AMPK Methods and Protocols

Chapter 3

Biophysical Interactions of Direct AMPK Activators

Ravi G. Kurumbail, Graham M. West, Venkatasubramanian Dharmarajan,

Kris A. Borzilleri, Jane M. Withka, Jessica Ward, Allan R. Reyes,

Francis Rajamohan, Patrick R. Griffin, and Matthew F. Calabrese

Abstract

Protein-ligand interactions can be evaluated by a number of different biophysical methods. Here we
describe some of the experimental methods that we have used to generate AMPK protein reagents and
characterize its interactions with direct synthetic activators. Recombinant heterotrimeric AMPK complexes
were generated using standard molecular biology methods by expression either in insect cells via infection
with three different viruses or more routinely inEscherichia coliwith a tricistronic expression vector.
Hydrogen/deuterium exchange (HDX) coupled with mass spectrometry was used to probe protein
conformational changes and potential binding sites of activators on AMPK. X-ray crystallographic studies
were carried out on crystals of AMPK with bound ligands to reveal detailed molecular interactions formed
by AMPK activators at near-atomic resolution. In order to gain insights into the mechanism of enzyme
activation and to probe the effects of AMPK activators on kinetic parameters such as Michaelis-Menten
constant (Km) or maximal reaction velocity (Vmax), we performed classical enzyme kinetic studies using
radioactive^33 P–ATP-based filter assay. Equilibrium dissociation constants (KD) and on and off rates of
ligand binding were obtained by application of surface plasmon resonance (SPR) technique.

Key wordsX-ray, Crystallography, HDX, Hydrogen/deuterium exchange, SPR, Enzymology, Kinet-

ics, AMPK, Activation, Baculovirus, Tricistronic

1 Introduction

Molecular interactions of small molecules with proteins define the

fundamental processes involved in recognition and discrimination

of substrates, cofactors, and products in endogenous systems. It is

often the ligand-induced conformational changes that are respon-

sible for modulation of functional activity of proteins. Quantifying

and carefully evaluating these biophysical interactions are critical for

the discovery and development of novel therapeutics. Methods

based on NMR, X-ray crystallography, cryo-electron microscopy,

and mass spectrometry have evolved over the years to characterize

binding sites and interactions between protein and ligands.

Dietbert Neumann and Benoit Viollet (eds.),AMPK:MethodsandProtocols, Methods in Molecular Biology, vol. 1732,
https://doi.org/10.1007/978-1-4939-7598-3_3,©Springer Science+Business Media, LLC 2018

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