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Crystal structure of a functional dimer of the PhoQ sensor domain.

Cheung J, Bingman CA, Reyngold M, Hendrickson WA, Waldburger CD - J. Biol. Chem. (2008)

Bottom Line: Analysis of the wild-type structure reveals a dimer that allows for the formation of a salt bridge across the dimer interface between Arg-50' and Asp-179 and with nickel ions bound to aspartate residues in the acidic cluster.The physiological importance of the salt bridge to in vivo PhoQ function has been confirmed by mutagenesis.The mutant structure has an alternative, non-physiological dimeric association.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biophysics, Howard Hughes Medical Institute, Columbia University, New York, New York 10032, USA.

ABSTRACT
The PhoP-PhoQ two-component system is a well studied bacterial signaling system that regulates virulence and stress response. Catalytic activity of the histidine kinase sensor protein PhoQ is activated by low extracellular concentrations of divalent cations such as Mg2+, and subsequently the response regulator PhoP is activated in turn through a classic phosphotransfer pathway that is typical in such systems. The PhoQ sensor domains of enteric bacteria contain an acidic cluster of residues (EDDDDAE) that has been implicated in direct binding to divalent cations. We have determined crystal structures of the wild-type Escherichia coli PhoQ periplasmic sensor domain and of a mutant variant in which the acidic cluster was neutralized to conservative uncharged residues (QNNNNAQ). The PhoQ domain structure is similar to that of DcuS and CitA sensor domains, and this PhoQ-DcuS-CitA (PDC) sensor fold is seen to be distinct from the superficially similar PAS domain fold. Analysis of the wild-type structure reveals a dimer that allows for the formation of a salt bridge across the dimer interface between Arg-50' and Asp-179 and with nickel ions bound to aspartate residues in the acidic cluster. The physiological importance of the salt bridge to in vivo PhoQ function has been confirmed by mutagenesis. The mutant structure has an alternative, non-physiological dimeric association.

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In vivo activity and relative expression levels of PhoQ. A, E. coli strain CSH26ΔQ/F′lacIQkan, containing the pNL3 reporter plasmid and pLPQ2 for expressing the wild-type and mutant PhoQ proteins, were assayed for β-galactosidase activity following growth in N-media (0 μm MgCl2) or as supplemented with 40 μm or 10 mm MgCl2. Specific activity is shown in Miller units. Standard deviations are <10% of the mean, calculated from at least three individual experiments. B, the relative expression levels of the wild-type and mutant PhoQ proteins are shown by Western blotting, probed using an antibody specific for the cytoplasmic domain of PhoQ. The PhoQ variants are labeled above each lane accordingly. The control is the E. coli strain containing the “empty” pLPQ2 vector lacking the phoP-phoQ operon.
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fig4: In vivo activity and relative expression levels of PhoQ. A, E. coli strain CSH26ΔQ/F′lacIQkan, containing the pNL3 reporter plasmid and pLPQ2 for expressing the wild-type and mutant PhoQ proteins, were assayed for β-galactosidase activity following growth in N-media (0 μm MgCl2) or as supplemented with 40 μm or 10 mm MgCl2. Specific activity is shown in Miller units. Standard deviations are <10% of the mean, calculated from at least three individual experiments. B, the relative expression levels of the wild-type and mutant PhoQ proteins are shown by Western blotting, probed using an antibody specific for the cytoplasmic domain of PhoQ. The PhoQ variants are labeled above each lane accordingly. The control is the E. coli strain containing the “empty” pLPQ2 vector lacking the phoP-phoQ operon.

Mentions: Measurements of Activity in Vivo—To examine the importance of the dimer interface, and in particular the Arg-50′ → Asp-179 salt bridge, to the function of PhoQ, R50D and D179R single mutants, which would disrupt the salt bridge, and an R50D/D179R double mutant, which could recreate the salt bridge in the opposite direction, were constructed. An additional G54D mutant was constructed in which we reasoned the insertion of a charged residue in the hydrophobic interface would be disruptive. The abilities of such mutants to activate PhoP-mediated transcription of a phoN-lacZ reporter gene in vivo were compared with the wild-type protein. As shown in Fig. 4A, phoN-lacZ expression is induced to roughly 3400 units by MgCl2 limitation when wild-type PhoQ protein is present, but it is only induced to only ∼900 and 245 units, respectively, when the PhoQ-R50D and PhoQ-D179R mutants are present. The effect of the double mutant is indeed compensatory rather than additive; reporter expression is activated to 2225 units for PhoQ-R50D/D179R, >50% of the wild-type level. The effect of the dimer-disrupting PhoQ-G54D mutant results in reporter gene induction to roughly 142 units in the magnesium limiting condition, an effect that most closely matches that of the PhoQ-D179R mutant. The mutations do not significantly affect the relative expression levels of PhoQ (Fig. 4B). These results indicate that the salt bridge observed in the crystal structure is physiologically important in the function of the protein and suggest that the integrity of the interface is crucial for the formation of the active state.


Crystal structure of a functional dimer of the PhoQ sensor domain.

Cheung J, Bingman CA, Reyngold M, Hendrickson WA, Waldburger CD - J. Biol. Chem. (2008)

In vivo activity and relative expression levels of PhoQ. A, E. coli strain CSH26ΔQ/F′lacIQkan, containing the pNL3 reporter plasmid and pLPQ2 for expressing the wild-type and mutant PhoQ proteins, were assayed for β-galactosidase activity following growth in N-media (0 μm MgCl2) or as supplemented with 40 μm or 10 mm MgCl2. Specific activity is shown in Miller units. Standard deviations are <10% of the mean, calculated from at least three individual experiments. B, the relative expression levels of the wild-type and mutant PhoQ proteins are shown by Western blotting, probed using an antibody specific for the cytoplasmic domain of PhoQ. The PhoQ variants are labeled above each lane accordingly. The control is the E. coli strain containing the “empty” pLPQ2 vector lacking the phoP-phoQ operon.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2376233&req=5

fig4: In vivo activity and relative expression levels of PhoQ. A, E. coli strain CSH26ΔQ/F′lacIQkan, containing the pNL3 reporter plasmid and pLPQ2 for expressing the wild-type and mutant PhoQ proteins, were assayed for β-galactosidase activity following growth in N-media (0 μm MgCl2) or as supplemented with 40 μm or 10 mm MgCl2. Specific activity is shown in Miller units. Standard deviations are <10% of the mean, calculated from at least three individual experiments. B, the relative expression levels of the wild-type and mutant PhoQ proteins are shown by Western blotting, probed using an antibody specific for the cytoplasmic domain of PhoQ. The PhoQ variants are labeled above each lane accordingly. The control is the E. coli strain containing the “empty” pLPQ2 vector lacking the phoP-phoQ operon.
Mentions: Measurements of Activity in Vivo—To examine the importance of the dimer interface, and in particular the Arg-50′ → Asp-179 salt bridge, to the function of PhoQ, R50D and D179R single mutants, which would disrupt the salt bridge, and an R50D/D179R double mutant, which could recreate the salt bridge in the opposite direction, were constructed. An additional G54D mutant was constructed in which we reasoned the insertion of a charged residue in the hydrophobic interface would be disruptive. The abilities of such mutants to activate PhoP-mediated transcription of a phoN-lacZ reporter gene in vivo were compared with the wild-type protein. As shown in Fig. 4A, phoN-lacZ expression is induced to roughly 3400 units by MgCl2 limitation when wild-type PhoQ protein is present, but it is only induced to only ∼900 and 245 units, respectively, when the PhoQ-R50D and PhoQ-D179R mutants are present. The effect of the double mutant is indeed compensatory rather than additive; reporter expression is activated to 2225 units for PhoQ-R50D/D179R, >50% of the wild-type level. The effect of the dimer-disrupting PhoQ-G54D mutant results in reporter gene induction to roughly 142 units in the magnesium limiting condition, an effect that most closely matches that of the PhoQ-D179R mutant. The mutations do not significantly affect the relative expression levels of PhoQ (Fig. 4B). These results indicate that the salt bridge observed in the crystal structure is physiologically important in the function of the protein and suggest that the integrity of the interface is crucial for the formation of the active state.

Bottom Line: Analysis of the wild-type structure reveals a dimer that allows for the formation of a salt bridge across the dimer interface between Arg-50' and Asp-179 and with nickel ions bound to aspartate residues in the acidic cluster.The physiological importance of the salt bridge to in vivo PhoQ function has been confirmed by mutagenesis.The mutant structure has an alternative, non-physiological dimeric association.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biophysics, Howard Hughes Medical Institute, Columbia University, New York, New York 10032, USA.

ABSTRACT
The PhoP-PhoQ two-component system is a well studied bacterial signaling system that regulates virulence and stress response. Catalytic activity of the histidine kinase sensor protein PhoQ is activated by low extracellular concentrations of divalent cations such as Mg2+, and subsequently the response regulator PhoP is activated in turn through a classic phosphotransfer pathway that is typical in such systems. The PhoQ sensor domains of enteric bacteria contain an acidic cluster of residues (EDDDDAE) that has been implicated in direct binding to divalent cations. We have determined crystal structures of the wild-type Escherichia coli PhoQ periplasmic sensor domain and of a mutant variant in which the acidic cluster was neutralized to conservative uncharged residues (QNNNNAQ). The PhoQ domain structure is similar to that of DcuS and CitA sensor domains, and this PhoQ-DcuS-CitA (PDC) sensor fold is seen to be distinct from the superficially similar PAS domain fold. Analysis of the wild-type structure reveals a dimer that allows for the formation of a salt bridge across the dimer interface between Arg-50' and Asp-179 and with nickel ions bound to aspartate residues in the acidic cluster. The physiological importance of the salt bridge to in vivo PhoQ function has been confirmed by mutagenesis. The mutant structure has an alternative, non-physiological dimeric association.

Show MeSH
Related in: MedlinePlus