scholarly journals Characterization of Nucleocapsid Binding by the Measles Virus and Mumps Virus Phosphoproteins

2004 ◽  
Vol 78 (16) ◽  
pp. 8630-8640 ◽  
Author(s):  
Richard L. Kingston ◽  
Walter A. Baase ◽  
Leslie S. Gay
Keyword(s):  
Amino Acids ◽  
Binding Site ◽  
Measles Virus ◽  
Expression System ◽  
Spectroscopic Studies ◽  
Mumps Virus ◽  
Binding Domain ◽  
Titration Calorimetry ◽  
N Protein ◽  
P Protein

ABSTRACT We report an analysis of the interaction between the P protein and the RNA-associated N protein (N-RNA) for both measles and mumps viruses with proteins produced in a bacterial expression system. During this study, we verified that the C-terminal tail of the N protein is not required for nucleocapsid formation. For both measles and mumps virus N, truncated proteins encompassing amino acids 1 to 375 assemble into nucleocapsid-like particles within the bacterial cell. For measles virus N, the binding site for the P protein maps to residues 477 to 505 within the tail of the molecule, a sequence relatively conserved among the morbilliviruses. For mumps virus N, a binding site for the P protein maps to the assembly domain of N (residues 1 to 398), while no strong binding of the P protein to the tail of N was detected. These results suggest that the site of attachment for the polymerase varies among the paramyxoviruses. Pulldown experiments demonstrate that the last 50 amino acids of both measles virus and mumps virus P (measles virus P, 457 to 507; mumps virus P, 343 to 391) by themselves constitute the nucleocapsid-binding domain (NBD). Spectroscopic studies show that the NBD is predominantly α-helical in both viruses. However, only in measles virus P is the NBD stable and folded, having a lesser degree of tertiary organization in mumps virus P. With isothermal titration calorimetry, we demonstrate that the measles virus P NBD binds to residues 477 to 505 of measles virus N with 1:1 stoichiometry. The dissociation constant (Kd ) was determined to be 13 μM at 20°C and 35 μM at 37°C. Our data are consistent with a model in which an α-helical nucleocapsid binding domain, located at the C terminus of P, is responsible for tethering the viral polymerase to its template yet also suggest that, in detail, polymerase binding in morbilliviruses and rubulaviruses differs significantly.

scholarly journals Inhibition of Ubiquitination and Stabilization of Human Ubiquitin E3 Ligase PIRH2 by Measles Virus Phosphoprotein

2005 ◽  
Vol 79 (18) ◽  
pp. 11824-11836 ◽  
Author(s):  
Mingzhou Chen ◽  
Jean-Claude Cortay ◽  
Ian R. Logan ◽  
Vasileia Sapountzi ◽  
Craig N. Robson ◽  
...  
Keyword(s):  
Binding Site ◽  
Measles Virus ◽  
Fluorescent Protein ◽  
E3 Ligase ◽  
Yeast Two Hybrid ◽  
Ubiquitin E3 Ligase ◽  
P Protein ◽  
Two Hybrid

ABSTRACT Using a C-terminal domain (PCT) of the measles virus (MV) phosphoprotein (P protein) as bait in a yeast two-hybrid screen, a cDNA identical to the recently described human p53-induced-RING-H2 (hPIRH2) cDNA was isolated. A glutathione S-transferase-hPIRH2 fusion protein expressed in bacteria was able to pull down P protein when mixed with an extract from P-expressing HeLa cells in vitro, and myc-tagged hPIRH2 could be reciprocally coimmunoprecipitated with MV P protein from human cells. Additionally, immunoprecipitation experiments demonstrated that hPIRH2-myc, MV P, and nucleocapsid (N) proteins form a ternary complex. The hPIRH2 binding site was mapped to the C-terminal X domain region of the P protein by using a yeast two-hybrid assay. The PCT binding site was mapped on hPIRH2 by using a novel yeast two-hybrid tagged PCR approach and by coimmunoprecipitation of hPIRH2 cysteine mutants and mouse/human PIRH2 chimeras. The hPIRH2 C terminus could mediate the interaction with MV P which was favored by the RING-H2 motif. When coexpressed with an enhanced green fluorescent protein-tagged hPIRH2 protein, MV P alone or in a complex with MV N was able to redistribute hPIRH2 to outside the nucleus, within intracellular aggregates. Finally, MV P efficiently stabilized hPIRH2-myc expression and prevented its ubiquitination in vivo but had no effect on the stability or ubiquitination of an alternative ubiquitin E3 ligase, Mdm2. Thus, MV P protein is the first protein from a pathogen that is able to specifically interact with and stabilize the ubiquitin E3 ligase hPIRH2 by preventing its ubiquitination.

scholarly journals Ankyrin binds to the 15th repetitive unit of erythroid and nonerythroid beta-spectrin.

1991 ◽  
Vol 115 (1) ◽  
pp. 267-277 ◽  
Author(s):  
S P Kennedy ◽  
S L Warren ◽  
B G Forget ◽  
J S Morrow
Keyword(s):  
Binding Site ◽  
Binding Capacity ◽  
Repeat Unit ◽  
Expression System ◽  
Membrane Vesicles ◽  
Enzymatic Digestion ◽  
Binding Domain ◽  
Amino Terminal ◽  
Prokaryotic Expression System

Ankyrin mediates the attachment of spectrin to transmembrane integral proteins in both erythroid and nonerythroid cells by binding to the beta-subunit of spectrin. Previous studies using enzymatic digestion, 2-nitro-5-thiocyanobenzoic acid cleavage, and rotary shadowing techniques have placed the spectrin-ankyrin binding site in the COOH-terminal third of beta-spectrin, but the precise site is not known. We have used a glutathione S-transferase prokaryotic expression system to prepare recombinant erythroid and nonerythroid beta-spectrin from cDNA encoding approximately the carboxy-terminal half of these proteins. Recombinant spectrin competed on an equimolar basis with 125I-labeled native spectrin for binding to erythrocyte membrane vesicles (IOVs), and also bound ankyrin in vitro as measured by sedimentation velocity experiments. Although full length beta-spectrin could inhibit all spectrin binding to IOVs, recombinant beta-spectrin encompassing the complete ankyrin binding domain but lacking the amino-terminal half of the molecule failed to inhibit about 25% of the binding capacity of the IOVs, suggesting that the ankyrin-independent spectrin membrane binding site must lie in the amino-terminal half of beta-spectrin. A nested set of shortened recombinants was generated by nuclease digestion of beta-spectrin cDNAs from ankyrin binding constructs. These defined the ankyrin binding domain as encompassing the 15th repeat unit in both erythroid and nonerythroid beta-spectrin, amino acid residues 1,768-1,898 in erythroid beta-spectrin. The ankyrin binding repeat unit is atypical in that it lacks the conserved tryptophan at position 45 (1,811) within the repeat and contains a nonhomologous 43 residue segment in the terminal third of the repeat. It also appears that the first 30 residues of this repeat, which are highly conserved between the erythroid and nonerythroid beta-spectrins, are critical for ankyrin binding activity. We hypothesize that ankyrin binds directly to the nonhomologous segment in the 15th repeat unit of both erythroid and nonerythroid beta-spectrin, but that this sequence must be presented in the context of a properly folded spectrin "repeat unit" structure. Future studies will identify which residues within the repeat unit are essential for activity, and which residues determine the specificity of various spectrins for different forms of ankyrin.

scholarly journals Domains of the Measles Virus N Protein Required for Binding to P Protein and Self-Assembly

Virology ◽  
1996 ◽  
Vol 216 (1) ◽  
pp. 272-277 ◽  
Author(s):  
BETTINA BANKAMP ◽  
SANDRA M. HORIKAMI ◽  
P.DAVID THOMPSON ◽  
MARION HUBER ◽  
MARTIN BILLETER ◽  
...  
Keyword(s):  
Self Assembly ◽  
Measles Virus ◽  
N Protein ◽  
P Protein

scholarly journals Identification and Characterization of a Regulatory Domain on the Carboxyl Terminus of the Measles Virus Nucleocapsid Protein

2002 ◽  
Vol 76 (17) ◽  
pp. 8737-8746 ◽  
Author(s):  
Xinsheng Zhang ◽  
Candace Glendening ◽  
Hawley Linke ◽  
Christopher L. Parks ◽  
Charles Brooks ◽  
...  
Keyword(s):  
Gene Expression ◽  
Amino Acids ◽  
Binding Affinity ◽  
Reporter Gene ◽  
Nucleocapsid Protein ◽  
Measles Virus ◽  
Reporter Gene Expression ◽  
Regulatory Domain ◽  
N Protein ◽  
Basal Activity

ABSTRACT The paramyxovirus template for transcription and genome replication consists of the RNA genome encapsidated by the nucleocapsid protein (N protein). The activity of the complex, consisting of viral polymerase plus template, can be measured with minireplicons in which the genomic coding sequence is replaced by chloramphenical acetyltransferase (CAT) antisense RNA. Using this approach, we showed that the C-terminal 24 amino acids of the measles virus N protein are dispensable for transcription and replication, based upon the truncation of N proteins used to support minireplicon reporter gene expression. Truncation at the C-terminal or penultimate amino acid 524 resulted in no change in CAT expression, whereas larger truncations spanning residues 523 to 502 were accompanied by an approximately twofold increase in basal activity. Reporter gene expression was enhanced by supplementation with the major inducible 70-kDa heat shock protein (Hsp72) for minireplicons with the N protein or the N protein truncated at position 525 or 524 but not in systems with a truncation at position 523 or 522. Naturally occurring sequence variants of the N protein with variations at positions 522 and 523 were also shown to lack Hsp72 responsiveness independent of changes in basal activity. Since these residues lie within a linear sequence predicting a direct Hsp72 interaction, N protein-Hsp72 binding reactions were analyzed by using surface plasmon resonance technology. Truncation of the C-terminal portion of the N protein by protease digestion resulted in a reduced binding affinity between Hsp72 and the N protein. Furthermore, with synthetic peptides, we established a correlation between the functional responsiveness and the binding affinity for Hsp72 of C-terminal N protein sequences. Collectively, these results show that the C-terminal 24 amino acids of the N protein represent a regulatory domain containing a functional motif that mediates a direct interaction with Hsp72.

scholarly journals Expression and Characterization of the Chitin-Binding Domain of Chitinase A1 from Bacillus circulans WL-12

2000 ◽  
Vol 182 (11) ◽  
pp. 3045-3054 ◽  
Author(s):  
Masayuki Hashimoto ◽  
Takahisa Ikegami ◽  
Shizuka Seino ◽  
Nobuhumi Ohuchi ◽  
Harumi Fukada ◽  
...  
Keyword(s):  
Hydrophobic Interactions ◽  
Expression System ◽  
Binding Activity ◽  
Binding Domain ◽  
Bacillus Circulans ◽  
Titration Calorimetry ◽  
Affinity Column ◽  
Chitin Binding Domain ◽  
Wide Range ◽  
Hydrolysis Of

ABSTRACT Chitinase A1 from Bacillus circulans WL-12 comprises an N-terminal catalytic domain, two fibronectin type III-like domains, and a C-terminal chitin-binding domain (ChBD). In order to study the biochemical properties and structure of the ChBD, ChBDChiA1 was produced in Escherichia coliusing a pET expression system and purified by chitin affinity column chromatography. Purified ChBDChiA1 specifically bound to various forms of insoluble chitin but not to other polysaccharides, including chitosan, cellulose, and starch. Interaction of soluble chitinous substrates with ChBDChiA1 was not detected by means of nuclear magnetic resonance and isothermal titration calorimetry. In addition, the presence of soluble substrates did not interfere with the binding of ChBDChiA1 to regenerated chitin. These observations suggest that ChBDChiA1recognizes a structure which is present in insoluble or crystalline chitin but not in chito-oligosaccharides or in soluble derivatives of chitin. ChBDChiA1 exhibited binding activity over a wide range of pHs, and the binding activity was enhanced at pHs near its pI and by the presence of NaCl, suggesting that the binding of ChBDChiA1 is mediated mainly by hydrophobic interactions. Hydrolysis of β-chitin microcrystals by intact chitinase A1 and by a deletion derivative lacking the ChBD suggested that the ChBD is not absolutely required for hydrolysis of β-chitin microcrystals but greatly enhances the efficiency of degradation.

scholarly journals Substitutions of Two Amino Acids in the Nucleotide-Binding Site Domain of a Resistance Protein Enhance the Hypersensitive Response and Enlarge the PM3F Resistance Spectrum in Wheat

2014 ◽  
Vol 27 (3) ◽  
pp. 265-276 ◽  
Author(s):  
Daniel Stirnweis ◽  
Samira Désiré Milani ◽  
Tina Jordan ◽  
Beat Keller ◽  
Susanne Brunner
Keyword(s):  
Amino Acids ◽  
Binding Site ◽  
Hypersensitive Response ◽  
Expression System ◽  
Coiled Coil ◽  
Nucleotide Binding ◽  
Nucleotide Binding Site ◽  
Allelic Series ◽  
Resistance Protein ◽  
Resistance Spectrum

Proteins with nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domains are major components of the plant immune system. They usually mediate resistance against a subgroup of races of a specific pathogen. For the allelic series of the wheat powdery mildew resistance gene Pm3, alleles with a broad and a narrow resistance spectrum have been described. Here, we show that a broad Pm3 spectrum range correlates with a fast and intense hypersensitive response (HR) in a Nicotiana transient-expression system and this activity can be attributed to two particular amino acids in the ARC2 subdomain of the NBS. The combined substitution of these amino acids in narrow-spectrum PM3 proteins enhances their capacity to induce an HR in Nicotiana benthamiana, and we demonstrate that these substitutions also enlarge the resistance spectrum of the Pm3f allele in wheat. Finally, using Bph14, we show that the region carrying the relevant amino acids also plays a role in the HR regulation of another coiled-coil NBS-LRR resistance protein. These results highlight the importance of an optimized NBS-‘molecular switch’ for the conversion of initial pathogen perception by the LRR into resistance-protein activation, and we describe a possible approach to extend the effectiveness of resistance genes via minimal targeted modifications in the NBS domain.

scholarly journals Localization and function of a calmodulin–apocalmodulin-binding domain in the N-terminal part of the type 1 inositol 1,4,5-trisphosphate receptor

2002 ◽  
Vol 365 (1) ◽  
pp. 269-277 ◽  
Author(s):  
Ilse SIENAERT ◽  
Nael NADIF KASRI ◽  
Sara VANLINGEN ◽  
Jan B. PARYS ◽  
Geert CALLEWAERT ◽  
...  
Keyword(s):  
Amino Acids ◽  
Recombinant Protein ◽  
Binding Site ◽  
Synthetic Peptides ◽  
Critical Role ◽  
Binding Domain ◽  
Terminal Part ◽  
Inositol 1,4,5 Trisphosphate ◽  
Presence And Absence

Calmodulin (CaM) is a ubiquitous protein that plays a critical role in regulating cellular functions by altering the activity of a large number of proteins, including the d-myo-inositol 1,4,5-trisphosphate (IP3) receptor (IP3R). CaM inhibits IP3 binding in both the presence and absence of Ca2+ and IP3-induced Ca2+ release in the presence of Ca2+. We have now mapped and characterized a Ca2+-independent CaM-binding site in the N-terminal part of the type 1 IP3R (IP3R1). This site could be responsible for the inhibitory effects of CaM on IP3 binding. We therefore expressed the N-terminal 581 amino acids of IP3R1 as a His-tagged recombinant protein, containing the functional IP3-binding pocket. We showed that CaM, both in the presence and absence of Ca2+, inhibited IP3 binding to this recombinant protein with an IC50 of approx. 2μM. Deletion of the N-terminal 225 amino acids completely abolished the effects of both Ca2+ and CaM on IP3 binding. We mapped the Ca2+-independent CaM-binding site to a recombinant glutathione S-transferase fusion protein containing the first 159 amino acids of IP3R1 and then made different synthetic peptides overlapping this region. We demonstrated that two synthetic peptides matching amino acids 49–81 and 106–128 bound CaM independently of Ca2+ and could reverse the inhibition of IP3 binding caused by CaM. This suggests that these sequences are components of a discontinuous Ca2+-independent CaM-binding domain, which is probably involved in the inhibition of IP3 binding by CaM.

Tyr199 in transmembrane domain 5 of the β2-adrenergic receptor interacts directly with the pharmacophore of a unique fluorenone-based antagonist

2001 ◽  
Vol 354 (3) ◽  
pp. 485-491 ◽  
Author(s):  
Zhongren WU ◽  
David S. THIRIOT ◽  
Arnold E. RUOHO
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Binding Site ◽  
Adrenergic Receptor ◽  
Transmembrane Domain ◽  
Expression System ◽  
Factor Xa ◽  
Fall Armyworm ◽  
New Information ◽  
Β2 Adrenergic Receptor

Mutagenesis of the β2-adrenergic receptor (β2AR) has suggested that amino acids in transmembrane domain 5 (TMD 5) play an important role in the interaction of the receptor with the catechol end of adrenergic agonists. However, little direct biochemical evidence for the interaction of any β2AR agonist or antagonist with TMD 5 has been reported. To identify receptor amino acids that contribute to the β2AR antagonist binding site, we identified the precise amino acid photoinsertion site of a novel carazolol-like fluorenone antagonist photoaffinity label, [125I]iodoaminoflisopolol ([125I]IAmF). A unique property of this photolabel is that the photoreactive centre is also the binding pharmacophore, which corresponds to the catechol end of related β2AR agonists. [125I]IAmF specifically photolabels membrane-bound and purified β2AR from a baculovirus/Spodoptera frugiperda (fall armyworm) (‘Sf9’) expression system. When the photolabelled β2AR was cleaved by trypsin or Factor Xa, 30kDa labelled peptides were generated. On the basis of concanavalin A binding and amino acid sequencing, these contain the N-terminus of the β2AR, including TMDs 1–5. Further cleavage of the 30kDa peptides with endoproteinase Lys-C generated a 4kDa labelled peptide with an N-terminal amino acid sequence between TMDs 4 and 5. Radiosequencing of this peptide demonstrated that the precise [125I]IAmF photoinsertion site was Tyr199 in TMD 5. Since the photoreactive centre and the binding pharmacophore of IAmF are the same, these data demonstrate that Tyr199 interacts with the planar fluorenone moiety of a carazolol-like β2AR antagonist, and contributes significant new information regarding the binding site for β2AR antagonists.

scholarly journals The unignorable N protein of COVID-19

2020 ◽  
Author(s):  
Zehua Zeng ◽  
Zhi Luo ◽  
Yubang Shan ◽  
Jingjie Yang ◽  
Chuan Chen ◽  
...  
Keyword(s):  
Protein Structure ◽  
Binding Site ◽  
Expression Profile ◽  
Rna Binding ◽  
Clinical Manifestations ◽  
Binding Domain ◽  
S Protein ◽  
N Protein ◽  
Ctl Epitope ◽  
Mirror Structure

Abstract With more and more people infected with COVID-19, it was found that the SARS-CoV-2 virus was quite different from SARS-CoV. Most researches have focused on ACE2 and S protein, however, the known variations of these proteins are not enough to explain why there were so many different clinical manifestations between patients infected with such two viruses. Here, the N protein of the two coronaviruses was parallelly analyzed. Through the analysis of N protein structure, protein conserved binding domain, binding site and ligand, and CTL epitope, it was found that N protein may have a unique expression profile in the SARS-CoV-2. For example, mirror structure and RNA binding tendency.

scholarly journals SAT-712 Computational Study of the Effect of Androgen Receptor BF 3 Site Mutations on DDE Binding

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Muniruzzaman Chowdhury ◽  
Frank Dean ◽  
Evangelia Kotsikorou
Keyword(s):  
Amino Acids ◽  
Androgen Receptor ◽  
Ligand Binding ◽  
Binding Site ◽  
Computational Study ◽  
Binding Domain ◽  
Ligand Binding Domain ◽  
Experimental Results ◽  
Wild Type ◽  
Surface Binding

Abstract Exposure to endocrine disrupting chemicals (EDCs) affects the function of the androgen receptor (AR) causing reproductive system problems such as reduced sperm counts, increased infertility, testicular dysgenesis syndrome, and testicular and prostate cancers, as well as reduced bone mass and diabetes mellitus in males. Experimental results have shown that the presence of EDCs such as the diphenyl compound DDT and its analogue DDE, allosterically cause the release of the stably bound dihydrotestosterone (DHT) from the steroid binding site of the AR ligand binding domain. It was hypothesized that EDCs mediate this effect via binding to the Binding Function 3 (BF 3) surface binding site. Five mutations of three BF 3 amino acids (F673K, F673W, G724R, G724M, and L830D) showed that the ability of DDE to inhibit AR activity was reduced, suggesting that DDE binds to the BF 3 site and allosterically regulates AR activity. In this study, the Induced Fit Docking protocol of the Schrodinger software was used to dock DDE into the BF 3 site of the wild type AR ligand binding domain as well as the five mutant BF 3 sites. The docking poses generated for each receptor were clustered and representative structures were selected. The receptor-ligand complexes were energy minimized using the Schrodinger module Macromodel. Finally, the energy of interaction between DDE and the BF 3 site amino acids was evaluated for each of the selected docks of the wild type and mutant receptors. The relationship between the energies of interaction and the experimental results for DDE inhibition of the mutant AR activities will be discussed.

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