ATPase site architecture is required for self-assembly and remodeling activity of a hexameric AAA+ transcriptional activator

Abstract

AAA+ proteins (ATPases associated with various cellular activities) are oligomeric ATPases that use ATP hydrolysis to remodel their substrates. By similarity with GTPases, a dynamic organization of the nucleotide-binding pockets between ATPase protomers is proposed to regulate functionality. Using the transcription activator PspF as an AAA+ model, we investigated contributions of conserved residues for roles in ATP hydrolysis and intersubunit communication. We determined the R-finger residue and revealed that it resides in a conserved "R-hand" motif (R(x)D(xxx)R) needed for its "trans-acting" activity. Further, a divergent Walker A glutamic acid residue acts synergistically with a tyrosine residue to function in ADP-dependent subunit-subunit coordination, forming the "ADP-switch" motif. Another glutamic acid controls hexamer formation in the presence of nucleotides. Together, these results lead to a "residue-nucleotide" interaction map upon which to base AAA+ core regulation.

Figure 1

Sequence Alignment, Localization, and Functional Activities of PspF Variants (A) Localization of “cis” and “trans” residues on the PspF structure (pdb 2C9C) (Rappas et al., 2006). Figure prepared using PyMol software. (B) Sequence alignment of PspF from Escherichia coli, NtrC from E. coli, ZraR from E. coli, AtoC from E. coli, NtrC1 from Aquifex aeolicus, NifA from Sinorhizobium meliloti, NorR from E. coli, AcoR from Pseudomonas aeruginosa, HrpR and HrpS from Pseudomonas syringae pv. tomato str. DC3000, and DctD from Rhodobacter capsulatus. Numbering is based on PspF sequence. Black triangles: amino acid substitution of the variants studied. (C–E) Side-chain orientation in the presence of ATP or ADP (i), substrate interaction activity by quantifying the amount of stable σ⁵⁴-PspF complex formed in the presence of ADP-AlF (ii), ATPase activity (iii) (see Table 1 for detail), and substrate remodeling activity by using RP_o formation assay (iv) were tested for L9, E43, L44, and Y126 variants (C); R162, D164, and R168 variants (D); and E118, R122, and E125 variants (E). Experiments were performed at least in triplicate, and the maximal variation observed was lower than 10%.

Figure 2

The Presence of Nucleotide ATP or ADP Inhibits E125 Variant Hexamer Formation We compared the elution profile obtained for E125 variants and WT in the absence or presence of nucleotide (ATP or ADP). Gel filtration on Superdex 200 was performed at 4°C. The scale bars give the scale of ordinate axis; absorption units (AU) correspond to an A_280nm of 1.

Figure 3

A Complex Interaction Network Is Occurring at the Level of the Nucleotide and PspF Interface (A) ATPase activity of the mixed cis (N64, D107, and E108) and trans (R162, D164, and R168) defective variants. Histograms represent the k_cat observed for the different variants mixed at equimolar concentration. Experiment was performed three times independently, and the maximal error observed was below 10%. (B and C) Schematic of PspF-ATP (B, pdb 2C9C) and PspF-ADP (C, pdb 2C98) interaction networks. Dashed black lanes represent polar contact between side chains, and purple dashed lanes represent polar contact with the solvent. Note that R122 side chain is not resolved in the ATP soaked crystal. Figure was generated using PyMol software.