ATP ground- and transition states of bacterial enhancer binding AAA+ ATPases support complex formation with their target protein, sigma54

Abstract

Transcription initiation by the sigma54 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-BeF(x) (ground-state mimics), ADP-AlF(x) (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-BeF(x) state, and from the ADP-AlF(x) state to the ADP state. Binding of the ATP analogs stabilizes the oligomeric form of the ATPase and its binding to sigma54, with ADP-AlF(x) having the largest effect. These data indicate that ATP binding promotes a conformational change that stabilizes complexes between EBPs and sigma54, while subsequent hydrolysis and phosphate release drive the conformational change needed to open the polymerase/promoter complex.

Fig. 1. Combined SAXS/WAXS data for NtrC1^C in various nucleotide states

(A) Scattering profiles (arbitrarily offset for clarity, with error bars - enlarged in inset), (B) the dashed region in panel A scaled to identical intensity at Q = 0.5 Å⁻¹; (C) p(R) functions (normalized to a common total probability), and (D) Guinier plots for NtrC1^C with 5 mM MgCl₂ (cyan), or with MgCl₂ plus 5 mM ATPγS (blue), AMPPNP (pink), repurified AMPPNP (red), ADP-BeF_x (yellow), ADP-AlF_x (green) or ADP (orange).

Fig. 2. Negative stain EM images of NtrC1^C bound to ADP-BeF_x

(A) Eigenimages after the first round of reference-free translational alignment (no symmetry or rotational alignment) and (B) class averages from the full dataset, representing 87% heptamers, 11% hexamers and 2% other forms. The number of raw particles contributing to each class-average is not indicated and may vary.

Fig. 3. Excluded volume surface models of the NtrC1^C heptamer

Top, side and bottom views of the filtered, averaged structure from 32 independently derived, chain-compatible solutions (cyan) for various nucleotide states are shown superimposed on the ADP-bound crystal structure of NtrC1^C (dark blue) with highlighted GAFTGA loops (red).

Fig. 4. ADP-BeF_x and AMPPNP stabilize a GAFTGA-loop dependent complex of σ54 with the ATPase domains of NtrC1 or PspF, or the full-length activated form of NtrC

(A) Size exclusion chromatograms developed in the presence of ADP-BeF_x or AMPPNP for NtrC1^C alone (solid gray line), σ54 alone (solid black line), or a mixture of the two with excess σ54 (dashed black and gray lines) are aligned below SDS PAGE analyses of (left to right): the purified proteins, the loading mixture, elution fractions for the ADP-BeF_x experiment (aligned with the chromatogram), and the loading mixture and aliquots of peaks P1 and P2 for the AMPPNP experiment. Same as for the ADP-BeF_x experiment in ‘a’ but for (B) the ATPase domain of PspF, (C) the full length form of NtrC (S160F, 3Ala) activated by binding Mg²⁺/BeF₃⁻, and (D) the T217A substitution variant of NtrC1^C in both ADP-BeF_x and ADP-AlF_x conditions (for the latter only fractions for peaks P1 and P2 were analyzed by SDS-PAGE).

Fig. 5. Stability of ATPase rings and σ54 complexes stabilized by ADP-BeF_x versus ADP-AlF_x

(A) Large-zone gel filtration of NtrC1^C in the presence of ADP-BeF_x (solid line) and ADP-AlF_x (dashed line). (B) The indicated portions of a native gel (lacking ADPMg and metal fluoride) were excised and subjected to SDS-PAGE to assess the presence of NtrC1^C and σ54 after being mixed with ADPMg and BeF_x or AlF_x. (C) Serial, 2-fold dilutions of 10 μg of PspF_(1−275) mixed with 10 μg of σ54 were subject to native gel electrophoresis with ADPMg-BeF_x or ADPMg-AlF_x present in the sample, buffer and gel.

Fig. 6. SAS-based model of the ATPase cycle and interactions with σ54

Binding of ATP extends the GAFTGA loops (one example in red) to permit initial binding to σ54. Conformational changes in the ATPase that accompany entry into the transition state for ATP hydrolysis keep the loops (one example in blue) extended but tighten association with σ54 in preparation for its being remodeled. Upon P_i release the loops (one example in gray) are retracted from above the ring surface, and contact with σ54 is lost. ADP is then release to repeat the cycle.