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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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(a) Predicted domains in the native MobM protein. The three conserved motifs located in the N-terminal moiety are indicated: (i) HxxR (unknown function), (ii) NYEL (proposed catalytic region) and (iii) HxDExxPHuH (metal ion coordination). The position of the putative Leu zipper in the C-terminal moiety is also indicated. (b) Stages in the purification of MobMN199. Fractions of the different purification steps were analysed by electrophoresis on 15% SDS–Tris–glycine–PAA gels. Samples loaded were: uninduced cultures (lane 1); cultures induced with IPTG and rifampicin (lane 2); supernatant of a total cell lysate (lane 3); supernatant after PEI precipitation (lane 4); supernatant of the ammonium sulphate precipitation step before (lane 5) and after (lane 6) dialysis against buffer A. The sample was loaded onto a heparin–agarose column and the proteins retained were eluted by a salt gradient (covered by lane 7). Fractions containing the peak of MobMN199 were pooled, dialysed against buffer A and concentrated. M indicates the molecular weight standards (in kDa). (c) Purified MobMN199 was injected onto a gel filtration column, and its elution profile was recorded; the inset shows the SDS–PAGE gel with the purified protein and the molecular size markers. (d) Relaxation assays with wild-type MobM and the MobMN199 protein. Supercoiled pMV158 DNA samples (300 ng; 8 nM) were incubated with and without (−) full-length MobM (left) or with the short MobMN199 fragment (right) in the presence of 15 mM MnCl2 at 30°C, 20 min. Protein concentrations used were 120, 240 and 480 nM. Generation of relaxed forms (FII) from supercoiled DNA forms (FI) was analysed by electrophoresis on 1% agarose gels without prior staining with EtBr, conditions in which forms FI′ are not resolved. The amounts of relaxed DNA forms generated by treatment of MobM and by MobMN199 were calculated by subtracting the amount of already nicked molecules (faint FII band in the untreated samples generated by mechanical shearing) from the FII-forms generated by protein treatment. The values of the protein-relaxed molecules were 25, 42 and 62%, and 28, 40 and 63% for samples treated with MobM and MobMN199, respectively. The weak band above relaxed forms FII has been observed before (20) and might correspond to relaxed DNA dimers.
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Figure 1: (a) Predicted domains in the native MobM protein. The three conserved motifs located in the N-terminal moiety are indicated: (i) HxxR (unknown function), (ii) NYEL (proposed catalytic region) and (iii) HxDExxPHuH (metal ion coordination). The position of the putative Leu zipper in the C-terminal moiety is also indicated. (b) Stages in the purification of MobMN199. Fractions of the different purification steps were analysed by electrophoresis on 15% SDS–Tris–glycine–PAA gels. Samples loaded were: uninduced cultures (lane 1); cultures induced with IPTG and rifampicin (lane 2); supernatant of a total cell lysate (lane 3); supernatant after PEI precipitation (lane 4); supernatant of the ammonium sulphate precipitation step before (lane 5) and after (lane 6) dialysis against buffer A. The sample was loaded onto a heparin–agarose column and the proteins retained were eluted by a salt gradient (covered by lane 7). Fractions containing the peak of MobMN199 were pooled, dialysed against buffer A and concentrated. M indicates the molecular weight standards (in kDa). (c) Purified MobMN199 was injected onto a gel filtration column, and its elution profile was recorded; the inset shows the SDS–PAGE gel with the purified protein and the molecular size markers. (d) Relaxation assays with wild-type MobM and the MobMN199 protein. Supercoiled pMV158 DNA samples (300 ng; 8 nM) were incubated with and without (−) full-length MobM (left) or with the short MobMN199 fragment (right) in the presence of 15 mM MnCl2 at 30°C, 20 min. Protein concentrations used were 120, 240 and 480 nM. Generation of relaxed forms (FII) from supercoiled DNA forms (FI) was analysed by electrophoresis on 1% agarose gels without prior staining with EtBr, conditions in which forms FI′ are not resolved. The amounts of relaxed DNA forms generated by treatment of MobM and by MobMN199 were calculated by subtracting the amount of already nicked molecules (faint FII band in the untreated samples generated by mechanical shearing) from the FII-forms generated by protein treatment. The values of the protein-relaxed molecules were 25, 42 and 62%, and 28, 40 and 63% for samples treated with MobM and MobMN199, respectively. The weak band above relaxed forms FII has been observed before (20) and might correspond to relaxed DNA dimers.

Mentions: The full-length MobM protein has a predicted size of 57 874 Da (494 residues) and exhibits three conserved motifs located in the N-terminal moiety (Figure 1a): (i) motif I (HxxR), of unknown function; (ii) motif II (NYD/EL), which contains the putative catalytic tyrosine; and (iii) motif III (HxDExxPHuH), also known as the 3H motif, probably involved in coordination of a divalent metal (13). To uncouple the N- and C-terminal domains of MobM, a strategy to obtain a protein containing only the first 199 N-terminal residues (MobMN199) was designed. The strategy was based on alignment of the relaxases belonging to the MOBV family (24), of which pMV158 is the prototype, revealed that roughly the first 200 amino acids were highly conserved among all the relaxases. Inspection of the DNA sequence around this region indicated that generation of a stop codon after the first 199 residues was relatively simple. Thus, the 5′-region of the pMV158 mobM gene (encoding the first 199 residues followed by a stop codon) was cloned into a suitable expression vector. To purify MobMN199, the protocol employed to obtain the full-length MobM protein (19) was improved by introducing one step of precipitation with polyethyleneimine (to precipitate nucleic acids), followed by an affinity (heparin-agarose) chromatography, and a final gel-filtration chromatography step (Figure 1b and c, and Supplementary Table S1). This new procedure allowed us to scale up the purification, so that the final yield of the MobMN199 protein was ∼4.5 mg/l of cell culture and purity was >98%. Under denaturing conditions, MobMN199 migrated between 30 and 20.1 kDa reference bands (Figure 1c), which agrees with the size of the protein predicted from its DNA sequence (23 261 Da; 199 residues). Determination of the N-terminal amino acid sequence showed that residue Met1 was removed, and the mass determined by MALDI-TOF (23 128 Da, not shown) was in agreement with the calculated molar mass (23 129 Da without Met1).Figure 1.


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)

(a) Predicted domains in the native MobM protein. The three conserved motifs located in the N-terminal moiety are indicated: (i) HxxR (unknown function), (ii) NYEL (proposed catalytic region) and (iii) HxDExxPHuH (metal ion coordination). The position of the putative Leu zipper in the C-terminal moiety is also indicated. (b) Stages in the purification of MobMN199. Fractions of the different purification steps were analysed by electrophoresis on 15% SDS–Tris–glycine–PAA gels. Samples loaded were: uninduced cultures (lane 1); cultures induced with IPTG and rifampicin (lane 2); supernatant of a total cell lysate (lane 3); supernatant after PEI precipitation (lane 4); supernatant of the ammonium sulphate precipitation step before (lane 5) and after (lane 6) dialysis against buffer A. The sample was loaded onto a heparin–agarose column and the proteins retained were eluted by a salt gradient (covered by lane 7). Fractions containing the peak of MobMN199 were pooled, dialysed against buffer A and concentrated. M indicates the molecular weight standards (in kDa). (c) Purified MobMN199 was injected onto a gel filtration column, and its elution profile was recorded; the inset shows the SDS–PAGE gel with the purified protein and the molecular size markers. (d) Relaxation assays with wild-type MobM and the MobMN199 protein. Supercoiled pMV158 DNA samples (300 ng; 8 nM) were incubated with and without (−) full-length MobM (left) or with the short MobMN199 fragment (right) in the presence of 15 mM MnCl2 at 30°C, 20 min. Protein concentrations used were 120, 240 and 480 nM. Generation of relaxed forms (FII) from supercoiled DNA forms (FI) was analysed by electrophoresis on 1% agarose gels without prior staining with EtBr, conditions in which forms FI′ are not resolved. The amounts of relaxed DNA forms generated by treatment of MobM and by MobMN199 were calculated by subtracting the amount of already nicked molecules (faint FII band in the untreated samples generated by mechanical shearing) from the FII-forms generated by protein treatment. The values of the protein-relaxed molecules were 25, 42 and 62%, and 28, 40 and 63% for samples treated with MobM and MobMN199, respectively. The weak band above relaxed forms FII has been observed before (20) and might correspond to relaxed DNA dimers.
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Related In: Results  -  Collection

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Figure 1: (a) Predicted domains in the native MobM protein. The three conserved motifs located in the N-terminal moiety are indicated: (i) HxxR (unknown function), (ii) NYEL (proposed catalytic region) and (iii) HxDExxPHuH (metal ion coordination). The position of the putative Leu zipper in the C-terminal moiety is also indicated. (b) Stages in the purification of MobMN199. Fractions of the different purification steps were analysed by electrophoresis on 15% SDS–Tris–glycine–PAA gels. Samples loaded were: uninduced cultures (lane 1); cultures induced with IPTG and rifampicin (lane 2); supernatant of a total cell lysate (lane 3); supernatant after PEI precipitation (lane 4); supernatant of the ammonium sulphate precipitation step before (lane 5) and after (lane 6) dialysis against buffer A. The sample was loaded onto a heparin–agarose column and the proteins retained were eluted by a salt gradient (covered by lane 7). Fractions containing the peak of MobMN199 were pooled, dialysed against buffer A and concentrated. M indicates the molecular weight standards (in kDa). (c) Purified MobMN199 was injected onto a gel filtration column, and its elution profile was recorded; the inset shows the SDS–PAGE gel with the purified protein and the molecular size markers. (d) Relaxation assays with wild-type MobM and the MobMN199 protein. Supercoiled pMV158 DNA samples (300 ng; 8 nM) were incubated with and without (−) full-length MobM (left) or with the short MobMN199 fragment (right) in the presence of 15 mM MnCl2 at 30°C, 20 min. Protein concentrations used were 120, 240 and 480 nM. Generation of relaxed forms (FII) from supercoiled DNA forms (FI) was analysed by electrophoresis on 1% agarose gels without prior staining with EtBr, conditions in which forms FI′ are not resolved. The amounts of relaxed DNA forms generated by treatment of MobM and by MobMN199 were calculated by subtracting the amount of already nicked molecules (faint FII band in the untreated samples generated by mechanical shearing) from the FII-forms generated by protein treatment. The values of the protein-relaxed molecules were 25, 42 and 62%, and 28, 40 and 63% for samples treated with MobM and MobMN199, respectively. The weak band above relaxed forms FII has been observed before (20) and might correspond to relaxed DNA dimers.
Mentions: The full-length MobM protein has a predicted size of 57 874 Da (494 residues) and exhibits three conserved motifs located in the N-terminal moiety (Figure 1a): (i) motif I (HxxR), of unknown function; (ii) motif II (NYD/EL), which contains the putative catalytic tyrosine; and (iii) motif III (HxDExxPHuH), also known as the 3H motif, probably involved in coordination of a divalent metal (13). To uncouple the N- and C-terminal domains of MobM, a strategy to obtain a protein containing only the first 199 N-terminal residues (MobMN199) was designed. The strategy was based on alignment of the relaxases belonging to the MOBV family (24), of which pMV158 is the prototype, revealed that roughly the first 200 amino acids were highly conserved among all the relaxases. Inspection of the DNA sequence around this region indicated that generation of a stop codon after the first 199 residues was relatively simple. Thus, the 5′-region of the pMV158 mobM gene (encoding the first 199 residues followed by a stop codon) was cloned into a suitable expression vector. To purify MobMN199, the protocol employed to obtain the full-length MobM protein (19) was improved by introducing one step of precipitation with polyethyleneimine (to precipitate nucleic acids), followed by an affinity (heparin-agarose) chromatography, and a final gel-filtration chromatography step (Figure 1b and c, and Supplementary Table S1). This new procedure allowed us to scale up the purification, so that the final yield of the MobMN199 protein was ∼4.5 mg/l of cell culture and purity was >98%. Under denaturing conditions, MobMN199 migrated between 30 and 20.1 kDa reference bands (Figure 1c), which agrees with the size of the protein predicted from its DNA sequence (23 261 Da; 199 residues). Determination of the N-terminal amino acid sequence showed that residue Met1 was removed, and the mass determined by MALDI-TOF (23 128 Da, not shown) was in agreement with the calculated molar mass (23 129 Da without Met1).Figure 1.

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