Sulochanadevi Baskaran

Architecture and dynamics of the autophagic phosphatidylinositol 3-kinase complex

The class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) that functions in early autophagy consists of the lipid kinase VPS34, the scaffolding protein VPS15, the tumor suppressor BECN1, and the autophagy-specific subunit ATG14. The structure of the ATG14-containing PI3KC3-C1 was determined by single-particle EM, revealing a V-shaped architecture. All of the ordered domains of VPS34, VPS15, and BECN1 were mapped by MBP tagging. The dynamics of the complex were defined using hydrogen–deuterium exchange, revealing a novel 20-residue ordered region C-terminal to the VPS34 C2 domain. VPS15 organizes the complex and serves as a bridge between VPS34 and the ATG14:BECN1 subcomplex. Dynamic transitions occur in which the lipid kinase domain is ejected from the complex and VPS15 pivots at the base of the V. The N-terminus of BECN1, the target for signaling inputs, resides near the pivot point. These observations provide a framework for understanding the allosteric regulation of lipid kinase activity. DOI: http://dx.doi.org/10.7554/eLife.05115.001

Structural basis for glucose-6-phosphate activation of glycogen synthase

Regulation of the storage of glycogen, one of the major energy reserves, is of utmost metabolic importance. In eukaryotes, this regulation is accomplished through glucose-6-phosphate levels and protein phosphorylation. Glycogen synthase homologs in bacteria and archaea lack regulation, while the eukaryotic enzymes are inhibited by protein kinase mediated phosphorylation and activated by protein phosphatases and glucose-6-phosphate binding. We determined the crystal structures corresponding to the basal activity state and glucose-6-phosphate activated state of yeast glycogen synthase-2. The enzyme is assembled into an unusual tetramer by an insertion unique to the eukaryotic enzymes, and this subunit interface is rearranged by the binding of glucose-6-phosphate, which frees the active site cleft and facilitates catalysis. Using both mutagenesis and intein-mediated phospho-peptide ligation experiments, we demonstrate that the enzyme’s response to glucose-6-phosphate is controlled by Arg583 and Arg587, while four additional arginine residues present within the same regulatory helix regulate the response to phosphorylation.

The protein-vesicle network of autophagy

The biogenesis of autophagosomes entails the nucleation and growth of a double-membrane sheet, the phagophore, which engulfs cytosol for delivery to the lysosome. Genetic studies have identified a class of Atg proteins that are essential for the process, yet the molecular mechanism of autophagosome biogenesis has been elusive. Proteomic, structural, super-resolution imaging, and biochemical reconstitution experiments have begun to fill in some of the gaps. This review describes progress and prospects for obtaining a four-dimensional network model of the nucleation and growth of the phagophore.

Discovery of a Novel Class of Covalent Inhibitor for Aldehyde Dehydrogenases

Background: ALDH enzymes metabolize aldehydes in many pathways, including the inactivation of cyclophosphamide. Results: Covalent inhibitors against ALDH were discovered, and their mechanism of action was determined. Conclusion: Covalent inhibitors against ALDH potentiate cell killing in cyclophosphamide-resistant cells. Significance: These inhibitors represent novel research tools and can serve as leads toward therapeutics where increased ALDH activity is associated with disease. Human aldehyde dehydrogenases (ALDHs) comprise a family of 17 homologous enzymes that metabolize different biogenic and exogenic aldehydes. To date, there are relatively few general ALDH inhibitors that can be used to probe the contribution of this class of enzymes to particular metabolic pathways. Here, we report the discovery of a general class of ALDH inhibitors with a common mechanism of action. The combined data from kinetic studies, mass spectrometric measurements, and crystallographic analyses demonstrate that these inhibitors undergo an enzyme-mediated β-elimination reaction generating a vinyl ketone intermediate that covalently modifies the active site cysteine residue present in these enzymes. The studies described here can provide the basis for rational approach to design ALDH isoenzyme-specific inhibitors as research tools and perhaps as drugs, to address diseases such as cancer where increased ALDH activity is associated with a cellular phenotype.

Multiple Glycogen-binding Sites in Eukaryotic Glycogen Synthase Are Required for High Catalytic Efficiency toward Glycogen

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 Vmax/S0.5 for glycogen by 40- and 70-fold, respectively. Combined mutation of site-1 and site-2 decreased the Vmax/S0.5 for glycogen by >3000-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.

How Atg18 and the WIPIs sense phosphatidylinositol 3-phosphate

The key autophagic lipid sensors are Atg18 in yeast and the WIPI proteins in mammals. Atg18 and the WIPIs belong to the PROPPIN family of proteins. PROPPINs are seven- bladed β-propellers that bind to phosphatidylinositol 3-phosphate (PtdIns3P) and phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P2]. In order to understand how PROPPINs bind phosphoinositides, we have determined the crystal structure of a representative, biochemically tractable PROPPIN, Hsv2 of Kluveromyces lactis. The structure revealed that PROPPINs contain two phosphoinositide binding sites which cooperate with a hydrophobic anchoring loop in membrane binding. These three binding elements cooperate in function, as demonstrated by the incremental loss of function in Atg18 mutants impaired in combinations of the two phosphoinositide binding sites and the hydrophobic loop.

Structural basis for 2′-phosphate incorporation into glycogen by glycogen synthase

Significance Glycogen is a branched glucose polymer found in most animals, fungi, bacteria, and archaea as an osmotically neutral means of energy storage. Glycogen also contains minor amounts of phosphate which can be removed by a dual specificity phosphatase, laforin. Accumulation of phosphate results in highly insoluble glycogen deposits and underlies Lafora disease, a devastating form of myoclonus epilepsy. In this paper, we present structural and kinetic data that support a plausible mechanism by which phosphate is directly incorporated into glycogen by glycogen synthase. Glycogen is a glucose polymer that contains minor amounts of covalently attached phosphate. Hyperphosphorylation is deleterious to glycogen structure and can lead to Lafora disease. Recently, it was demonstrated that glycogen synthase catalyzes glucose–phosphate transfer in addition to its characteristic glucose transfer reaction. Glucose-1,2-cyclic-phosphate (GCP) was proposed to be formed from UDP-Glc breakdown and subsequently transferred, thus providing a source of phosphate found in glycogen. To gain further insight into the molecular basis for glucose–phosphate transfer, two structures of yeast glycogen synthase were determined; a 3.0-Å resolution structure of the complex with UMP/GCP and a 2.8-Å resolution structure of the complex with UDP/glucose. Structural superposition of the complexes revealed that the bound ligands and most active site residues are positioned similarly, consistent with the use of a common transfer mechanism for both reactions. The N-terminal domain of the UDP⋅glucose complex was found to be 13.3° more closed compared with a UDP complex. However, the UMP⋅GCP complex was 4.8° less closed than the glucose complex, which may explain the low efficiency of GCP transfer. Modeling of either α- or β-glucose or a mixture of both anomers can account for the observed electron density of the UDP⋅glucose complex. NMR studies of UDP-Glc hydrolysis by yeast glycogen synthase were used to verify the stereochemistry of the product, and they also showed synchronous GCP accumulation. The similarities in the active sites of glycogen synthase and glycogen phosphorylase support the idea of a common catalytic mechanism in GT-B enzymes independent of the specific reaction catalyzed.

Unveiling the role of VPS34 kinase domain dynamics in regulation of the autophagic PI3K complex

ABSTRACT The class III PI 3-kinase, VPS34 forms distinct complexes essential for cargo sorting and membrane trafficking in endocytosis as well as for autophagosome nucleation and maturation. We used integrative structural biology approach to provide insights into the conformational dynamics of the complex and mechanisms that regulate VPS34 activity at the membrane.

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 (G-6-P). 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 G-6-P. 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. On the basis of 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.