Heterogeneous nucleotide occupancy stimulates functionality of phage shock protein F, an AAA plus transcriptional activator

The catalytic AAA+ domain (PspF(1-275)) of an enhancer-binding protein is necessary and sufficient to contact sigma 54-RNA polymerase holoenzyme (E sigma(54)), remodel it, and in so doing catalyze open promoter complex formation. Whether ATP binding and hydrolysis is coordinated between subunits of PspF and the precise nature of the nucleotide(s) bound to the oligomeric forms responsible for substrate remodeling are unknown. We demonstrate that ADP stimulates the intrinsic ATPase activity of PspF(1-275) and propose that this heterogeneous nucleotide occupancy in a PspF(1-275) hexamer is functionally important for specific activity. Binding of ADP and ATP triggers the formation of functional PspF(1-275) hexamers as shown by a gain of specific activity. Furthermore, ATP concentrations congruent with stoichiometric ATP binding to PspF(1-275) inhibit ATP hydrolysis and E sigma(54)-promoter open complex formation. Demonstration of a heterogeneous nucleotide-bound state of a functional PspF(1-275)center dot E sigma(54) complex provides clear biochemical evidence for heterogeneous nucleotide occupancy in this AAA+ protein. Based on our data, we propose a stochastic nucleotide binding and a coordinated hydrolysis mechanism in PspF(1-275) hexamers.