The Pvc15 D2-Pnf SP Interaction Mediates AAA ATPase Activity, Payload Stability, and Translocation into the PVC Tube Lumen.

The Photorhabdus virulence cassette (PVC) is an elegant, multi-protein, contractile nanostructure which injects functionally bioactive polypeptides into the eukaryotic cytosol. Signal peptides (SPs) are N-terminal amino acid motifs from native payload proteins which can also associate wide range of heterologous proteins to the PVC tube lumen. In addition, Pvc15 is a classic AAA ATPase encoded within the PVC operon which encodes an N-domain and tandem AAA domains D1 and D2. This work finds that Pvc15s ATPase activity is mediated by the E555 residue situated in the Walker B motif of the sole functional ATPase domain, D2. Pvc15 multimerisation may be required to translocate payload into the PVC tube lumen, which is facilitated by intact N and D1 domains. In addition, ATPase activity requires the presence of other PVC operon components which hints at a PVC-regulated mode of action for loading. Furthermore, D2 is a chaperone for the native Pnf SP, though C-terminal truncations of the Pnf SP confers Pvc15-independent stability to the payload. These findings provide insight into the nuanced roles of the Pvc15-SP interaction for payload-PVC association and the effects that SP length and type has on payload stability and PVC-loading ability.