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

S. oneidensis MR-1 HipAso is regulated conformationally upon autophosphorylation. (a) Structural detail of the AMPPMP (atom colored) and Mg2+ (green spheres) binding site in HipAso (blue). Residues involved are shown in stick representation. (b) Detailed view of the ejection of the pLoop of HipAso upon autophosphorylation. Ser147 and phosphate represented as spheres. (c) Identification of the phosphorylated peptide by nanoLCMS. The peptide LSVAGVQPK was observed at charge state 2+ in two forms differing by 80 Da in molecular weight. Sequence alignment of the pLoop regions in S. oneidensis MR-1 and E. coli.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: S. oneidensis MR-1 HipAso is regulated conformationally upon autophosphorylation. (a) Structural detail of the AMPPMP (atom colored) and Mg2+ (green spheres) binding site in HipAso (blue). Residues involved are shown in stick representation. (b) Detailed view of the ejection of the pLoop of HipAso upon autophosphorylation. Ser147 and phosphate represented as spheres. (c) Identification of the phosphorylated peptide by nanoLCMS. The peptide LSVAGVQPK was observed at charge state 2+ in two forms differing by 80 Da in molecular weight. Sequence alignment of the pLoop regions in S. oneidensis MR-1 and E. coli.

Mentions: In E. coli, HipA kinase activity is essential for growth arrest and MDT (12). To further investigate the mechanism of HipAso in S. oneidensis MR-1, we determined the structure of HipAso in complex with the non-hydrolysable ATP analogue AMPPNP and Mg2+ to 1.83 Å resolution (Table 1). This structural analysis at high resolution revealed binding of AMPPNP and Mg2+ at high occupancy and delineation of the protein-nucleotide interaction landscape. The ATP binding site in HipAso is engulfed by the N- and C-terminal domains and most interacting residues are contributed by five loop regions (Figure 4a and Supplementary Figure S4a). All residues involved in Mg2+ and ATP binding are highly conserved in S. oneidensis MR-1 and E. coli (Figure 4a and Supplementary Figure S1). Given that the physiological ATP concentration in the cell is enough to drive the autophosphorylation of HipA, we propose that phosphorylation of HipAso is essential for the formation of the ternary HipABso:DNA complex. The HipAso pLoop consists of residues 129–152 and is ejected out of the pocket compared to the non-phosphorylated form (Figure 4b). Furthermore, the pLoop in autophosphorylated HipAso, as observed in the HipABso:DNA complex, projects to the surface. Such activation mechanism involving ejection of the pLoop upon phosphorylation has also been proposed for E. coli HipA (43). This could be a common feature for the HipA kinase family. To investigate the autophosphorylation capacity of HipAso, we incubated freshly purified HipAso with ATP and Mg2+ overnight and observed a dramatic increase of the corresponding phosphorylated peptide intensity from approximately 5% to almost 45% relative to the signal of the non-phosphorylated peptide in LCMS (Figure 4c).


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)

S. oneidensis MR-1 HipAso is regulated conformationally upon autophosphorylation. (a) Structural detail of the AMPPMP (atom colored) and Mg2+ (green spheres) binding site in HipAso (blue). Residues involved are shown in stick representation. (b) Detailed view of the ejection of the pLoop of HipAso upon autophosphorylation. Ser147 and phosphate represented as spheres. (c) Identification of the phosphorylated peptide by nanoLCMS. The peptide LSVAGVQPK was observed at charge state 2+ in two forms differing by 80 Da in molecular weight. Sequence alignment of the pLoop regions in S. oneidensis MR-1 and E. coli.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: S. oneidensis MR-1 HipAso is regulated conformationally upon autophosphorylation. (a) Structural detail of the AMPPMP (atom colored) and Mg2+ (green spheres) binding site in HipAso (blue). Residues involved are shown in stick representation. (b) Detailed view of the ejection of the pLoop of HipAso upon autophosphorylation. Ser147 and phosphate represented as spheres. (c) Identification of the phosphorylated peptide by nanoLCMS. The peptide LSVAGVQPK was observed at charge state 2+ in two forms differing by 80 Da in molecular weight. Sequence alignment of the pLoop regions in S. oneidensis MR-1 and E. coli.
Mentions: In E. coli, HipA kinase activity is essential for growth arrest and MDT (12). To further investigate the mechanism of HipAso in S. oneidensis MR-1, we determined the structure of HipAso in complex with the non-hydrolysable ATP analogue AMPPNP and Mg2+ to 1.83 Å resolution (Table 1). This structural analysis at high resolution revealed binding of AMPPNP and Mg2+ at high occupancy and delineation of the protein-nucleotide interaction landscape. The ATP binding site in HipAso is engulfed by the N- and C-terminal domains and most interacting residues are contributed by five loop regions (Figure 4a and Supplementary Figure S4a). All residues involved in Mg2+ and ATP binding are highly conserved in S. oneidensis MR-1 and E. coli (Figure 4a and Supplementary Figure S1). Given that the physiological ATP concentration in the cell is enough to drive the autophosphorylation of HipA, we propose that phosphorylation of HipAso is essential for the formation of the ternary HipABso:DNA complex. The HipAso pLoop consists of residues 129–152 and is ejected out of the pocket compared to the non-phosphorylated form (Figure 4b). Furthermore, the pLoop in autophosphorylated HipAso, as observed in the HipABso:DNA complex, projects to the surface. Such activation mechanism involving ejection of the pLoop upon phosphorylation has also been proposed for E. coli HipA (43). This could be a common feature for the HipA kinase family. To investigate the autophosphorylation capacity of HipAso, we incubated freshly purified HipAso with ATP and Mg2+ overnight and observed a dramatic increase of the corresponding phosphorylated peptide intensity from approximately 5% to almost 45% relative to the signal of the non-phosphorylated peptide in LCMS (Figure 4c).

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