AMPK Methods and Protocols

Chapter 2

Visualizing AMPK Drug Binding Sites Through

Crystallization of Full-Length Phosphorylatedα 2 β 1 γ 1

Heterotrimer

Christopher G. Langendorf, Jonathan S. Oakhill, and Bruce E. Kemp

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 A ̊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 theγ-subunit via co-crystallization with phosphorylated AMPK (pT172)α 2 β 1 γ1 isoform orα2/
1 β 1 γ1 chimera. Drugs with binding affinities above 500 nM fail to co-crystallize with AMPK using these
parameters.

Key wordsAMPK heterotrimer, Protein crystallization, Allosteric activation, Recombinant protein,

Expression and purification

1 Introduction

AMP-activated protein kinase (AMPK) has become an attractive

drug target for therapeutics aimed at type 2 diabetes, obesity,

nonalcoholic fatty liver disease (NAFLD), cardiovascular disease,

and some cancers. The AMPK complex is anαβγheterotrimer

containing a catalyticα-subunit and regulatoryβ- andγ-subunits.

Multiple isoforms of each subunit exist in mammals (α1/2,β1/2,

γ1/2/3) with each displaying cell/tissue-specific variation in

expression. The potential importance of AMPK in metabolic dis-

ease treatment is well illustrated by the discovery that the glucose-

lowering drug metformin, used to treat over 120 million people

globally with type 2 diabetes, is an indirect activator of AMPK [1].

The first direct allosteric activator of AMPK, A-769662, was

identified by Cool and colleagues in 2006 [2]. The discovery of a

direct allosteric activator increased the urgency for AMPK struc-

tural information, which at this stage was limited to an isolated

Dietbert Neumann and Benoit Viollet (eds.),AMPK: Methods and Protocols, Methods in Molecular Biology, vol. 1732,
https://doi.org/10.1007/978-1-4939-7598-3_2,©Springer Science+Business Media, LLC 2018

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