Limits...
The bacterial antitoxin HipB establishes a ternary complex with operator DNA and phosphorylated toxin HipA to regulate bacterial persistence.

Wen Y, Behiels E, Felix J, Elegheert J, Vergauwen B, Devreese B, Savvides SN - Nucleic Acids Res. (2014)

Bottom Line: The structure of HipASO in complex with a non-hydrolyzable ATP analogue shows that HipASO autophosphorylation is coupled to an unusual conformational change of its phosphorylation loop.However, HipASO is unable to phosphorylate the translation factor Elongation factor Tu, contrary to previous reports, but in agreement with more recent findings.Our studies suggest that the phosphorylation state of HipA is an important factor in persistence and that the structural and mechanistic diversity of HipAB modules as regulatory factors in bacterial persistence is broader than previously thought.

View Article: PubMed Central - PubMed

Affiliation: Unit for Biological Mass Spectrometry and Proteomics, Laboratory for Protein Biochemistry and Biomolecular Engineering (L-ProBE), Ghent University, K.L. Ledeganckstraat 35, 9000 Ghent, Belgium Unit for Structural Biology, Laboratory for Protein Biochemistry and Biomolecular Engineering (L-ProBE), Ghent University, K.L. Ledeganckstraat 35, 9000 Ghent, Belgium.

Show MeSH

Related in: MedlinePlus

Biochemical reconstitution and characterization of HipBso (SO0705) and HipAso (SO0706) complexes. (a) SEC profiles of HipBso (orange), HipAso (blue), HipABso complex (red) and HipABso:DNA complex (green). SDS-PAGE analysis of peak fraction is accompanied with the SEC profile. (b) Thermodynamic characterization of the interaction between HipAso and HipBso. (c) Thermodynamic characterization of the interaction between HipBso and single hipBASO operator DNA. (d) Thermodynamic characterization of the interaction between the HipBso:DNA complex and the non-phosphorylated active site mutant HipAsoD306Q. (e) Thermodynamic characterization of the interaction between the HipBso:DNA complex and recombinant HipAso as a mixture of phosphorylated (∼10%) and non-phosphorylated HipAso. The derived interaction stoichiometry (N) and equilibrium dissociation constant (KD) are shown together with ΔH, −ΔTS and ΔG.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4150777&req=5

Figure 1: Biochemical reconstitution and characterization of HipBso (SO0705) and HipAso (SO0706) complexes. (a) SEC profiles of HipBso (orange), HipAso (blue), HipABso complex (red) and HipABso:DNA complex (green). SDS-PAGE analysis of peak fraction is accompanied with the SEC profile. (b) Thermodynamic characterization of the interaction between HipAso and HipBso. (c) Thermodynamic characterization of the interaction between HipBso and single hipBASO operator DNA. (d) Thermodynamic characterization of the interaction between the HipBso:DNA complex and the non-phosphorylated active site mutant HipAsoD306Q. (e) Thermodynamic characterization of the interaction between the HipBso:DNA complex and recombinant HipAso as a mixture of phosphorylated (∼10%) and non-phosphorylated HipAso. The derived interaction stoichiometry (N) and equilibrium dissociation constant (KD) are shown together with ΔH, −ΔTS and ΔG.

Mentions: To enable studies toward obtaining structural and mechanistic insights into the role of HipAso and HipBso in persistence, we overexpressed and purified recombinant HipAso and HipBso. Elution profiles during SEC showed that HipAso could be isolated as a monomer with an apparent molecular weight of 50 kDa, while HipBso could be purified as a dimer with an apparent molecular weight of 30 kDa. Simple mixing of the proteins resulted in the formation of a complex between HipAso and HipBso with an apparent molecular mass of 130 kDa, consistent with 2:2 stoichiometry. Furthermore, the HipABso complex can bind a single operator DNA sequence to form a complex with a distinct chromatographic behavior (Figure 1a). Molecular weights obtained for HipBso, HipBso:DNA HipABso and HipABso:DNA by SAXS correlate well with the apparent sizes estimated from the retention volumes in SEC (Supplementary Table S2).


The bacterial antitoxin HipB establishes a ternary complex with operator DNA and phosphorylated toxin HipA to regulate bacterial persistence.

Wen Y, Behiels E, Felix J, Elegheert J, Vergauwen B, Devreese B, Savvides SN - Nucleic Acids Res. (2014)

Biochemical reconstitution and characterization of HipBso (SO0705) and HipAso (SO0706) complexes. (a) SEC profiles of HipBso (orange), HipAso (blue), HipABso complex (red) and HipABso:DNA complex (green). SDS-PAGE analysis of peak fraction is accompanied with the SEC profile. (b) Thermodynamic characterization of the interaction between HipAso and HipBso. (c) Thermodynamic characterization of the interaction between HipBso and single hipBASO operator DNA. (d) Thermodynamic characterization of the interaction between the HipBso:DNA complex and the non-phosphorylated active site mutant HipAsoD306Q. (e) Thermodynamic characterization of the interaction between the HipBso:DNA complex and recombinant HipAso as a mixture of phosphorylated (∼10%) and non-phosphorylated HipAso. The derived interaction stoichiometry (N) and equilibrium dissociation constant (KD) are shown together with ΔH, −ΔTS and ΔG.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150777&req=5

Figure 1: Biochemical reconstitution and characterization of HipBso (SO0705) and HipAso (SO0706) complexes. (a) SEC profiles of HipBso (orange), HipAso (blue), HipABso complex (red) and HipABso:DNA complex (green). SDS-PAGE analysis of peak fraction is accompanied with the SEC profile. (b) Thermodynamic characterization of the interaction between HipAso and HipBso. (c) Thermodynamic characterization of the interaction between HipBso and single hipBASO operator DNA. (d) Thermodynamic characterization of the interaction between the HipBso:DNA complex and the non-phosphorylated active site mutant HipAsoD306Q. (e) Thermodynamic characterization of the interaction between the HipBso:DNA complex and recombinant HipAso as a mixture of phosphorylated (∼10%) and non-phosphorylated HipAso. The derived interaction stoichiometry (N) and equilibrium dissociation constant (KD) are shown together with ΔH, −ΔTS and ΔG.
Mentions: To enable studies toward obtaining structural and mechanistic insights into the role of HipAso and HipBso in persistence, we overexpressed and purified recombinant HipAso and HipBso. Elution profiles during SEC showed that HipAso could be isolated as a monomer with an apparent molecular weight of 50 kDa, while HipBso could be purified as a dimer with an apparent molecular weight of 30 kDa. Simple mixing of the proteins resulted in the formation of a complex between HipAso and HipBso with an apparent molecular mass of 130 kDa, consistent with 2:2 stoichiometry. Furthermore, the HipABso complex can bind a single operator DNA sequence to form a complex with a distinct chromatographic behavior (Figure 1a). Molecular weights obtained for HipBso, HipBso:DNA HipABso and HipABso:DNA by SAXS correlate well with the apparent sizes estimated from the retention volumes in SEC (Supplementary Table S2).

Bottom Line: The structure of HipASO in complex with a non-hydrolyzable ATP analogue shows that HipASO autophosphorylation is coupled to an unusual conformational change of its phosphorylation loop.However, HipASO is unable to phosphorylate the translation factor Elongation factor Tu, contrary to previous reports, but in agreement with more recent findings.Our studies suggest that the phosphorylation state of HipA is an important factor in persistence and that the structural and mechanistic diversity of HipAB modules as regulatory factors in bacterial persistence is broader than previously thought.

View Article: PubMed Central - PubMed

Affiliation: Unit for Biological Mass Spectrometry and Proteomics, Laboratory for Protein Biochemistry and Biomolecular Engineering (L-ProBE), Ghent University, K.L. Ledeganckstraat 35, 9000 Ghent, Belgium Unit for Structural Biology, Laboratory for Protein Biochemistry and Biomolecular Engineering (L-ProBE), Ghent University, K.L. Ledeganckstraat 35, 9000 Ghent, Belgium.

Show MeSH
Related in: MedlinePlus