Structural basis for effector recognition by an antibacterial type IV secretion system.

Abstract

Many soil-, water-, and plant-associated bacterial species from the orders Xanthomonadales, Burkholderales, and Neisseriales carry a

type IV secretion system (T4SS) specialized in translocating effec- tor proteins into other gram-negative species, leading to target

cell death. These effectors, known as X-Tfes, carry a carboxyl- terminal domain of ∼120 residues, termed XVIPCD, characterized

by several conserved motifs and a glutamine-rich tail. Previous studies showed that the XVIPCD is required for interaction with

the T4SS coupling protein VirD4 and for T4SS-dependent translo- cation. However, the structural basis of the XVIPCD–VirD4 interac- tion is unknown. Here, we show that the XVIPCD interacts with

the central all-alpha domain of VirD4 (VirD4AAD). We used solution NMR spectroscopy to solve the structure of the XVIPCD of X-TfeXAC2609 from Xanthomonas citri and to map its interaction surface with VirD4AAD. Isothermal titration calorimetry and in vivo Xanthomonas citri versus Escherichia coli competition assays using wild-type and mutant X-TfeXAC2609 and X-TfeXAC3634 indicate that XVIPCDs can be divided into two regions with distinct functions: the well-folded N-terminal region contains specific conserved motifs that are responsible for interactions with VirD4AAD, while both N- and carboxyl-terminal regions are required for effective

X-Tfe translocation into the target cell. The conformational stabil- ity of the N-terminal region is reduced at and below pH 7.0, a prop- erty that may facilitate X-Tfe unfolding and translocation through

the more acidic environment of the periplasm.