AMPK Methods and Protocols

laboratories have utilized tricistronic expression vectors to produce

recombinant enzyme for full-length AMPK crystallization [6, 7, 11].

Full-length active AMPK is purified via a two-step method

involving NiNTA IMAC affinity resin and size-exclusion chroma-

tography. CaMKK2 purified from insect cells is then used to acti-

vate AMPK by phosphorylation of Thr172 in the activation loop.

This is one of the most critical steps as over-phosphorylation can

inhibit crystallization (seeNote 1). Before crystallization experi-

ments, the protein is further purified with another size-

exclusion step.

1.2 AMPK
Crystallization

The structure of full-lengthα 2 β 1 γ1 bound to C2 revealed theα2-

RIM was disengaged. The added flexibility of theα2-RIM inhibited

C2-α 2 β 1 γ1 co-crystallization; therefore, we employed two meth-

ods to reduce the impact of theα-RIM on crystallization. (1) AMP

bound to theγ-subunit strengthens theα2-RIM/γ-subunit inter-

action; therefore, we used a combination of AMP-bound and

C2-bound AMPK heterotrimer for crystal formation. In this sys-

tem we are using the AMP-bound molecule to add structural

integrity to the crystal, which allows for the more flexible

Fig. 1Cartoon representation of AMPK heterotrimer.α 2 β 1 γ1 (PDB/4CFE [6])

showing theα-subunit (green),β-subunit (cyan), andγ-subunit (magenta). Key

structural features have been labeled as follows:α-kinase domain,α-auto-

inhibitory domain (α-AID), α-regulatory interacting motif (α-RIM) 1 and

2,β-carbohydrate binding module (β-CBM), allosteric drug and metabolite

(ADaM) binding site, and theαβγ-scaffold domain. Allosteric modulators have

been labeled, including staurosporine, AMP, A-769662, and C2, the latter

overlaid from PDB 4ZHX [10]

Visualization of Drug Binding Sites 17