Abstract
Mammalian AMP-activated protein kinase (AMPK) acts as an important sensor of cellular energy homeostasis related with AMP/ADP to ATP ratio. The overall architecture of AMPK has been determined in either homotrimer or monomer form by electron microscopy (EM) and X-ray crystallography successively. Accordingly proposed models have consistently revealed a key role of the α subunit linker in sensing adenosine nucleoside binding on the γ subunit and mediating allosteric regulation of kinase domain (KD) activity, whereas there are vital differences in orienting N-terminus of α subunit and locating carbohydrate-binding module (CBM) of β subunit. Given that Mg2+, an indispensable cofactor of AMPK was present in the EM sample preparation buffer however absent when forming crystals, here we carried out further reconstructions without Mg2+ to expectably inspect if this ion may contribute to this difference. However, no essential alteration has been found in this study compared to our early work. Further analyses indicate that the intra-molecular movement of the KD and CBM are most likely due to the flexible linkage of the disordered linkers with the rest portion as well as a contribution from the plasticity in the inter-molecular assembly mode, which might ulteriorly reveal an architectural complication of AMPK.
Highlights
AMP-activated protein kinase (AMPK), an α β γ heterotrimeric complex, plays an essential role in regulating cellular metabolism and maintaining energy homeostasis by sensing AMP/ADP to ATP ratio in mammals[1,2,3]
In spite of showing consistent characteristics such as kinase domain (KD)-autoinhibitory domain (AID) is adjacent to the scaffold region not the regulation core complex, AID is located behind KD hinge to inhibit its activity until α -Thr[172] is phosphorylated, architectural analyses based on the structures generated from electron microscopy (EM) and X-ray crystallography have revealed its structural flexibility
AMPK appeared as multidispersed particles in solution, with monomers coexisting with certain proportional homo-oligomers[20,28,29]
Summary
AMP-activated protein kinase (AMPK), an α β γ heterotrimeric complex, plays an essential role in regulating cellular metabolism and maintaining energy homeostasis by sensing AMP/ADP to ATP ratio in mammals[1,2,3]. In the past five years, several groups have reported the overall architecture of mammalian AMPK in either homotrimer or monomer form by electron microscopy (EM) and X-ray crystallography successively Those structures were determined in varied functional preparations, primarily focusing on the threonine 172 (rat α 1, human α 2) within the activation loop of KD, whose phosphorylation by the upstream kinases[14,15,16,17,18,19] defines the prerequisite of an active enzyme, the CBM and its activators, and binding of AMP/ADP to the CBS-3 site and CBS-4 site of γ subunit for an allosteric modulation or protecting α -Thr[172] against dephosphorylation[5,9,20,21]. Further analyses have revealed the disordered linker sequences together with the intra- and inter-molecular interactions may contribute to a certain plasticity of AMPK architecture, which in turn demonstrates an architectural complication of this enzyme complex
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