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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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Secondary-structure analysis of MobMN199. (a) Computational analyses. The amino acid sequence of MobMN199 is depicted as well as a summary of the most reliable predictions of secondary structure (using PSIPred, Jpred, NPS@, SABLE and PredictProtein programs). Boxes and arrows below the amino acid sequence of MobMN199 correspond to helices and β-strands, respectively. Boldface letters indicate the conserved residues in the MobM family (Figure 1a). (b) CD spectra of MobMN199 (15 µM) protein in buffer CD1, at 10°C. The solid line represents the fit of the experimental curve by the CONTIN method. Experimental data (diamonds) were acquired using 0.02-cm optical path-length quartz cells.
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Figure 3: Secondary-structure analysis of MobMN199. (a) Computational analyses. The amino acid sequence of MobMN199 is depicted as well as a summary of the most reliable predictions of secondary structure (using PSIPred, Jpred, NPS@, SABLE and PredictProtein programs). Boxes and arrows below the amino acid sequence of MobMN199 correspond to helices and β-strands, respectively. Boldface letters indicate the conserved residues in the MobM family (Figure 1a). (b) CD spectra of MobMN199 (15 µM) protein in buffer CD1, at 10°C. The solid line represents the fit of the experimental curve by the CONTIN method. Experimental data (diamonds) were acquired using 0.02-cm optical path-length quartz cells.

Mentions: The predicted secondary-structure of MobMN199 obtained by computational methods (Figure 3a) indicated a distribution of α-helices alternating with β-strands (the so-called α/β-fold), which is a typical feature of the Rep/Mob family of relaxases with known structures, including RepB_pMV158 (47), TraI_F (6), TrwC_R388 (7) and MobA_R1162 (22). The results were tested experimentally by CD in the far-UV region (Figure 3b). The CD spectrum of MobMN199 showed two minima at 208 and 222 nm, a characteristic feature of α-helical structures. Estimation of the secondary-structure yielded similar results when two different deconvolution methods were used (Table 1). The average of α-helices and β-strands content provided by these two methods agrees with the predicted secondary structure. The reduction of the total α-helical content (∼40%) of MobMN199 as compared to the full-length protein (∼60%) is consistent with the high content in α-helices predicted for the C-terminal moiety of the MobM protein (19).Figure 3.


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)

Secondary-structure analysis of MobMN199. (a) Computational analyses. The amino acid sequence of MobMN199 is depicted as well as a summary of the most reliable predictions of secondary structure (using PSIPred, Jpred, NPS@, SABLE and PredictProtein programs). Boxes and arrows below the amino acid sequence of MobMN199 correspond to helices and β-strands, respectively. Boldface letters indicate the conserved residues in the MobM family (Figure 1a). (b) CD spectra of MobMN199 (15 µM) protein in buffer CD1, at 10°C. The solid line represents the fit of the experimental curve by the CONTIN method. Experimental data (diamonds) were acquired using 0.02-cm optical path-length quartz cells.
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Related In: Results  -  Collection

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Figure 3: Secondary-structure analysis of MobMN199. (a) Computational analyses. The amino acid sequence of MobMN199 is depicted as well as a summary of the most reliable predictions of secondary structure (using PSIPred, Jpred, NPS@, SABLE and PredictProtein programs). Boxes and arrows below the amino acid sequence of MobMN199 correspond to helices and β-strands, respectively. Boldface letters indicate the conserved residues in the MobM family (Figure 1a). (b) CD spectra of MobMN199 (15 µM) protein in buffer CD1, at 10°C. The solid line represents the fit of the experimental curve by the CONTIN method. Experimental data (diamonds) were acquired using 0.02-cm optical path-length quartz cells.
Mentions: The predicted secondary-structure of MobMN199 obtained by computational methods (Figure 3a) indicated a distribution of α-helices alternating with β-strands (the so-called α/β-fold), which is a typical feature of the Rep/Mob family of relaxases with known structures, including RepB_pMV158 (47), TraI_F (6), TrwC_R388 (7) and MobA_R1162 (22). The results were tested experimentally by CD in the far-UV region (Figure 3b). The CD spectrum of MobMN199 showed two minima at 208 and 222 nm, a characteristic feature of α-helical structures. Estimation of the secondary-structure yielded similar results when two different deconvolution methods were used (Table 1). The average of α-helices and β-strands content provided by these two methods agrees with the predicted secondary structure. The reduction of the total α-helical content (∼40%) of MobMN199 as compared to the full-length protein (∼60%) is consistent with the high content in α-helices predicted for the C-terminal moiety of the MobM protein (19).Figure 3.

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