Structural and functional studies on nucleotide binding to
AMP-activated protein kinase.
Doctoral thesis, UCL (University College London).
AMP-activated protein kinase (AMPK) is an enzyme that senses and regulates cellular energy balance thus playing a key role in homeostasis. As such it is a target for treatment of metabolic disorders such as type II diabetes. AMPK is a hetero-trimeric complex composed of an α, β and γ subunit. α contains the catalytic kinase domain, β is a scaffolding subunit that enables complex formation and γ monitors cellular energy via nucleotide binding to its CBS domains. AMPK is primarily activated by phosphorylation at Thr-172 on the activation loop in the kinase domain. It exerts its cellular effects via phosphorylation of a range of downstream targets involved in different aspects of energy production & utilization. The aim of this thesis is to characterize the mechanistic basis of energy regulation of mammalian AMPK via structural and binding measurements. Fluorescence studies have been facilitated by the use of N-methylanthraniloyl (mant) labelled AMP and of β-Nicotinamide adenine dinucleotide 2’-phosphate (NADPH) to monitor competition with AMP, ADP and ATP. A number of mutations in the γ subunit, which interfere with the normal function of AMPK and cause Wolff-Parkinson- White (WPW) syndrome, have also assessed for changes in nucleotide binding affinities and potential implications for the regulation of kinase activity. Thermal denaturation experiments are used to investigate the stabilizing effects of nucleotides and other small molecule ligands. This method was used in a low throughput screen against an enriched list of compounds selected from an in silico screen to try to identify novel activators. I have also determined the structure of the regulatory fragment of the enzyme bound to 5-aminoimidazole-4-carboximide riboside monophosphate (ZMP), an intermediate on the biosynthetic route to AMP, the fluorescence reporter mant-AMP and the WPW mutants Arg298\rightarrowGly, Arg69\rightarrowGln and His150\rightarrowArg. The structures of the mutants have revealed that nucleotide binding is impaired due to a reduced affinity for the nucleotides thus affecting the regulation of the kinase.
|Title:||Structural and functional studies on nucleotide binding to AMP-activated protein kinase|
|Open access status:||An open access version is available from UCL Discovery|
|Additional information:||The project was fully funded by the MRC and carried out at the National Institute for Medical Research|
|UCL classification:||UCL > School of Life and Medical Sciences > Faculty of Life Sciences > Biosciences (Division of) > Structural and Molecular Biology|
Archive Staff Only