Molecular interactions between the VirD4 coupling protein and T4SS effectors

Type IV secretion systems (T4SS) translocate proteins or DNA-protein complexes into target cells. T4SSs can be conjugative, specialized in horizontal DNA transfer, or effector protein translocators, found in various pathogenic bacteria. The Xanthomonas citri T4SS (T4SS_XAC) injects toxins into competing bacteria via contact-dependent mechanisms. The T4SS_XAC is formed by 11 VirB subunits (VirB1-VirB11) and the hexameric ATPase VirD4, a membrane protein that recruits T4SS substrates (X-Tfes). It was already shown that the VirD4 All-Alpha-Domain (VirD4_AAD) recognizes the X-Tfe by binding to the flexible Xanthomonas VirD4 Interacting Protein Conserved Domain (XVIPCD, ~120 aa). However, the mechanism of interaction between the hexameric VirD4 with the XVIPCD or the full-length X-Tfe is unknown. This limitation is in part due to the VirD4 poor solubility and instability in solution. Here, we designed three new VirD4 truncated constructs: Δ22 lacking the signal peptide; Δ94 lacking the N-terminal transmembrane helix; and Δ112, which contains a deletion of the transmembrane helix and of a small cytosolic alpha-helix. Protein expression tests in Escherichia coli showed that the Δ94 is the most soluble construct, regardless of the plasmid expression system used. However, it precipitates quickly after purification. We observed that VirD4-Δ94 co-elutes with the X-Tfe^XAC1918 in the gel filtration column. Furthermore, although VirD4-Δ94 is highly unstable, in the complex with X-Tfe^XAC1918 and in the absence of ATP it remains stable for several days in solution. The complex displays various oligomeric states, which are detected by gel filtration. Addition of ATP to the complex solution leads to quick precipitation. We now aim to better characterize the VirD4-Δ94 ATPase activity and the interaction with the X-Tfes using biophysical and structural biology methods.