Abstract
The Type IV Secretion System (T4SS) is the only bacterial secretion system known to translocate both DNA and protein substrates. The VirB/D4 system from Agrobacterium tumefaciens is a typical T4SS. It facilitates the bacteria to translocate the VirD2-T-DNA complex to the host cell cytoplasm. In addition to protein-DNA complexes, the VirB/D4 system is also involved in the translocation of several effector proteins, including VirE2, VirE3 and VirF into the host cell cytoplasm. These effector proteins aid in the proper integration of the translocated DNA into the host genome. The VirD2-binding protein (VBP) is a key cytoplasmic protein that recruits the VirD2–T-DNA complex to the VirD4-coupling protein (VirD4 CP) of the VirB/D4 T4SS apparatus. Here, we report the crystal structure and associated functional studies of the C-terminal domain of VBP. This domain mainly consists of α-helices, and the two monomers of the asymmetric unit form a tight dimer. The structural analysis of this domain confirms the presence of a HEPN (higher eukaryotes and prokaryotes nucleotide-binding) fold. Biophysical studies show that VBP is a dimer in solution and that the HEPN domain is the dimerization domain. Based on structural and mutagenesis analyses, we show that substitution of key residues at the interface disrupts the dimerization of both the HEPN domain and full-length VBP. In addition, pull-down analyses show that only dimeric VBP can interact with VirD2 and VirD4 CP. Finally, we show that only Agrobacterium harboring dimeric full-length VBP can induce tumors in plants. This study sheds light on the structural basis of the substrate recruiting function of VBP in the T4SS pathway of A. tumefaciens and in other pathogenic bacteria employing similar systems.
Highlights
The Type IV Secretion System (T4SS) has an unmatched versatility among the seven different secretion systems known in bacteria
The T4SS shares a common ancestry with bacterial conjugation systems [1], with three types of T4SS described to date: (1) conjugation systems, defined as machines that translocate DNA substrates to recipient cells by a contactdependent process; (2) effector translocation systems, functioning to deliver proteins or other effector molecules to eukaryotic target cells; and (3) DNA release or uptake systems, which translocate DNA to or from the extracellular milieu [2]
A. tumefaciens affects more than 140 species of dicots [19], instigating infection through the efficient translocation of the VirD2–T-DNA complex [2], a prerequisite for the integration of T-DNA into the plant genome and eventual tumor formation in plants [23]
Summary
The Type IV Secretion System (T4SS) has an unmatched versatility among the seven different secretion systems known in bacteria. Conjugation systems within the T4SS facilitate the translocation of protein-DNA complexes from the bacterium into the cytoplasm of the host cell. A. tumefaciens translocates to the host cell a segment of the Ti (tumor inducing) plasmid between the right and left borders (T-DNA) in complex with a cytoplasmic relaxase protein (VirD2) [3]. Class II comprises 11 VirB proteins that form the T4SS apparatus which is responsible for the translocation of the VirD2–T-DNA complex and the effector proteins into the host cell cytoplasm [4,8]. VirD4 CP, the coupling protein in the A. tumefaciens VirB/D4 system, is an inner membrane protein with a large cytoplasmic domain essential for the transfer of both the T-DNA strand and VirE2 to host cells [10,11,12]. The VirD2–T-DNA complex and VirE2 are Author Summary
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