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Architecture and Allostery of Full Length AMP-activated Protein Kinase by Single Particle Electron Microscopy

Author: ZhuLi
Tutor: WuYi
School: Lanzhou University
Course: Biophysical
Keywords: AMP-activated protein kinase Negative staining Three-dimensional reconstruction of electron microscopy single particle Oligomeric
CLC: Q51
Type: PhD thesis
Year: 2011
Downloads: 82
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Abstract

Mammalian AMP-activated protein kinase (AMPK) by α (two subtypes), β (two subtypes), γ (three subtypes) three subunits of heterotrimeric protein complex, can be sensitive to perceive the changes in intracellular AMP / ATP levels, plays a very important role in the maintenance of eukaryotic cells and organisms metabolism and energy homeostasis. Where α is the catalytic subunit, including the N-terminal serine / threonine kinase domain (KD), presumably having a self-suppression function AID domain and the C-terminus of the β subunit binding domain. β is a scaffolding subunit and the N-terminal myristoyl group can be mediated by AMPK and membrane interactions, the middle of the glycogen-binding domain (GBD), and the adoption of the C-terminal and the combination of the α and γ subunits, mediated by the whole The AMPK assembly. γ is a regulatory subunit, by four series CBS domain composed of two pairs of Bateman domain (s) have a small disk-shaped structure of central soluble channel, and contains three adenosine binding sites, two of which can be competitive binds ATP · Mg2 or AMP, to the third loci firmly combined with the part of non-commutative AMP. The structural integrity of the three subunits premise is the AMPK to play normal kinase activity. The AMPK may upstream protein kinase the LKB1-MO25α-STRADα or the CaMKKβ the α subunit activation loop conserved Thr172 sites of phosphorylation and activation, plus AMP and gamma subunits allosteric activation of the kinase activity increase 1000 times. Despite most of AMPK protein sequence fragment crystal structure resolved by X-ray crystallography technology, but because of lack of the complete structure of the complex information, so there are some problems not yet solved. First, the framework of the full-length AMPK three subunits is unknown; Secondly, adenosine and γ subunits combine to produce in the crystal structure a minor conformational change is transmitted to the α subunit and the regulation of its kinase activity remains unclear; also oligomeric forms, as well as the factors that mediate its oligomerization of the AMPK homologous oligomers exist and function have been reported in the literature, but still worthy of further investigation. In this thesis, we first reported murine full-length AMPK protein resolved by electron microscopy single particle three-dimensional reconstruction techniques (subtype composition α1β1γ1) homotrimer combined with the three-dimensional structure of the state in the ground state and the different adenosine to discuss the AMPK formed trimer oligomeric conditions and mediates its oligomeric factors. Punctured build AMPK kinase domain (KD) and glycogen binding domain (GBD) mutant, auxiliary fitting of the crystal structure, we have identified the molecular architecture of the full-length AMPK. After the comparison on the model of AMPK ATP and AMPK AMP model, we observed a series of conformational changes in the binding of adenosine and γ subunits of the kinase domain of the α subunit is inhibiting or activating the process. AMPK ATP model with AMPK yl state model comparison conclusions, AMPK No combination of 1,2 adenosine binding site, its conformation tends to be in the excited state of the allosteric. Finally, we give AMPK structure and regulation of the tri-state model. The findings of this paper is not only to make the current of AMPK assembly model with a larger correction, but also reveal the α subunit connection sequence (LS) in the perception of the gamma subunit ligand binding state and pass / zoom subunits long-range allosteric effectors The key and its possible mechanisms for further study of the AMPK subunits within each domain or subunit may exist between the interactions at the same time provides a certain idea.

Architecture and Allostery of Full Length AMP-activated Protein Kinase by Single Particle Electron Microscopy

Abstract

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