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Visualizing AMPK Drug Binding Sites Through Crystallization of Full-Length Phosphorylated α2β1γ1 Heterotrimer.

Authors
  • Langendorf, Christopher G1
  • Oakhill, Jonathan S2, 3
  • Kemp, Bruce E4, 3
  • 1 Protein Chemistry & Metabolism Unit, St. Vincent's Institute of Medical Research, Fitzroy, VIC, Australia. [email protected] , (Australia)
  • 2 Metabolic Signalling Laboratory, St. Vincent's Institute of Medical Research, Fitzroy, VIC, Australia. , (Australia)
  • 3 Mary MacKillop Institute for Health Research, Australian Catholic University, Melbourne, VIC, Australia. , (Australia)
  • 4 Protein Chemistry & Metabolism Unit, St. Vincent's Institute of Medical Research, Fitzroy, VIC, Australia. , (Australia)
Type
Published Article
Journal
Methods in molecular biology (Clifton, N.J.)
Publication Date
Jan 01, 2018
Volume
1732
Pages
15–27
Identifiers
DOI: 10.1007/978-1-4939-7598-3_2
PMID: 29480466
Source
Medline
Keywords
Language
English
License
Unknown

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.

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