Now showing 1 - 1 of 1
ItemElucidating the mechanism of myristoylated AMP-activated protein kinase by hydrogen deuterium exchange mass spectrometry (HDX-MS)Ali, Nada ( 2015)The heterotrimeric AMP-activated protein kinase (AMPK), consisting of α, β, and subunits, is a stress-sensing enzyme that plays a crucial role in regulating cellular energy metabolism. AMPK can differentially regulate catabolic and anabolic pathways in the cell and is therefore a target for Type 2 diabetes treatment. AMPK activity can be regulated (i) post-translationally via Thr 172 phosphorylation of the α-kinase domain by upstream kinases and/or via N-terminal myristoylation of the β-subunit, and (ii) allosterically by -subunit sensing of the AMP/ATP ratio. This implies a dynamic behaviour involving local and/or global conformational changes within and between subunits. Protein dynamics can occur over many timescales, from sub-picoseconds to seconds. Hydrogen-deuterium exchange (HDX) has a long history of being employed to investigate dynamics, as increases in exchange imply increased flexibility or reduce solvent access. Over the last two decades, mass spectrometry (MS) has become a dominant technique for measuring protein conformational changes, via HDX, due to its sensitivity, speed, and ability to work with large protein masses. HDX-MS is a new technique to our laboratory and institute, however, we successfully developed an MS system and pieced together a ‘home-built’ apparatus. After the success of this approach, we used HDX global analysis to investigate the structural and dynamic properties of truncated AMPK. The results showed that AMP and ATP.Mg2+ bound the γ-subunit and reduced its exchange rate, causing overall rigid body movement in the γ-subunit. However, nucleotide binding had no impact on the deuterium exchange level of truncated α-and β-subunits. Then we used HDX-MS to investigate the structural and dynamics properties of the mammalian myristoylated and non-myristoylated kinase-dead AMPK (D139→A) in the presence and absence of nucleotides. The global-analysis results showed increased deuterium exchange along the entire β- and γ-subunits of apo myr AMPK, suggesting more dynamic which was unexpected. HDX-MS local analysis data suggested that the myristoyl group binds near the first helix of the C-terminal lobe of the kinase domain similar to other myristoylated kinases. Our data, however, also showed that ATP.Mg2+ results in a global stabilization of myristoylated AMPK, and most notably for peptides of the β-CBM, the activation loop of the α-kinase domain and peptides of the AID. AMP did not have that effect such that HDX for myristoylated and non-myristoylated AMPK are similar. These differences in dynamics may account for a reduced basal rate of phosphorylation of Thr172 in myristoylated AMPK.