Structural basis for regulated inhibition and substrate selection in yeast glycogen synthase

Indiana University-Purdue University Indianapolis (IUPUI)Glycogen synthase (GS) is the rate limiting enzyme in the synthesis of glycogen. Eukaryotic GS catalyzes the transfer of glucose from UDP-glucose to the non-reducing ends of glycogen and its activity is negatively regulated by phosphorylation and allosterically activated by glucose-6-phosphate (G6P). A highly conserved cluster of six arginine residues on the C-terminal domain controls the responses toward these opposing signals. Previous studies had shown that tetrameric enzyme exists in three conformational states which are linked to specific structural changes in the regulatory helices that carry the cluster of arginines. These helices are found opposite and anti-parallel to one another at one of the subunit interfaces. The binding of G6P beneath the regulatory helices induces large scale conformational changes which open up the catalytic cleft for better substrate access. We solved the crystal structure of the enzyme in its inhibited state and found that the tetrameric and regulatory interfaces are more compacted compared to other states. The structural consequence of the tighter interfaces within the inhibited state of the tetramer is to lower the ability of glycogen chains to access to the catalytic cleft. Based on these observations, we developed a novel regulatory feature in yeast GS by substituting two of its conserved arginine residues on the regulatory helix with cysteines that permits its activity to be controlled by reversible oxidation/reduction of the cysteine residues which mimics the effects of reversible phosphorylation. In addition to defining the structural changes that give rise to the inhibited states, we also used X-ray crystallography to define the mechanism by which the enzyme discriminates between different UDP-sugar donors to be used as substrates in the catalytic mechanism of yeast GS. We found that only donor substrates can adopt the catalytically favorable bent conformation for donor transfer to a growing glycogen chain

Discovery of a series of aromatic lactones as ALDH1/2-directed inhibitors

In humans, the aldehyde dehydrogenase superfamily consists of 19 isoenzymes which mostly catalyze the NAD(P)(+)-dependent oxidation of aldehydes. Many of these isoenzymes have overlapping substrate specificities and therefore their potential physiological functions may overlap. Thus the development of new isoenzyme-selective probes would be able to better delineate the function of a single isoenzyme and its individual contribution to the metabolism of a particular substrate. This specific study was designed to find a novel modulator of ALDH2, a mitochondrial ALDH isoenzyme most well-known for its role in acetaldehyde oxidation. 53 compounds were initially identified to modulate the activity of ALDH2 by a high-throughput esterase screen from a library of 63,000 compounds. Of these initial 53 compounds, 12 were found to also modulate the oxidation of propionaldehyde by ALDH2. Single concentration measurements at 10μM compound were performed using ALDH1A1, ALDH1A2, ALDH1A3, ALDH2, ALDH1B1, ALDH3A1, ALDH4A1, and/or ALDH5A1 to determine the selectivity of these 12 compounds toward ALDH2. Four of the twelve compounds shared an aromatic lactone structure and were found to be potent inhibitors of the ALDH1/2 isoenzymes, but have no inhibitory effect on ALDH3A1, ALDH4A1 or ALDH5A1. Two of the aromatic lactones show selectivity within the ALDH1/2 class, and one appears to be selective for ALDH2 compared to all other isoenzymes tested

Redox Switch for the Inhibited State of Yeast Glycogen Synthase Mimics Regulation by Phosphorylation

Glycogen synthase (GS) is the rate limiting enzyme in the synthesis of glycogen. Eukaryotic GS is negatively regulated by covalent phosphorylation and allosterically activated by glucose-6-phosphate (G6P). To gain structural insights into the inhibited state of the enzyme, we solved the crystal structure of yGsy2-R589A/R592A to a resolution of 3.3 Å. The double mutant has an activity ratio similar to the phosphorylated enzyme and also retains the ability to be activated by G6P. When compared to the 2.88 Å structure of the wild-type G-6-P activated enzyme, the crystal structure of the low-activity mutant showed that the N-terminal domain of the inhibited state is tightly held against the dimer-related interface thereby hindering acceptor access to the catalytic cleft. Based on these two structural observations, we developed a reversible redox regulatory feature in yeast GS by substituting cysteine residues for two highly conserved arginine residues. When oxidized, the cysteine mutant enzyme exhibits activity levels similar to the phosphorylated enzyme, but cannot be activated by G-6-P. Upon reduction, the cysteine mutant enzyme regains normal activity levels and regulatory response to G-6-P activation

Structural insights into the inhibited state of Glycogen Synthase

poster abstractGlycogen is an osmotically inert polymer of glucose, synthesized during times of nutritional sufficiency so that it can be rapidly catabolized when there is an energy demand1. Glycogen synthase (GS) is responsible for the bulk of its synthesis by transferring glucose from UDPG to an existing glucose polymer1. Eukaryotic GS is allosterically activated by glucose-6-phosphate (G6P) and negatively regulated by covalent phosphorylation2. A cluster of six arginine residues are conserved across all eukaryotic species which determine the enzyme’s ability to respond to these activating and inhibitory signals2. Prior structural studies from our lab had shed light on the dephosphorylated and activated state of the enzyme3. However, little is known on the phosphorylated state of the enzyme. For structural studies on the inhibited state, we used the yGsy2R589/592A mutant as a surrogate since it has a basal activity state similar to the inhibited phosphorylated state. We solved the structure of the mutant to a resolution of 3.3 Å. While the overall structural arrangement of the tetramer is similar to the basal state enzyme, the interfaces are more closed. In particular, the N-terminal Rossmann-fold domain is rotated toward the interface by 5.9°, limiting access to the active site by the acceptor end of the glycogen chain. Coincident with this domain closure, we also observed that the the distance between the regulatory helices of adjacent monomers are moved closer to one another. Based on this observation, we hypothesized we could develop a reversible redox regulatory feature in the enzyme by substituting cysteine residues for arginines 581 and 592, which lie across from each other at the interface. Consistent with our hypothesis, the yGsy2R581/592C double mutant exhibited very low activity, and could not be activated by G6P. However, normal function of the enzyme could be restored in the presence of reducing agents like DTT, BME and TCEP. Taken together, our mutational work demonstrates that the conserved arginine cluster in the regulatory helix, both regulates the enzyme’s response to signaling inputs and keeps the enzyme in a basal state conformation that is poised to respond to the activating and inhibitory inputs

Incorporation of phosphate into glycogen by glycogen synthase

The storage polymer glycogen normally contains small amounts of covalently attached phosphate as phosphomonoesters at C2, C3 and C6 atoms of glucose residues. In the absence of the laforin phosphatase, as in the rare childhood epilepsy Lafora disease, the phosphorylation level is elevated and is associated with abnormal glycogen structure that contributes to the pathology. Laforin therefore likely functions in vivo as a glycogen phosphatase. The mechanism of glycogen phosphorylation is less well-understood. We have reported that glycogen synthase incorporates phosphate into glycogen via a rare side reaction in which glucose-phosphate rather than glucose is transferred to a growing polyglucose chain (Tagliabracci et al. (2011) Cell Metab13, 274-282). We proposed a mechanism to account for phosphorylation at C2 and possibly at C3. Our results have since been challenged (Nitschke et al. (2013) Cell Metab17, 756-767). Here we extend the evidence supporting our conclusion, validating the assay used for the detection of glycogen phosphorylation, measurement of the transfer of (32)P from [β-(32)P]UDP-glucose to glycogen by glycogen synthase. The (32)P associated with the glycogen fraction was stable to ethanol precipitation, SDS-PAGE and gel filtration on Sephadex G50. The (32)P-signal was not affected by inclusion of excess unlabeled UDP before analysis or by treatment with a UDPase, arguing against the signal being due to contaminating [β-(32)P]UDP generated in the reaction. Furthermore, [(32)P]UDP did not bind non-covalently to glycogen. The (32)P associated with glycogen was released by laforin treatment, suggesting that it was present as a phosphomonoester. The conclusion is that glycogen synthase can mediate the introduction of phosphate into glycogen, thereby providing a possible mechanism for C2, and perhaps C3, phosphorylation

Discovery and Development of Small-Molecule Inhibitors of Glycogen Synthase

The overaccumulation of glycogen appears as a hallmark in various glycogen storage diseases (GSDs), including Pompe, Cori, Andersen, and Lafora disease. Accumulating evidence suggests that suppression of glycogen accumulation represents a potential therapeutic approach for treating these GSDs. Using a fluorescence polarization assay designed to screen for inhibitors of the key glycogen synthetic enzyme, glycogen synthase (GS), we identified a substituted imidazole, (rac)-2-methoxy-4-(1-(2-(1-methylpyrrolidin-2-yl)ethyl)-4-phenyl-1H-imidazol-5-yl)phenol (H23), as a first-in-class inhibitor for yeast GS 2 (yGsy2p). Data from X-ray crystallography at 2.85 Å, as well as kinetic data, revealed that H23 bound within the uridine diphosphate glucose binding pocket of yGsy2p. The high conservation of residues between human and yeast GS in direct contact with H23 informed the development of around 500 H23 analogs. These analogs produced a structure–activity relationship profile that led to the identification of a substituted pyrazole, 4-(4-(4-hydroxyphenyl)-3-(trifluoromethyl)-1H-pyrazol-5-yl)pyrogallol, with a 300-fold improved potency against human GS. These substituted pyrazoles possess a promising scaffold for drug development efforts targeting GS activity in GSDs associated with excess glycogen accumulation