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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.

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The flexible C-terminal tail of HipBso is accommodated by a hydrophobic pocket in HipAso. (a) Structural detail of the interaction site between HipBso (yellow) and HipAso (blue). (b) Structure-based sequence alignment of S. oneidensis and E. coli HipB (18% identity) showing the conserved W96 residue. (c) Crystal structure (2.35 A) of the HipBso dimer showing the different conformations of the N-termini forming a α0′ helix and β0 sheet, respectively. (d) SAXS modeling showing both HipBso and HipBso:DNA complex have a disordered N- and C-terminus. Five CORAL models, aligned with the N- and C-termini, are colored differently and shown in sphere representation.
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Figure 3: The flexible C-terminal tail of HipBso is accommodated by a hydrophobic pocket in HipAso. (a) Structural detail of the interaction site between HipBso (yellow) and HipAso (blue). (b) Structure-based sequence alignment of S. oneidensis and E. coli HipB (18% identity) showing the conserved W96 residue. (c) Crystal structure (2.35 A) of the HipBso dimer showing the different conformations of the N-termini forming a α0′ helix and β0 sheet, respectively. (d) SAXS modeling showing both HipBso and HipBso:DNA complex have a disordered N- and C-terminus. Five CORAL models, aligned with the N- and C-termini, are colored differently and shown in sphere representation.

Mentions: An additional striking difference between E. coli and S. oneidensis HipAB:DNA complexes concerns the structural order of the C-terminal segment of HipB. We posit that this could have functional implications in terms of the post-translational regulation. E. coli HipB also displays structural disorder at its C-terminal end in the HipAB:DNA complex and it has been demonstrated that the C-terminal tryptophan is proteolytically cleaved by the ATP-dependent Lon protease (40). Interestingly, in our structure of HipABso:DNA at 3.8 Å resolution, we can trace the C terminus of HipBso into a hydrophobic pocket of HipAso. Moreover, in our second structure of HipABso:DNA to 3.4 Å resolution, we see clear density in the same pocket for the last three C terminal residues (Gly95, Trp96 and Tyr97) of HipBso (Figure 3a and Supplementary Figure S3c).


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)

The flexible C-terminal tail of HipBso is accommodated by a hydrophobic pocket in HipAso. (a) Structural detail of the interaction site between HipBso (yellow) and HipAso (blue). (b) Structure-based sequence alignment of S. oneidensis and E. coli HipB (18% identity) showing the conserved W96 residue. (c) Crystal structure (2.35 A) of the HipBso dimer showing the different conformations of the N-termini forming a α0′ helix and β0 sheet, respectively. (d) SAXS modeling showing both HipBso and HipBso:DNA complex have a disordered N- and C-terminus. Five CORAL models, aligned with the N- and C-termini, are colored differently and shown in sphere representation.
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Related In: Results  -  Collection

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Figure 3: The flexible C-terminal tail of HipBso is accommodated by a hydrophobic pocket in HipAso. (a) Structural detail of the interaction site between HipBso (yellow) and HipAso (blue). (b) Structure-based sequence alignment of S. oneidensis and E. coli HipB (18% identity) showing the conserved W96 residue. (c) Crystal structure (2.35 A) of the HipBso dimer showing the different conformations of the N-termini forming a α0′ helix and β0 sheet, respectively. (d) SAXS modeling showing both HipBso and HipBso:DNA complex have a disordered N- and C-terminus. Five CORAL models, aligned with the N- and C-termini, are colored differently and shown in sphere representation.
Mentions: An additional striking difference between E. coli and S. oneidensis HipAB:DNA complexes concerns the structural order of the C-terminal segment of HipB. We posit that this could have functional implications in terms of the post-translational regulation. E. coli HipB also displays structural disorder at its C-terminal end in the HipAB:DNA complex and it has been demonstrated that the C-terminal tryptophan is proteolytically cleaved by the ATP-dependent Lon protease (40). Interestingly, in our structure of HipABso:DNA at 3.8 Å resolution, we can trace the C terminus of HipBso into a hydrophobic pocket of HipAso. Moreover, in our second structure of HipABso:DNA to 3.4 Å resolution, we see clear density in the same pocket for the last three C terminal residues (Gly95, Trp96 and Tyr97) of HipBso (Figure 3a and Supplementary Figure S3c).

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.

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Related in: MedlinePlus