The cofactor-induced pre-active conformation in PhoB

PhoB is an Escherichia coli transcription factor from a two-component signal transduction system that is sensitive to limiting environmental phosphate conditions. It consists of an N-­terminal receiver domain (RD) and a C-terminal DNA-binding domain. The protein is activated upon phosphoryl­ation at the RD, an event that depends on Mg²⁺ binding. The structure of PhoB RD in complex with Mg²⁺ is presented, which shows three protomers in the asymmetric unit that interact across two different surfaces. One association is symmetric and has been described as a non-active dimerization contact; the other involves the α4-β5-α5 interface and recalls the contact found in activated PhoB. However, here this last interaction is not perfectly symmetric and helix α4, which in the activated molecule undergoes a helical shift, becomes strongly destabilized in one of the interacting monomers. All protomers bind the cation in a similar manner but, interestingly, at the prospective binding site for the phosphate moiety the side chains of either Glu88 (in helix α4) or Trp54 alternate and interact with active-site atoms. When Glu88 is inside the cavity, helix α4 is arranged similarly to the unliganded wild-type structure. However, if Trp54 is present, the helix loses its contacts with the active-site cavity and vanishes. Accordingly, the presence of Trp54 in the active site induces a flexible state in helix α4, potentially allowing a helical shift that phosphorylation would eventually stabilize.