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Visualizing AMPK drug binding sites through crystallization of full-length phosphorylated α2β1γ1 heterotrimer
Langendorf, Christopher G. Oakhill, Jonathan S. Kemp, Bruce E.
Langendorf, Christopher G.
Oakhill, Jonathan S.
Kemp, Bruce E.
Abstract
Here, we describe the crystallization protocol for AMPK, including protein production and purification. AMPK can be readily crystallized in the presence of PEG to give diffracting crystals to a resolution of between 2.5 and 3.5 Å using synchrotron radiation. This method allows for visualization of drugs or small molecules that bind to the ADaM site, CBS sites, ATP binding site, and the newly identified C2 binding sites in the γ-subunit via co-crystallization with phosphorylated AMPK (pT172) α2β1γ1 isoform or α2/1β1γ1 chimera. Drugs with binding affinities above 500 nM fail to co-crystallize with AMPK using these parameters.
Keywords
AMPK heterotrimer, protein crystallization, allosteric activation, recombinant protein expression and purification
Date
2018
Type
Book chapter
Journal
Book
AMPK: Methods and protocol
Volume
Issue
Page Range
15-27
Article Number
ACU Department
Faculty of Health Sciences
Collections
Relation URI
Source URL
Event URL
Open Access Status
License
File Access
Controlled