A role for the carbohydrate-binding module (CBM) in regulatory SnRK1 subunits: the effect of maltose on SnRK1 activity.

SnRK1 is a protein kinase complex that is involved in several aspects of plant growth and development. There are published data indicative of a participation of SnRK1 in the regulation of the synthesis and degradation of starch, although the molecular mechanism is not known. In this work, we performed electron microscopy to explore the in vivo localization of the regulatory and catalytic subunits that constitute the SnRK1 complex. The results indicated that all the subunits are present in the chloroplast and, in particular, the SnRK1 ßγ and SnRK1 ß3 subunits are associated with starch. Furthermore, the regulatory subunits bind maltose, a relevant product of starch degradation. The kinase activity of immunoprecipitated complexes containing the ßγ regulatory subunit was positively regulated by maltose only in the complexes obtained from Arabidopsis leaves collected at dusk. Recombinant complexes with the SnRK1α1 catalytic subunit, SnRK1ßγ and three different ß subunits showed that maltose only had an effect on a complex formed with the ß3 subunit. Truncation of the CBM domain form SnRK1 ßγ abolished the maltose activation of the complex and the activity was significantly reduced, indicating that the CBM is a positive regulator of SnRK1. A model of the SnRK1α1/ßγ/ß3 complex suggests the presence of two putative maltose-binding sites, both involving ligand interactions with the ßγ subunit and the α subunit.

Expression of recombinant SnRK1 in E. coli. Characterization of adenine nucleotide binding to the SnRK1.1/AKINßγ-ß3 complex.

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/AKINßγ-ß3 was modeled by homology modeling and Molecular Dynamics simulations (MD). The model predicted an intimate and extensive contact between a hydrophobic region of AKINßγ and the ß3 subunit. While the AKINßγ 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 AKINßγ-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.

Structural and functional basis for starch binding in the SnRK1 subunits AKINß2 and AKINßγ.

Specialized carbohydrate-binding domains, the Starch-Binding Domain (SBD) and the Glycogen Binding Domain (GBD), are motifs of approximately 100 amino acids directly or indirectly associated with starch or glycogen metabolism. Members of the regulatory ß subunit of the heterotrimeric complex AMPK/SNF1/SnRK1 contain an SBD or GBD. In Arabidopsis thaliana, the ß regulatory subunit AKINß2 and a γ-type subunit, AKINßγ, also have an SBD. In this work, we compared the SBD of AKINß2 and AKINßγ with the GBD present in rat AMPKß1 and demonstrated that they conserved the same overall topology. The majority of the amino acids identified in the protein-carbohydrate interactions in the rat AMPKß1 are conserved in the two plant proteins. In AKINßγ, there is an insertion of three amino acids that creates a loop adjacent to one of the conserved tryptophan residues. Functionally, the SBD from AKINßγ and AKINß2 could bind starch, but there was an important difference in the association when an amylose/amylopectin (A/A) mixture was used. The physiological relevance of binding to starch was clear for AKINßγ, because immunolocalization experiments identified this protein inside the chloroplast. SnRK1 activity was not affected by the addition of A/A to the reaction mixture. However, addition of starch inhibited the activity 85%. Furthermore, proteins associated with A/A and starch in an in vitro-binding assay accounted for 10-20% of total SnRK1 kinase activity. Interestingly, the identification of the SnRK1 subunits associated to the protein-carbohydrate complex indicated that only the catalytic subunits, AKIN10 and AKIN11, and the regulatory subunit AKINßγ were present. These results suggest that a dimer formed between either catalytic subunit and AKINßγ could be associated with the A/A mixture in its active form but the same subunits are inactivated when binding to starch.