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1.
Proc Natl Acad Sci U S A ; 118(44)2021 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-34716271

RESUMO

Plants and animals use cell surface receptors to sense and interpret environmental signals. In legume symbiosis with nitrogen-fixing bacteria, the specific recognition of bacterial lipochitooligosaccharide (LCO) signals by single-pass transmembrane receptor kinases determines compatibility. Here, we determine the structural basis for LCO perception from the crystal structures of two lysin motif receptor ectodomains and identify a hydrophobic patch in the binding site essential for LCO recognition and symbiotic function. We show that the receptor monitors the composition of the amphiphilic LCO molecules and uses kinetic proofreading to control receptor activation and signaling specificity. We demonstrate engineering of the LCO binding site to fine-tune ligand selectivity and correct binding kinetics required for activation of symbiotic signaling in plants. Finally, the hydrophobic patch is found to be a conserved structural signature in this class of LCO receptors across legumes that can be used for in silico predictions. Our results provide insights into the mechanism of cell-surface receptor activation by kinetic proofreading of ligands and highlight the potential in receptor engineering to capture benefits in plant-microbe interactions.


Assuntos
Fabaceae/genética , Lipopolissacarídeos/metabolismo , Simbiose/fisiologia , Fabaceae/metabolismo , Expressão Gênica/genética , Regulação da Expressão Gênica de Plantas/genética , Cinética , Lipopolissacarídeos/genética , Micorrizas/fisiologia , Proteínas de Plantas/genética , Plantas/metabolismo , Rhizobium/fisiologia , Transdução de Sinais , Simbiose/genética
2.
Nat Commun ; 9(1): 3307, 2018 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-30120230

RESUMO

Methods for site-selective chemistry on proteins are in high demand for the synthesis of chemically modified biopharmaceuticals, as well as for applications in chemical biology, biosensors and more. Inadvertent N-terminal gluconoylation has been reported during expression of proteins with an N-terminal His tag. Here we report the development of this side-reaction into a general method for highly selective N-terminal acylation of proteins to introduce functional groups. We identify an optimized N-terminal sequence, GHHHn- for the reaction with gluconolactone and 4-methoxyphenyl esters as acylating agents, facilitating the introduction of functionalities in a highly selective and efficient manner. Azides, biotin or a fluorophore are introduced at the N-termini of four unrelated proteins by effective and selective acylation with the 4-methoxyphenyl esters. This Gly-Hisn tag adds the unique capability for highly selective N-terminal chemical acylation of expressed proteins. We anticipate that it can find wide application in chemical biology and for biopharmaceuticals.


Assuntos
Dipeptídeos/metabolismo , Peptídeos/metabolismo , Proteínas/metabolismo , Acilação , Sequência de Aminoácidos , Azidas/química , Biotina/metabolismo , Ésteres/metabolismo , Corantes Fluorescentes/química , Gluconatos/metabolismo , Lactonas/metabolismo , Peptídeos/química , Polietilenoglicóis/química , Processamento de Proteína Pós-Traducional
4.
Bioconjug Chem ; 29(4): 1219-1230, 2018 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-29437382

RESUMO

The reaction of unprotected carbohydrates with aminooxy reagents to provide oximes is a key method for the construction of glycoconjugates. Aniline and derivatives serve as organocatalysts for the formation of oximes from simple aldehydes, and we have previously reported that aniline also catalyzes the formation of oximes from the more complex aldehydes, carbohydrates. Here, we present a comprehensive study of the effect of aniline analogues on the formation of carbohydrate oximes and related glycoconjugates depending on organocatalyst structure, pH, nucleophile, and carbohydrate, covering more than 150 different reaction conditions. The observed superiority of the 1,4-diaminobenzene (PDA) catalyst at neutral pH is rationalized by NMR analyses and DFT studies of reaction intermediates. Carbohydrate oxime formation at pH 7 is demonstrated by the formation of a bioactive glycoconjugate from a labile, decorated octasaccharide originating from exopolysaccharides of the soil bacterium Mesorhizobium loti. This study of glycoconjugate formation includes the first direct comparison of aniline-catalyzed reaction rates and equilibrium constants for different classes of nucleophiles, including primary oxyamines, secondary N-alkyl oxyamines, as well as aryl and arylsulfonyl hydrazides. We identified 1,4-diaminobenzene as a superior catalyst for the construction of oxime-linked glycoconjugates under mild conditions.


Assuntos
Glicoconjugados/química , Oximas/química , Fenilenodiaminas/química , Catálise , Glicoconjugados/síntese química , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Mesorhizobium/química , Oximas/síntese química , Fenilenodiaminas/síntese química , Polissacarídeos Bacterianos/síntese química , Polissacarídeos Bacterianos/química
5.
Nat Protoc ; 12(11): 2411-2422, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29072708

RESUMO

Glycobiology, in particular the study of carbohydrate-protein interactions and the events that follow, has become an important research focus in recent decades. To study these interactions, many assays require homogeneous glycoconjugates in suitable amounts. Their synthesis is one of the methodological challenges of glycobiology. Here, we describe a versatile, three-stage protocol for the formation of glycoconjugates from unprotected carbohydrates, including those purified from natural sources, as exemplified here by rhizobial Nod factors and exopolysaccharide fragments. The first stage is to add an oligo(ethylene glycol) linker (OEG-linker) that has a terminal triphenylmethanethiol group to the reducing end of the oligosaccharide by oxime formation catalyzed by aniline. The triphenylmethyl (trityl) tag is then removed from the linker to expose a thiol (stage 2) to allow a conjugation reaction at the thiol group (stage 3). There are many possible conjugation reactions, depending on the desired application. Examples shown in this protocol are as follows: (i) coupling of the oligosaccharide to a support for surface plasmon resonance (SPR) studies, (ii) fluorescence labeling for microscale thermophoresis (MST) or bioimaging, and (iii) biotinylation for biolayer interferometry (BLI) studies. This protocol starts from unprotected carbohydrates and provides glycoconjugates in milligram amounts in just 2 d.


Assuntos
Técnicas de Química Sintética , Glicoconjugados/síntese química , Glicômica/métodos , Lipopolissacarídeos/química , Compostos de Sulfidrila/química , Compostos de Tritil/química , Compostos de Anilina/química , Biotinilação , Catálise , Interferometria , Imagem Óptica , Oximas/química , Polietilenoglicóis/química , Ligação Proteica , Ressonância de Plasmônio de Superfície
6.
J Biol Chem ; 291(40): 20946-20961, 2016 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-27502279

RESUMO

In the symbiosis formed between Mesorhizobium loti strain R7A and Lotus japonicus Gifu, rhizobial exopolysaccharide (EPS) plays an important role in infection thread formation. Mutants of strain R7A affected in early exopolysaccharide biosynthetic steps form nitrogen-fixing nodules on L. japonicus Gifu after a delay, whereas mutants affected in mid or late biosynthetic steps induce uninfected nodule primordia. Recently, it was shown that a plant receptor-like kinase, EPR3, binds low molecular mass exopolysaccharide from strain R7A to regulate bacterial passage through the plant's epidermal cell layer (Kawaharada, Y., Kelly, S., Nielsen, M. W., Hjuler, C. T., Gysel, K., Muszynski, A., Carlson, R. W., Thygesen, M. B., Sandal, N., Asmussen, M. H., Vinther, M., Andersen, S. U., Krusell, L., Thirup, S., Jensen, K. J., et al. (2015) Nature 523, 308-312). In this work, we define the structure of both high and low molecular mass exopolysaccharide from R7A. The low molecular mass exopolysaccharide produced by R7A is a monomer unit of the acetylated octasaccharide with the structure (2,3/3-OAc)ß-d-RibfA-(1→4)-α-d-GlcpA-(1→4)-ß-d-Glcp-(1→6)-(3OAc)ß-d-Glcp-(1→6)-*[(2OAc)ß-d-Glcp-(1→4)-(2/3OAc)ß-d-Glcp-(1→4)-ß-d-Glcp-(1→3)-ß-d-Galp]. We propose it is a biosynthetic constituent of high molecular mass EPS polymer. Every new repeating unit is attached via its reducing-end ß-d-Galp to C-4 of the fourth glucose (asterisked above) of the octasaccharide, forming a branch. The O-acetylation occurs on the four glycosyl residues in a non-stoichiometric ratio, and each octasaccharide subunit is on average substituted with three O-acetyl groups. The availability of these structures will facilitate studies of EPR3 receptor binding of symbiotically compatible and incompatible EPS and the positive or negative consequences on infection by the M. loti exo mutants synthesizing such EPS variants.


Assuntos
Lotus/metabolismo , Mesorhizobium/metabolismo , Mutação , Epiderme Vegetal/metabolismo , Polissacarídeos Bacterianos/metabolismo , Simbiose/fisiologia , Configuração de Carboidratos , Lotus/genética , Lotus/microbiologia , Mesorhizobium/genética , Epiderme Vegetal/genética , Epiderme Vegetal/microbiologia , Polissacarídeos Bacterianos/genética
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