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SummaryAMP-activated protein kinase (AMPK) monitors cellular energy, regulates genes involved in ATP synthesis and consumption, and is allosterically activated by nucleotides and synthetic ligands. Analysis of the intact enzyme with hydrogen/deuterium exchange mass spectrometry reveals conformational perturbations of AMPK in response to binding of nucleotides, cyclodextrin, and a synthetic small molecule activator, A769662. Results from this analysis clearly show that binding of AMP leads to conformational changes primarily in the γ subunit of AMPK and subtle changes in the α and β subunits. In contrast, A769662 causes profound conformational changes in the glycogen binding module of the β subunit and in the kinase domain of the α subunit, suggesting that the molecular binding site of the latter resides between the α and β subunits. The distinct short- and long-range perturbations induced upon binding of AMP and A769662 suggest fundamentally different molecular mechanisms for activation of AMPK by these two ligands.

Glycogen synthase is a rate-limiting enzyme in the biosynthesis of glycogen and has an essential role in glucose homeostasis. The three-dimensional structures of yeast glycogen synthase (Gsy2p) complexed with maltooctaose identified four conserved maltodextrin-binding sites distributed across the surface of the enzyme. Site-1 is positioned on the N-terminal domain, site-2 and site-3 are present on the C-terminal domain, and site-4 is located in an interdomain cleft adjacent to the active site. Mutation of these surface sites decreased glycogen binding and catalytic efficiency toward glycogen. Mutations within site-1 and site-2 reduced the V(max)/S(0.5) for glycogen by 40- and 70-fold, respectively. Combined mutation of site-1 and site-2 decreased the V(max)/S(0.5) for glycogen by3000-fold. Consistent with the in vitro data, glycogen accumulation in glycogen synthase-deficient yeast cells (Δgsy1-gsy2) transformed with the site-1, site-2, combined site-1/site-2, or site-4 mutant form of Gsy2p was decreased by up to 40-fold. In contrast to the glycogen results, the ability to utilize maltooctaose as an in vitro substrate was unaffected in the site-2 mutant, moderately affected in the site-1 mutant, and almost completely abolished in the site-4 mutant. These data show that the ability to utilize maltooctaose as a substrate can be independent of the ability to utilize glycogen. Our data support the hypothesis that site-1 and site-2 provide a "toehold mechanism," keeping glycogen synthase tightly associated with the glycogen particle, whereas site-4 is more closely associated with positioning of the nonreducing end during catalysis.

AMPK beta subunits contain a conserved domain that causes association with glycogen. Although glycogen availability is known to affect AMPK regulation in vivo, the molecular mechanism for this has not been clear. We now show that AMPK is inhibited by glycogen, particularly preparations with high branching content. We synthesized a series of branched oligosaccharides and show that those with a single alpha1-->6 branch are allosteric inhibitors that also inhibit phosphorylation by upstream kinases. Removal of the outer chains of glycogen using phosphorylase, thus exposing the outer branches, renders inhibition of AMPK more potent. Inhibition by all carbohydrates tested was dependent on the glycogen-binding domain being abolished by mutation of residues required for carbohydrate binding. Our results suggest the hypothesis that AMPK, as well as monitoring immediate energy availability by sensing AMP/ATP, may also be able to sense the status of cellular energy reserves in the form of glycogen.

The SNF1/AMP-activated protein kinase (AMPK) family is required for adaptation to metabolic stress and energy homeostasis. The γ subunit of AMPK binds AMP and ATP, and mutations that affect binding cause human disease. We have here addressed the role of the Snf4 (γ) subunit in regulating SNF1 protein kinase in response to glucose availability in Saccharomyces cerevisiae. Previous studies of mutant cells lacking Snf4 suggested that Snf4 counteracts autoinhibition by the C-terminal sequence of the Snf1 catalytic subunit but is dispensable for glucose regulation, and AMP does not activate SNF1 in vitro. We first introduced substitutions at sites that, in AMPK, contribute to nucleotide binding and regulation. Mutations at several sites relieved glucose inhibition of SNF1, as judged by catalytic activity, phosphorylation of the activation-loop Thr-210, and growth assays, although analogs of the severe human mutations R531G/Q had little effect. We further showed that alterations of Snf4 residues that interact with the glycogen-binding domain (GBD) of the β subunit strongly relieved glucose inhibition. Finally, substitutions in the GBD of the Gal83 β subunit that are predicted to disrupt interactions with Snf4 and also complete deletion of the GBD similarly relieved glucose inhibition of SNF1. Analysis of mutant cells lacking glycogen synthase showed that regulation of SNF1 is normal in the absence of glycogen. These findings reveal novel roles for Snf4 and the GBD in regulation of SNF1.

AMP-activated protein kinase (AMPK) coordinates cellular metabolism in response to energy demand as well as to a variety of stimuli. The AMPK beta subunit acts as a scaffold for the alpha catalytic and gamma regulatory subunits and targets the AMPK heterotrimer to glycogen. We have determined the structure of the AMPK beta glycogen binding domain in complex with beta-cyclodextrin. The structure reveals a carbohydrate binding pocket that consolidates all known aspects of carbohydrate binding observed in starch binding domains into one site, with extensive contact between several residues and five glucose units. beta-cyclodextrin is held in a pincer-like grasp with two tryptophan residues cradling two beta-cyclodextrin glucose units and a leucine residue piercing the beta-cyclodextrin ring. Mutation of key beta-cyclodextrin binding residues either partially or completely prevents the glycogen binding domain from binding glycogen. Modeling suggests that this binding pocket enables AMPK to interact with glycogen anywhere across the carbohydrate's helical surface.

The cysteine-specific reagent 5,5'-dithiobis(2-nitrobenzoic acid) inactivates the Escherichia coli glycogen synthase (Holmes, E., and Preiss, J. (1982) Arch. Biochem. Biophys. 216, 736-740). To find the responsible residue, all cysteines, Cys(7), Cys(379), and Cys(408), were substituted combinatorially by Ser. 5,5'-Dithiobis(2-nitrobenzoic acid) modified and inactivated the enzyme if and only if Cys(379) was present and it was prevented by the substrate ADP-glucose (ADP-Glc). Mutations C379S and C379A increased the S(0.5) for ADP-Glc 40- and 77-fold, whereas the specific activity was decreased 5.8- and 4.3-fold, respectively. Studies of inhibition by glucose 1-phosphate and AMP indicated that Cys(379) was involved in the interaction of the enzyme with the phosphoglucose moiety of ADP-Glc. Other mutations, C379T, C379D, and C379L, indicated that this site is intolerant for bulkier side chains. Because Cys(379) is in a conserved region, other residues were scanned by mutagenesis. Replacement of Glu(377) by Ala and Gln decreased V(max) more than 10,000-fold without affecting the apparent affinity for ADP-Glc and glycogen binding. Mutation of Glu(377) by Asp decreased V(max) only 57-fold indicating that the negative charge of Glu(377) is essential for catalysis. The activity of the mutation E377C, on an enzyme form without other Cys, was chemically restored by carboxymethylation. Other conserved residues in the region, Ser(374) and Gln(383), were analyzed by mutagenesis but found not essential. Comparison with the crystal structure of other glycosyltransferases suggests that this conserved region is a loop that is part of the active site. The results of this work indicate that this region is critical for catalysis and substrate binding.

The AMP-activated protein kinase (AMPK) is an alphabetagamma heterotrimer that is activated by low cellular energy status and affects a switch away from energy-requiring processes and toward catabolism. While it is primarily regulated by AMP and ATP, high muscle glycogen has also been shown to repress its activation. Mutations in the gamma2 and gamma3 subunit isoforms lead to arrhythmias associated with abnormal glycogen storage in human heart and elevated glycogen in pig muscle, respectively. A putative glycogen binding domain (GBD) has now been identified in the beta subunits. Coexpression of truncated beta subunits lacking the GBD with alpha and gamma subunits yielded complexes that were active and normally regulated. However, coexpression of alpha and gamma with full-length beta caused accumulation of AMPK in large cytoplasmic inclusions that could be counterstained with anti-glycogen or anti-glycogen synthase antibodies. These inclusions were not affected by mutations that increased or abolished the kinase activity and were not observed by using truncated beta subunits lacking the GBD. Our results suggest that the GBD binds glycogen and can lead to abnormal glycogen-containing inclusions when the kinase is overexpressed. These may be related to the abnormal glycogen storage bodies seen in heart disease patients with gamma2 mutations.

Lafora disease (progressive myoclonus epilepsy of Lafora type) is an autosomal recessive neurodegenerative disorder resulting from defects in the EPM2A gene. EPM2A encodes a 331-amino acid protein containing a carboxyl-terminal phosphatase catalytic domain. We demonstrate that the EPM2A gene product also contains an amino-terminal carbohydrate binding domain (CBD) and that the CBD is critical for association with glycogen both in vitro and in vivo. The CBD domain localizes the phosphatase to specific subcellular compartments that correspond to the expression pattern of glycogen processing enzyme, glycogen synthase. Mutations in the CBD result in mis-localization of the phosphatase and thereby suggest that the CBD targets laforin to intracellular glycogen particles where it is likely to function. Thus naturally occurring mutations within the CBD of laforin likely result in progressive myoclonus epilepsy due to mis-localization of phosphatase expression.

In this study we demonstrate that recombinant rabbit muscle protein phosphatase-1 immobilized on CH-Sepharose is an efficient affinity chromatography support for the isolation of subunits of phosphatase-1. The support was used to isolate the glycogen binding subunit of phosphatase-1 from muscle and nonmuscle rat tissues. Examination of the affinity-purified material from rat heart and liver showed that the major component was a 160-kDa polypeptide on SDS-PAGE. The identity of the purified liver 160-kDa polypeptide as the glycogen binding subunit was confirmed by the demonstration that it is capable of binding to glycogen, and is phosphorylated by the catalytic subunit of PKA. The novel observation was made that the phosphorylation was dependent on the presence of glycogen. Examination of the material from heart, lung, liver, kidney, and brain showed a similar phenomenon. Our studies show that this subunit is widely distributed in tissues. The affinity support was also efficient in the isolation of the NIPP-1 (nuclear inhibitor of protein phosphatase-1) proteins from calf thymus. Examination of heat-treated extracts of rat liver led to the isolation of a novel 19-kDa inhibitory protein which could also be phosphorylated by PKA and may represent the rat liver homolog of calf thymus NIPP-1.

Reaction of four of the eight sulfhydryl groups per subunit of rat liver glycogen synthetase D through mixed disulfide formation with oxidized glutathione leads to inactivation of the enzyme and its dissociation from glycogen. The differential protection offered by glucose 6-phosphate against inactivation and dissociation permits determination of the number of sulfhydryl groups involved in each process. Modification of the first sulfhydryl group had no effect on enzyme activity or glycogen binding. Reaction of the second sulfhydryl group led to complete inactivation of the enzyme and was accompanied by an increase in the Ka for glucose-6-P. Modification of two more sulfhydryl groups per subunit resulted in release of the enzyme from glycogen. Gel filtration experiments revealed that disruption of the glycogen-glycogen synthetase D complex was a consequence of dissociation of the enzyme itself into a subunit (mol wt 86,000) which could not bind glycogen. The enzyme-glycogen complex could be reconstituted from glycogen and glycogen-free, inactive glycogen synthetase D by treatment with glucose-6-P. Reduction with dithiothreitol, which by itself failed to restore the complex, significantly lowered the concentration of glucose-6-P required to fully achieve reconstitution. Treatment with glucose-6-P, but not reduction with dithiothreitol, reassociated the inactive subunit into a high molecular weight species which is the form capable of binding glycogen. These results suggest that glucose-6-P plays an important role in maintaining the glycogen-glycogen synthetase D complex.

Crystallographic binding studies of various metabolites to phosphorylase b in the presence of 2 m m -IMP have been carried out at low resolution (8.7 A) with three-dimensional data and at high resolution (3 a) with two-dimensional data. From correlation of peaks observed in difference Fourier syntheses based on these two sets of data, the following binding sites have been identified: (1) the “active” site to which the substrate, glucose 1-phosphate, and the substrate analogues, maltotriose and arsenate, bind and which is close to the subunit-subunit interface of the phosphorylase dimer; (2) the allosteric adenine-nucleotide binding site to which the allosteric activator AMP and the allosteric inhibitor ATP bind and which is very close to the active site; (3) the inhibitor binding site for glucose 6-phosphate, which is also close to the active site. Glucose 6-phosphate causes extensive conformational changes in the protein, which are the largest observed for all the metabolites studied so far; (4) a glycogen binding site on the surface of the molecule to which maltotriose binds. The distance over the surface of the phosphorylase molecule from this site to the active site is 50 to 60 A; (5) a second glucose 1-phosphate binding site situated in the interior of the molecule. The significance of this site is not yet understood but its position in the centre of the molecule suggests that it may have a key role in the control and catalysis of phosphorylase.