RESUMO
Agrobacterium tumefaciens infects its plant hosts by a mechanism of horizontal gene transfer. This capability has led to its widespread use in artificial genetic transformation. In addition to DNA, the bacterium delivers an abundant ssDNA binding protein, VirE2, whose roles in the host include protection from cytoplasmic nucleases and adaptation for nuclear import. In Agrobacterium, VirE2 is bound to its acidic chaperone VirE1. When expressed in vitro in the absence of VirE1, VirE2 is prone to oligomerization and forms disordered filamentous aggregates. These filaments adopt an ordered solenoidal form in the presence of ssDNA, which was characterized previously by electron microscopy and three-dimensional image processing. VirE2 coexpressed in vitro with VirE1 forms a soluble heterodimer. VirE1 thus prevents VirE2 oligomerization and competes with its binding to ssDNA. We present here a crystal structure of VirE2 in complex with VirE1, showing that VirE2 is composed of two independent domains presenting a novel fold, joined by a flexible linker. Electrostatic interactions with VirE1 cement the two domains of VirE2 into a locked form. Comparison with the electron microscopy structure indicates that the VirE2 domains adopt different relative orientations. We suggest that the flexible linker between the domains enables VirE2 to accommodate its different binding partners.
Assuntos
Agrobacterium tumefaciens/química , Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , Canais Iônicos/química , Chaperonas Moleculares/química , Complexos Multiproteicos/química , Fatores de Virulência/química , Transporte Ativo do Núcleo Celular/fisiologia , Agrobacterium tumefaciens/metabolismo , Proteínas de Bactérias/metabolismo , Citoplasma/enzimologia , DNA Bacteriano/química , DNA Bacteriano/metabolismo , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Desoxirribonucleases/metabolismo , Dimerização , Transferência Genética Horizontal/fisiologia , Canais Iônicos/metabolismo , Chaperonas Moleculares/metabolismo , Complexos Multiproteicos/metabolismo , Plantas/enzimologia , Plantas/genética , Plantas/microbiologia , Ligação Proteica/fisiologia , Dobramento de Proteína , Estrutura Quaternária de Proteína/fisiologia , Estrutura Terciária de Proteína/fisiologia , Eletricidade Estática , Fatores de Virulência/metabolismoRESUMO
Agrobacterium tumefaciens infects plant cells by the transfer of DNA. A key factor in this process is the bacterial virulence protein VirE2, which associates stoichiometrically with the transported single-stranded (ss) DNA molecule (T-strand). As observed in vitro by transmission electron microscopy, VirE2-ssDNA readily forms an extended helical complex with a structure well suited to the tasks of DNA protection and nuclear import. Here we have elucidated the role of the specific molecular chaperone VirE1 in regulating VireE2-VirE2 and VirE2-ssDNA interactions. VirE2 alone formed functional filamentous aggregates capable of ssDNA binding. In contrast, co-expression with VirE1 yielded monodisperse VirE1-VirE2 complexes. Cooperative binding of VirE2 to ssDNA released VirE1, resulting in a controlled formation mechanism for the helical complex that is further promoted by macromolecular crowding. Based on this in vitro evidence, we suggest that the constrained volume of the VirB channel provides a natural site for the exchange of VirE2 binding from VirE1 to the T-strand.
Assuntos
Agrobacterium tumefaciens/fisiologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/fisiologia , DNA Bacteriano/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Canais Iônicos/antagonistas & inibidores , Chaperonas Moleculares/fisiologia , Plantas/genética , Plantas/microbiologia , Transformação Genética , Agrobacterium tumefaciens/genética , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/ultraestrutura , DNA Bacteriano/química , DNA de Cadeia Simples/química , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/ultraestrutura , Canais Iônicos/biossíntese , Canais Iônicos/metabolismo , Canais Iônicos/ultraestrutura , Chaperonas Moleculares/biossíntese , Chaperonas Moleculares/genética , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Ligação Proteica/genética , Desnaturação Proteica/genética , Estrutura Secundária de Proteína/genéticaRESUMO
The principal goal of the Israel Structural Proteomics Center (ISPC) is to determine the structures of proteins related to human health in their functional context. Emphasis is on the solution of structures of proteins complexed with their natural partner proteins and/or with DNA. To date, the ISPC has solved the structures of 14 proteins, including two protein complexes. It has adopted automated high-throughput (HTP) cloning and expression techniques and is now expressing in Escherichia coli, Pichia pastoris and baculovirus, and in a cell-free E. coli system. Protein expression in E. coli is the primary system of choice in which different parameters are tested in parallel. Much effort is being devoted to development of automated refolding of proteins expressed as inclusion bodies in E. coli. The current procedure utilizes tagged proteins from which the tag can subsequently be removed by TEV protease, thus permitting streamlined purification of a large number of samples. Robotic protein crystallization screens and optimization utilize both the batch method under oil and vapour diffusion. In order to record and organize the data accumulated by the ISPC, a laboratory information-management system (LIMS) has been developed which facilitates data monitoring and analysis. This permits optimization of conditions at all stages of protein production and structure determination. A set of bioinformatics tools, which are implemented in our LIMS, is utilized to analyze each target.