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Bacterial genome partitioning: N-terminal domain of IncC protein encoded by broad-host-range plasmid RK2 modulates oligomerisation and DNA binding.

Batt SM, Bingle LE, Dafforn TR, Thomas CM - J. Mol. Biol. (2008)

Bottom Line: ParA proteins normally occur in one of two forms, differing by their N-terminal domain (NTD) of approximately 100 aa, which is generally associated with site-specific DNA binding.The IncC1 NTD does not dimerise or bind DNA alone, but it does bind IncC2 in the presence of nucleotides.Mixing IncC1 and IncC2 improved polymerisation and DNA binding.

View Article: PubMed Central - PubMed

Affiliation: School of Biosciences, University of Birmingham, Edgbaston, Birmingham, UK.

ABSTRACT
ParA Walker ATPases form part of the machinery that promotes better-than-random segregation of bacterial genomes. ParA proteins normally occur in one of two forms, differing by their N-terminal domain (NTD) of approximately 100 aa, which is generally associated with site-specific DNA binding. Unusually, and for as yet unknown reasons, parA (incC) of IncP-1 plasmids is translated from alternative start codons producing two forms, IncC1 (364 aa) and IncC2 (259 aa), whose ratio varies between hosts. IncC2 could be detected as an oligomeric form containing dimers, tetramers and octamers, but the N-terminal extension present in IncC1 favours nucleotide-stimulated dimerisation as well as high-affinity and ATP-dependent non-specific DNA binding. The IncC1 NTD does not dimerise or bind DNA alone, but it does bind IncC2 in the presence of nucleotides. Mixing IncC1 and IncC2 improved polymerisation and DNA binding. Thus, the NTD may modulate the polymerisation interface, facilitating polymerisation/depolymerisation and DNA binding, to promote the cycle that drives partitioning.

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Velocity sedimentation of IncC proteins. (a-i) ATP titrated onto IncC1 (raw analysis). (a-ii) Graph of the area under the IncC1 dimer curve plotted against ATP concentration (micromolar). (b-i) ADP titrated onto IncC1 (raw analysis). (b-ii) Graph of the area under the IncC1 dimer curve plotted against ADP concentration (micromolar). (c) Comparison of the S values for the IncC1-ATP dimer and the IncC1-ADP dimer. (d) IncC2 in the presence and that in the absence of 2 mM ATP.
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fig3: Velocity sedimentation of IncC proteins. (a-i) ATP titrated onto IncC1 (raw analysis). (a-ii) Graph of the area under the IncC1 dimer curve plotted against ATP concentration (micromolar). (b-i) ADP titrated onto IncC1 (raw analysis). (b-ii) Graph of the area under the IncC1 dimer curve plotted against ADP concentration (micromolar). (c) Comparison of the S values for the IncC1-ATP dimer and the IncC1-ADP dimer. (d) IncC2 in the presence and that in the absence of 2 mM ATP.

Mentions: Velocity sedimentation experiments with 30 μM IncC1 and varying ATP concentrations confirmed that IncC1 forms a dimer (mass = 80 kDa, 2.3S) in the presence of this nucleotide; in the absence of ATP, the soluble IncC1 that is detectable is exclusively monomeric (mass = 40 kDa, 1.6S) (Fig. 3ai). Plotting the dimer concentration at increasing ATP concentrations (Fig. 3aii) enabled the apparent dissociation constant (Kapp) of IncC1 binding ATP to be calculated as 7.7 μM, considerably lower than the apparent Kd values for other ParA proteins, which are in the range of 33–100 μM.13,14,19,22 Magnesium was important for coordinating the nucleotide, with the Kapp being 130-fold higher (940 μM for 20 μM IncC1) when Mg2+ was absent (data not shown). With magnesium, all of IncC1 is dimeric at or above 30 μM ATP, approximately one ATP molecule per IncC1 monomer. ADP also caused the formation of IncC1 dimers (Fig. 3bi), but with a higher Kapp (Fig. 3bii; 54 μM). Hill analysis29 of the data indicated that there was no cooperativity with ATP, whereas the ADP-induced dimerisation was highly cooperative involving two sites, the second of which was calculated to have a 1000-fold higher affinity.


Bacterial genome partitioning: N-terminal domain of IncC protein encoded by broad-host-range plasmid RK2 modulates oligomerisation and DNA binding.

Batt SM, Bingle LE, Dafforn TR, Thomas CM - J. Mol. Biol. (2008)

Velocity sedimentation of IncC proteins. (a-i) ATP titrated onto IncC1 (raw analysis). (a-ii) Graph of the area under the IncC1 dimer curve plotted against ATP concentration (micromolar). (b-i) ADP titrated onto IncC1 (raw analysis). (b-ii) Graph of the area under the IncC1 dimer curve plotted against ADP concentration (micromolar). (c) Comparison of the S values for the IncC1-ATP dimer and the IncC1-ADP dimer. (d) IncC2 in the presence and that in the absence of 2 mM ATP.
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fig3: Velocity sedimentation of IncC proteins. (a-i) ATP titrated onto IncC1 (raw analysis). (a-ii) Graph of the area under the IncC1 dimer curve plotted against ATP concentration (micromolar). (b-i) ADP titrated onto IncC1 (raw analysis). (b-ii) Graph of the area under the IncC1 dimer curve plotted against ADP concentration (micromolar). (c) Comparison of the S values for the IncC1-ATP dimer and the IncC1-ADP dimer. (d) IncC2 in the presence and that in the absence of 2 mM ATP.
Mentions: Velocity sedimentation experiments with 30 μM IncC1 and varying ATP concentrations confirmed that IncC1 forms a dimer (mass = 80 kDa, 2.3S) in the presence of this nucleotide; in the absence of ATP, the soluble IncC1 that is detectable is exclusively monomeric (mass = 40 kDa, 1.6S) (Fig. 3ai). Plotting the dimer concentration at increasing ATP concentrations (Fig. 3aii) enabled the apparent dissociation constant (Kapp) of IncC1 binding ATP to be calculated as 7.7 μM, considerably lower than the apparent Kd values for other ParA proteins, which are in the range of 33–100 μM.13,14,19,22 Magnesium was important for coordinating the nucleotide, with the Kapp being 130-fold higher (940 μM for 20 μM IncC1) when Mg2+ was absent (data not shown). With magnesium, all of IncC1 is dimeric at or above 30 μM ATP, approximately one ATP molecule per IncC1 monomer. ADP also caused the formation of IncC1 dimers (Fig. 3bi), but with a higher Kapp (Fig. 3bii; 54 μM). Hill analysis29 of the data indicated that there was no cooperativity with ATP, whereas the ADP-induced dimerisation was highly cooperative involving two sites, the second of which was calculated to have a 1000-fold higher affinity.

Bottom Line: ParA proteins normally occur in one of two forms, differing by their N-terminal domain (NTD) of approximately 100 aa, which is generally associated with site-specific DNA binding.The IncC1 NTD does not dimerise or bind DNA alone, but it does bind IncC2 in the presence of nucleotides.Mixing IncC1 and IncC2 improved polymerisation and DNA binding.

View Article: PubMed Central - PubMed

Affiliation: School of Biosciences, University of Birmingham, Edgbaston, Birmingham, UK.

ABSTRACT
ParA Walker ATPases form part of the machinery that promotes better-than-random segregation of bacterial genomes. ParA proteins normally occur in one of two forms, differing by their N-terminal domain (NTD) of approximately 100 aa, which is generally associated with site-specific DNA binding. Unusually, and for as yet unknown reasons, parA (incC) of IncP-1 plasmids is translated from alternative start codons producing two forms, IncC1 (364 aa) and IncC2 (259 aa), whose ratio varies between hosts. IncC2 could be detected as an oligomeric form containing dimers, tetramers and octamers, but the N-terminal extension present in IncC1 favours nucleotide-stimulated dimerisation as well as high-affinity and ATP-dependent non-specific DNA binding. The IncC1 NTD does not dimerise or bind DNA alone, but it does bind IncC2 in the presence of nucleotides. Mixing IncC1 and IncC2 improved polymerisation and DNA binding. Thus, the NTD may modulate the polymerisation interface, facilitating polymerisation/depolymerisation and DNA binding, to promote the cycle that drives partitioning.

Show MeSH
Related in: MedlinePlus