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Structural basis of the selective activation of enzyme isoforms

Structural basis of the selective activation of enzyme isoforms: Allosteric

response to activators of b1- and b2-containing AMPK complexes


AMP-activated protein kinase (AMPK) is a key energy sensor regulating the cell metabolism in response to energy supply and demand. The evolutionary adaptation of AMPK to different tissues is accomplished through the expression of distinct isoforms that can form up to 12 complexes, which exhibit notable differences in the sensitivity to allosteric activators. To shed light into the molecular determinants of the allosteric regulation of this energy sensor, they have examined the structural and dynamical properties of b1- and b2-containing AMPK complexes formed with small molecule activators A-769662 and SC4, and dissected the mechanical response leading to active-like enzyme conformations through the analysis of interaction networks between structural domains. The results reveal the mechanical sensitivity of the a2b1 complex, in contrast with a larger resilience of the a2b2 species, especially regarding modulation by A-769662. Furthermore, binding of activators to a2b1 consistently promotes the pre-organization of the ATP-binding site, favoring the adoption of activated states of the enzyme. These findings are discussed in light of the changes in the residue content of b-subunit isoforms, particularly regarding the b1Asn111 b2Asp111 substitution as a key factor in modulating the mechanical sensitivity of b1- and b2-containing AMPK complexes.Their studies pave the way for the design of activators tailored for improving the treatment of tissue-specific metabolic disorders.


Protein isoforms provide complexity to the structural and functional space of the human proteome. Diverse mechanisms, such as genetic changes in protein-coding regions, alternative splicing, and post-translational alterations, mediate the formation of isoforms, enriching the functional diversity by modulating enzymatic activities, molecular interactions, and subcellular localizations In this context, disclosing the molecular basis of the structural variation between isoforms is critical for understanding their functional adaptation to different tissues and organs, and for the design of therapeutic approaches tailored to the Spatio-temporal context of diseases.


A) Superposition of the energy-minimized averaged holo + ATP structures for α2β1 and α2β2 complexes with A-769662 and SC4 (ligands are not shown for the sake of clarity). The backbone of α and β subunits is shown as green and blue cartoon, respectively (P-loop highlighted in yellow). The X-ray structures of α2β1 and α2β2 (PDB IDs 4CFF and 6B2E, respectively) are shown in magenta cartoon. B) RMSF (Å) average of the residues determined along the last 500 ns of the three independent replicas run for apo (red), holo and holo + ATP (green and black, respectively). Complexes bound to A-769662 and SC4 are shown in middle and bottom panels. The highlighted bars denote regions corresponding to P-loop (purple), activation loop (cyan), CBM domain (orange), and C-interacting helix (green).


The evolutionary adaptation of protein isoforms is a key aspect of mammalian adenosine monophosphate-activated protein kinase (AMPK), which can form up to 12 different combinations according to the tissue-dependent expression of isoforms identified for the three structural components required for full activity. This can be understood from the role played as a fuel-sensing enzyme in preserving the cellular energy homeostasis, since AMPK activation reduces the rate of anabolic pathways and up-regulates catabolic processes, resulting in increased levels of ATP. Furthermore, AMPK is implicated in metabolic disorders such as obesity and type 2 diabetes, cardiovascular diseases and cancer, attracting widespread interest as a therapeutic target.


AMPK is a heterotrimeric complex consisting of a catalytic asubunit and two regulatory (b and c) components, which are encoded by multiple genes, including two a (a1, a2), two b (b1, b2), and three c (c1, c2, c3) isoforms. The catalytic subunit has a Ser/Thr kinase domain at the N-terminus and its C-terminus is necessary for the formation of the complex with the other components. The b-subunit has a central carbohydrate-binding module (CBM) that mediates AMPK interaction with glycogen, and the Cterminal region acts as a scaffold for the heterotrimeric assembly.


The c-subunit has four tandem repeats of the cystathionine bsynthase (CBS) domain and contains four adenine nucleotide binding sites, which mediate the allosteric activation of the kinase activity by AMP, enabling AMPK to react to subtle fluctuations in the AMP/ATP ratio. Due to its role in energy homeostasis, AMPK is finely regulated by different mechanisms, such as phosphorylation of a2Thr172 in the activation loop of the kinase domain. Thus, binding of AMP to the c-subunit, in conjunction with Thr172 phosphorylation by upstream kinases such as LKB1 and CaMKKb, converts the inactive enzyme into an active species, which is several thousand-fold more active. AMPK can also be indirectly activated by compounds such as metformin, phenformin and olygomycin, which increase the intracellular levels of AMP. Finally, a direct activation of AMPK can be triggered by modulators such as the thienopyridone drug A-769662, which binds to the so-called allosteric drug and metabolite (ADaM) site located at the interface between a- and b-subunits, promoting an activation mechanism independent of Thr172 phosphorylation. Indeed, binding of A-769662 can increase the AMPK activity >90-fold when Ser108 in the CBM domain of the bsubunit is phosphorylated, and may also protect against dephosphorylation of Thr172.


Several direct AMPK activators have been reported in the last few years, showing in some cases a marked isoform selectivity, as can be noticed upon inspection of the biochemical data collected in Table 1 for selected small molecule AMPK activators. A-769662 is active in a2b1c1 but not in a2b2c1, at least up to concentrations of 10 lM, demonstrating selectivity toward b1-containing AMPK complexes. Likewise, the enzyme activation is ~7-fold larger in a2b1c1 relative to a2b2c1. A mild b1-selective activation is found for 991 and SC4, as they activate b1- and b2-containing AMPK complexes, although a higher (~2-fold) activation is observed in the former case. The activation also seems to be slightly larger for the enzyme containing the a2 isoform. While PF-249 is an activator selective for b1-containing complexes, PF-739 is a pan-activator that activates a2b1c1 and a2b2c1 complexes, though it still exhibits a larger affinity for the b1-containing isoforms (EC50 ratio of ~ 8 and ~ 15 between a2b1c1/a2b2c1 and a1b1c1/a1b2c1, respectively) [30]. PF-739 seems also to have a larger effect on the a2-containing AMPK complexes, as the ratio of the EC50 values is 1.8–3.2 larger for a1b1c1 and a1b2c1 relative to the a2-containing enzymes. Finally, MT47-100, a structural analogue of A-769662 possessing a dihydroquinoline ring instead of the thienopyridone core, activates both a1b1c1 and a2b1c1, but promotes the inhibition of b2-containing complexes.


Understanding the molecular mechanisms that underlie the regulatory effect of direct activators, particularly targeting selectively a given isoform, is of utmost relevance for gaining insight into the puzzling tissue-dependent modulation of AMPK, and to disclose drugs active against specific pathological disorders. Given the sensitivity of the AMPK enzyme to the precise combination of different isoforms, as exemplified by the distinctive trends observed in the activation of complexes with a1/a2 and b1/b2 isoforms (see above), they have adopted a ’divide-and-conquer’ strategy in order to explore the molecular basis of the selective isoform activation of AMPK, specifically focusing on the role played by b1- and b2-subunits. To this end, extended molecular dynamics (MD) simulations have been performed for the apo forms, the holo complexes formed with A-769662 and SC4 bound to the ADaM site, and the ternary (holo + ATP) complexes formed by the enzyme bound to both activator (in the ADaM site) and ATP (in the ATPbinding site). The analysis of the structural and dynamical properties of AMPK complexes and the identification of the interaction networks between a- and b-subunits have unveiled distinctive molecular determinants of the allosteric regulation exerted by direct activators on b1- and b2-containing enzymes.


Representation of the first essential motion derived from the ED analysis of the protein backbone for A) the α2β1 and α2β2 apo species, and their B) holo and C) holo + ATP complexes with A-769662 and SC4 determined from the snapshots sampled along the last 500 ns of simulations. The P-loop is shown in yellow, the helices formed by residues 100–110 and 220–229 in the α-subunit in green, and the CBM domain in magenta.

  1. Aledavood, E., Forte, A., Estarellas, C. ve Javier Luque, F.. (2021). Structural basis of the selective activation of enzyme isoforms: Allosteric response to activators of β1- and β2-containing AMPK complexes. Computational and Structural Biotechnology Journal, 19, 3394–3406. doi:10.1016/j.csbj.2021.05.056