicking assays (19), Mg2+ addition only induced a small thermal up-shift in the final part of the denaturation profile (Figure 4a; orange triangles).Figure 4.


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)

Temperature-associated changes in the secondary-structure of MobMN199. (a) Thermal denaturation profiles of the protein (15 µM) measured at 218 nm in presence or absence of divalent metals (free protein, black; 15 mM MgCl2, orange; 0.015 mM MnCl2, dark blue; 0.2 mM MnCl2, red; 1.5 mM MnCl2, green; 8 mM MnCl2, purple; and 15 mM MnCl2, blue). Data fit (continuous lines) assumed the superimposition of two apparently independent transitions whose thermodynamic parameters are shown in Table 2. The inset compares the thermal denaturation profiles of MobMN199 in CD2 (solid symbols) and ITC (open symbols) buffers in the absence (black) and in the presence (purple) of 8 mM MnCl2. (b) Far-UV CD spectra registered before denaturation (10°C, solid lines) or under renaturing conditions (heated samples cooled to 10°C, dashed lines) without metal (black), and in the presence of 15 mM Mg2+ (orange) or 15 mM Mn2+ (blue). A spectrum of MobMN199 at 85°C (solid grey line) is also depicted.
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Figure 4: Temperature-associated changes in the secondary-structure of MobMN199. (a) Thermal denaturation profiles of the protein (15 µM) measured at 218 nm in presence or absence of divalent metals (free protein, black; 15 mM MgCl2, orange; 0.015 mM MnCl2, dark blue; 0.2 mM MnCl2, red; 1.5 mM MnCl2, green; 8 mM MnCl2, purple; and 15 mM MnCl2, blue). Data fit (continuous lines) assumed the superimposition of two apparently independent transitions whose thermodynamic parameters are shown in Table 2. The inset compares the thermal denaturation profiles of MobMN199 in CD2 (solid symbols) and ITC (open symbols) buffers in the absence (black) and in the presence (purple) of 8 mM MnCl2. (b) Far-UV CD spectra registered before denaturation (10°C, solid lines) or under renaturing conditions (heated samples cooled to 10°C, dashed lines) without metal (black), and in the presence of 15 mM Mg2+ (orange) or 15 mM Mn2+ (blue). A spectrum of MobMN199 at 85°C (solid grey line) is also depicted.
Mentions: To determine the influence of Mn2+ and Mg2+ on MobMN199 structural stability, CD spectroscopic studies were performed in the absence or presence of either cation (Figure 4a). Changes in the ellipticity associated with protein denaturation were monitored at 218 nm while the temperature was increased from 10 to 90°C at the rate of 50°C/h or 20°C/h with similar results. Denaturation of the unbound protein started around 15°C (Figure 4a; black triangles). The ellipticity strongly decreased as the temperature was increased, showing a denaturation profile that slightly deviated from the behaviour expected for a two-state transition (Figure 4). Addition of Mn2+ at one-to-one stoichiometry strongly modified MobMN199 denaturation, shifting the apparent transition temperature by ∼5°C. A further increase in Mn2+ concentration clearly showed the presence of at least two different processes, particularly at the higher Mn2+ concentrations tested. A very different picture was observed when the cation used was Mg2+: even at 15 mM, the metal concentration typically used in DNA-nicking assays (19), Mg2+ addition only induced a small thermal up-shift in the final part of the denaturation profile (Figure 4a; orange triangles).Figure 4.

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