Nicotiana attenuata LECTIN RECEPTOR KINASE1 suppresses the insect-mediated inhibition of induced defense responses during Manduca sexta herbivory

Nicotiana attenuata has the capacity to respond specifically to herbivory by its natural herbivore, Manduca sexta, through the perception of elicitors in larval oral secretions. We demonstrate that Lectin receptor kinase 1 (LecRK1) functions during M. sexta herbivory to suppress the insect-mediated inhibition of jasmonic acid (JA)-induced defense responses. Gene function analysis performed by reducing LecRK1 expression in N. attenuata by both virus-induced gene silencing and inverted repeated RNA interference (ir-lecRK1 plants) revealed that LecRK1 was essential to mount a full defense response against M. sexta folivory; larvae growing on ir-lecRK1 plants were 40 to 100% larger than those growing on wild-type plants. The insect-induced accumulation of nicotine, diterpene-glucosides, and trypsin protease inhibitors, as well as the expression of Thr deaminase, was severalfold reduced in ir-lecRK1 plants compared with the wild type. The accumulation of JA and JA-Ile was unaffected during herbivory in ir-lecRK1 plants; however, salicylic acid (SA) accumulation was increased by twofold. The expression of nahG in ir-lecRK1 plants prevented the increased accumulation of SA and restored the defense response against M. sexta herbivory. The results suggest that LecRK1 inhibits the accumulation of SA during herbivory, although other mechanisms may also be affected.     

Multimerization domains are associated with apparent strand exchange activity in BLM and WRN DNA helicases

BLM and WRN are members of the RecQ family of DNA helicases that act to suppress genome instability and cancer predisposition. In addition to a RecQ helicase domain, each of these proteins contains an N-terminal domain of approximately 500 amino acids (aa) that is incompletely characterized. Previously, we showed that the N-terminus of Sgs1, the yeast ortholog of BLM, contains a physiologically important 200 aa domain (Sgs1_103–322) that displays single-stranded DNA (ssDNA) binding, strand annealing (SA), and apparent strand-exchange (SE) activities in vitro. Here we used a genetic assay to search for heterologous proteins that could functionally replace this domain of Sgs1 in vivo. In contrast to Rad59, the oligomeric Rad52 protein provided in vivo complementation, suggesting that multimerization is functionally important. An N-terminal domain of WRN was also identified that could replace Sgs1_103–322 in yeast. This domain, WRN_235–526, contains a known coiled coil and displays the same SA and SE activities as Sgs1_103–322. The coiled coil domain of WRN_235–526 is required for both its in vivo activity and its in vitro SE activity_. Based on this result, a potential coiled coil was identified within Sgs1_103–322. This 25 amino acid region was similarly essential for wt Sgs1 activity in vivo and was replaceable by a heterologous coiled coil. Taken together, the results indicate that a coiled coil and a closely linked apparent SE activity are conserved features of the BLM and WRN DNA helicases.     

Cloning and Characterization of the Polyhydroxybutyrate Depolymerase Gene of Pseudomonas stutzeri and Analysis of the Function of Substrate-Binding Domains

The extracellular polyhydroxybutyrate (PHB) depolymerase gene (phaZ_Pst) of Pseudomonas stutzeri was cloned and sequenced. phaZ_Pst was composed of 1,728 bp encoding a protein of 576 amino acids. Analyses of the N-terminal amino acid sequence and the matrix-assisted laser desorption/ionization–time-of-flight (MALDI-TOF) mass spectrum of the purified enzyme showed that the mature enzyme consisted of 538 amino acids with a deduced molecular mass of 57,506 Da. Analysis of the deduced amino acid sequence of the protein revealed a domain structure containing a catalytic domain, putative linker region, and two putative substrate-binding domains (SBDI and SBDII). The putative linker region was similar to the repeating units of the cadherin-like domain of chitinase A from Vibrio harveyi and chitinase B from Clostridium paraputrificum. The binding characteristics of SBDs to poly([R]-3-hydroxybutyrate) [P(3HB)] and chitin granules were characterized by using fusion proteins of SBDs with glutathione S-transferase (GST). These GST fusion proteins with SBDII and SBDI showed binding activity toward P(3HB) granules but did not bind on chitin granules. It has been suggested that the SBDs of the depolymerase interact specifically with the surface of P(3HB). In addition, a kinetic analysis for the enzymatic hydrolysis of 3-hydroxybutyrate oligomers of various sizes has suggested that the catalytic domain of the enzyme recognizes at least two monomeric units as substrates.     

Newly Characterized Region of CP190 Associates with Microtubules and Mediates Proper Spindle Morphology in Drosophila Stem Cells

CP190 is a large, multi-domain protein, first identified as a centrosome protein with oscillatory localization over the course of the cell cycle. During interphase it has a well-established role within the nucleus as a chromatin insulator. Upon nuclear envelope breakdown, there is a striking redistribution of CP190 to centrosomes and the mitotic spindle, in addition to the population at chromosomes. Here, we investigate CP190 in detail by performing domain analysis in cultured Drosophila S2 cells combined with protein structure determination by X-ray crystallography, in vitro biochemical characterization, and in vivo fixed and live imaging of cp190 mutant flies. Our analysis of CP190 identifies a novel N-terminal centrosome and microtubule (MT) targeting region, sufficient for spindle localization. This region consists of a highly conserved BTB domain and a linker region that serves as the MT binding domain. We present the 2.5 Å resolution structure of the CP190 N-terminal 126 amino acids, which adopts a canonical BTB domain fold and exists as a stable dimer in solution. The ability of the linker region to robustly localize to MTs requires BTB domain-mediated dimerization. Deletion of the linker region using CRISPR significantly alters spindle morphology and leads to DNA segregation errors in the developing Drosophila brain neuroblasts. Collectively, we highlight a multivalent MT-binding architecture in CP190, which confers distinct subcellular cytoskeletal localization and function during mitosis.     

Structures of PHR Domains from Mus musculus Phr1 (Mycbp2) Explain the Loss-of-Function Mutation (Gly1092 → Glu) of the C. elegans Ortholog RPM-1

PHR [PAM (protein associated with Myc)-HIW (Highwire)-RPM-1 (regulator of presynaptic morphology 1)] proteins are conserved, large multi-domain E3 ubiquitin ligases with modular architecture. PHR proteins presynaptically control synaptic growth and axon guidance and postsynaptically regulate endocytosis of glutamate receptors. Dysfunction of neuronal ubiquitin-mediated proteasomal degradation is implicated in various neurodegenerative diseases. PHR proteins are characterized by the presence of two PHR domains near the N-terminus, which are essential for proper localization and function. Structures of both the first and second PHR domains of Mus musculus (mouse) Phr1 (MYC binding protein 2, Mycbp2) have been determined, revealing a novel β sandwich fold composed of 11 antiparallel β-strands. Conserved loops decorate the apical side of the first PHR domain (MmPHR1), yielding a distinct conserved surface feature. The surface of the second PHR domain (MmPHR2), in contrast, lacks significant conservation. Importantly, the structure of MmPHR1 provides insights into a loss-of-function mutation, Gly1092 → Glu, observed in the Caenorhabditis elegans ortholog RPM-1.     

The N-terminal region is crucial for the thermostability of the G-domain of Bacillus stearothermophilus EF-Tu

Bacterial elongation factor Tu (EF-Tu) is a model monomeric G protein composed of three covalently linked domains. Previously, we evaluated the contributions of individual domains to the thermostability of EF-Tu from the thermophilic bacterium Bacillus stearothermophilus. We showed that domain 1 (G-domain) sets up the basal level of thermostability for the whole protein. Here we chose to locate the thermostability determinants distinguishing the thermophilic domain 1 from a mesophilic domain 1. By an approach of systematically swapping protein regions differing between G-domains from mesophilic Bacillus subtilis and thermophilic B. stearothermophilus, we demonstrate that a small portion of the protein, the N-terminal 12 amino acid residues, plays a key role in the thermostability of this domain. We suggest that the thermostabilizing effect of the N-terminal region could be mediated by stabilizing the functionally important effector region. Finally, we demonstrate that the effect of the N-terminal region is significant also for the thermostability of the full-length EF-Tu.     

Three dimensional modeling of C-terminal loop of CssA subunit in CS6 of Enterotoxigenic Escherichia coli and its interaction with the 70 KDa domain of Fibronectin

Colonization factor CS6 of enterotoxigenic Escherichia coli (ETEC) helps to establish the adherence of CS6-expressing ETEC in the intestinal wall. CS6 is composed of two structural subunits, known as CssA and CssB. During CS6-expressing ETEC adherence in intestinal wall, 15 amino acid residues containing Cterminal region of CssA subunit, help to bind with N-terminal 70kDa domain of fibronectin (Fn). In this study, we have predicted a theoretical structural model for C-terminal domain of CssA by homology modelling using protein data bank (PDB) file, 1NTY-A as template (66.67% sequence identity) in Discovery Studio. The structural model of N-terminal region of Fn was also determined by homology modelling using PDB files 1FBR and 1E88 as templates. The structure of the model was also validated by Ramachandran plot. The energy minimization for Fn was performed in standard dynamic cascade using Steepest Descent algorithm followed by Adopted Basis NR algorithm in Discovery studio. The docking model between C-terminal domain and fibronectin were generated by using ClusPro algorithm. This docking study would be help for better understanding how CS6 interacts with fibronectin of intestinal extracellular matrix in the host during infection, and would be of great help towards subunit vaccine generation.     

The Middle Region of an HP1-binding Protein, HP1-BP74, Associates with Linker DNA at the Entry/Exit Site of Nucleosomal DNA

In higher eukaryotic cells, DNA molecules are present as chromatin fibers, complexes of DNA with various types of proteins; chromatin fibers are highly condensed in metaphase chromosomes during mitosis. Although the formation of the metaphase chromosome structure is essential for the equal segregation of replicated chromosomal DNA into the daughter cells, the mechanism involved in the organization of metaphase chromosomes is poorly understood. To identify proteins involved in the formation and/or maintenance of metaphase chromosomes, we examined proteins that dissociated from isolated human metaphase chromosomes by 0.4 m NaCl treatment; this treatment led to significant chromosome decondensation, but the structure retained the core histones. One of the proteins identified, HP1-BP74 (heterochromatin protein 1-binding protein 74), composed of 553 amino acid residues, was further characterized. HP1-BP74 middle region (BP74Md), composed of 178 amino acid residues (Lys⁹⁷–Lys²⁷⁴), formed a chromatosome-like structure with reconstituted mononucleosomes and protected the linker DNA from micrococcal nuclease digestion by ∼25 bp. The solution structure determined by NMR revealed that the globular domain (Met¹⁵³–Thr²³⁷) located within BP74Md possesses a structure similar to that of the globular domain of linker histones, which underlies its nucleosome binding properties. Moreover, we confirmed that BP74Md and full-length HP1-BP74 directly binds to HP1 (heterochromatin protein 1) and identified the exact sites responsible for this interaction. Thus, we discovered that HP1-BP74 directly binds to HP1, and its middle region associates with linker DNA at the entry/exit site of nucleosomal DNA in vitro.     

Salicylic Acid and a Chitin Elicitor Both Control Expression of the CAD1 Gene Involved in the Plant Immunity of Arabidopsis

The Arabidopsis mutant cad1 (constitutively activated cell death 1) shows a phenotype that mimics hypersensitive response (HR)-like cell death. The CAD1 gene, which encodes a protein containing a domain with significant homology to the MACPF (membrane attach complex and perforin) domain of complement components and perforin, is likely to control plant immunity negatively and has a W-box cis-element in its promoter region. We found that expression of the CAD1 gene and other W-box containing genes, such as NPR1 and PR2, was promoted by salicylic acid (SA) and benzothiadiazole (BTH) as a SA agonist. The CAD1 gene was also stimulated by a purified chitin oligosaccharide elicitor (degree of polymerization = 8). This latter control was not under SA, because CAD1 expression was not suppressed in 35SnahG transgenic plants, which are unable to accumulate SA. These expression profiles were confirmed by promoter analysis using pCAD1::GUS transgenic plants. The CAD1 expression promoted by BTH and the chitin elicitor was not suppressed in the npr1 mutant, which is insensitive to SA signaling. These results indicate that the CAD1 gene is regulated by two distinct pathways involving SA and a chitin elicitor: viz., SA signaling mediated through an NPR1-independent pathway, and chitin elicitor signaling, through an SA-independent pathway. Three CAD1 homologs that have multiple W-box elements in their promoters were also found to be under the control of SA.     

Armadillo: domain boundary prediction by amino acid composition

The identification and annotation of protein domains provides a critical step in the accurate determination of molecular function. Both computational and experimental methods of protein structure determination may be deterred by large multi-domain proteins or flexible linker regions. Knowledge of domains and their boundaries may reduce the experimental cost of protein structure determination by allowing researchers to work on a set of smaller and possibly more successful alternatives. Current domain prediction methods often rely on sequence similarity to conserved domains and as such are poorly suited to detect domain structure in poorly conserved or orphan proteins. We present here a simple computational method to identify protein domain linkers and their boundaries from sequence information alone. Our domain predictor, Armadillo (http://armadillo.blueprint.org), uses any amino acid index to convert a protein sequence to a smoothed numeric profile from which domains and domain boundaries may be predicted. We derived an amino acid index called the domain linker propensity index (DLI) from the amino acid composition of domain linkers using a non-redundant structure dataset. The index indicates that Pro and Gly show a propensity for linker residues while small hydrophobic residues do not. Armadillo predicts domain linker boundaries from Z-score distributions and obtains 35% sensitivity with DLI in a two-domain, single-linker dataset (within +/-20 residues from linker). The combination of DLI and an entropy-based amino acid index increases the overall Armadillo sensitivity to 56% for two domain proteins. Moreover, Armadillo achieves 37% sensitivity for multi-domain proteins, surpassing most other prediction methods. Armadillo provides a simple, but effective method by which prediction of domain boundaries can be obtained with reasonable sensitivity. Armadillo should prove to be a valuable tool for rapidly delineating protein domains in poorly conserved proteins or those with no sequence neighbors. As a first-line predictor, domain meta-predictors could yield improved results with Armadillo predictions.     

Quantitative phosphoproteomics of lectin receptor‐like kinase VI.4 dependent abscisic acid response in Arabidopsis thaliana

Lectin receptor‐like kinases (LecRKs) play important roles in the responses to adverse environment stress. Abscisic acid (ABA) is a plant hormone involved in plant growth, development and adverse environmental stress responses. Although some studies of ABA response LecRK genes have been reported, the molecular mechanisms of LecRKs regulation of downstream pathways under ABA induction are not well understood. The present study showed that LecRK‐VI.4 responded to ABA and negatively regulated stomatal closure. Here, a quantitative phosphoproteomics approach based on mass spectrometry was employed to study the roles of LecRK‐VI.4 in the ABA signaling pathway. Metal oxide affinity beads and C18 chromatography were used for phosphopeptide enrichment and separation. The isobaric tags for relative and absolute quantitation were used for profiling the phosphoproteome of mutant lecrk‐vi.4‐1 and wild‐type Col‐0 Arabidopsis under normal growth conditions or ABA treatments. In total, 475 unique phosphopeptides were quantified, including 81 phosphopeptides related to LecRK‐VI.4 regulation. Gene ontology, protein–protein interaction and motif analysis were performed. The bioinformatics data showed that phosphorylated proteins regulated by LecRK‐VI.4 had close relations with factors of stomatal function, which included aquaporin activity, H⁺ pump activity and the Ca²⁺ concentration in the cytoplasm. These data have expanded our understanding of how LecRK‐VI.4 regulates ABA‐mediated stomatal movements.     

Classification of Protein Kinases on the Basis of Both Kinase and Non-Kinase Regions

Background

Protein phosphorylation is a generic way to regulate signal transduction pathways in all kingdoms of life. In many organisms, it is achieved by the large family of Ser/Thr/Tyr protein kinases which are traditionally classified into groups and subfamilies on the basis of the amino acid sequence of their catalytic domains. Many protein kinases are multi-domain in nature but the diversity of the accessory domains and their organization are usually not taken into account while classifying kinases into groups or subfamilies.

Methodology

Here, we present an approach which considers amino acid sequences of complete gene products, in order to suggest refinements in sets of pre-classified sequences. The strategy is based on alignment-free similarity scores and iterative Area Under the Curve (AUC) computation. Similarity scores are computed by detecting common patterns between two sequences and scoring them using a substitution matrix, with a consistent normalization scheme. This allows us to handle full-length sequences, and implicitly takes into account domain diversity and domain shuffling. We quantitatively validate our approach on a subset of 212 human protein kinases. We then employ it on the complete repertoire of human protein kinases and suggest few qualitative refinements in the subfamily assignment stored in the KinG database, which is based on catalytic domains only. Based on our new measure, we delineate 37 cases of potential hybrid kinases: sequences for which classical classification based entirely on catalytic domains is inconsistent with the full-length similarity scores computed here, which implicitly consider multi-domain nature and regions outside the catalytic kinase domain. We also provide some examples of hybrid kinases of the protozoan parasite Entamoeba histolytica.

Conclusions

The implicit consideration of multi-domain architectures is a valuable inclusion to complement other classification schemes. The proposed algorithm may also be employed to classify other families of enzymes with multi-domain architecture.     

植物萜类合成酶1-脱氧-D-木酮糖-5-磷酸还原异构酶的分子结构特征与功能预测分析

采用生物信息学的方法和工具对已在GenBank上注册的拟南芥、玉米、岩蔷薇、水稻、黄花蒿、亚麻等植物的萜类合成酶1-脱氧-D-木酮糖-5-磷酸还原异构酶的核酸及氨基酸序列进行分析,并对其组成成分、转运肽、跨膜拓朴结构域、疏水性/亲水性、蛋白质二级及三级结构、分子系统进化关系等进行预测和推断。结果表明：该类酶基因的全长包括5′、3′非翻译区和一个开放阅读框,无跨膜结构域,是一个具转运肽的亲水性蛋白,包括两个功能DXR结合motif及两个功能NADPH结合motif,α-螺旋和不规则卷曲是蛋白质二级结构最大量的结构元件,β-转角和β-折叠散布于整个蛋白质中,蛋白质的功能域在空间结构上折叠成“V”形,“V”形的两臂由N-端与C-端构成,“V”形的底部,是N 端臂与C-端臂的结合域。