The heterotrimeric AMP-activated protein kinase (AMPK), consisting of α, β and γ subunits, is a stress-sensing enzyme that plays a crucial role for regulating cellular energy metabolism. AMPK can differentially regulate essential metabolic pathways in the cell and is therefore a target for type II diabetes treatment. The activation of AMPK switches on catabolic and represses anabolic pathways. AMPK activity can be regulated post-translationally via phosphorylation of Thr 172 α-kinase domain by upstream kinases and allosterically by γ-subunit sensing of AMP/ATP ratio. Recently, it has been reported that myristoylation of the β-subunit enhances AMPK to reach maximal activity in response to AMP signals. The myrisoyl group enhances AMP to allosterically activate the enzyme and to trigger Thr 172 phosphorylation by upstream kinases. This implies a dynamic behaviour involving local and/or global conformational changes within and between subunits. Notably, the crystal structure of AMPK that represents the N-terminal region of the β subunit, that would include the myristoyl group has not been solved and therefore where the myristoyl group binds is not known. It has been suggested that the binding pocket could be located in the α-catalytic kinase domain in a similar position as shown by the crystal structure of protein kinase A (PKA).
The aim of this project is to study the conformational effects of myristoylation on AMPK structure and to locate where the myristoyl group binds. In addition myristoylation-dependent differences in the peptide residues known to have significant roles in interactions with nucleotides is investigated. Applying hydrogen deuterium exchange mass spectrometry (HDX-MS) provides fundamental information about conformational change of AMPK in nonmyristoylation, myristoylation, or nucleotide-bound forms and the degree of localization of these differences. Results from this study revealed that myristoylation destabilizes the β subunit. The nucleotide allosteric effects on myristoylated protein showed conformational changes primarily in the AMPK γ subunit and subtle changes in the α and β subunits.