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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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Structural studies of the HipABso:DNA complex. (a) Crystal structure of the HipABso:DNA complex shown in different orientations. A dimer of HipBso (yellow) interacts with the C-terminal helical bundles (dark blue) of two HipAso; the N-terminus (light blue) of HipAso does not take part in the interaction with HipBso. (b) X-ray crystal structure of the E.coli HipAB:DNA complex (PDB: 3DNV) in top view oriented based on a superposition with duplex DNA and HipBso shown in panel (a). (c) SAXS analysis of the HipABso complex and comparison with the crystal structure of HipABso (χ2 = 1.5) and that of E. coli HipAB (χ2 = 5.7). (d) Detail of the residues involved in the interaction between HipAso and HipBso. (e) Detail of the interaction interface between HipBso and single hipABso operator DNA. A positively charged patch on HipAso defined by R380, R383, R384 and R429 forms a complementary interaction site with the negatively charged phosphodiester backbone of the operator DNA. Further interaction details are given in Supplementary Figure S3b. (f) Bending of the operator DNA upon interaction with the helix-turn-helix motif of HipBso. (g) Structural detail of the interactions stabilizing the phosphorylated pLoop of HipAso in the ternary complex.
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Figure 2: Structural studies of the HipABso:DNA complex. (a) Crystal structure of the HipABso:DNA complex shown in different orientations. A dimer of HipBso (yellow) interacts with the C-terminal helical bundles (dark blue) of two HipAso; the N-terminus (light blue) of HipAso does not take part in the interaction with HipBso. (b) X-ray crystal structure of the E.coli HipAB:DNA complex (PDB: 3DNV) in top view oriented based on a superposition with duplex DNA and HipBso shown in panel (a). (c) SAXS analysis of the HipABso complex and comparison with the crystal structure of HipABso (χ2 = 1.5) and that of E. coli HipAB (χ2 = 5.7). (d) Detail of the residues involved in the interaction between HipAso and HipBso. (e) Detail of the interaction interface between HipBso and single hipABso operator DNA. A positively charged patch on HipAso defined by R380, R383, R384 and R429 forms a complementary interaction site with the negatively charged phosphodiester backbone of the operator DNA. Further interaction details are given in Supplementary Figure S3b. (f) Bending of the operator DNA upon interaction with the helix-turn-helix motif of HipBso. (g) Structural detail of the interactions stabilizing the phosphorylated pLoop of HipAso in the ternary complex.

Mentions: The structure of the HipABso:DNA ternary complex was determined in two crystal forms at 3.4 Å and 3.8 Å resolution (Table 1). The S. oneidensis MR-1 HipABso:DNA ternary complex assembles as a compact HipBso dimer bound by a 25 bp duplex operator DNA, with each of the HipBso subunits interacting with a separate HipAso subunit (Figure 2a). Unexpectedly, the overall HipABso:DNA complex assembly is remarkably distinct from the previously characterized E. coli HipABso:DNA ternary complex (PDB:3DNV) (14) (Figure 2b). Notably, E. coli HipA uses both its N- and C-terminal domains to contact the entire side of the HipB dimer, without making any contacts with operator DNA. Thus, viewed from the top of the DNA double helix, HipAso emerges as a toxin that employs dramatically different structural principles to engage in a ternary complex with HipB:DNA compared to the prototypic counterpart in E. coli (Figure 2a and b). We have cross-validated the observed configuration of the HipABso:DNA complex by SAXS experiments of the complex in solution, which revealed that the crystallographically observed complex agrees well with the scattering data in solution. At the same time, a model based on the E. coli HipAB:DNA complex configuration strongly contradicts our SAXS data (Figure 2c). Together this analysis lends orthogonal support to the crystallographically observed HipABso:DNA complex.


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)

Structural studies of the HipABso:DNA complex. (a) Crystal structure of the HipABso:DNA complex shown in different orientations. A dimer of HipBso (yellow) interacts with the C-terminal helical bundles (dark blue) of two HipAso; the N-terminus (light blue) of HipAso does not take part in the interaction with HipBso. (b) X-ray crystal structure of the E.coli HipAB:DNA complex (PDB: 3DNV) in top view oriented based on a superposition with duplex DNA and HipBso shown in panel (a). (c) SAXS analysis of the HipABso complex and comparison with the crystal structure of HipABso (χ2 = 1.5) and that of E. coli HipAB (χ2 = 5.7). (d) Detail of the residues involved in the interaction between HipAso and HipBso. (e) Detail of the interaction interface between HipBso and single hipABso operator DNA. A positively charged patch on HipAso defined by R380, R383, R384 and R429 forms a complementary interaction site with the negatively charged phosphodiester backbone of the operator DNA. Further interaction details are given in Supplementary Figure S3b. (f) Bending of the operator DNA upon interaction with the helix-turn-helix motif of HipBso. (g) Structural detail of the interactions stabilizing the phosphorylated pLoop of HipAso in the ternary complex.
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Figure 2: Structural studies of the HipABso:DNA complex. (a) Crystal structure of the HipABso:DNA complex shown in different orientations. A dimer of HipBso (yellow) interacts with the C-terminal helical bundles (dark blue) of two HipAso; the N-terminus (light blue) of HipAso does not take part in the interaction with HipBso. (b) X-ray crystal structure of the E.coli HipAB:DNA complex (PDB: 3DNV) in top view oriented based on a superposition with duplex DNA and HipBso shown in panel (a). (c) SAXS analysis of the HipABso complex and comparison with the crystal structure of HipABso (χ2 = 1.5) and that of E. coli HipAB (χ2 = 5.7). (d) Detail of the residues involved in the interaction between HipAso and HipBso. (e) Detail of the interaction interface between HipBso and single hipABso operator DNA. A positively charged patch on HipAso defined by R380, R383, R384 and R429 forms a complementary interaction site with the negatively charged phosphodiester backbone of the operator DNA. Further interaction details are given in Supplementary Figure S3b. (f) Bending of the operator DNA upon interaction with the helix-turn-helix motif of HipBso. (g) Structural detail of the interactions stabilizing the phosphorylated pLoop of HipAso in the ternary complex.
Mentions: The structure of the HipABso:DNA ternary complex was determined in two crystal forms at 3.4 Å and 3.8 Å resolution (Table 1). The S. oneidensis MR-1 HipABso:DNA ternary complex assembles as a compact HipBso dimer bound by a 25 bp duplex operator DNA, with each of the HipBso subunits interacting with a separate HipAso subunit (Figure 2a). Unexpectedly, the overall HipABso:DNA complex assembly is remarkably distinct from the previously characterized E. coli HipABso:DNA ternary complex (PDB:3DNV) (14) (Figure 2b). Notably, E. coli HipA uses both its N- and C-terminal domains to contact the entire side of the HipB dimer, without making any contacts with operator DNA. Thus, viewed from the top of the DNA double helix, HipAso emerges as a toxin that employs dramatically different structural principles to engage in a ternary complex with HipB:DNA compared to the prototypic counterpart in E. coli (Figure 2a and b). We have cross-validated the observed configuration of the HipABso:DNA complex by SAXS experiments of the complex in solution, which revealed that the crystallographically observed complex agrees well with the scattering data in solution. At the same time, a model based on the E. coli HipAB:DNA complex configuration strongly contradicts our SAXS data (Figure 2c). Together this analysis lends orthogonal support to the crystallographically observed HipABso:DNA complex.

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