Crystal Structure of a Functional Dimer of the PhoQ Sensor Domain

Jonah Cheung,Craig A Bingman,Marsha Reyngold,Wayne A Hendrickson,Carey D Waldburger

doi:10.1074/jbc.m710592200

Jonah Cheung, Craig A Bingman + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m710592200

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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.

Highlights

Cell survival in a changing environment requires constant adaptation
PhoQ is found in non-pathogenic Gram-negative bacteria such as Escherichia coli where it controls a regulon that includes several of the same genes that are regulated in S. typhimurium and many that are not [10, 11]
This, in turn, has led others to identify PDC sensor domains as PAS domains; there are no actual similarities outside the ␤-sheet, whereas PDC and PAS domains each have their own distinguishing characteristics

Summary

EXPERIMENTAL PROCEDURES

Bacterial Strains and Plasmids—pLPQ2 [12] is a pSC101derived plasmid in which expression of the E. coli phoP-phoQ operon is driven by the lacUV5 promoter. pNL3 [12] is a pBR322-derived reporter plasmid for assaying PhoP-mediated transcriptional activation that contains the phoN promoter fused to lacZ. pAED4Q [12] is a pUC19-derived plasmid in which the PhoQ sensor domain (residues 43–190) is expressed from the phage T7 ␾10 promoter [13]. ␤-Galactosidase Assays—␤-Galactosidase assays were performed as described [14] in N-media with additions of MgCl2 as indicated using strain CSH26⌬Q/FЈ lacIQkan/ pNL3 containing derivatives of pLPQ2 with wild-type phoQ or mutant variants. Preparation of Cell Membranes and Western Blotting—E. coli CSH26⌬Q/FЈ lacIQkan/pNL3 strains containing derivatives of pLPQ2 with wild-type phoQ or mutant variants were grown to mid-log phase in N-media with appropriate antibiotics as described previously [14] and induced for 2 h at 37 °C with the addition of 1 mM isopropyl-␤-D-thiogalactopyranoside. Protein Purification and Crystallization—The E. coli PhoQ wild-type sensor domain construct (residues 43–190), and the acid mutant construct (residues 43–190) in which residues 148 –154 were changed from EDDDDAE to QNNNNAQ were expressed and purified from E. coli strain X90(DE3) as previously described [12]. The atomic coordinates and structure factors for both wild-type and acid mutant E. coli PhoQ sensor domains have been deposited

RESULTS

Findings

DISCUSSION