fragment of theβ-subunit [3], the carbohydrate binding module
(CBM), and a truncated kinase domain from the orthologous yeast
kinase, SNF1 [4]. The fission yeast coreαβγstructure comprising
fragments of theα- andβ-subunits associated withγwas the first to
reveal the nucleotide binding architecture [5]. From this point on
there was incremental increase in our AMPK structural knowledge
until the landmark report from the Gamblin laboratory of the full-
length α 2 β 1 γ1 heterotrimer [6]. This structure was followed
quickly by the full-length structures ofα 1 β 1 γ1 andα 1 β 2 γ1[ 7]
heterotrimers (for a comprehensive review of the structural history
of AMPK,seeKurumbail and Calabrese [8]). It should be noted
that while full-length proteins were crystallized, the resulting struc-
tures lacked density for the first ~70 residues of theβ-subunit and
approximately 80 residues from two flexible loops in the
C-terminus of theα-subunit. The initialα 2 β 1 γ1 crystallization
conditions were identified and optimized using the second-
generation high-affinity drug 991 [6], which were then used to
obtain the co-crystal structure of A-769662 bound to AMPK at
3.92 A ̊ resolution. The three full-length AMPK heterotrimer
structures all have a differentα-/β-subunit combination in complex
withγ1.
The full-length AMPK structures revealed the A-769662 allo-
steric binding site, later termed the allosteric drug and metabolite
(ADaM) binding site (Fig.1)[ 9]. The ADaM site is a hydrophobic
pocket formed between theα-N-lobe of the kinase domain and the
β-CBM (Fig.1)[ 6, 10]. The allosteric activation by ADaM site
drugs is thought to occur through stabilization of theα-C-helix by
the β-C-interacting helix, which is directly C-terminal to the
β-CBM [6]. The X-ray crystal structures of AMPK have been
critical for identifying and understanding the allosteric binding
sites. Recently we used X-ray crystallography to discover novel
allosteric sites in the γ-subunit, distinct from the AMP sites
[10]. We solved the structure of AMPK, co-crystallized in the
presence of the AMP-mimetic C2 (5-(5-hydroxyl-isoxazol-3-yl)-
furan-2-phosphonic acid); the resulting structure revealed two
molecules of C2 bound to the solvent accessible core of the
γ-subunit (Fig.1). Here in we described the techniques that will
allow for visualization of compounds targeted to all of the known
AMPK allosteric sites via X-ray crystallography.1.1 Expression
and Purification
of AMPK for X-Ray
Crystallography
The heterotrimeric constructs utilized here are full-lengthα 2 β 1 γ 1
(UniProt: P54646, Q9Y478, and P54619, respectively) and a chi-
meric enzyme with theα-regulatory interacting motif (α-RIM,
Fig. 1) from α1 interchanged with α2(α2(1–347)/α 1
(349–401)/α2(397–end)β 1 γ1;α1 UniProt: Q13131) [10].
We use dual AMPK expression vectors, cloningα2/γ1 into
pETDuet™-1 andβ1intopRSFDuet™-1, resulting in incorpora-
tion of an N-terminal hexa-histidine tag onto α2[ 10]. Most16 Christopher G. Langendorf et al.