ATP ground- and transition states of bacterial enhancer binding AAA plus ATPases support complex formation with their target protein, σ54

Transcription initiation by the sigma 54 form of bacterial RNA polymerase requires hydrolysis of ATP by an enhancer binding protein (EBP). We present SAS-based solution structures of the ATPase domain of the EBP NtrC1 from Aquifex aeolicus in different nucleotide states. Structures of apo protein and that bound to AMPPNP or ADP-BeFx (ground-state mimics), or ADP-AlFx (a transition-state mimic), or ADP (product) show substantial changes in the position of the GAFTGA loops that contact polymerase, particularly upon conversion from the apo state to the ADP-BeFx state, and from the ADP-AlFx state to the ADP state. Binding of the ATP analogs stabilizes the oligomeric form of the ATPase and its binding to sigma 54, with ADP-AIF, having the largest effect. These data indicate that ATP binding promotes a conformational change that stabilizes complexes between EBPs and sigma 54, while subsequent hydrolysis and phosphate release drive the conformational change needed to open the polymerase/promoter complex.