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Journal of Bacteriology, January 2003, p. 262-273, Vol. 185, No. 1
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.1.262-273.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Residue R113 Is Essential for PhoP Dimerization and Function: a Residue Buried in the Asymmetric PhoP Dimer Interface Determined in the PhoPN Three-Dimensional Crystal Structure

Yinghua Chen,1 Catherine Birck,2,{dagger} Jean-Pierre Samama,2,{dagger} and F. Marion Hulett1*

Laboratory for Molecular Biology, Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois 60607,1 Groupe de Cristallographie Biologique, CNRS-IPBS, 31077, Toulouse, France2

Received 9 July 2002/ Accepted 4 October 2002

Bacillus subtilis PhoP is a member of the OmpR/PhoB family of response regulators that is directly required for transcriptional activation or repression of Pho regulon genes in conditions under which Pi is growth limiting. Characterization of the PhoP protein has established that phosphorylation of the protein is not essential for PhoP dimerization or DNA binding but is essential for transcriptional regulation of Pho regulon genes. DNA footprinting studies of PhoP-regulated promoters showed that there was cooperative binding between PhoP dimers at PhoP-activated promoters and/or extensive PhoP oligomerization 3' of PhoP-binding consensus repeats in PhoP-repressed promoters. The crystal structure of PhoPN described in the accompanying paper revealed that the dimer interface between two PhoP monomers involves nonidentical surfaces such that each monomer in a dimer retains a second surface that is available for further oligomerization. A salt bridge between R113 on one monomer and D60 on another monomer was judged to be of major importance in the protein-protein interaction. We describe the consequences of mutation of the PhoP R113 codon to a glutamate or alanine codon and mutation of the PhoP D60 codon to a lysine codon. In vivo expression of either PhoPR113E, PhoPR113A, or PhoPD60K resulted in a Pho-negative phenotype. In vitro analysis showed that PhoPR113E was phosphorylated by PhoR (the cognate histidine kinase) but was unable to dimerize. Monomeric PhoPR113E~P was deficient in DNA binding, contributing to the PhoPR113E in vivo Pho-negative phenotype. While previous studies emphasized that phosphorylation was essential for PhoP function, data reported here indicate that phosphorylation is not sufficient as PhoP dimerization or oligomerization is also essential. Our data support the physiological relevance of the residues of the asymmetric dimer interface in PhoP dimerization and function.

* Corresponding author. Mailing address: Laboratory for Molecular Biology, Department of Biological Sciences, University of Illinois at Chicago, 900 S. Ashland Avenue (M/C 567), Chicago, IL 60607. Phone: (312) 996-5460. Fax: (312) 413-2691. E-mail: Hulett{at}uic.edu.

{dagger} Present address: Département de Biologie et de Génomique Structuales, IGBMC, 67404 Illkirch, France.

Journal of Bacteriology, January 2003, p. 262-273, Vol. 185, No. 1
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.1.262-273.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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