The RavA-ViaA chaperone complex modulates bacterial persistence through its association with the fumarate reductase enzyme

Abstract

Regulatory ATPase variant A (RavA) is a MoxR AAA+ protein that functions together with a partner protein termed von Willebrand factor type A interacting with AAA+ ATPase (ViaA). RavA-ViaA are functionally associated with anaerobic respiration in Escherichia coli through interactions with the fumarate reductase (Frd) electron transport complex. Through this association, RavA and ViaA modulate the activity of the Frd complex and, hence, are proposed to have chaperone-like activity. However, the functional role of RavA-ViaA in the cell is not yet well established. We had demonstrated that RavA-ViaA can sensitize E. coli cells to sublethal concentrations of the aminoglycoside class of antibiotics. Since Frd has been associated with bacterial persistence against antibiotics, the relationship of RavA-ViaA and Frd was explored within this context. Experiments performed here reveal a function of RavA-ViaA in bacterial persistence upon treatment with antibiotics through the association of the chaperone complex with Frd. As part of this work, the NMR structure of the N-terminal domain of ViaA was solved. The structure reveals a novel alpha helical fold, which we name the VAN fold, that has not been observed before. We show that this domain is required for the function of the chaperone complex. We propose that modulating the levels of RavA-ViaA could enhance the susceptibility of Gram-negative bacteria to antibiotics.

Keywords: AAA+ proteins; MoxR; NMR structure; RavA; ViaA; bacterial persistence; von Willebrand factor type A domain.

Figure 1

Domain organization and structures of RavA and ViaA.A, top shows the domain organization of RavA. Domain boundaries are indicated along with the amino acid residues marking them. Bottom shows the structure of a RavA protomer (PDB ID: 3NBX), with the three domains colored in the same manner as the domain organization figure above, as well as of the RavA hexamer side (left) and top (right) views (PDB ID: 6SZA). All structures were drawn using PyMOL (https://pymol.org/2/). B, top shows the domain organization of ViaA. Domain boundaries are indicated along with the amino acid residues marking them. The term “previous NTV” refers to our definition of the NTV in our previous work (12). Bottom shows the predicted structure of full-length ViaA derived from AlphaFold (https://alphafold.ebi.ac.uk/entry/P0ADN0) with the domains colored in the same way as the domain organization figure above. RavA, regulatory ATPase variant A; ViaA, von Willebrand factor type A interacting with AAA+ ATPase; NTV, N-terminal ViaA domain.

Figure 2

Mapping the RavA–ViaA interaction.A, SDS-PAGE gel showing the 311-residue ViaA N-terminal segment on the right and the same protein fragment at different time points after it has undergone trypsin digestion. B, MALDI-TOF of the trypsin digested fragment (NTV, ViaA residues 1–191). C–E, ATPase activity for 0.5 μM of RavA (C), RavAΔLARA (D), or RavA-AAA+ (E) are shown in the presence of equimolar concentrations of various constructs of the ViaA protein. For all graphs shown, ∗ is indicative of significant changes in ATPase activity of RavA or RavA mutants upon addition of a given protein or domain with a p-value < 0.05. RavA, regulatory ATPase variant A; ViaA, von Willebrand factor type A interacting with AAA+ ATPase; NTV, N-terminal ViaA domain.

Figure 3

Effect of RavA-ViaA on persistence.A–D, CFU counts for the indicated strains of Escherichia coli MG1655 grown in LB-fumarate media tested for persistence in the presence of 30 μg/ml kanamycin. For all the plasmids listed, the genes are expressed under their native promoter. p11, empty vector; pRavAViaA, p11-based construct expressing RavA and ViaA; pFrd, p11-based construct expressing FrdABCD; pRavA, p11-based construct expressing RavA; pRavA_mViaA, p11-based construct expressing functional ViaA but an ATPase-deficient K52Q mutant of RavA; pRavAViaAΔNTV, p11-based construct expressing RavA and ViaA deleted of NTV; pRavAViaAΔMD, p11-based construct expressing RavA and ViaA deleted of MD; pRavAViaAΔCTV, p11-based construct expressing RavA and ViaA deleted of CTV. For all graphs shown, ∗ and ∗∗ are indicative of counts that were found to be significant with a p-value < 0.05 and p-value < 0.01, respectively. ViaA, von Willebrand factor type A interacting with AAA+ ATPase; CFU, colony-forming unit; NTV, N-terminal ViaA domain; MD, middle domain; CTV, C-terminal domain of ViaA; RavA, regulatory ATPase variant A; Frd, fumarate reductase.

Figure 4

Structure of NTV.A, shown is the NMR structural ensemble of 20 lowest energy conformations. Each helix is indicated by a different color. The disordered C terminus is shown in light blue. B, top shows the lowest energy conformation of NTV without the disordered C terminus. Bottom shows a plot of ¹³C_α secondary chemical shifts (difference from random coil) as a function of sequence position with location of helices highlighted with the same colors used in the structures. C, surface potential of NTV without the disordered C terminus in different views as indicated. The surface electrostatics were calculated considering the effects of solvation using the Adaptive Poisson–Boltzmann Solver (APBS) (49) plugin in ChimeraX (50). No counter-ions were added during this calculation. D, the three hydrophobic cores observed in NTV with core residues labeled as indicated. NTV, N-terminal ViaA domain.

Figure 5

Comparison of NTV structure with other structures.A, alignment of top VAST hit SP18 (PDB ID 1GAK, gray) (45) to fragment 1 of the NTV NMR structure (blue). The NMR structure of NTV is divided into two fragments based on VAST domain identification and structure comparisons. Fragment 1 (blue) is comprised of residues 1 to 65 and 140 to 165, while fragment 2 (red) spans residues 66 to 139. The RMSD value comparisons are given in the figures. B, on the left is shown an alignment of top VAST hit HPt (PDB ID 3US6, gray) (46) to fragment 2 of the NTV NMR structure (red). An overlay of the hydrophobic core around C76 in NTV with that of HPt around C97 is shown on the right. C, Cα alignment of AlphaFold structure of NTV (gray) to the solved NMR structure (blue, red). ViaA, von Willebrand factor type A interacting with AAA+ ATPase; HPt, histidine-containing phosphotransfer; NTV, N-terminal ViaA domain; VAST, Vector Alignment Search Tool.

Figure 6

The interactions of RavA and ViaA. Domain organization of RavA and ViaA highlighting the domains mediating the interactions between the two proteins, as well as with LdcI and FrdA. Frd, fumarate reductase; LdcI, inducible lysine decarboxylase; RavA, regulatory ATPase variant A; ViaA, von Willebrand factor type A interacting with AAA+ ATPase.