The X-ray crystal structures of two constitutively active mutants of the Escherichia coli PhoB receiver domain give insights into activation

The PhoR/PhoB two-component system is a key regulatory protein network enabling Escherichia coli to respond to inorganic phosphate (Pi) starvation conditions by turning on Pho regulon genes for more efficient Pi uptake and use of alternative phosphorus sources. Under environmental Pi depletion, the response regulator (RR) component, PhoB, is phosphorylated at the receiver domain (RD), a process that requires Mg2+ bound at the active site. Phosphorylation of the RD relieves the inhibition of the PhoB effector domain (ED), a DNA-binding region that binds to Pho regulon promoters to activate transcription. The molecular details of the activation are proposed to involve dimerization of the RD and a conformational change in the RD detected by the ED. The structure of the PhoB RD shows a symmetrical interaction involving alpha 1, loop beta 5 alpha 5 and N terminus of alpha 5 elements, also seen in the complex of PhoB RD with Mg2+, in which helix alpha 4 highly increases its flexibility. PhoB RD in complex with Mg2+ and BeF3- (an emulator of the phosphate moiety) undergoes a dramatic conformational change on helix a4 and shows another interaction involving a4, (35 and a5 segments. We have selected a series of constitutively active PhoB mutants (PhoB(CA)) that are able to turn on the Pho regulon promoters in the absence phosphorylation and, as they cannot be inactivated, should therefore mimic the active RD state of PhoB and its functional oligomerisation. We have analysed the PhoBCA RD crystal structures of two such mutants, Asp53Ala/Tyr102Cys and Asp10Ala/Asp53Glu. Interestingly, both mutants reproduce the homodimeric arrangement through the symmetric interface encountered in the unbound and magnesium-bound wild-type PhoB RD structures. Besides, the mutant RD structures show a modified active site organization as well as changes at helix a4 that correlate with repositioning of surrounding residues, like the active-site events indicator Trp54, putatively redifining the interaction with the ED in the full-length protein. (c) 2006 Elsevier Ltd. All rights reserved.