Heterogeneous nucleotide occupancy stimulates functionality of phage shock protein F, an AAA+ transcriptional activator.
The catalytic AAA+ domain (PspF1-275) of an enhancer-binding protein is necessary and sufficient to contact sigma54-RNA polymerase holoenzyme (Esigma54), 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 PspF1-275 and propose that this heterogeneous nucleotide occupancy in a PspF1-275 hexamer is functionally important for specific activity. Binding of ADP and ATP triggers the formation of functional PspF1-275 hexamers as shown by a gain of specific activity. Furthermore, ATP concentrations congruent with stoichiometric ATP binding to PspF1-275 inhibit ATP hydrolysis and Esigma54-promoter open complex formation. Demonstration of a heterogeneous nucleotide-bound state of a functional PspF1-275.Esigma54 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 PspF1-275 hexamers.
Joly
Nicolas
N
Division of Biology, Sir Alexander Fleming Building, Imperial College London, London SW7 2AZ, United Kingdom.
Schumacher
Jörg
J
Buck
Martin
M
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism*; D000266:Adenylyl Imidodiphosphate / Q000378:metabolism*; D020134:Catalytic Domain; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D010957:Plasmids; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism*; D014158:Transcription, Genetic
D029968:Escherichia coli Proteins; C098954:PspF protein, E coli; D015534:Trans-Activators; D000266:Adenylyl Imidodiphosphate; C022571:adenosine 5'-O-(3-thiotriphosphate); D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases