RESUMEN
The plant glucan phosphatases Starch EXcess 4 (SEX4) and Like Sex Four2 (LSF2) apply different starch binding mechanisms. SEX4 contains a carbohydrate binding module, and LSF2 has two surface binding sites (SBSs). We determined KDapp for amylopectin and amylose, and KD for ß-cyclodextrin and validated binding site mutants deploying affinity gel electrophoresis (AGE) and surface plasmon resonance. SEX4 has a higher affinity for amylopectin; LSF2 prefers amylose and ß-cyclodextrin. SEX4 has 50-fold lower KDapp for amylopectin compared to LSF2. Molecular dynamics simulations and AGE data both support long-distance mutual effects of binding at SBSs and the active site in LSF2.
Asunto(s)
Amilopectina/metabolismo , Amilosa/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Fosfatasas de Especificidad Dual/metabolismo , Modelos Moleculares , Hojas de la Planta/enzimología , Sustitución de Aminoácidos , Amilopectina/química , Amilosa/química , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Sitios de Unión , Conformación de Carbohidratos , Gránulos Citoplasmáticos/química , Gránulos Citoplasmáticos/enzimología , Gránulos Citoplasmáticos/metabolismo , Fosfatasas de Especificidad Dual/química , Fosfatasas de Especificidad Dual/genética , Cinética , Simulación de Dinámica Molecular , Mutación , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Resonancia por Plasmón de Superficie , beta-Ciclodextrinas/química , beta-Ciclodextrinas/metabolismoRESUMEN
Glucan phosphatases are central to the regulation of starch and glycogen metabolism. Plants contain two known glucan phosphatases, Starch EXcess4 (SEX4) and Like Sex Four2 (LSF2), which dephosphorylate starch. Starch is water-insoluble and reversible phosphorylation solubilizes its outer surface allowing processive degradation. Vertebrates contain a single known glucan phosphatase, laforin, that dephosphorylates glycogen. In the absence of laforin, water-soluble glycogen becomes insoluble, leading to the neurodegenerative disorder Lafora Disease. Because of their essential role in starch and glycogen metabolism glucan phosphatases are of significant interest, yet a comparative analysis of their activities against diverse glucan substrates has not been established. We identify active site residues required for specific glucan dephosphorylation, defining a glucan phosphatase signature motif (CζAGΨGR) in the active site loop. We further explore the basis for phosphate position-specific activity of these enzymes and determine that their diverse phosphate position-specific activity is governed by the phosphatase domain. In addition, we find key differences in glucan phosphatase activity toward soluble and insoluble polyglucan substrates, resulting from the participation of ancillary glucan-binding domains. Together, these data provide fundamental insights into the specific activity of glucan phosphatases against diverse polyglucan substrates.
Asunto(s)
Proteínas de Arabidopsis/química , Arabidopsis/enzimología , Fosfatasas de Especificidad Dual/química , Glucógeno/química , Almidón/química , Secuencias de Aminoácidos , Humanos , Estructura Terciaria de Proteína , Proteínas Tirosina Fosfatasas no Receptoras/químicaRESUMEN
Glycogen is the major mammalian glucose storage cache and is critical for energy homeostasis. Glycogen synthesis in neurons must be tightly controlled due to neuronal sensitivity to perturbations in glycogen metabolism. Lafora disease (LD) is a fatal, congenital, neurodegenerative epilepsy. Mutations in the gene encoding the glycogen phosphatase laforin result in hyperphosphorylated glycogen that forms water-insoluble inclusions called Lafora bodies (LBs). LBs induce neuronal apoptosis and are the causative agent of LD. The mechanism of glycogen dephosphorylation by laforin and dysfunction in LD is unknown. We report the crystal structure of laforin bound to phosphoglucan product, revealing its unique integrated tertiary and quaternary structure. Structure-guided mutagenesis combined with biophysical and biochemical analyses reveal the basis for normal function of laforin in glycogen metabolism. Analyses of LD patient mutations define the mechanism by which subsets of mutations disrupt laforin function. These data provide fundamental insights connecting glycogen metabolism to neurodegenerative disease.