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

Crystal Structure of a Functional Dimer of the PhoQ Sensor Domain^*

^‡Department of Biochemistry and Molecular Biophysics, the ^§Department of Microbiology, and the ^¶Howard Hughes Medical Institute, Columbia University, New York, New York 10032

↵³ To whom correspondence may be addressed: Dept. of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, 10032. Tel.: 212-305-3456; Fax: 212-205-7379; E-mail: wayne{at}convex.hhmi.columbia.edu. ⁴ To whom correspondence may be addressed: Dept. of Biology, William Paterson University, Wayne, NJ 07470. Tel.: 973-720-2486; Fax: 973-720-2338; E-mail: WaldburgerC{at}wpunj.edu.

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.

Footnotes

The atomic coordinates and structure factors (codes 3BQ8 and 3BQA) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
↵* This work was supported, in whole or in part, by National Institutes of Health Grants AI41566 (to C. D. W.) and GM34102 (to W. A. H.). Beamline X4A of the National Synchrotron Light Source at Brookhaven National Laboratory, a Department of Energy facility, is supported by the New York Structural Biology Center. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵ The on-line version of this article (available at http://www.jbc.org) contains supplemental text and references.
↵ Author's Choice—Final version full access.
↵1 Both authors contributed with equivalent importance to this work.
↵2 Present address: Dept. of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706.