Mechanism Of Allosteric Regulation Of AMPK By Nucleotides

AMP-activated protein kinase (AMPK) is a heterotrimer, highly conserved in all eukaryotes, AMPK is a serine/threonine protein kinase. AMPK functions as a cellular energy sensor and thus is a key player in regulating energy balance at both the cellular and whole body levels.AMPK is comprised of a catalytic subunit (a) and two regulatory subunits (β and γ). Catalytic subunit has two isoforms (al and a2), regulatory subunits totally have five isoforms (β1,β2, γ1, γ2and γ3), comprising up to12isoenzyme combinations, each with varying tissue and subcellular expression. The a subunit consists of N-terminal serine/threonine catalytic domain (KD), auto-inhibitory domain (AID) and β-subunit binding domain. The β subunit acts as a molecular scaffold, binding both a and y subunits via the C-terminal subunit binding sequence, the N-terminus is the myristoylated domain, following by the glycogen binding domain. The y subunit contains two pairs of cystathionine β-synthase-like (CBS) domains that bind to AMP/ADP/ATP to regulate the activity of AMPK.The activity of AMPK is regulated by increasing cellular AMP/ATP ratio. So far, there are at least three upstream kinases that can activate AMPK, including tumor repressor kinase (LKB1), TGF-β activated kinase-1(TAK1) and calmodulin dependent protein kinase kinase (CaMKK). LKB1activates AMPK by direct phosphorylation at Thr172of AMPK a, CaMKK phosphorylates Thr172regardless whether there is an increase in cellular AMP concentration, and instead, its activity is dependent on the concentration of Ca2+. Current studies suggeste that AMP binds to AMPKy subunit to induce conformational changes of AMPK, one conformation is to increase the activity directly while the other is to make the conformation accessibility to upstream kinases for phosphorylation to enhance the activity. Once activated, it mobilizes the catabolic pathways to generate ATP, and at the same time, shuts down ATP-consuming anabolic processes to regulate energy homeostasis.AMPK has been found to play a key role in the regulation of carbohydrate and lipid metabolism, tumor cell growth, transcription and translation based on previous studies. So AMPK becomes an important pharmacological target for the treatment of diabetes, obesity and cancer. In spite of AMPK’s prominent roles in metabolic diseases and cancer, the mechanisms of the allosteric regulation of its kinase activity are still not clear. The critical barrier towards understanding these mechanisms is the lack of high resolution structures of holo-AMPK in AMP-bound, ATP-bound and glycogen-bound states.In addition, the mechanism of inbition of AID also doesn’t clear. To understand this mechanism, we need clearly know the structure of KD-AID. So far, there is only one structure of KD-AID reported which is an open, inactivated KD-AID structure of S. pombe. In this structure AID binds to the hinge of KD, in turn inhibits the activity of KD. In addition, AID is also very important for the regulation of AMPK activity by AMP. From the alignment of AID, it is clear that human and S. pombe are quite different. Therefore it is not clear if the human KD-AID shares similar structural homology and mode of autoinhibition to that of the yeast homolog. To address this issue, we want to obtain the structural information about the human a subunit that comprises the catalytic domain and AID.For human KD-AID, we tried to express and crystallize different KD-AID fragments and mutations. Eventually, we got the crystal of human KD-AID [α1(11-353)K43A], based on the data of X-ray, we got a2.95A structure of the human kinase domain bound to the adjacent auto-inhibitory domain in the inactive state after many failed attempts. Our studies revealed that human AMPK AID binds to the hinge region of the kinase domain and binding of the AID decreases kinase activity analogous to the yeast homolog. Mutants at the KD-AID interface not only abolish the inhibition for KD, also abolish AMP allosteric activation for AMPK complex; however, not abolish AMP protection for the dephosphorylation of AMPK.Alpha screen is a method to detect the interaction, used this methods, we found conformational/allosteric changes in AMPK upon nucleotide binding. Based on our biochemical data, we proposed a model:AMP binding to the y subunit, increases the interaction between αRIM and CBS-3, makes AID dissociate from KD, then active AMPK; while binding of ATP, decreases the interaction between α-RIM and CBS-3, makes AID bind to KD, then inhibit AMPK. In combination with biochemical and mutational analysis, the results revealed an underlying mechanism of AMPK regulation by AMP and ATP, the binding changes the interaction between CBS-3and α-RIM, then, this signal changes the position of AID to modulate AMPK activity. In addition, ATP not only competes with activator AMP for binding, but may also actively inhibit AMPK.In summary, this study focused on the structure of KD-AID and functions of KD-AID and AMPK complex, these results provided further insight for the regulatory mechanism of AMPK.