AMPK Methods and Protocols

Chapter 1

Production and Crystallization of Full-Length Human

AMP-Activated Protein Kinase (α 1 β 1 γ1)

Julia A. Hubbard, Bing Xiao, and Jon R. Wilson

Abstract

Determination of the crystal structure of AMP-activated protein kinase (AMPK) is fundamental to under-
standing its biological function and role in a number of diseases related to energy metabolism including type
2 diabetes, obesity, and cancer. We describe methods for the expression and purification of a human full-
length active AMPK complex that is suitable for biochemical and structural analyses, followed by methods
for its crystallization in complex with small molecule activators. Quality control of the purified protein by
functional and biophysical analysis was an essential part of the process enabling the achievement of crystals
of the full-length protein capable of being used for high-resolution structure determination by X-ray
diffraction. X-ray structures have been determined of both phosphorylated and non-phosphorylated
forms of full-length human AMPKα 1 β 1 γ1.

Key wordsAMPK, Crystallization, ADaM activator, Nucleotide binding, Kinase, Protein purification

1 Introduction

AMP-activated protein kinase (AMPK) has a key role in regulating

metabolism and is therefore an important therapeutic target for

metabolic disorders. Initial structural studies of AMPK, which

focused on the structure of the regulatory sub-complex of AMPK

(core domain: C-terminal domain ofα-subunit, C-terminal domain

ofβ-subunit, full-lengthγ-subunit) [1], facilitated an understand-

ing of its activation by AMP, ADP, and ATP [2]. Detailed protocols

for the crystallization of the core domain of AMPK can be obtained

from these publications [1, 2] and from the thesis of Underwood,

E[3].

Here we describe the purification and crystallization of the

active full-length human AMPK (isoformα 1 β 1 γ1), which extended

the work on the core domain and allowed for the first time an

understanding of the protein’s activation by potential drug mole-

cules [4], the surprising feature of which was the identification and

characterization of the binding site now referred to as the allosteric

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_1,©Springer Science+Business Media, LLC 2018

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