drug and metabolite (ADaM)-binding pocket. Ligand binding in
the ADaM pocket occurs at the interface of the kinase and
glycogen-binding domain (GBD).
Crystallization of full-length human AMPKα 1 β 1 γ1 took over
a decade of iterative experimentation and was enabled by the close
collaboration of protein chemists, structural biologists, protein
biophysicists and biologists, as well as synthetic chemists and
computational biochemists. This approach employed standard
methods for the identification and removal of some disordered
regions from the protein by limited proteolysis and structure pre-
diction, mutation, optimization of construct design for expression
and folding, optimization of growth conditions for yield of soluble
protein and optimization of purification and protein storage, and
crystal screening. An unusual but critical key feature that enabled
crystallization of the full-length complex was the design of a suit-
able small molecule ligand, which had the properties of solubility
and affinity required for stabilization of the full-length complex.
Once the crystallization conditions were established, this enabled
structure-activity relationships of a diverse set of chemistries to be
explored with other small molecule ligands [5–7]. Recently crystal
structures have been reported for full-length AMPK without small
molecule activators in the ADaM site [8]; however, these are diffi-
cult to interpret in detail due to the lower resolution>4A ̊obtained
with this form of the protein.
In this way, direct knowledge of AMPK’s structure has con-
tributed to the development of new potential drug candidates. This
is further evidenced by the identification of a new AMPK activator
for treatment of lung cancer [9], which was based on the full-length
human AMPKα 1 β 1 γ1 structure (Protein Data Bank, PDB ID:
4CFE). This chapter aims to enable the reader to rapidly progress
to their own experimental biophysical and structural studies of full-
length human AMPKα 1 β 1 γ1 protein.2 Materials
2.1 Reagents
and Equipment
Prepare all solutions using ultrapure water (prepared by purifying
deionized water, to attain a sensitivity of 18 MΩcm at 25C) and
analytical grade reagents. Prepare and store all reagents at room
temperature (unless indicated otherwise). Diligently follow local
regulations when disposing waste materials.- Microbiological incubator.
- Basic spectrophotometer to measure cell culture optical
density. - NanoDrop spectrophotometer to measure protein
concentration.
2 Julia A. Hubbard et al.