Structural Mechanism of GAF-Regulated σ54 Activators from Aquifex aeolicus

The σ subunits of bacterial RNA polymerase occur in many variant forms and confer promoter specificity to the holopolymerase. Members of the σ54 family of σ subunits require the action of a ‘transcriptional activator’ protein to open the promoter and initiate transcription. The activator proteins undergo regulated assembly from inactive dimers to hexamers that are active ATPases. These contact σ54 directly and, through ATP hydrolysis, drive a conformational change that enables promoter opening. σ54 activators use several different kinds of regulatory domains to respond to a wide variety of intracellular signals. One common regulatory module, the GAF domain, is used by σ54 activators to sense small-molecule ligands. The structural basis for GAF domain regulation in σ54 activators has not previously been reported. Here, we present crystal structures of GAF regulatory domains for Aquifex aeolicus σ54 activators NifA-like homolog (Nlh)2 and Nlh1 in three functional states—an ‘open’, ATPase-inactive state; a ‘closed’, ATPase-inactive state; and a ‘closed’, ligand-bound, ATPase-active state. We also present small-angle X-ray scattering data for Nlh2-linked GAF-ATPase domains in the inactive state. These GAF domain dimers regulate σ54 activator proteins by holding the ATPase domains in an inactive dimer conformation. Ligand binding of Nlh1 dramatically remodels the GAF domain dimer interface, disrupting the contacts with the ATPase domains. This mechanism has strong parallels to the response to phosphorylation in some two-component regulated σ54 activators. We describe a structural mechanism of GAF-mediated enzyme regulation that appears to be conserved among humans, plants, and bacteria.