VirE1 is a specific molecular chaperone for the exported single-stranded-DNA-binding protein VirE2 in Agrobacterium

Agrobacterium tumefaciens induces tumours on plants by transferring a nucleoprotein complex, the T-complex, from the bacterium to the plant cell. The T-complex consists of a single-stranded DNA (ssDNA) segment, the T-DNA, and VirD2, an endonuclease covalently attached to the 5' end of the T-DNA. A type IV secretion system encoded by the virB operon and virD4 is required for the entry of the T-complex and VirE2, a ssDNA-binding protein, into plant cells. The VirE1 protein is specifically required for the export of the VirE2 protein, as demonstrated by extracellular complementation and tumour formation. In this report, using a yeast two-hybrid system, we demonstrated that the VirE1 and VirE2 proteins interact and confirmed this interaction by in vitro binding assays. Although VirE2 is a ssDNA-binding protein, addition of ssDNA into the binding buffer did not interfere with the interaction of VirE1 and VirE2. VirE2 also interacts with itself, but the interaction between VirE1 and VirE2 is stronger than the VirE2 self-interaction, as measured in a lacZ reporter gene assay. In addition, the interaction of VirE2 with itself is inhibited by VirE1, indicating that VirE2 binds VirE1 preferentially. Analysis of various virE2 deletions indicated that the VirE1 interaction domain of VirE2 overlaps the VirE2 self-interaction domain. Incubation of extracts from Escherichia coli overexpressing His-VirE1 with the extracts of E. coli overexpressing His-VirE2 increased the yield of His-VirE2 in the soluble fraction. In a similar purified protein solubility assay, His-VirE1 increased the amount of His-VirE2 partitioning into the soluble fraction. In Agrobacterium, VirE2 was undetectable in the soluble protein fraction unless VirE1 was co-expressed. When urea was added to solubilize any large protein aggregates, a low level of VirE2 was detected. These results indicate that VirE1 prevents VirE2 from aggregating, enhances the stability of VirE2 and, perhaps, maintains VirE2 in an export-competent state. Analysis of the deduced amino acid sequence of the VirE1 protein revealed that the VirE1 protein shares a number of properties with molecular chaperones that are involved in the transport of specific proteins into animal and plant cells using type III secretion systems. We suggest that VirE1 functions as a specific molecular chaperone for VirE2, the first such chaperone linked to the presumed type IV secretion system.     

Bacterial protein translocase: a unique molecular machine with an army of substrates

Secretion of most polypeptides across the bacterial plasma membrane is catalyzed by the Sec protein translocase. This complex molecular machine comprises a flexible transmembrane conduit coupled to a motor-like component and displays four activities: (a) it is a specific receptor at its cytoplasmic side for all secretory polypeptides, (b) it converts metabolic energy from ATP and proton gradients into mechanical motion, (c) it prevents substrates from folding in statu translocanti and (d) it binds and releases short segments of the polymeric substrate sequentially. Combination of these activities allows translocase to move processively along the length of the substrate. Substrates are thus gradually expelled from the membrane and are released for subsequent extracytoplasmic folding.     

Virus assembly: Imaging a molecular machine

A recent structural study of a double-stranded DNA bacteriophage has provided remarkable new insights into the assembly of a complex virus particle and ushers in a new era in the imaging of non-icosahedral viruses.     

The VirE1VirE2 complex of Agrobacterium tumefaciens interacts with single-stranded DNA and forms channels

The VirE2 protein is crucial for the transfer of single-stranded DNA (ssDNA) from Agrobacterium tumefaciens to the nucleus of the plant host cell because of its ssDNA binding activity, assistance in nuclear import and putative ssDNA channel activity. The native form of VirE2 in Agrobacterium's cytoplasm is in complex with its specific chaperone, VirE1. Here, we describe the ability of the VirE1VirE2 complex to both bind ssDNA and form channels. The affinity of the VirE1VirE2 complex for ssDNA is slightly reduced compared with VirE2, but the kinetics of binding to ssDNA are unaffected by the presence of VirE1. Upon binding of VirE1VirE2 to ssDNA, similar helical structures to those reported for the VirE2-ssDNA complex were observed by electron microscopy. The VirE1VirE2 complex can release VirE1 once the VirE2-ssDNA complexes assembled. VirE2 exhibits a low affinity for small unilamellar vesicles composed of bacterial lipids and a high affinity for lipid vesicles containing sterols and sphingolipids, typical components of animal and plant membranes. In contrast, the VirE1VirE2 complex associated similarly with all kind of lipids. Finally, black lipid membrane experiments revealed the ability of the VirE1VirE2 complex to form channels. However, the majority of the channels displayed a conductance that was a third of the conductance of VirE2 channels. Our results demonstrate that the binding of VirE1 to VirE2 does not inhibit VirE2 functions and that the effector-chaperone complex is multifunctional.     

Structures of LRP2 reveal a molecular machine for endocytosis

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Ribosome as a molecular machine

General principles of structure and function of the ribosome are surveyed, and the translating ribosome is regarded as a molecular conveying machine. Two coupled conveying processes, the passing of compact tRNA globules and the drawing of linear mRNA chain through intraribosomal channel, are considered driven by discrete acts of translocation during translation. Instead of mechanical transmission mechanisms and power-stroke 'motors', thermal motion and chemically induced changes in affinities of ribosomal binding sites for their ligands (tRNAs, mRNA, elongation factors) are proposed to underlie all the directional movements within the ribosomal complex. The GTP-dependent catalysis of conformational transitions by elongation factors during translation is also discussed.     

VirE1 protein mediates export of the single-stranded DNA-binding protein VirE2 from Agrobacterium tumefaciens into plant cells.

Agrobacterium tumefaciens transfers single-stranded DNAs (T strands) into plant cells. VirE1 and VirE2, which is a single-stranded DNA binding protein, are important for tumorigenesis. We show that T strands and VirE2 can enter plant cells independently and that export of VirE2, but not of T strands, depends on VirE1.     

RNA polymerase as a molecular machine

Structure and function of DNA-dependent RNA polymerase is considered in terms of a conveying molecular machine. The use of mechanical energy and mechanical devices, such as "power-stroke motor", is supposed unlikely in the conveying function of RNA polymerase, as well as other molecular machines. Brownian motion and thermal mobility of macromolecules and their parts are postulated as the only motive impulse at the molecular level. Binding of substrates and subsequent chemical reaction as the energy input may provide successive selection and fixation of alternative conformational states of the enzyme complex thus providing the directionality of the conveyance ("Brownian ratchet mechanism"). The following sequence of events "substrate binding--fixation of a certain conformational state--chemical reaction--fixation of an alternative conformational state--translocation (dissociation and downstream reassociation) of product-template duplex" is proposed as the principal scheme of the forward movement of RNA polymerase along DNA template.     

The SCF ubiquitin ligase: insights into a molecular machine

Ubiquitin ligases are well suited to regulate molecular networks that operate on a post-translational timescale. The F-box family of proteins - which are the substrate-recognition components of the Skp1-Cul1-F-box-protein (SCF) ubiquitin ligase - are important players in many mammalian functions. Here we explore a unifying and structurally detailed view of SCF-mediated proteolytic control of cellular processes that has been revealed by recent studies.     

Escherichia coli translocase: the unravelling of a molecular machine

Protein translocation across the bacterial cytoplasmic membrane has been studied extensively in Escherichia coli. The identification of the components involved and subsequent reconstitution of the purified translocation reaction have defined the minimal constituents that allowed extensive biochemical characterization of the so-called translocase. This functional enzyme complex consists of the SecYEG integral membrane protein complex and a peripherally bound ATPase, SecA. Under translocation conditions, four SecYEG heterotrimers assemble into one large protein complex, forming a putative protein-conducting channel. This tetrameric arrangement of SecYEG complexes and the highly dynamic SecA dimer together form a proton-motive force- and ATP-driven molecular machine that drives the stepwise translocation of targeted polypeptides across the cytoplasmic membrane. Recent findings concerning the translocase structure and mechanism of protein translocation are discussed and shine new light on controversies in the field.     

A unique structural feature of a phospholipase A2 is probed by molecular dynamics

The unusual catalytic network, revealed by the crystal structure of one of the two phospholipases A₂ (PLA₂) from the venom of the crotalid A.p.piscivorus has been probed using molecular dynamics. The catalytic network has been remodeled to a conformation similar to that found in all other PLA₂, and the modeled structure has been submitted to energy minimization and molecular dynamics simulation, to explore the conformational space of the network. The calculations have yielded a large reorganization of the catalytic network, which gets a conformation close to that of the crystal structure. These results suggest that the unusual catalytic network observed in the studied PLA₂ is a structural feature of the protein and not a crystal artifact.     

Kits and their unique role in molecular biology: a brief retrospective

Since their initial development nearly 20 years ago, molecular biology kits have evolved from simple protocols and reagents for cloning of DNA to the more recent complex reagent sets that enable whole genomic sequencing. Initially met with resistance by some who felt that using them deprived researchers of the basic knowledge of how to create reagents, molecular biology kits have taken on an important role in the biological sciences. In this article we describe kit development, why kits have succeeded in molecular biology, and how they have paved the way for the more recent widespread use of core facilities.     

Arp2/3 complex: advances on the inner workings of a molecular machine.

Several new papers report progress on the structure and function of Arp2/3 complex. A crystal structure, a cryo-EM structure, and a reconstitution of the complex from subunits have been reported. New results also address the nucleation mechanism and the role of bound nucleotide.     

The Bam machine: A molecular cooper

The bacterial outer membrane (OM) is an exceptional biological structure with a unique composition that contributes significantly to the resiliency of Gram-negative bacteria. Since all OM components are synthesized in the cytosol, the cell must efficiently transport OM-specific lipids and proteins across the cell envelope and stably integrate them into a growing membrane. In this review, we discuss the challenges associated with these processes and detail the elegant solutions that cells have evolved to address the topological problem of OM biogenesis. Special attention will be paid to the Bam machine, a highly conserved multiprotein complex that facilitates OM β-barrel folding. This article is part of a Special Issue entitled: Protein Folding in Membranes.     

VIP1, an Arabidopsis protein that interacts with Agrobacterium VirE2, is involved in VirE2 nuclear import and Agrobacterium infectivity.

T-DNA nuclear import is a central event in genetic transformation of plant cells by Agrobacterium. This event is thought to be mediated by two bacterial proteins, VirD2 and VirE2, which are associated with the transported T-DNA molecule. While VirD2 is imported into the nuclei of plant, animal and yeast cells, nuclear uptake of VirE2 occurs most efficiently in plant cells. To understand better the mechanism of VirE2 action, a cellular interactor of VirE2 was identified and its encoding gene cloned from Arabidopsis. The identified plant protein, designated VIP1, specifically bound VirE2 and allowed its nuclear import in non-plant systems. In plants, VIP1 was required for VirE2 nuclear import and Agrobacterium tumorigenicity, participating in early stages of T-DNA expression.     

Plant formins: diverse isoforms and unique molecular mechanism

The completed genome from the model plant Arabidopsis thaliana reveals the presence of a diverse multigene family of formin-like sequences, comprising more than 20 isoforms. This review highlights recent findings from biochemical, cell biological and reverse-genetic analyses of this family of actin nucleation factors. Important advances in understanding cellular function suggest major roles for plant formins during cytokinesis and cell expansion. Biochemical studies on a subset of plant formins emphasize the need to examine molecular mechanisms outside of mammalian and yeast systems. Notably, a combination of solution-based assays for actin dynamics and timelapse, single-filament imaging with TIRFM provide evidence for the first non-processive formin (AtFH1) in eukaryotes. Despite these advances it remains difficult to generate a consensus view of plant formin activities and cellular functions. One limitation to summarizing formin properties relates to the enormous variability in domain organization among the plant formins. Generating homology-based predictions that depend on conserved domains outside of the FH1 and FH2 will be virtually impossible for plant formins. A second major drawback is the lack of facile techniques for examining dynamics of individual actin filaments within live plant cells. This constraint makes it extremely difficult to bridge the gap between biochemical characterization of particular formin and its specific cellular function. There is promise, however, that recent technical advances in engineering appropriate fluorescent markers and new fluoresence imaging techniques will soon allow the direct visualization of cortical actin filament dynamics. The emergence of other model systems for studying actin cytoskeleton in vivo, such as the moss Physcomitrella patens, may also enhance our knowledge of plant formins.     

Three-dimensional reconstruction of Agrobacterium VirE2 protein with single-stranded DNA

Agrobacterium tumefaciens infects plant cells by a unique mechanism involving an interkingdom genetic transfer. A single-stranded DNA substrate is transported across the two cell walls along with the bacterial virulence proteins VirD2 and VirE2. A single VirD2 molecule covalently binds to the 5'-end of the single-stranded DNA, while the VirE2 protein binds stoichiometrically along the length of the DNA, without sequence specificity. An earlier transmission/scanning transmission electron microscopy study indicated a solenoidal ("telephone coil") organization of the VirE2-DNA complex. Here we report a three-dimensional reconstruction of this complex using electron microscopy and single-particle image-processing methods. We find a hollow helical structure of 15.7-nm outer diameter, with a helical rise of 51.5 nm and 4.25 VirE2 proteins/turn. The inner face of the protein units contains a continuous wall and an inward protruding shelf. These structures appear to accommodate the DNA binding. Such a quaternary arrangement naturally sequesters the DNA from cytoplasmic nucleases and suggests a mechanism for its nuclear import by decoration with host cell factors. Coexisting with the helices, we also found VirE2 tetrameric ring structures. A two-dimensional average of the latter confirms the major features of the three-dimensional reconstruction.