Activation of AMP-activated protein kinase revealed by hydrogen/deuterium exchange mass spectrometry.
AMP-activated protein kinase (AMPK) monitors cellular energy, regulates genes involved in ATP synthesis and consumption, and is allosterically activated by nucleotides and synthetic ligands. Analysis of the intact enzyme with hydrogen/deuterium exchange mass spectrometry reveals conformational perturbations of AMPK in response to binding of nucleotides, cyclodextrin, and a synthetic small molecule activator, A769662. Results from this analysis clearly show that binding of AMP leads to conformational changes primarily in the γ subunit of AMPK and subtle changes in the α and β subunits. In contrast, A769662 causes profound conformational changes in the glycogen binding module of the β subunit and in the kinase domain of the α subunit, suggesting that the molecular binding site of the latter resides between the α and β subunits. The distinct short- and long-range perturbations induced upon binding of AMP and A769662 suggest fundamentally different molecular mechanisms for activation of AMPK by these two ligands.
Department of Molecular Therapeutics, The Scripps Research Institute, Scripps Florida, Jupiter, FL 33458, USA.
Rachelle R
RR
Landgraf
Devrishi
D
Goswami
Francis
F
Rajamohan
Melissa S
MS
Harris
Matthew F
MF
Calabrese
Lise R
LR
Hoth
Rachelle
R
Magyar
Bruce D
BD
Pascal
Michael J
MJ
Chalmers
Scott A
SA
Busby
Ravi G
RG
Kurumbail
Patrick R
PR
Griffin
D055372:AMP-Activated Protein Kinases / Q000737:chemistry*; D000494:Allosteric Regulation; D001713:Biphenyl Compounds; D020134:Catalytic Domain; D041961:Deuterium Exchange Measurement; D004789:Enzyme Activation; D020536:Enzyme Activators / Q000737:chemistry; D006801:Humans; D008958:Models, Molecular; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011753:Pyrones / Q000737:chemistry; D013876:Thiophenes / Q000737:chemistry
D001713:Biphenyl Compounds; D020536:Enzyme Activators; D011753:Pyrones; D013876:Thiophenes; D055372:AMP-Activated Protein Kinases; C512408:4-hydroxy-3-(4-(2-hydroxyphenyl)phenyl)-6-oxo-7H-thieno(2,3-b)pyridine-5-carbonitrile