Expression of recombinant SnRK1 in E. coli. Characterization of adenine nucleotide binding to the SnRK1.1/AKINβγ-β3 complex

- SnRK1.1 and SnRK1.2 subunits are active after being phosphorylated by SnAK2 and their activity increased by the union of the regulatory subunits.
- SnRK1.1 and SnRK1.2 phosphomimetic subunits are not active.
- The model for the plant complex SnRK1.1/AKINβγ-β3 predicts contacts between the catalytic subunit and the dimer AKINβγ-β3.
- In silico docking events and Molecular Dynamic simulations identified one site for AMP binding.
- Experimental approaches using the AKINβγ-β3 dimer confirmed the binding of adenine nucleotides.

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/AKINβγ-β3 was modeled by homology modeling and Molecular Dynamics simulations (MD). The model predicted an intimate and extensive contact between a hydrophobic region of AKINβγ and the β3 subunit. While the AKINβγ 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 AKINβγ-βs 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.

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