The Crystal Structure of the Phosphorylation Domain in PhoP Reveals a Functional Tandem Association Mediated by an Asymmetric Interface

Abstract

PhoP from Bacillus subtilis belongs to the OmpR subfamily of response regulators. It regulates the transcription of several operons and participates in a signal transduction network that controls adaptation of the bacteria to phosphate deficiency. The receiver domains of two members of this subfamily, PhoB from Escherichia coli and DrrD from Thermotoga maritima, have been structurally characterized. These modules have similar overall folds but display remarkable differences in the conformation of the ␤4-␣4 and ␣4 regions. The crystal structure of the receiver domain of PhoP (PhoPN) described in this paper illustrates yet another geometry in this region. Another major issue of the structure determination is the dimeric state of the protein and the novel mode of association between receiver domains. The protein-protein interface is provided by two different surfaces from each protomer, and the tandem unit formed through this asymmetric interface leaves free interaction surfaces. This design is well suited for further association of PhoP dimers to form oligomeric structures. The interprotein interface buries 970 Å 2 from solvent and mostly involves interactions between charged residues. As described in the accompanying paper, mutations of a single residue in one salt bridge shielded from solvent prevented dimerization of the unphosphorylated and phosphorylated response regulator and had drastic functional consequences. The three structurally documented members of the OmpR family (PhoB, DrrD, and PhoP) provide a framework to consider possible relationships between structural features and sequence signatures in critical regions of the receiver domains.

Structural Analysis and Solution Studies of the Activated Regulatory Domain of the Response Regulator ArcA: A Symmetric Dimer Mediated by the α4-β5-α5 Face

“…This would leave the α4-β5-α5 face of the regulatory domain available for formation of an active symmetric dimer and the effector domain free to bind DNA with a different symmetry. Additionally, the formation of the α4-β5-α5 dimer interface juxtaposes the linkers of the RRs on the same plane, as opposed to the dimers formed in inactive E. coli PhoB 64 and B. subtilis PhoP, 50 facilitating association of the effector domains into dimers for DNA binding. While this model is compatible with tandem binding to direct repeats, it does not exclude the possibility of other binding modes such as symmetric binding to inverted repeats as has been postulated for some OmpR/PhoB subfamily members.…”

“…A multiple sequence alignment of the regulatory domains of all members of this subfamily from E. coli, 20 in addition to subfamily members from other organisms for which structures of the regulatory domain are available, 33,34,49,50 revealed that the residues involved in key dimer interface interactions are highly conserved within this subfamily (Fig. 5).…”

“…Such interactions might include, for example, the formation of active higher order oligomers, or the formation of alternate inactive dimer interfaces as seen in E. coli PhoB 64 and B. subtilis PhoP. 50 …”

Structure of the DNA-Binding Domain of the Response Regulator PhoP from Mycobacterium tuberculosis,

“…Currently, the only available DNA complex structure is the DNA-binding domain of PhoB from E. coli in complex with its pho box DNA (16), which shows a tandem dimer. The N-terminal domain of PhoP from B. subtilis also has a tandem association in the crystal structure (8). However, crystal structures of the N-terminal domain of E. coli ArcA, TorR, and KdpE show that they form a symmetric dimer mediated by the α4-β5-α5 face (17,18).…”

“…The phosphoryl group is then transferred to a conserved aspartate residue of PhoP (7), a response regulator that regulates the expression of particular genes (5). In Bacillus subtilis, the PhoP-PhoR system senses phosphate, being activated under limited phosphate conditions, and it regulates the expression of 31 genes that are involved in phosphate utilization (8). The related PhoP-PhoQ system in Salmonella enterica typhimurium, however, senses Mg 2+ (9), low pH (10,11), and antibacterial peptides (12,13); it regulates genes involved in Mg 2+ transport and acquisition and also controls the expression of several virulence factors (14).…”