Intersubunit complementation of sugar signal transduction in VirA heterodimers and posttranslational regulation of VirA activity in Agrobacterium tumefaciens

The VirA/VirG two-component regulatory system of Agrobacterium tumefaciens regulates expression of the virulence (vir) genes that control the infection process leading to crown gall tumor disease on susceptible plants. VirA, a membrane-bound homodimer, initiates vir gene induction by communicating the presence of molecular signals found at the site of a plant wound through phosphorylation of VirG. Inducing signals include phenols, monosaccharides, and acidic pH. While sugars are not essential for gene induction, their presence greatly increases vir gene expression when levels of the essential phenolic signal are low. Reception of the sugar signal depends on a direct interaction between ChvE, a sugar-binding protein, and VirA. Here we show that the sugar signal received in the periplasmic region of one subunit within a VirA heterodimer can enhance the kinase function of the second subunit. However, sugar enhancement of vir gene expression was vector dependent. virA alleles expressed from pSa-derived vectors inhibited signal transduction by endogenous VirA. Inhibition was conditional, depending on the induction medium and the virA allele tested. Moreover, constitutive expression of virG overcame the inhibitory effect of some but not all virA alleles, suggesting that there may be more than one inhibitory mechanism.     

Factors That Control the Chemistry of the LOV Domain Photocycle

Algae, plants, bacteria and fungi contain Light-Oxygen-Voltage (LOV) domains that function as blue light sensors to control cellular responses to light. All LOV domains contain a bound flavin chromophore that is reduced upon photon absorption and forms a reversible, metastable covalent bond with a nearby cysteine residue. In Avena sativa LOV2 (AsLOV2), the photocycle is accompanied by an allosteric conformational change that activates the attached phototropin kinase in the full-length protein. Both the conformational change and formation of the cysteinyl-flavin adduct are stabilized by the reduction of the N5 atom in the flavin’s isoalloxazine ring. In this study, we perform a mutational analysis to investigate the requirements for LOV2 to photocycle. We mutated all the residues that interact with the chromophore isoalloxazine ring to inert functional groups but none could fully inhibit the photocycle except those to the active-site cysteine. However, electronegative side chains in the vicinity of the chromophore accelerate the N5 deprotonation and the return to the dark state. Mutations to the N414 and Q513 residues identify a potential water gate and H₂O coordination sites. These residues affect the electronic nature of the chromophore and photocycle time by helping catalyze the N5 reduction leading to the completion of the photocycle. In addition, we demonstrate that dehydration leads to drastically slower photocycle times. Finally, to investigate the requirements of an active-site cysteine for photocycling, we moved the nearby cysteine to alternative locations and found that some variants can still photocycle. We propose a new model of the LOV domain photocycle that involves all of these components.     

植物类金属硫蛋白半胱氨酸富含区结构的建模

详细了解蛋白质的三级结构信息有助于理解其生物学功能。随着植物基因组研究的进展 ,已发现了 50多个植物类金属硫蛋白 (Metallothionein_Like ,MT_L)基因。但至今只有少数几个MT_L蛋白得到了纯化 ,而其结构尚无报道 ,因此有必要建立分析这类蛋白结构特征的方法。本研究根据已知的哺乳动物MT的结构数据 ,分析得出了CXC、CXXC模式和金属 硫络合簇结构原子间的距离限制条件 ,并用距离几何算法计算得出预测蛋白可能的构象 ;然后通过统计分析筛选出目标函数值显著较小、构象能低的结构作为这些蛋白半胱氨酸富含区的预测结构 ,由此建成了适合于植物类金属硫蛋白半胱氨酸富含区的结构预测方法。从应用该方法正确地预测出了已知结构的蓝蟹MT的结构来看 ,该方法是可行的。并用该方法预测了油菜MT_L蛋白的半胱氨酸富含区的结构。     

植物蛋白质合成延伸因子

蛋白质的生物合成是一个需要许多大分子如起动因子、延伸因子、终止因子、核糖体、信使RNA、氨酰合成酶和tR NA协同作用的复杂的生理生化过程。植物蛋白质合成延伸因子eEF1和eEF2通过在核糖体上催化氨基酸链的延伸而推动、控制蛋白质的合成。文章介绍植物蛋白质生物合成延伸因子的研究进展     

Mutational analysis of the input domain of the VirA protein of Agrobacterium tumefaciens.

The transmembrane sensor protein VirA activates VirG in response to high levels of acetosyringone (AS). In order to respond to low levels of AS, VirA requires the periplasmic sugar-binding protein ChvE and monosaccharides released from plant wound sites. To better understand how VirA senses these inducers, the C58 virA gene was randomly mutagenized, and 14 mutants defective in vir gene induction and containing mutations which mapped to the input domain of VirA were isolated. Six mutants had single missense mutatiions in three widely separated areas of the periplasmic domain. Eight mutants had mutations in or near an amphipathic helix, TM1, or TM2. Four of the mutations in the periplasmic domain, when introduced into the corresponding A6 virA sequence, caused a specific defect in the vir gene response to glucose. This suggests that most of the periplasmic domain is required for the interaction with, or response to, ChvE. Three of the mutations from outside the periplasmic domain, one from each transmembrane domain and one from the amphiphathic helix, were made in A6 virA. These mutants were defective in the vir gene response to AS. These mutations did not affect the stability or topology of VirA or prevent dimerization; therefore, they may interfere with detection of AS or transmission of the signals to the kinase domain. Characterization of C58 chvE mutants revealed that, unlike A6 VirA, C58 VirA requires ChvE for activation of the vir genes.     

High-Resolution X-Ray Structure and Functional Analysis of the Murine Norovirus 1 Capsid Protein Protruding Domain

Murine noroviruses (MNV) are closely related to the human noroviruses (HuNoV), which cause the majority of nonbacterial gastroenteritis. Unlike HuNoV, MNV grow in culture and in a small-animal model that represents a tractable model to study norovirus biology. To begin a detailed investigation of molecular events that occur during norovirus binding to cells, the crystallographic structure of the murine norovirus 1 (MNV-1) capsid protein protruding (P) domain has been determined. Crystallization of the bacterially expressed protein yielded two different crystal forms (Protein Data Bank identifiers [PDB ID], 3LQ6 and 3LQE). Comparison of the structures indicated a large degree of structural mobility in loops on the surface of the P2 subdomain. Specifically, the A′-B′ and E′-F′ loops were found in open and closed conformations. These regions of high mobility include the known escape mutation site for the neutralizing antibody A6.2 and an attenuation mutation site, which arose after serial passaging in culture and led to a loss in lethality in STAT1^−/− mice, respectively. Modeling of a Fab fragment and crystal structures of the P dimer into the cryoelectron microscopy three-dimensional (3D) image reconstruction of the A6.2/MNV-1 complex indicated that the closed conformation is most likely bound to the Fab fragment and that the antibody contact is localized to the A′-B′ and E′-F′ loops. Therefore, we hypothesize that these loop regions and the flexibility of the P domains play important roles during MNV-1 binding to the cell surface.Murine noroviruses (MNV) are members of the family Caliciviridae, which contains small icosahedral viruses with positive-sense, single-stranded RNA genomes (18). MNV is related to human noroviruses (HuNoV), which cause most of the sporadic cases and outbreaks of infectious nonbacterial gastroenteritis worldwide in people of all ages (4, 15, 28, 36, 38, 64). However, noroviruses are an understudied group of viruses due to the previous lack of a tissue culture system and small-animal model. Since its discovery in 2003 (23), MNV has become an increasingly important model to study norovirus biology (66). The availability of a small-animal model, cell culture, and reverse-genetics system, combined with many shared characteristics of human and murine noroviruses, allows detailed studies of norovirus biology (7, 23, 63, 65, 66).The norovirus genome is organized into 3 major open reading frames (ORFs), which encode the nonstructural polyprotein (∼200 kDa) and the major (VP1; ∼58-kDa) and minor (VP2; ∼20-kDa) capsid proteins (18). Recently, a putative ORF-4 was identified in MNV, but the existence of that product and its function remain unknown (60). Norovirus capsids are formed from 180 copies of VP1 arranged with T=3 icosahedral symmetry (9, 25, 46-48). Each capsid protein is divided into an N-terminal arm (N), a shell (S), and a C-terminal protruding (P) domain, with the last two domains connected by a short hinge. VP1 self-assembles into virus-like particles (VLPs) in baculovirus, mammalian, and plant expression systems (21, 22, 50, 57, 67). The S domain forms a smooth shell around the viral genome but is unable to bind to receptors (3, 55). The P domain dimerizes, forming arch-like structures on the capsid surface, and is subdivided into P1 (the stem of the arch) and P2 (the top of the arch) subdomains. The sequence of the P2 subdomain is the least conserved, followed by the P1 and S domains with the highest degree of conservation. While the S domain of Norwalk virus (NV) is required in order to form VLPs in a baculovirus expression system, the P domains contribute to stability by intermolecular interactions (3, 24). The homodimeric interactions of the HuNoV P domain, observed by crystallographic studies of VLPs, is retained when the protein region is expressed in a bacterial expression system (55). In addition, the norovirus P domain, specifically the P2 subdomain, contains the sites for antigenicity, immune-driven evolution, and cell binding (13a, 20, 25, 32, 41, 51, 56). For MNV-1, the Fab fragment of the neutralizing antibody A6.2 binds to the outermost tip of the P2 subdomain and is thought to prevent infection by blocking capsid-receptor interaction (25).Early steps in the norovirus life cycle are determinants of norovirus tropism (19) and thereby determine the outcome of a viral infection. While the tropism of HuNoV remains unknown, MNV-1 has a tropism for murine macrophages and dendritic cells in vitro and in vivo (62, 65). Recent studies from our laboratory demonstrated that MNV-1 binds to sialic acid on murine macrophages, in particular on the ganglioside GD1a (58). It subsequently enters murine macrophages and dendritic cells in a pH-independent manner (43). To better understand MNV-cell surface binding, we expressed, purified, and determined the high-resolution structure of the MNV-1 P domain at 2.0-Å resolution. Here, we show that, similar to HuNoV P domains (10, 55), recombinant MNV-1 P domains can be expressed and fold in a biologically correct manner. This was shown by the ability of the recombinant MNV-1 P domain to bind murine macrophages, to competitively inhibit MNV-1 infection, and to be recognized by the neutralizing antibody A6.2, which interferes with macrophage binding. Expressed P domain yielded different crystal forms with significant structural differences in the outermost loops of the P2 subdomains. Overall, the MNV-1 P-domain crystal structures show tertiary structures similar to those of HuNoV P domains, with the greatest structural variation in the polypeptide loops on the outer surface of the P domain corresponding to the mobile regions among the various crystal forms. In particular, one of these loops, E′-F′, was observed in “open” and “closed” conformations. Modeling of a Fab fragment and the crystal structures of the P domain into the cryoelectron microscopy three-dimensional (3D) reconstruction of the Fab/MNV-1 complex indicated that the “closed” conformation is the form likely being bound by the neutralizing antibody A6.2. Two sequences located in the A′-B′ and E′-F′ loops were identified as epitopes for A6.2. Biological support for the in silico modeling data comes from a recombinant MNV-1 in which amino acids of the Norwalk virus E′-F′ loop replaced those of MNV-1 and that was no longer neutralized by A6.2. We hypothesize that flexibility in the E′-F′ loop is important for virus-cell interaction and that A6.2 might sterically block viral binding to the cell surface and/or prevent structural changes in the viral capsid required during receptor interaction. In addition, a channel at the interphase of the P dimer was identified that is stabilized by an “ionic lock” (i.e., a bridge formed by two sets of opposing arginine and glutamic acid residues). We hypothesize that the ionic lock may act as a trigger for structural changes important during infection, possibly at the level of host cell entry. Together, these data identify several potential movements within the MNV-1 P domain, which points to the flexibility of the MNV-1 capsid.     

Genetic analysis of the signal-sensing region of the histidine protein kinase VirA of Agrobacterium tumefaciens

The membrane-bound sensor protein kinase VirA of Agrobacterium tumefaciens detects plant phenolic substances, which induce expression of vir genes that are essential for the formation of the crown gall tumor. VirA also responds to specific monosaccharides, which enhance vir expression. These sugars are sensed by the periplasmic domain of VirA that includes the region homologous to the chemoreceptor Trg, and the phenolics are thought to be detected by a part of the cytoplasmic linker domain, while the second transmembrane domain (TM2) is reported to be nonessential. To define regions of VirA that are essential for signal sensing, we introduced base-substitution and deletion mutations into coding regions that are conserved among the respective domains of VirA proteins from various Agrobacterium strains, and examined the effects of these mutations on vir induction and tumorigenicity. The results show that the Trg-homologous region in the periplasmic domain is not essential for the enhancement of vir gene expression by sugars. Most mutations in the TM2 domain also failed to influence enhancement by sugars and reduced the level of vir induction, but a mutation in the TM2 region adjacent to the cytoplasmic linker abolished induction of the vir genes. In the linker domain, sites essential for vir induction by phenolics were scattered over the entire region. We propose that a topological feature formed by the linker domain and at least part of the TM2 may be crucial for activation of a membrane-anchored VirA protein. Complementation analysis with two different VirA mutants suggested that intermolecular phosphorylation between VirA molecules occurs in vivo, and that two intact periplasmic regions in a VirA dimer are required for the enhancement of vir induction by sugars. Received: 14 December 1999 / Accepted: 10 April 2000     

稻瘟病菌分泌蛋白MoCDIE2诱导植物细胞死亡的功能分析

由稻瘟病菌(Magnaporthe oryzae)引起的稻瘟病是全球最严重的植物真菌病害之一。稻瘟病菌通过分泌效应蛋白进入与植物相互作用界面或转运到植物细胞内,抑制寄主植物的免疫防卫反应,使病原菌成功侵染。通过农杆菌介导的异源表达策略,筛选到能引起非寄主植物烟草细胞死亡的候选效应蛋白MoCDIE2(Cell Death-Inducing Effector)。序列分析表明:MoCDIE2基因编码一个蓖麻毒素B凝集素蛋白;系统发育树构建结果表明MoCDIE2同源蛋白保守存在于丝状真菌中;利用基因敲除的方法获得MoCDIE2的敲除突变体,结果表明MoCDIE2的敲除突变体在菌丝生长和致病性方面与野生型菌株Guy11没有明显差异。     

Presence of a Fibronectin Type III Domain in a Plant Protein

A hidden Markov model (HMM) approach was used to identify potential candidates in sequence databases for fibronectin type III domains in plants, a kingdom heretofore bereft of these structures. Fortuitously, one of the proteins uncovered had already had a crystal structure published, allowing direct structural confirmation of the existence of this domain in plants. Received: 19 December 1997 / Accepted: 23 December 1997     

Cellular Localization and Functional Analysis of the Protein Encoded by the Chromosomal Virulence Gene(acvB) of Agrobacterium tumefaciens

A chromosomal virulence gene, acvB, of Agrobacterium tumefaciens [J. Bacteriol., 175, 3208–3212 (1993)] was over-expressed in Escherichia coil. A 47-kDa protein was produced and localized in the periplasmic space of E. coli. Amino acid sequence analysis of its N-terminal demonstrated that a signal peptide of 24 amino acids was cleaved from the pre AcvB protein to produce the mature 47-kDa protein. Western-blot analysis using the antiserum against the AcvB protein detected a 47-kDa protein in the periplasmic space only with strain A208 (acvB ⁺). The amount of AcvB protein synthesized was not increased in strain A208 by induction with acetosyringone (100 μm). There was observed no significant difference in induction by acetosyringone of virB:: lacZ, virD:: lacZ, and virE:: lacZ fusion genes regardless of the presence or absence of the acvB gene. The T-strand (lower strand of T-DNA) was detected in strains A208 as well as B119 (acvB^?) which were cultured in induction medium containing acetosyringone. AcvB protein bound to single-stranded DNAs with no apparent sequence specificity. The results suggest that AcvB protein binds to the T-strand in periplasm and mediates the transfer of the T-strand from A. tumefaciens to the host plant cell.     

Functional Domain Analysis of the Cell Division Inhibitor EzrA

The precise spatial and temporal control of bacterial cell division is achieved through the balanced actions of factors that inhibit assembly of the tubulin-like protein FtsZ at aberrant subcellular locations or promote its assembly at the future sites of division. In Bacillus subtilis, the membrane anchored cell division protein EzrA, interacts directly with FtsZ to prevent aberrant FtsZ assembly at cell poles and contributes to the inherently dynamic nature of the cytokinetic ring. Recent work suggests EzrA also serves as a scaffolding protein to coordinate lateral growth with cell wall biosynthesis through interactions with a host of proteins, a finding consistent with EzrA''s four extensive coiled-coil domains. In a previous study we identified a conserved patch of residues near EzrA''s C-terminus (the QNR motif) that are critical for maintenance of a dynamic cytokinetic ring, but dispensable for EzrA-mediated inhibition of FtsZ assembly at cell poles. In an extension of this work, here we report that EzrA''s two C-terminal coiled-coils function in concert with the QNR motif to mediate interactions with FtsZ and maintain the dynamic nature of the cytokinetic ring. In contrast, EzrA''s two N-terminal coiled-coils are dispensable for interaction between EzrA and FtsZ in vitro and in vivo, but required for EzrA mediated inhibition of FtsZ assembly at cell poles. Finally, chimeric analysis indicates that EzrA''s transmembrane anchor plays a generic role: concentrating EzrA at the plasma membrane where presumably it can most effectively modulate FtsZ assembly.     

Environmental conditions differentially affect vir gene induction in different Agrobacterium strains. Role of the VirA sensor protein

The induction of vir gene expression in different types of Agrobacterium strains shows different pH sensitivity profiles. The pH sensitivity pattern demonstrated by octopine Ti strains was similar to that of a supervirulent leucinopine Ti strain, whereas this was different from that shown by nopaline Ti strains and agropine Ri strains. Data are given which indicate that these differences are due to different properties of the virA genes of these wild types. An exceptional case was formed by strains with the limited-host-range plasmid pTiAG57 which showed AS-dependent vir induction only if reduced inoculum sizes were used and the temperature was 28°C or below.     

Functional Mapping of the DNA Binding Domain of Bovine Papillomavirus E1 Protein

Bovine papillomavirus type 1 (BPV-1) requires viral proteins E1 and E2 for efficient DNA replication in host cells. E1 functions at the BPV origin as an ATP-dependent helicase during replication initiation. Previously, we used alanine mutagenesis to identify two hydrophilic regions of the E1 DNA binding domain (E1DBD), HR1 (E1(179-191)) and HR3 (E1(241-252)), which are critical for sequence-specific recognition of the papillomavirus origin. Based on sequence and structure, these regions are similar in spacing and location to DNA binding regions A and B2 of T antigen, the DNA replication initiator of simian virus 40 (SV40). HR1 and A are both part of extended loops which are supported by residues from the HR3 and B2 alpha-helices. Both elements contain basic residues which may contact DNA, although lack of cocrystal structures for both E1 and T antigen make this uncertain. To better understand how E1 interacts with origin DNA, we used random mutagenesis and a yeast one-hybrid screen to select mutations of the E1DBD which disrupt sequence-specific DNA interactions. From the screen we selected seven single point mutants and one double point mutant (F175S, N184Y/K288R, D185G, V193M, F237L, K241E, R243K, and V246D) for in vitro analysis. All mutants tested in electrophoretic mobility shift assays displayed reduced sequence-specific DNA binding compared to the wild-type E1DBD. Mutants D185G, F237L, and R243K were rescued in vitro for DNA binding by the replication enhancer protein E2. We also tested the eight mutations in full-length E1 for the ability to support DNA replication in Chinese hamster ovary cells. Only mutants D185G, F237L, and R243K supported significant DNA replication in vivo which highlights the importance of E1DBD-E2 interactions for papillomavirus DNA replication. Based on the specific point mutations examined, we also assigned putative roles to individual residues in DNA binding. Finally, we discuss sequence and spacing similarities between E1 HR1 and HR3 and short regions of two other DNA tumor virus origin-binding proteins, SV40 T antigen and Epstein-Barr virus nuclear antigen 1 (EBNA1). We propose that all three proteins use a similar DNA recognition mechanism consisting of a loop structure which makes base-specific contacts (HR1) and a helix which primarily contacts the DNA backbone (HR3).     

Domain Analysis of the FliM Protein of Escherichia coli

The FliM protein of Escherichia coli is required for the assembly and function of flagella. Genetic analyses and binding studies have shown that FliM interacts with several other flagellar proteins, including FliN, FliG, phosphorylated CheY, other copies of FliM, and possibly MotA and FliF. Here, we examine the effects of a set of linker insertions and partial deletions in FliM on its binding to FliN, FliG, CheY, and phospho-CheY and on its functions in flagellar assembly and rotation. The results suggest that FliM is organized into multiple domains. A C-terminal domain of about 90 residues binds to FliN in coprecipitation experiments, is most stable when coexpressed with FliN, and has some sequence similarity to FliN. This C-terminal domain is joined to the rest of FliM by a segment (residues 237 to 247) that is poorly conserved, tolerates linker insertion, and may be an interdomain linker. Binding to FliG occurs through multiple segments of FliM, some in the C-terminal domain and others in an N-terminal domain of 144 residues. Binding of FliM to CheY and phospho-CheY was complex. In coprecipitation experiments using purified FliM, the protein bound weakly to unphosphorylated CheY and more strongly to phospho-CheY, in agreement with previous reports. By contrast, in experiments using FliM in fresh cell lysates, the protein bound to unphosphorylated CheY about as well as to phospho-CheY. Determinants for binding CheY occur both near the N terminus of FliM, which appears most important for binding to the phosphorylated protein, and in the C-terminal domain, which binds more strongly to unphosphorylated CheY. Several different deletions and linker insertions in FliM enhanced its binding to phospho-CheY in coprecipitation experiments with protein from cell lysates. This suggests that determinants for binding phospho-CheY may be partly masked in the FliM protein as it exists in the cytoplasm. A model is proposed for the arrangement and function of FliM domains in the flagellar motor.     

Efficient mRNA Polyadenylation Requires a Ubiquitin-Like Domain,a Zinc Knuckle,and a RING Finger Domain,All Contained in the Mpe1 Protein

Almost all eukaryotic mRNAs must be polyadenylated at their 3′ ends to function in protein synthesis. This modification occurs via a large nuclear complex that recognizes signal sequences surrounding a poly(A) site on mRNA precursor, cleaves at that site, and adds a poly(A) tail. While the composition of this complex is known, the functions of some subunits remain unclear. One of these is a multidomain protein called Mpe1 in the yeast Saccharomyces cerevisiae and RBBP6 in metazoans. The three conserved domains of Mpe1 are a ubiquitin-like (UBL) domain, a zinc knuckle, and a RING finger domain characteristic of some ubiquitin ligases. We show that mRNA 3′-end processing requires all three domains of Mpe1 and that more than one region of Mpe1 is involved in contact with the cleavage/polyadenylation factor in which Mpe1 resides. Surprisingly, both the zinc knuckle and the RING finger are needed for RNA-binding activity. Consistent with a role for Mpe1 in ubiquitination, mutation of Mpe1 decreases the association of ubiquitin with Pap1, the poly(A) polymerase, and suppressors of mpe1 mutants are linked to ubiquitin ligases. Furthermore, an inhibitor of ubiquitin-mediated interactions blocks cleavage, demonstrating for the first time a direct role for ubiquitination in mRNA 3′-end processing.     

Pleiotropic phenotypes caused by genetic ablation of the receiver module of the Agrobacterium tumefaciens VirA protein.

The VirA protein of Agrobacterium tumefaciens is a transmembrane sensory kinase that phosphorylates the VirG response regulator in response to chemical signals released from plant wound sites. VirA contains both a two-component kinase module and, at its carboxyl terminus, a receiver module. We previously provided evidence that this receiver module inhibited the activity of the kinase module and that inhibition might be neutralized by phosphorylation. In this report, we provide additional evidence for this model by showing that overexpressing the receiver module in trans can restore low-level basal activity to a VirA mutant protein lacking the receiver module. We also show that ablation of the receiver module restores activity to the inactive VirA (delta324-413) mutant, which has a deletion within a region designated the linker module. This indicates that deletion of the linker module does not denature the kinase module, but rather locks the kinase into a phenotypically inactive conformation, and that this inactivity requires the receiver module. These data provide genetic evidence that the kinase and receiver modules of VirA attain their native conformations autonomously. The receiver module also restricts the variety of phenolic compounds that have stimulatory activity, since removal of this module causes otherwise nonstimulatory phenolic compounds such as 4-hydroxyacetophenone to stimulate vir gene expression.