Structural and functional basis for starch binding in the SnRK1 subunits AKINβ2 and AKINβγ

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 beta subunit of the heterotrimeric complex AMPK/SNF1/SnRK1 contain an SBD or GBD. In Arabidopsis thaliana, the beta regulatory subunit AKIN beta 2 and a gamma-type subunit, AKIN beta gamma, also have an SBD. In this work, we compared the SBD of AKIN beta 2 and AKIN beta gamma with the GBD present in rat AMPK beta 1 and demonstrated that they conserved the same overall topology. The majority of the amino acids identified in the protein-carbohydrate interactions in the rat AMPK beta 1 are conserved in the two plant proteins. In AKIN beta gamma, there is an insertion of three amino acids that creates a loop adjacent to one of the conserved tryptophan residues. Functionally, the SBD from AKIN beta gamma and AKIN beta 2 could bind starch, but there was an important difference in the association when an amylose/amylopectin (NA) mixture was used. The physiological relevance of binding to starch was clear for AKIN beta gamma, 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, AKIN 10 and AKIN11, and the regulatory subunit AKIN beta gamma were present. These results suggest that a dimer formed between either catalytic subunit and AKIN beta gamma could be associated with the A/A mixture in its active form but the same subunits are inactivated when binding to starch.