Overall view of RavA protomer structure. (A) Sequence of E. coli RavA showing secondary structure and conserved motifs. (B) X-ray structure of RavA protomer. αβα subdomain is shown in brown, all-α subdomain is shown in wheat, the linker between the two subdomains is shown in green, triple-helical bundle domain is shown in blue, the LARA domain is shown in dark blue, and bound ADP is shown in violet. The α-helices and β-strands are labeled sequentially except for β_a and β_b of the Pre-Sensor 1 β-Hairpin insertion. Residues 88–97 and 438–441 were not observed in the X-ray structure and are indicated by a dashed line. The figure was generated using PYMOL. (C) Shown is a topological diagram of the LARA domain drawn using TopDraw (25) and its electrostatic surface potential calculated using Delphi (26). Colors are according to the calculated electrostatic surface potential and range from red (potential of -5 kT) to blue (+5 kT). The hydrophobic core of the domain is made by the side chains of hydrophobic residues from each of the β-strands (β1: L362, L364, L366, L372; β2: V377, I380, F382; β3: I397, L401; β4: L410, L412; β5: L420, V422; β6: L432) as well as residues L387, W390 and L391 from the α14 helix.

RavA-ADP hexamer structure (A, Top) Characteristic class averages of the negatively stained RavA-ADP hexamer; (Bottom) projections of the final 3D reconstruction at similar orientations. (B) Top and side views of the EM 3D reconstruction of the RavA-ADP hexamer. An atomic model of RavA hexamer was generated from the X-ray structure of the RavA protomer by docking into the EM envelope of the hexamer and comparison with the X-ray structure of the HslU hexamer (PDB ID code 1DO0). (C) Ribbon representation of RavA AAA+ module (Left) and HslU AAA+ module (Right, PDB ID code 1DO0). Different subdomains are colored as in Fig. 1 and conserved motifs are shown; the Sensor 2 motif is colored in dark blue and the nucleotide is shown in violet. The I-domain of HslU has been omitted for clarity. (D, Left) Schematic representation of RavA AAA+ domain from the hexameric model viewed along the sixfold axis. (Right) X-ray structure of HslU hexamer AAA+ domain. For each structure, the αβα and all-α subdomains of one protomer are colored in brown and wheat, respectively. The other protomers are colored in light and dark gray for the αβα and all-α subdomains, respectively. (E) Space-filling and ribbon models of a representative dimer of each hexamer in D. Nucleotide is shown in violet, whereas the blue circle indicates the location of the Sensor 2 motif.

The LARA domain mediates RavA-LdcI and RavA-RavA interactions. (A) Fit of the RavA hexameric model and LdcI decamer into the EM envelope of the LdcI-RavA complex (12) viewed from the side (Left) and the top (Right). One LdcI dimer is colored in red (the upper monomer) and green (the lower monomer). ppGpp bound to LdcI is drawn as blue spheres. (B) Biacore sensorgrams and equilibrium binding curves showing the interaction between LdcI (on chip) and WT RavA in the absence of nucleotide (Left), or WT RavA in the presence of ATP (Middle), or the LARA domain (Right). (C) Biacore sensorgram showing the lack of interaction between LdcI (on chip) and RavAΔLARA at 15 μM. (D) Biacore sensorgrams and equilibrium binding curves showing the interaction between WT RavA and LARA domain (Left) and the very weak interaction between RavAΔLARA and LARA domain (Right).

RavA binding to LdcI antagonizes the inhibitory effect of ppGpp on LdcI activity. (A) The ATPase activity of RavA measured by ITC in the presence of different nucleotides. Error bars represent the standard deviation of the average of three experiments. (B) LdcI activity measured by ITC in the presence of RavA or RavAΔLARA and/or ppGpp. Note that the concentrations of proteins, substrate, and inhibitor are the final concentrations after mixing. In this panel, the RavA-LdcI complex is preformed in the syringe before adding ppGpp. (C) LdcI activity measured by ITC. In this experiment, LdcI-ppGpp complex is preformed in the well before adding RavA. (D) The effect of RavA overexpression on LdcI activity in the cell. ΔcadBA knockout strains and WT cells overexpressing RavA, RavAΔLARA, or RavA(K52Q) were grown to log phase in defined rich media buffered at pH 5. RavA, RavAΔLARA, or RavA(K52Q) were induced and cells were then shifted to minimal media weakly buffered at pH 5 containing no amino acids to induce ppGpp production and supplemented with 30 mM lysine. The OD₆₀₀ of the cells is shown (Top); the pH of the culture media is shown (Bottom Left). (Bottom Right) The increase in pH/OD₆₀₀ normalized to the value at 0+ (right after shift). Each time point is the result of at least three replicates. Error bars represent the standard deviations of the measurements.