Flagellin-sensing 2 (FLS2) is a leucine-rich repeat/transmembrane domain/protein kinase (LRR-RLK) that is the plant receptor for bacterial flagellin or the flagellin-derived flg22 peptide. Previous ...work has shown that after flg22 binding, FLS2 releases BIK1 kinase and homologs and associates with BAK1 kinase, and that FLS2 kinase activity is critical for FLS2 function. However, the detailed mechanisms for activation of FLS2 signaling remain unclear. The present study initially identified multiple FLS2 in vitro phosphorylation sites and found that Serine-938 is important for FLS2 function in vivo. FLS2-mediated immune responses are abolished in transgenic plants expressing FLS2(S938A), while the acidic phosphomimic mutants FLS2(S938D) and FLS2(S938E) conferred responses similar to wild-type FLS2. FLS2-BAK1 association and FLS2-BIK1 disassociation after flg22 exposure still occur with FLS2(S938A), demonstrating that flg22-induced BIK1 release and BAK1 binding are not sufficient for FLS2 activity, and that Ser-938 controls other aspects of FLS2 activity. Purified BIK1 still phosphorylated purified FLS2(S938A) and FLS2(S938D) mutant kinase domains in vitro. Phosphorylation of BIK1 and homologs after flg22 exposure was disrupted in transgenic Arabidopsis thaliana plants expressing FLS2(S938A) or FLS2(D997A) (a kinase catalytic site mutant), but was normally induced in FLS2(S938D) plants. BIK1 association with FLS2 required a kinase-active FLS2, but FLS2-BAK1 association did not. Hence FLS2-BIK1 dissociation and FLS2-BAK1 association are not sufficient for FLS2-mediated defense activation, but the proposed FLS2 phosphorylation site Ser-938 and FLS2 kinase activity are needed both for overall defense activation and for appropriate flg22-stimulated phosphorylation of BIK1 and homologs.
Structure and Mechanism of Mouse Cysteine Dioxygenase McCoy, Jason G.; Bailey, Lucas J.; Bitto, Eduard ...
Proceedings of the National Academy of Sciences - PNAS,
02/2006, Letnik:
103, Številka:
9
Journal Article
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Odprti dostop
Cysteine dioxygenase (CDO) catalyzes the oxidation of L-cysteine to cysteine sulfinic acid. Deficiencies in this enzyme have been linked to autoimmune diseases and neurological disorders. The x-ray ...crystal structure of CDO from Mus musculus was solved to a nominal resolution of 1.75 Å. The sequence is 91% identical to that of a human homolog. The structure reveals that CDO adopts the typical β-barrel fold of the cupin superfamily. The NE2 atoms of His-86, -88, and -140 provide the metal binding site. The structure further revealed a covalent linkage between the side chains of Cys-93 and Tyr-157, the cysteine of which is conserved only in eukaryotic proteins. Metal analysis showed that the recombinant enzyme contained a mixture of iron, nickel, and zinc, with increased iron content associated with increased catalytic activity. Details of the predicted active site are used to present and discuss a plausible mechanism of action for the enzyme.
The Ebola Virus is a causative agent of viral hemorrhagic fever outbreaks and a potential global health risk. The outbreak in West Africa (2013–2016) led to 11,000+ deaths and 30,000+ Ebola infected ...individuals. The current outbreak in the Democratic Republic of Congo (DRC) with 3000+ confirmed cases and 2000+ deaths attributed to Ebola virus infections provides a reminder that innovative countermeasures are still needed. Ebola virus encodes 7 open reading frames (ORFs). Of these, the nucleocapsid protein (eNP) encoded by the first ORF plays many significant roles, including a role in viral RNA synthesis. Here we describe efforts to target the C-terminal domain of eNP (eNP-CTD) that contains highly conserved residues 641–739 as a pan-Ebola antiviral target. Interactions of eNP-CTD with Ebola Viral Protein 30 (eVP30) and Viral Protein 40 (eVP40) have been shown to be crucial for viral RNA synthesis, virion formation, and virion transport. We used nuclear magnetic response (NMR)-based methods to screened the eNP-CTD against a fragment library. Perturbations of 1D 1H NMR spectra identified of 48 of the 439 compounds screened as potential eNP CTD interactors. Subsequent analysis of these compounds to measure chemical shift perturbations in 2D 1H,15N NMR spectra of 15N-labeled protein identified six with low millimolar affinities. All six perturbed an area consisting mainly of residues at or near the extreme C-terminus that we named “Site 1” while three other sites were perturbed by other compounds. Our findings here demonstrate the potential utility of eNP as a target, several fragment hits, and provide an experimental pipeline to validate viral-viral interactions as potential panfiloviral inhibitor targets.
•Performed a high throughput screen (HTS) to identify binders of Ebola NP.•Identified several binders that target Ebola NP C-terminal domain.•Validated binding and propose structure-activity relationships.•Highlight the utility of Ebola NP C-terminus as a potential therapeutic target.
Human rhinovirus strains differ greatly in their virulence, and this has been correlated with the differing substrate specificity of the respective 2A protease (2Apro). Rhinoviruses use their 2Apro ...to cleave a spectrum of cellular proteins important to virus replication and anti-host activities. These enzymes share a chymotrypsin-like fold stabilized by a tetra-coordinated zinc ion. The catalytic triad consists of conserved Cys (C105), His (H34), and Asp (D18) residues. We used a semi-automated NMR protocol developed at NMRFAM to determine the solution structure of 2Apro (C105A variant) from an isolate of the clinically important rhinovirus C species (RV-C). The backbone of C2 2Apro superimposed closely (1.41-1.81 Å rmsd) with those of orthologs from RV-A2, coxsackie B4 (CB4), and enterovirus 71 (EV71) having sequence identities between 40% and 60%. Comparison of the structures suggest that the differential functional properties of C2 2Apro stem from its unique surface charge, high proportion of surface aromatics, and sequence surrounding the di-tyrosine flap.
Ebola viral infections have resulted in several deadly epidemics in recent years in West and Central Africa. Because only one of the seven proteins encoded by the viral genome possesses enzymatic ...activity, disruption of protein–protein interactions is a promising route for antiviral drug development. We carried out a screening campaign to identify small, drug-like compounds that bind to the C-terminal region of the multifunctional Ebola nucleoprotein (eNP) with the objective of discovering ones that disrupt its binding to other Ebola proteins or to the single-stranded RNA genome. In the course of this effort we assigned the backbone
1
H,
15
N, and
13
C resonances of residues 600‒739, the region that contains the critical eVP30 binding region 600‒615 targeted by host factors, and used the assigned chemical shifts to predict secondary structural features and peptide dynamics. This work supports and extends the previous X-ray crystal structures and NMR studies of residues 641‒739. We found that the 600‒739 domain consists of separate regions that are largely disordered and ordered.
Arabidopsis thaliana Regulator of G protein Signalling 1 (AtRGS1) is a protein with a predicted N-terminal 7-transmembrane (7TM) domain and a C-terminal cytosolic RGS1 box domain. The RGS1 box domain ...exerts GTPase activation (GAP) activity on Gα (AtGPA1), a component of heterotrimeric G protein signaling in plants. AtRGS1 may perceive an exogenous agonist to regulate the steady-state levels of the active form of AtGPA1. It is uncertain if the full-length AtRGS1 protein exerts any atypical effects on Gα, nor has it been established exactly how AtRGS1 contributes to perception of an extracellular signal and transmits this response to a G-protein dependent signaling cascade. Further studies on full-length AtRGS1 have been inhibited due to the extreme low abundance of the endogenous AtRGS1 protein in plants and lack of a suitable heterologous system to express AtRGS1. Here, we describe methods to produce full-length AtRGS1 by cell-free synthesis into unilamellar liposomes and nanodiscs. The cell-free synthesized AtRGS1 exhibits GTPase activating activity on Gα and can be purified to a level suitable for biochemical analyses.
•Two protocols are provided for AtRGS1 in vitro synthesis and purification.•AtRGS1 solubilized better in nanodisc than in unilamellar liposomes.•Incorporation into nanodisc conserves the GAP activity of AtRGS1.
Protocols have been developed and applied in the high-throughput production of selenomethionine labeled fusion proteins using the conditional Met auxotroph
Escherichia coli B834. The large-scale ...growth and expression uses a chemically defined auto-induction medium containing 125
mg
L
−1 selenomethionine, salts and trace metals, other amino acids including 10
mg
L
−1 of methionine, vitamins except vitamin B
12, and glucose, glycerol, and
α-lactose. A schematic for a shaker rack that can hold up to twenty-four 2-L polyethylene terephthalate beverage bottles in a standard laboratory refrigerated floor shaker is provided. The growth cycle from inoculation of the culture bottle through the growth, induction, and expression was timed to take ∼24
h. Culture growth in the auto-induction medium gave an average final optical density at 600
nm of ∼6 and an average wet cell mass yield of ∼14
g from 2
L of culture in greater than 150 expression trials. A simple method for visual scoring of denaturing electrophoresis gels for total protein expression, solubility, and effectiveness of fusion protein proteolysis was developed and applied. For the favorably scored expression trials, the average yield of purified, selenomethionine-labeled target protein obtained after proteolysis of the fusion protein was ∼30
mg. Analysis by mass spectrometry showed greater than 90% incorporation of selenomethionine over a ∼8-fold range of selenomethionine concentrations in the growth medium, with higher growth rates observed at the lower selenomethionine concentrations. These protein preparations have been utilized to solve X-ray crystal structures by multiwavelength anomalous diffraction phasing.
Protocols have been developed and applied for the high-throughput production of
U-
15N- or
U-
13C-,
U-
15N-labeled proteins using the conditional methionine auxotroph
Escherichia coli B834. The ...large-scale growth and expression uses a chemically defined auto-induction medium containing salts and trace metals, vitamins including vitamin B
12, and glucose, glycerol, and lactose. The results from nine expression trials in 2-L of the auto-induction medium (500
mL in each of four polyethylene terephthalate beverage bottles) gave an average final optical density at 600
nm of ∼5, an average wet cell mass yield of ∼9.5
g
L
−1, and an average yield of ∼20
mg of labeled protein in the six instances in which proteolysis of the fusion protein was observed. Correlations between the cell mass recovered, the level of protein expression, and the relative amounts of glucose, glycerol, and lactose in the auto-induction medium were noted. Mass spectral analysis showed that the purified proteins contained both
15N and
13C at levels greater than 95%.
1H–
15N heteronuclear single quantum correlation spectroscopy as well as
13C;
15N-edited spectroscopy showed that the purified
U-
15N- and
U-
13C,
U-
15N-labeled proteins were suitable for structure analysis.
The product of gene At3g16450.1 from Arabidopsis thaliana is a 32 kDa, 299-residue protein classified as resembling a myrosinase-binding protein (MyroBP). MyroBPs are found in plants as part of a ...complex with the glucosinolate-degrading enzyme myrosinase, and are suspected to play a role in myrosinase-dependent defense against pathogens. Many MyroBPs and MyroBP-related proteins are composed of repeated homologous sequences with unknown structure. We report here the three-dimensional structure of the At3g16450.1 protein from Arabidopsis, which consists of two tandem repeats. Because the size of the protein is larger than that amenable to high-throughput analysis by uniform ¹³C/¹⁵N labeling methods, we used stereo-array isotope labeling (SAIL) technology to prepare an optimally ²H/¹³C/¹⁵N-labeled sample. NMR data sets collected using the SAIL protein enabled us to assign ¹H, ¹³C and ¹⁵N chemical shifts to 95.5% of all atoms, even at a low concentration (0.2 m m) of protein product. We collected additional NOESY data and determined the three-dimensional structure using the cyana software package. The structure, the first for a MyroBP family member, revealed that the At3g16450.1 protein consists of two independent but similar lectin-fold domains, each composed of three β-sheets.
Brazzein is a small, heat-stable, intensely sweet protein consisting of 54 amino acid residues. Based on the wild-type brazzein, 25 brazzein mutants have been produced to identify critical regions ...important for sweetness. To assess their sweetness, psychophysical experiments were carried out with 14 human subjects. First, the results suggest that residues 29–33 and 39–43, plus residue 36 between these stretches, as well as the C-terminus are involved in the sweetness of brazzein. Second, charge plays an important role in the interaction between brazzein and the sweet taste receptor.
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