10.1371/journal.ppat.1003948PLoS Pathogens103

A Pitzschke

Abhilash Padavannil

Adrian Velazquez-Campoy

CE Alvarez-Martinez

Chacko Jobichen

CM Hamilton

DJ Oliver

E Cascales

G Kozlov

GN Murshudov

J. Sivaraman

Jayaraman Seetharaman

JR Zupan

K Atmakuri

K Veluthambi

L Holm

Liu Yang

LW Moore

M Grynberg

M Guo

M Lessl

P Emsley

PH Brown

PJ Christie

RB Kumar

S Doublié

S Okamoto

SF Altschul

Shen Q. Pan

SN Ho

T Terwilliger

T Tzfira

TA Dang

TC Terwilliger

V Citovsky

Wenbo Ma

Yang Qinghua

PubMed

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. © 2014 Padavannil et al.Peer Reviewe

Padavannil, A.

Jobichen, C.

Qinghua, Y.

Seetharaman, J.

Velázquez-Campoy, Adrián

Yang, L.

Pan, S.Q.

Sivaraman, J.

Digital.CSIC

Dimerization of VirD2 Binding Protein Is Essential for Agrobacterium Induced Tumor Formation in Plants

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 a-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. 
Author Summary: 
Agrobacterium tumefaciens causes crown gall disease (tumors) in agriculturally important plant species. It initiates infection through its Ti plasmid, which integrates a portion of its own DNA (T-DNA) into that of the host genome. The T-DNA is bound to VirD2 relaxase, and this complex is required for the efficient translocation and integration of the T-DNA into the plant genome for tumor formation. Two additional proteins, among others, are also required for Agrobacterium tumorigenesis: VirD4-coupling protein (CP) and VirD2-binding protein (VBP). VBP is responsible for recruiting VirD2–T-DNA to VirD4 CP to help localize T-DNA to the Type IV Secretion System apparatus for transfer. However, it is still unclear how VBP recruits the complex to VirD4 CP. Here, we report the crystal structure and associated functional studies of the C-terminal domain of VBP. We show that the C-terminal domain is the dimerization domain of VBP and only dimeric VBP is functional and essential for the induction of tumor in plants. This study enhances the understanding of the role of VBP in recruiting VirD2–T-DNA in A. tumefaciens prior to its transfer into the host plant. This mode of action can be extended to other pathogenic bacteria employing similar secretion systems

Velazquez-Campoy, A.

Repositorio Universidad de Zaragoza

English

Padavannil Abhilash

Jobichen Chacko

Qinghua Yang

Seetharaman Jayaraman

Velazquez-Campoy Adrian

Yang Liu

Pan Shen Q.

Sivaraman J.

Public Library of Science (PLOS)

 Induced Tumor Formation in Plants

<div>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.</div

Abhilash Padavannil (535017)

Chacko Jobichen (43238)

Yang Qinghua (535018)

Jayaraman Seetharaman (455444)

Adrian Velazquez-Campoy (1974124)

Liu Yang (23752)

Shen Q. Pan (535019)

J. Sivaraman (31440)

The Francis Crick Institute

Dimerization of VirD2 Binding Protein Is Essential for Agrobacterium Induced Tumor Formation in Plants

Crossref

PLoS Pathogens

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

Shen Q Pan

J Sivaraman

Directory of Open Access Journals

Dimerization of VirD2 binding protein is essential for Agrobacterium induced tumor formation in plants.

https://figshare.com/articles/Dimerization_of_VirD2_Binding_Protein_Is_Essential_for_Agrobacterium_Induced_Tumor_Formation_in_Plants/960917

Dimerization of VirD2 Binding Protein Is Essential for Agrobacterium Induced Tumor Formation in Plants

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Digital.CSIC

Repositorio Universidad de Zaragoza

Public Library of Science (PLOS)

The Francis Crick Institute

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The Francis Crick Institute

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