Structural and molecular basis of O-GlcNAcylation-phosphorylation crosstalk in protein Kinase-B (Akt2).

Abstract

Post Translational Modifications significantly influence conformational preferences and activity of proteins. In Protein Kinase-B (Akt2), numerous residues undergo phosphorylation and O-GlcNAcylation, in same region. This results in a crosstalk mechanism modulating Akt2 activity, and is dysregulated in metabolic diseases such as type-II diabetes. However, atomic level details of how such modifications influence Akt2 dynamics and activity is poorly understood. In this work, we employed in silico approaches to explore the conformational preferences and functional consequences of Akt2 in O-GlcNAcylated, phosphorylated and unmodified states. Multi-microsecond simulations, resulting in total sampling of 7.6μs were performed on Akt2 in all the above states, and the structural effects of such modifications were analyzed. We found that such diverse modifications result in considerable difference in Akt2 dynamics. In phosphorylated state, the activation loop of Akt2 is highly flexible and has high solvent accessibility, in contrast with the buried nature of O-GlcNAcylated Akt2. This involved contacts between the O-GlcNAc residues and the pleckstrein homology domain, resulting in its burial. In the phosphorylated state, an elaborate hydrogen bonded network was observed, where phosphorylated threonine residues and active site residues formed hydrogen bonds with the phosphatidylinositol (3,4,5)-trisphosphate (PIP3) binding residues, forming the structural basis of autoinhibition and phosphatase shielding by Akt2. We further sampled for first time, the conformational changes associated with the PIP3 induced Akt2 activation from the Akt2 simulations in presence of PIP3, ATP and Mg2+ cofactor. This work demonstrated the structural effects of varied post-translational modifications, which form a crosstalk mechanism regulating Akt2 activity.