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 effector 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 translocation. However, the structural basis of the XVIPCD-VirD4 interaction is unknown. Here, we show that the XVIPCD interacts with the central all-alpha domain of VirD4 (VirD4_AAD). We used solution NMR spectroscopy to solve the structure of the XVIPCD of X-Tfe^XAC2609 from <i>Xanthomonas citri</i> and to map its interaction surface with VirD4_AAD Isothermal titration calorimetry and in vivo <i>Xanthomonas citri</i> versus <i>Escherichia coli</i> competition assays using wild-type and mutant X-Tfe^XAC2609 and X-Tfe^XAC3634 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 VirD4_AAD, while both N- and carboxyl-terminal regions are required for effective X-Tfe translocation into the target cell. The conformational stability of the N-terminal region is reduced at and below pH 7.0, a property that may facilitate X-Tfe unfolding and translocation through the more acidic environment of the periplasm.