Expression of recombinant SnRK1 in E. coli. Characterization of adenine nucleotide binding to the SnRK1.1/AKINß?-ß3 complex
Por:
Maya-Bernal J.L., Ávila A., Ruiz-Gayosso A., Trejo-Fregoso R., Pulido N., Sosa-Peinado A., Zúñiga-Sánchez E., Martínez-Barajas E., Rodríguez-Sotres R., Coello P.
Publicada:
1 oct 2017
Resumen:
The SnRK1 complexes in plants belong to the family of AMPK/SNF1 kinases, which have been associated with the control of energy balance, in addition to being involved in the regulation of other aspects of plant growth and development. Analysis of complex formation indicates that increased activity is achieved when the catalytic subunit is phosphorylated and bound to regulatory subunits. SnRK1.1 subunit activity is higher than that of SnRK1.2, which also exhibits reduced activation due to the regulatory subunits. The catalytic phosphomimetic subunits (T175/176D) do not exhibit high activity levels, which indicate that the amino acid change does not produce the same effect as phosphorylation. Based on the mammalian AMPK X-ray structure, the plant SnRK1.1/AKINß?-ß3 was modeled by homology modeling and Molecular Dynamics simulations (MD). The model predicted an intimate and extensive contact between a hydrophobic region of AKINß? and the ß3 subunit. While the AKINß? prediction retains the 4 CBS domain organization of the mammalian enzyme, significant differences are found in the putative nucleotide binding pockets. Docking and MD studies identified two sites between CBS 3 and 4 which may bind adenine nucleotides, but only one appears to be functional, as judging from the predicted binding energies. The recombinant AKINß?-ßs complexes were found to bind adenine nucleotides with dissociation constant (Kd) in the range of the AMP low affinity site in AMPK. The saturation binding data was consistent with a one-site model, in agreement with the in silico calculations. As has been suggested previously, the effect of AMP was found to slow down dephosphorylation but did not influence activity. © 2017 Elsevier B.V.
Filiaciones:
Maya-Bernal J.L.:
Departamento de Bioquímica, Facultad de Química, UNAM, Ciudad de México, 04510, Mexico
Ávila A.:
Departamento de Bioquímica, Facultad de Química, UNAM, Ciudad de México, 04510, Mexico
Ruiz-Gayosso A.:
Departamento de Bioquímica, Facultad de Química, UNAM, Ciudad de México, 04510, Mexico
Trejo-Fregoso R.:
Departamento de Bioquímica, Facultad de Química, UNAM, Ciudad de México, 04510, Mexico
Pulido N.:
Centro de Investigaciones Químicas, UAEMMorelos 62210, Mexico
Sosa-Peinado A.:
Departamento de Bioquímica, Facultad de Medicina, UNAM, Ciudad de México, 04510, Mexico
Zúñiga-Sánchez E.:
Departamento de Bioquímica, Facultad de Química, UNAM, Ciudad de México, 04510, Mexico
Martínez-Barajas E.:
Departamento de Bioquímica, Facultad de Química, UNAM, Ciudad de México, 04510, Mexico
Rodríguez-Sotres R.:
Departamento de Bioquímica, Facultad de Química, UNAM, Ciudad de México, 04510, Mexico
Coello P.:
Departamento de Bioquímica, Facultad de Química, UNAM, Ciudad de México, 04510, Mexico
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