TY - JOUR
T1 - The disordered region of arabidopsis VIP1 binds the agrobacterium VirE2 protein outside its DNA-binding site
AU - Maes, Michal
AU - Amit, Einav
AU - Danieli, Tsafi
AU - Lebendiker, Mario
AU - Loyter, Abraham
AU - Friedler, Assaf
AU - Schreiber, Gideon
N1 - Publisher Copyright:
© The Author 2014. Published by Oxford University Press. All rights reserved.
PY - 2014/11/1
Y1 - 2014/11/1
N2 - Agrobacterium is a pathogen that genetically transforms plants. The bacterial VirE2 protein envelopes the T-DNA of Agrobacterium and protects it from degradation. Within the transfected cells, VirE2 interacts with the plant VIP1 leading to nuclear transport of the T-DNA complex. Active VirE2 is an oligomer with a tendency to aggregate, hampering its studies at the molecular level. In addition, no structural or quantitative information is available regarding VIP1 or its interactions. The lack of information is mainly because both VIP1 and VirE2 are difficult to express and purify. Here, we present the development of efficient protocols that resulted in pure and stable Histagged VIP1 and VirE2. Circular dichroism spectroscopy and computational predictions indicated that VIP1 is mostly intrinsically disordered. This may explain the variety of protein-protein interactions it participates in. Size exclusion chromatography revealed that VirE2 exists in a two-state equilibrium between a monomer and an oligomeric form. Using the purified proteins, we performed peptide array screening and revealed the binding sites on both proteins. VirE2 binds the disordered regions of VIP1, while the site in VirE2 that binds VIP1 is different from the VirE2 DNA-binding site. Peptides derived from these sites may be used as lead compounds that block Agrobacterium infection of plants.
AB - Agrobacterium is a pathogen that genetically transforms plants. The bacterial VirE2 protein envelopes the T-DNA of Agrobacterium and protects it from degradation. Within the transfected cells, VirE2 interacts with the plant VIP1 leading to nuclear transport of the T-DNA complex. Active VirE2 is an oligomer with a tendency to aggregate, hampering its studies at the molecular level. In addition, no structural or quantitative information is available regarding VIP1 or its interactions. The lack of information is mainly because both VIP1 and VirE2 are difficult to express and purify. Here, we present the development of efficient protocols that resulted in pure and stable Histagged VIP1 and VirE2. Circular dichroism spectroscopy and computational predictions indicated that VIP1 is mostly intrinsically disordered. This may explain the variety of protein-protein interactions it participates in. Size exclusion chromatography revealed that VirE2 exists in a two-state equilibrium between a monomer and an oligomeric form. Using the purified proteins, we performed peptide array screening and revealed the binding sites on both proteins. VirE2 binds the disordered regions of VIP1, while the site in VirE2 that binds VIP1 is different from the VirE2 DNA-binding site. Peptides derived from these sites may be used as lead compounds that block Agrobacterium infection of plants.
KW - Agrobacterium
KW - Peptide arrays
KW - Protein expression and purification
KW - VIP1
KW - VirE2
UR - http://www.scopus.com/inward/record.url?scp=84931837565&partnerID=8YFLogxK
U2 - 10.1093/protein/gzu036
DO - 10.1093/protein/gzu036
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C2 - 25212215
AN - SCOPUS:84931837565
SN - 1741-0126
VL - 27
SP - 439
EP - 446
JO - Protein Engineering, Design and Selection
JF - Protein Engineering, Design and Selection
IS - 11
ER -