Asymmetric Structure of the Native Rhodobacter sphaeroides Dimeric LH1-RC Complex

2021 ◽  
Author(s):  
Kazutoshi Tani ◽  
Ryo Kanno ◽  
Riku Kikuchi ◽  
Saki Kawamura ◽  
Kenji V. P. Nagashima ◽  
...  
Keyword(s):  
Structural Integrity ◽  
Critical Role ◽  
Photosynthetic Bacterium ◽  
Structural Features ◽  
Asymmetric Structure ◽  
Wild Type ◽  
Core Complex ◽  
In The Wild ◽  
Dimeric Form ◽  
Biochemical Analyses

The light-harvesting-reaction center (LH1-RC) core complex of purple photosynthetic bacterium Rhodobacter (Rba.) sphaeroides is characterized by the presence of both a dimeric form and a monomeric form. Following structure determination of the monomeric LH1-RC including its previously unrecognized component designated protein-U (Nat. Common.12, 6300, 2021), here we present cryo-EM structures of the dimeric LH1-RC from native Rba. sphaeroides IL106 at 2.75 Å resolution and from an LH1-RC monomer lacking protein-U (ΔU) at 2.64 Å resolution. The native dimeric core complex reveals many asymmetric features in the arrangement of its two monomeric components including the structural integrity of protein-U, the overall LH1 organization, and the rigidities of the proteins and pigments that form the complex. PufX polypeptides play a critical role in connecting two monomers, with one PufX interacting at its N-terminus with another PufX and an LH1 β-polypeptide in another monomer, in good agreement with biochemical analyses. One of the proteins-U was only partially identified in the dimeric structure, signaling significantly different degrees of disorder in the two monomers. The ΔU LH1-RC monomer revealed a half-moon-shaped structure containing 11 α- and 10 β-polypeptides (compared with 14 of each in the wild type), indicating a critical role for protein-U in controlling the number of αβ-subunits required for correct assembly and stabilization of the LH1-RC dimer. The structural features are discussed in relation to the unusual topology of intracytoplasmic photosynthetic membranes and an assembly model proposed for the native Rba. sphaeroides dimeric LH1-RC complex in membranes of wild-type cells.

scholarly journals Cytosolic Phospholipase A2α–deficient Mice Are Resistant to Collagen-induced Arthritis

2003 ◽  
Vol 197 (10) ◽  
pp. 1297-1302 ◽  
Author(s):  
Martin Hegen ◽  
Linhong Sun ◽  
Naonori Uozumi ◽  
Kazuhiko Kume ◽  
Mary E. Goad ◽  
...  
Keyword(s):  
Critical Role ◽  
Wild Type ◽  
Collagen Induced Arthritis ◽  
Cytosolic Phospholipase ◽  
Severity Scores ◽  
In The Wild ◽  
Cytosolic Phospholipase A2α ◽  
Antibody Levels ◽  
Deficient Mice

Pathogenic mechanisms relevant to rheumatoid arthritis occur in the mouse model of collagen-induced arthritis (CIA). Cytosolic phospholipase A2α (cPLA2α) releases arachidonic acid from cell membranes to initiate the production of prostaglandins and leukotrienes. These inflammatory mediators have been implicated in the development of CIA. To test the hypothesis that cPLA2α plays a key role in the development of CIA, we backcrossed cPLA2α-deficient mice on the DBA/1LacJ background that is susceptible to CIA. The disease severity scores and the incidence of disease were markedly reduced in cPLA2α-deficient mice compared with wild-type littermates. At completion of the study, >90% of the wild-type mice had developed disease whereas none of the cPLA2α-deficient mice had more than one digit inflamed. Furthermore, visual disease scores correlated with severity of disease determined histologically. Pannus formation, articular fibrillation, and ankylosis were all dramatically reduced in the cPLA2α-deficient mice. Although the disease scores differed significantly between cPLA2α mutant and wild-type mice, anti-collagen antibody levels were similar in the wild-type mice and mutant littermates. These data demonstrate the critical role of cPLA2α in the pathogenesis of CIA.

scholarly journals Colonization and Inflammation Deficiencies in Mongolian Gerbils Infected by Helicobacter pylori Chemotaxis Mutants

2005 ◽  
Vol 73 (3) ◽  
pp. 1820-1827 ◽  
Author(s):  
David J. McGee ◽  
Melanie L. Langford ◽  
Emily L. Watson ◽  
J. Elliot Carter ◽  
Yu-Ting Chen ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Immune Cell ◽  
Response Regulator ◽  
Critical Role ◽  
Mongolian Gerbils ◽  
Wild Type ◽  
In The Wild ◽  
H Pylori

ABSTRACT Helicobacter pylori causes disease in the human stomach and in mouse and gerbil stomach models. Previous results have shown that motility is critical for H. pylori to colonize mice, gerbils, and other animal models. The role of chemotaxis, however, in colonization and disease is less well understood. Two genes in the H. pylori chemotaxis pathway, cheY and tlpB, which encode the chemotaxis response regulator and a methyl-accepting chemoreceptor, respectively, were disrupted. The cheY mutation was complemented with a wild-type copy of cheY inserted into the chromosomal rdxA gene. The cheY mutant lost chemotaxis but retained motility, while all other strains were motile and chemotactic in vitro. These strains were inoculated into gerbils either alone or in combination with the wild-type strain, and colonization and inflammation were assessed. While the cheY mutant completely failed to colonize gerbil stomachs, the tlpB mutant colonized at levels similar to those of the wild type. With the tlpB mutant, there was a substantial decrease in inflammation in the gerbil stomach compared to that with the wild type. Furthermore, there were differences in the numbers of each immune cell in the tlpB-mutant-infected stomach: the ratio of lymphocytes to neutrophils was about 8 to 1 in the wild type but only about 1 to 1 in the mutant. These results suggest that the TlpB chemoreceptor plays an important role in the inflammatory response while the CheY chemotaxis regulator plays a critical role in initial colonization. Chemotaxis mutants may provide new insights into the steps involved in H. pylori pathogenesis.

Conserved tyrosine-147 plays a critical role in the ligand-gated current of the epithelial cation/amino acid transporter/channel CAATCH1

2002 ◽  
Vol 205 (16) ◽  
pp. 2545-2553 ◽  
Author(s):  
Bruce R. Stevens ◽  
Daniel H. Feldman ◽  
Zhilin Liu ◽  
William R. Harvey
Keyword(s):  
Amino Acid ◽  
Critical Role ◽  
Amino Acid Transporter ◽  
Ionic Channel ◽  
Wild Type ◽  
Channel Conductance ◽  
Current Voltage ◽  
In The Wild ◽  
Inward Currents ◽  
Acid Transporter

SUMMARYCAATCH1 functions both as an amino-acid-gated cation channel and as a cation-dependent, proline-preferring, nutrient amino acid transporter in which the two functions are thermodynamically uncoupled. This study focuses on the ionic channel aspect, in which a Tyr147 (wild type) to Phe147 (Y147F) site-directed mutation was investigated by steady-state electrophysiological measurements in the Xenopus laevisoocyte expression system. This tyrosine residue is conserved within the third transmembrane domain in members of the Na+:neurotransmitter transporter family (SNF), where it plays a role in binding pharmacological ligands such as cocaine to the serotonin (SERT), dopamine (DAT) and norepinephrine (NET) transporters. Epithelial CAATCH1 is a member of the SNF family. The results show that amino acid ligand-gating selectivity and current magnitudes in Na+- and K+-containing media are differentially altered in CAATCH1 Y147F compared with the wild type. In the absence of amino acid ligands, the channel conductance of Na+,K+ and Li+ that is observed in the wild type was reduced to virtually zero in Y147F. In the wild type, proline binding increased conductance strongly in Na+-containing medium and moderately in K+-containing medium, whereas in Y147F proline failed to elicit any cation currents beyond those of N-methyl-D-glucamine- or water-injected oocytes. In the wild type, methionine binding strongly inhibited inward Na+ currents, whereas in Y147F it strongly stimulated inward currents in both Na+ and K+-containing media. Indeed, in Na+-containing medium, the relative potency ranking for inward current inhibition in the wild type(Met>Leu>Gly>Phe>Thr) was similar to the ranking of ligand-permissive gating of large inward currents in Y147F. In Na+-containing medium, current/voltage relationships elicited by ligands in the wild type were complex and reversing, whereas in Y147F they were linear and inwardly rectifying. In K+-containing medium,current/voltage relationships remained non-linear in Y147F. Both wild-type and Y147F currents were Cl--independent. Together, these data demonstrate a critical role for Tyr147 in ligand-binding selectivity and modulation of the ionic channel conductance in CAATCH1. The results support the argument that inhibition of the CAATCH1 conductance by free methionine shares some properties in common with ligand inhibition of DAT, SERT, NET and the γ-aminobutyric acid transporter (GAT1).

scholarly journals Identification and Characterization of the Dicarboxylate Uptake System DccT in Corynebacterium glutamicum

2008 ◽  
Vol 190 (19) ◽  
pp. 6458-6466 ◽  
Author(s):  
Jung-Won Youn ◽  
Elena Jolkver ◽  
Reinhard Krämer ◽  
Kay Marin ◽  
Volker F. Wendisch
Keyword(s):  
Corynebacterium Glutamicum ◽  
Tricarboxylic Acid Cycle ◽  
Carbon Sources ◽  
Mrna Levels ◽  
Uptake System ◽  
Dependent Manner ◽  
Wild Type ◽  
Prolonged Incubation ◽  
In The Wild ◽  
Biochemical Analyses

ABSTRACT Many bacteria can utilize C4-carboxylates as carbon and energy sources. However, Corynebacterium glutamicum ATCC 13032 is not able to use tricarboxylic acid cycle intermediates such as succinate, fumarate, and l-malate as sole carbon sources. Upon prolonged incubation, spontaneous mutants which had gained the ability to grow on succinate, fumarate, and l-malate could be isolated. DNA microarray analysis showed higher mRNA levels of cg0277, which subsequently was named dccT, in the mutants than in the wild type, and transcriptional fusion analysis revealed that a point mutation in the promoter region of dccT was responsible for increased expression. The overexpression of dccT was sufficient to enable the C. glutamicum wild type to grow on succinate, fumarate, and l-malate as the sole carbon sources. Biochemical analyses revealed that DccT, which is a member of the divalent anion/Na+ symporter family, catalyzes the effective uptake of dicarboxylates like succinate, fumarate, l-malate, and likely also oxaloacetate in a sodium-dependent manner.

scholarly journals Insulin Receptor Substrate-1 Is Required for Bone Anabolic Function of Parathyroid Hormone in Mice

Endocrinology ◽  
2005 ◽  
Vol 146 (6) ◽  
pp. 2620-2628 ◽  
Author(s):  
Masayuki Yamaguchi ◽  
Naoshi Ogata ◽  
Yusuke Shinoda ◽  
Toru Akune ◽  
Satoru Kamekura ◽  
...  
Keyword(s):  
Insulin Receptor ◽  
Insulin Receptor Substrate ◽  
Wild Type ◽  
Paracrine Factor ◽  
Protein Levels ◽  
Anabolic Action ◽  
Igf I ◽  
In The Wild ◽  
Biochemical Analyses

Abstract Bone anabolic action of PTH has been suggested to be mediated by induction of IGF-I in osteoblasts; however, little is known about the molecular mechanism by which IGF-I leads to bone formation under the PTH stimulation. This study initially confirmed in mouse osteoblast cultures that PTH treatment increased IGF-I mRNA and protein levels and alkaline phosphatase activity, which were accompanied by phosphorylations of IGF-I receptor, insulin receptor substrate (IRS)-1 and IRS-2, essential adaptor molecules for the IGF-I signaling. To learn the involvement of IRS-1 and IRS-2 in the bone anabolic action of PTH in vivo, IRS-1−/− and IRS-2−/− mice and their respective wild-type littermates were given daily injections of PTH (80 μg/kg) or vehicle for 4 wk. In the wild-type mice, the PTH injection increased bone mineral densities of the femur, tibia, and vertebrae by 10–20% without altering the serum IGF-I level. These stimulations were similarly seen in IRS-2−/− mice; however, they were markedly suppressed in IRS-1−/− mice. Although the PTH anabolic effects were stronger on trabecular bones than on cortical bones, the stimulations on both bones were blocked in IRS-1−/− mice but not in IRS-2−/− mice. Histomorphometric and biochemical analyses showed an increased bone turnover by PTH, which was also blunted by the IRS-1 deficiency, though not by the IRS-2 deficiency. These results indicate that the PTH bone anabolic action is mediated by the activation of IRS-1, but not IRS-2, as a downstream signaling of IGF-I that acts locally as an autocrine/paracrine factor.

scholarly journals Two Chalcone Synthase Isozymes Participate Redundantly in UV-Induced Sakuranetin Synthesis in Rice

2020 ◽  
Vol 21 (11) ◽  
pp. 3777
Author(s):  
Hye Lin Park ◽  
Youngchul Yoo ◽  
Seong Hee Bhoo ◽  
Tae-Hoon Lee ◽  
Sang-Won Lee ◽  
...  
Keyword(s):  
Chalcone Synthase ◽  
Developmental Stages ◽  
Rice Genome ◽  
The Other ◽  
Wild Type ◽  
Flavonoid Pathway ◽  
Key Enzyme ◽  
In The Wild ◽  
Bona Fide ◽  
Biochemical Analyses

Chalcone synthase (CHS) is a key enzyme in the flavonoid pathway, participating in the production of phenolic phytoalexins. The rice genome contains 31 CHS family genes (OsCHSs). The molecular characterization of OsCHSs suggests that OsCHS8 and OsCHS24 belong in the bona fide CHSs, while the other members are categorized in the non-CHS group of type III polyketide synthases (PKSs). Biochemical analyses of recombinant OsCHSs also showed that OsCHS24 and OsCHS8 catalyze the formation of naringenin chalcone from p-coumaroyl-CoA and malonyl-CoA, while the other OsCHSs had no detectable CHS activity. OsCHS24 is kinetically more efficient than OsCHS8. Of the OsCHSs, OsCHS24 also showed the highest expression levels in different tissues and developmental stages, suggesting that it is the major CHS isoform in rice. In oschs24 mutant leaves, sakuranetin content decreased to 64.6% and 80.2% of those in wild-type leaves at 2 and 4 days after UV irradiation, respectively, even though OsCHS24 expression was mostly suppressed. Instead, the OsCHS8 expression was markedly increased in the oschs24 mutant under UV stress conditions compared to that in the wild-type, which likely supports the UV-induced production of sakuranetin in oschs24. These results suggest that OsCHS24 acts as the main CHS isozyme and OsCHS8 redundantly contributes to the UV-induced production of sakuranetin in rice leaves.

scholarly journals Critical role of Arg433 in rat transketolase activity as probed by site-directed mutagenesis

1998 ◽  
Vol 333 (2) ◽  
pp. 367-372 ◽  
Author(s):  
Yunjo SOH ◽  
Byoung J. SONG ◽  
Jiingjau JENG ◽  
Abraham T. KALLARAKAL
Keyword(s):  
Critical Role ◽  
Immunoblot Analysis ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Hydrogen Bond Interactions ◽  
Apparent Homogeneity ◽  
Arginine Residues ◽  
Biochemical Analyses

It has been shown that one arginine per monomer at an unknown position is essential for enzyme activity of the homodimeric transketolase (TK) [Kremer, Egan and Sable (1980) J. Biol. Chem. 255, 2405–2410]. To identify the critical arginine, four highly conserved arginine residues of rat TK (Arg102, Arg350, Arg433 and Arg506) were replaced with alanine by site-directed mutagenesis. Wild-type and mutant TK proteins were produced in Escherichia coli and characterized. The Arg102 → Ala mutant exhibited similar catalytic activity to the wild-type enzyme, whereas Arg350 → Ala, Arg506 → Ala and Arg433 → Ala mutants exhibited 36.7, 37.0 and 6.1% of the wild-type activity respectively. Three recombinant proteins (wild-type, Arg350 → Ala and Arg433 → Ala) were purified to apparent homogeneity using Ni2+-affinity chromatography and further characterized. All these proteins were able to form homodimers (148 kDa), as shown by immunoblot analysis subsequent to non-denaturing gel electrophoresis. The Arg433 → Ala mutant protein was less stable than the wild-type and Arg350 → Ala proteins at 55 °C. Kinetic analyses revealed that both Vmax and Km values were markedly affected in the Arg433 → Ala mutant. The Km values for two substrates xylulose 5-phosphate and ribose 5-phosphate were 11.5- and 24.3-fold higher respectively. The kcat/Km values of the Arg433 → Ala mutant for the two substrates were less than 1% of those of the wild-type protein. Molecular modelling of the rat TK revealed that Arg433 of one monomer has three potential hydrogen-bond interactions with the catalytically important highly conserved loop of the other monomer. Thus, our biochemical analyses and modelling data suggest the critical role of the previously uncharacterized Arg433 in TK activity.

scholarly journals Invariant Thr244 is essential for the efficient acylation step of the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans

2006 ◽  
Vol 400 (3) ◽  
pp. 521-530 ◽  
Author(s):  
Arnaud Pailot ◽  
Katia D'Ambrosio ◽  
Catherine Corbier ◽  
François Talfournier ◽  
Guy Branlant
Keyword(s):  
Streptococcus Mutans ◽  
Ternary Complex ◽  
Critical Role ◽  
Hydride Transfer ◽  
Binary Complex ◽  
Wild Type ◽  
Hydrogen Bond Interaction ◽  
Methyl Group ◽  
Unit Increase ◽  
In The Wild

One of the most striking features of several X-ray structures of CoA-independent ALDHs (aldehyde dehydrogenases) in complex with NAD(P) is the conformational flexibility of the NMN moiety. However, the fact that the rate of the acylation step is high in GAPN (non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase) from Streptococcus mutans implies an optimal positioning of the nicotinamide ring relative to the hemithioacetal intermediate within the ternary GAPN complex to allow an efficient and stereospecific hydride transfer. Substitutions of serine for invariant Thr244 and alanine for Lys178 result in a drastic decrease of the efficiency of hydride transfer which becomes rate-limiting. The crystal structure of the binary complex T244S GAPN–NADP shows that the absence of the β-methyl group leads to a well-defined conformation of the NMN part, including the nicotinamide ring, clearly different from that depicted to be suitable for an efficient hydride transfer in the wild-type. The ∼0.6-unit increase in pKapp of the catalytic Cys302 observed in the ternary complex for both mutated GAPNs is likely to be due to a slight difference in positioning of the nicotinamide ring relative to Cys302 with respect to the wild-type ternary complex. Taken together, the data support a critical role of the Thr244 β-methyl group, held in position through a hydrogen-bond interaction between the Thr244 β-hydroxy group and the ϵ-amino group of Lys178, in permitting the nicotinamide ring to adopt a conformation suitable for an efficient hydride transfer during the acylation step for all the members of the CoA-independent ALDH family.

scholarly journals Strigolactone promotes cytokinin degradation through transcriptional activation ofCYTOKININ OXIDASE/DEHYDROGENASE 9in rice

2019 ◽  
Vol 116 (28) ◽  
pp. 14319-14324 ◽  
Author(s):  
Jingbo Duan ◽  
Hong Yu ◽  
Kun Yuan ◽  
Zhigang Liao ◽  
Xiangbing Meng ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Plant Hormone ◽  
Response Regulator ◽  
Critical Role ◽  
Primary Response ◽  
Cytokinin Oxidase ◽  
Wild Type ◽  
Shoot Architecture ◽  
In The Wild ◽  
F Box Protein

Strigolactones (SLs), a group of terpenoid lactones derived from carotenoids, are plant hormones that control numerous aspects of plant development. Although the framework of SL signaling that the repressor DWARF 53 (D53) could be SL-dependently degraded via the SL receptor D14 and F-box protein D3 has been established, the downstream response genes to SLs remain to be elucidated. Here we show that the cytokinin (CK) content is dramatically increased in shoot bases of the rice SL signaling mutantd53. By examining transcript levels of all the CK metabolism-related genes after treatment with SL analog GR24, we identifiedCYTOKININ OXIDASE/DEHYDROGENASE 9(OsCKX9) as a primary response gene significantly up-regulated within 1 h of treatment in the wild type but not ind53. We also found that OsCKX9 functions as a cytosolic and nuclear dual-localized CK catabolic enzyme, and that the overexpression ofOsCKX9suppresses the browning ofd53calli. Both the CRISPR/Cas9-generatedOsCKX9mutants andOsCKX9-overexpressing transgenic plants showed significant increases in tiller number and decreases in plant height and panicle size, suggesting that the homeostasis ofOsCKX9plays a critical role in regulating rice shoot architecture. Moreover, we identified the CK-inducible rice type-A response regulatorOsRR5as the secondary SL-responsive gene, whose expression is significantly repressed after 4 h of GR24 treatment in the wild type but not inosckx9. These findings reveal a comprehensive plant hormone cross-talk in which SL can induce the expression ofOsCKX9to down-regulate CK content, which in turn triggers the response of downstream genes.

scholarly journals Amyloid fibers provide structural integrity to Bacillus subtilis biofilms

2010 ◽  
Vol 107 (5) ◽  
pp. 2230-2234 ◽  
Author(s):  
Diego Romero ◽  
Claudio Aguilar ◽  
Richard Losick ◽  
Roberto Kolter
Keyword(s):  
Extracellular Matrix ◽  
Bacillus Subtilis ◽  
Biological Activity ◽  
Biofilm Formation ◽  
Structural Integrity ◽  
Wild Type ◽  
Amyloid Fibers ◽  
The Matrix ◽  
Biochemical Analyses ◽  
Main Components

Bacillus subtilis forms biofilms whose constituent cells are held together by an extracellular matrix. Previous studies have shown that the protein TasA and an exopolysaccharide are the main components of the matrix. Given the importance of TasA in biofilm formation, we characterized the physicochemical properties of this protein. We report that purified TasA forms fibers of variable length and 10–15 nm in width. Biochemical analyses, in combination with the use of specific dyes and microscopic analyses, indicate that TasA forms amyloid fibers. Consistent with this hypothesis, TasA fibers required harsh treatments (e.g., formic acid) to be depolymerized. When added to a culture of a tasA mutant, purified TasA restored wild-type biofilm morphology, indicating that the purified protein retained biological activity. We propose that TasA forms amyloid fibers that bind cells together in the biofilm.

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