In two-component signal transduction, an input triggers phosphorylation of receiver domains that regulate the status of output modules. One such module is the AAA+ ATPase domain in bacterial enhancer-binding proteins that remodel the σ54-form of RNA polymerase. We report X-ray solution scattering and electron microscopy structures of the activated, full-length nitrogen-regulatory protein C (NtrC) showing a novel mechanism for regulation of AAA+ ATPase assembly via the juxtaposition of the receiver domains and ATPase assembly via the juxtaposition of the receiver domains and ATPase ring. Accompanying the hydrolysis cycle that is required for transcriptional activation, we observed major order-disorder changes in the GAFTGA loops involved in σ54 binding, as well as in the DNA-binding domains.

We recently solved both SAXS/WAXS (small- and wide-angle X-ray scattering) and EM (electron microscopy) structures of the full-length, activated form of NtrC from Salmonella typhimurium (De Carlo, et al. 2006). Docking of atomic models for the individual domains into the structures reveals their organization within the activated ring and uncovers a novel mechanism for the use of two-component signal transduction to regulate the assembly of AAA+ ATPase domains.

Comparison of the ADP- versus ADP-AlFx bound structures shows that in the latter the DNA-binding domain becomes more ordered with respect to other domains, and the GAFTGA loop region extends outward on the other side of the ring. These two features are of potential importance in the activation process.

The structural model that we describe for activated NtrC, in which the activated receiver domain of one subunit is in contact with the ATPase domain of a second one to stabilize ring assembly, differs dramatically from the model proposed to explain how two-component signal transduction regulates assembly of AAA+ ATPase domains in the enhancer-binding proteins NtrC1 and DctD (see Figure). Consequently, the new model establishes the structural differences underlying positive versus negative regulation for this family of enhancer-binding proteins (see Figure).

Our structure of the activated NtrC suggests that phenotypes of substitutions in residues 149-156 of NtrC helix αN arise from specific changes in the interface between ATPase and receiver domains, and it suggests intermediates for the assembly process. Finally, we show that order-disorder transitions in the GAFTGA loop region and DNA-binding domains accompany the nucleotide hydrolysis cycle. Since coupling of hydrolysis, DNA binding, and σ54-factor remodeling can be dramatically perturbed (Yan and Kustu, 1999), these transitions must be understood to learn how these AAA+ ATPases perform mechanical work to remodel the σ54 form of RNA polymerase.