In two-component signal transduction systems (TCSs), responses to stimuli are
mediated through phosphotransfer between protein components. Canonical TCSs use
His → Asp phosphotransfer in which phosphoryl groups are transferred from a
conserved His on a sensory histidine kinase (HK) to a conserved Asp on a
response regulator (RR). RRs contain the catalytic core of His → Asp
phosphotransfer, evidenced by the ability of RRs to autophosphorylate with small
molecule analogues of phospho-His proteins. Phosphorelays are a more complex
variation of TCSs that additionally utilize Asp → His phosphotransfer through
the use of an additional component, the histidine-containing phosphotransfer
domain (Hpt), which reacts with RRs both as phosphodonors and phosphoacceptors.
Here we show that imidazole has features of a rudimentary Hpt. Imidazole acted
as a nucleophile and attacked phosphorylated RRs (RR-P) to produce
monophosphoimidazole (MPI) and unphosphorylated RR. Phosphotransfer from RR-P to
imidazole required the intact RR active site, indicating that the RR provided
the core catalytic machinery for Asp → His phosphotransfer. Imidazole
functioned in an artificial phosphorelay to transfer phosphoryl groups between
unrelated RRs. The X-ray crystal structure of an activated RR·imidazole complex
showed imidazole oriented in the RR active site similarly to the His of an Hpt.
Imidazole interacted with RR nonconserved active site residues, which influenced
the relative reactivity of RR-P with imidazole versus water. Rate constants for
reaction of imidazole or MPI with chimeric RRs suggested that the RR active site
contributes to the kinetic preferences exhibited by the YPD1 Hpt.
Links
