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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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DNA binding by N-terminal deletion mutants of IncC1. (a) Diagram of IncC1 N-terminal deletion mutants. All delta (Δ) N-terminal mutants continue to the end of IncC1 (not shown in diagram). Amino acids corresponding to the HTH domain on other ParA proteins are in boldface. The predicted secondary structure (labelled 2°) is shown above (cylinders correspond to α-helices).33 (b) Graphs of band shifts performed with the IncC1 N-terminal deletion mutants using supercoiled pSMB201 DNA. For band shifts with ATP, the percentage of change in mobility was calculated as the percentage of reduction in the distance of the DNA band from the well. For band shifts with no nucleotide (as well as IncC2 and NΔ60 with ATP), the percentage of retardation was calculated by the reduction of the DNA band's intensity, which was analysed using Quantity One (BioRad).
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fig5: DNA binding by N-terminal deletion mutants of IncC1. (a) Diagram of IncC1 N-terminal deletion mutants. All delta (Δ) N-terminal mutants continue to the end of IncC1 (not shown in diagram). Amino acids corresponding to the HTH domain on other ParA proteins are in boldface. The predicted secondary structure (labelled 2°) is shown above (cylinders correspond to α-helices).33 (b) Graphs of band shifts performed with the IncC1 N-terminal deletion mutants using supercoiled pSMB201 DNA. For band shifts with ATP, the percentage of change in mobility was calculated as the percentage of reduction in the distance of the DNA band from the well. For band shifts with no nucleotide (as well as IncC2 and NΔ60 with ATP), the percentage of retardation was calculated by the reduction of the DNA band's intensity, which was analysed using Quantity One (BioRad).

Mentions: Deletion mutants (Fig. 5a) were constructed in approximately 20-aa steps, with endpoints defined by the predicted secondary structures of the NTDs of other ParA proteins, ensuring that complete α-helices and β-sheets were included or deleted from each mutant derivative. The predicted structure of the IncC1-NTD was also consistent with the known structure of the Ffh signal recognition particle protein, which shows sequence similarities to ParA proteins.31,32 EMSAs with supercoiled plasmid DNA (Fig. 5b) showed that even removal of 23 aa from the N-terminus decreased efficiency of DNA binding: by 1.5-fold in the absence of any nucleotide and by 5-fold in the presence of ATP. Deletion of 41 aa had a slight additional effect (2-fold and 8-fold in the absence and in the presence of ATP, respectively), but the greatest decline in DNA binding was shown by IncC1-NΔ60 and IncC1-NΔ80, both of which no longer contain the region that in other ParA proteins encodes the putative HTH domain. These mutants both retarded the DNA at similar concentrations as with IncC2—a difference of 7-fold in the absence of any nucleotide and that of 18-fold in the presence of ATP as compared with wild-type IncC1. There was also a difference in the gradual reduction in DNA mobility in the presence of ATP: IncC1-NΔ23, IncC1-NΔ41 and IncC1-NΔ80 all behaved similarly to IncC1, while IncC1-NΔ60 behaved more like IncC2, retarding the DNA over a narrower protein concentration, which demonstrates that binding is more cooperative. A point mutation in the NTD of IncC1 (S55N) did not alter the basic affinity of IncC1 for supercoiled DNA but did slightly reduce the ability of ATP to stimulate binding (Fig. 5b). Tests for the dimerisation of the deletion derivatives revealed that even the smallest deletion (23 aa) resulted in complete loss of ATP-stimulated dimerisation possessed by IncC1 (data not shown). When the NTD of IncC1 was purified alone, it did not retard the DNA at all at any of the concentrations tested (data not shown). Thus, the influence of the NTD on IncC behaviour depends on the whole domain being intact and in the context of a complete IncC protein.


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)

DNA binding by N-terminal deletion mutants of IncC1. (a) Diagram of IncC1 N-terminal deletion mutants. All delta (Δ) N-terminal mutants continue to the end of IncC1 (not shown in diagram). Amino acids corresponding to the HTH domain on other ParA proteins are in boldface. The predicted secondary structure (labelled 2°) is shown above (cylinders correspond to α-helices).33 (b) Graphs of band shifts performed with the IncC1 N-terminal deletion mutants using supercoiled pSMB201 DNA. For band shifts with ATP, the percentage of change in mobility was calculated as the percentage of reduction in the distance of the DNA band from the well. For band shifts with no nucleotide (as well as IncC2 and NΔ60 with ATP), the percentage of retardation was calculated by the reduction of the DNA band's intensity, which was analysed using Quantity One (BioRad).
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fig5: DNA binding by N-terminal deletion mutants of IncC1. (a) Diagram of IncC1 N-terminal deletion mutants. All delta (Δ) N-terminal mutants continue to the end of IncC1 (not shown in diagram). Amino acids corresponding to the HTH domain on other ParA proteins are in boldface. The predicted secondary structure (labelled 2°) is shown above (cylinders correspond to α-helices).33 (b) Graphs of band shifts performed with the IncC1 N-terminal deletion mutants using supercoiled pSMB201 DNA. For band shifts with ATP, the percentage of change in mobility was calculated as the percentage of reduction in the distance of the DNA band from the well. For band shifts with no nucleotide (as well as IncC2 and NΔ60 with ATP), the percentage of retardation was calculated by the reduction of the DNA band's intensity, which was analysed using Quantity One (BioRad).
Mentions: Deletion mutants (Fig. 5a) were constructed in approximately 20-aa steps, with endpoints defined by the predicted secondary structures of the NTDs of other ParA proteins, ensuring that complete α-helices and β-sheets were included or deleted from each mutant derivative. The predicted structure of the IncC1-NTD was also consistent with the known structure of the Ffh signal recognition particle protein, which shows sequence similarities to ParA proteins.31,32 EMSAs with supercoiled plasmid DNA (Fig. 5b) showed that even removal of 23 aa from the N-terminus decreased efficiency of DNA binding: by 1.5-fold in the absence of any nucleotide and by 5-fold in the presence of ATP. Deletion of 41 aa had a slight additional effect (2-fold and 8-fold in the absence and in the presence of ATP, respectively), but the greatest decline in DNA binding was shown by IncC1-NΔ60 and IncC1-NΔ80, both of which no longer contain the region that in other ParA proteins encodes the putative HTH domain. These mutants both retarded the DNA at similar concentrations as with IncC2—a difference of 7-fold in the absence of any nucleotide and that of 18-fold in the presence of ATP as compared with wild-type IncC1. There was also a difference in the gradual reduction in DNA mobility in the presence of ATP: IncC1-NΔ23, IncC1-NΔ41 and IncC1-NΔ80 all behaved similarly to IncC1, while IncC1-NΔ60 behaved more like IncC2, retarding the DNA over a narrower protein concentration, which demonstrates that binding is more cooperative. A point mutation in the NTD of IncC1 (S55N) did not alter the basic affinity of IncC1 for supercoiled DNA but did slightly reduce the ability of ATP to stimulate binding (Fig. 5b). Tests for the dimerisation of the deletion derivatives revealed that even the smallest deletion (23 aa) resulted in complete loss of ATP-stimulated dimerisation possessed by IncC1 (data not shown). When the NTD of IncC1 was purified alone, it did not retard the DNA at all at any of the concentrations tested (data not shown). Thus, the influence of the NTD on IncC behaviour depends on the whole domain being intact and in the context of a complete IncC protein.

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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