Architecture And Allostery Of Full Length AMP-activated Protein Kinase By Single Particle Electron Microscopy

Mammalian AMP-activated protein kinase (AMPK) is a heterotrimeric protein complex, consisting ofα(two isoforms),β(two isoforms) andγ(three isoforms) subunits. It's a master intracellular sensor for the changes in the ratio of AMP/ATP, and has a crucial role in maintaining the metabolism and cellular energy homeostasis of eukaryotic cells and organisms. The a catalytic subunit contains an N-terminal Ser/Thr kinase domain (KD), a putative autoinhibitory domain (AID) and a C-terminal region required forβsubunit binding. Theβscaffolding subunit mediates AMPK assembly by binding with bothαandγsubunits through its C-terminal sequence and also contains an N-terminal myristoyl group which is responsible for targeting AMPK to the membrane and an internal glycogen-binding domain. Theγregulatory subunit, composed of four tandem cystathionineβ-synthase (CBS) domains which making up two pairs of Bateman domains, is shaped like a flattened disk with a small solvent-accessible channel around the center, and contains three adenosine binding sites, two of which are exchangeable for ATP·Mg2+ and AMP while the third site contains a tightly bound AMP that does not exchange. The integrity of all these three subunits is the precondition of AMPK to get optimal activity. AMPK is activated (～1000 fold) by phosphorylation of a conserved threonine (Thr172 in rat) in the activation loop of the KD by upstream kinases, including LKB1-MO25α-STRADαor CaMKKβ, and together with AMP binding to itsγsubunits.Although published X-ray crystal structures have covered most of AMPK's sequence, several fundamental issues on AMPK's function still remain elusive due to lacking structural information of the intact complex. First, the molecular architecture of the three multidomain subunits in AMPK is unknown. Second, it is unclear how the subtle structural changes seen in the crystal structure of theγsubunits upon adenosine binding can be ultimately translated into the regulation of the kinase activity in theαsubunit. Finally, whereas the existence and functional relevance of AMPK homo-oligomer is emerging, it is necessary to further dissect the structure and determinants of AMPK oligomerization. In our work, we report the first 3D reconstruction of full length rat AMPK (α1β1γ1) in basal and adenosine-bound states by single particle electron microscopy and have discussed the condition for AMPK monomers forming homotrimer and the possible electrostatic interaction mediating the oligomerization.With the aid of observation of the AMPK truncation mutants, and docking of the X-ray crystal structures of AMPK subunits or fragments, we are able to establish the molecular architecture of AMPK. By comparing the structures of AMPK in ATP-and AMP-bound states, we are able to visualize the sequential conformational changes induced kinase activation or inhibition that transmits from the adenosine binding sites in theγsubunit to the kinase domain of theαsubunit. And by comparing AMPK in ATP-bound state with its basal state, we conclude that AMPK in basal state may actually be in a priming configuration. Finally, we yield a three-state model for AMPK structure and regulation.Our findings reported here not only make substantial revision to the current model of AMPK assembly, but also highlight a key role of the linker sequence of theαsubunit in sensing the adenosine binding state of theγsubunit and the presumed mechanism in transmission and amplification of the long-term allostery of AMPK between the three subunits, meanwhile provide clues for further study of the inter- and intrasubunit cross-talks underlying the assembly and function of AMPK.