Publications (11) View all
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Dataset: supplementary-content-phk-2009
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Article: Protein structure determination by electron cryo-microscopy
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ABSTRACT: Transmission electron cryo-microscopy (cryoEM) is a versatile tool in the structural analysis of proteins and biological macromolecular assemblies. In this review, we present a brief survey of the methods used in cryoEM, and their current developments. These latest advances provide exciting opportunities for the three-dimensional structural determination of macromolecular complexes that are either too large or too heterogeneous to be investigated by conventional X-ray crystallography or nuclear magnetic resonance (NMR). The endeavour of understanding the function of protein or macromolecular complex is often helped by combining data from electron microscopy and X-ray crystallography. We will thus provide a brief overview of the computational techniques involved in combining data from different techniques for the interpretation of the EM structure.Current Opinion in Pharmacology 06/2009; · 6.86 Impact Factor -
Article: The structure of phosphorylase kinase holoenzyme at 9.9 angstroms resolution and location of the catalytic subunit and the substrate glycogen phosphorylase.
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ABSTRACT: Phosphorylase kinase (PhK) coordinates hormonal and neuronal signals to initiate the breakdown of glycogen. The enzyme catalyzes the phosphorylation of inactive glycogen phosphorylase b (GPb), resulting in the formation of active glycogen phosphorylase a. We present a 9.9 angstroms resolution structure of PhK heterotetramer (alphabetagammadelta)4 determined by cryo-electron microscopy single-particle reconstruction. The enzyme has a butterfly-like shape comprising two lobes with 222 symmetry. This three-dimensional structure has allowed us to dock the catalytic gamma subunit to the PhK holoenzyme at a location that is toward the ends of the lobes. We have also determined the structure of PhK decorated with GPb at 18 angstroms resolution, which shows the location of the substrate near the kinase subunit. The PhK preparation contained a number of smaller particles whose structure at 9.8 angstroms resolution was consistent with a proteolysed activated form of PhK that had lost the alpha subunits and possibly the gamma subunits.Structure 02/2009; 17(1):117-27. · 6.35 Impact Factor -
Article: 3D mapping of glycogenosis-causing mutations in the large regulatory alpha subunit of phosphorylase kinase.
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ABSTRACT: Mutations in the liver isoform of the Phosphorylase Kinase (PhK) alpha subunit (PHKA2 gene) cause X-linked liver glycogenosis (XLG), the most frequent type of PhK deficiency (glycogen-storage disease type IX). XLG patients can be divided in two subgroups, with similar clinical features but different activity of PhK (decreased in liver and blood cells for XLG-I and low in liver but normal or enhanced in blood cells for XLG-II). Here, we show that the PHKA2 missense mutations and small in-frame deletions/insertions are concentrated into two domains of the protein, which were recently described. In the N-terminal glucoamylase domain, mutations (principally leading to XLG-II) are clustered within the predicted glycoside-binding site, suggesting that they may have a direct impact on a possible hydrolytic activity of the PhK alpha subunit, which remains to be demonstrated. In the C-terminal calcineurin B-like domain (domain D), mutations (principally leading to XLG-I) are clustered in a region predicted to interact with the regulatory region of the PhK catalytic subunit and in a region covering this interaction site. Altogether, these results show that PHKA2 missense mutations or small in-frame deletions/insertions may have a direct impact on the PhK alpha functions and provide a framework for further experimental investigation.Biochimica et Biophysica Acta 11/2008; 1782(11):664-70. · 4.66 Impact Factor -
Article: The subnanometer resolution structure of the glutamate synthase 1.2-MDa hexamer by cryoelectron microscopy and its oligomerization behavior in solution: functional implications.
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ABSTRACT: The three-dimensional structure of the hexameric (alphabeta)(6) 1.2-MDa complex formed by glutamate synthase has been determined at subnanometric resolution by combining cryoelectron microscopy, small angle x-ray scattering, and molecular modeling, providing for the first time a molecular model of this complex iron-sulfur flavoprotein. In the hexameric species, interprotomeric alpha-alpha and alpha-beta contacts are mediated by the C-terminal domain of the alpha subunit, which is based on a beta helical fold so far unique to glutamate synthases. The alphabeta protomer extracted from the hexameric model is fully consistent with it being the minimal catalytically active form of the enzyme. The structure clarifies the electron transfer pathway from the FAD cofactor on the beta subunit, to the FMN on the alpha subunit, through the low potential [4Fe-4S](1+/2+) centers on the beta subunit and the [3Fe-4S](0/1+) cluster on the alpha subunit. The (alphabeta)(6) hexamer exhibits a concentration-dependent equilibrium with alphabeta monomers and (alphabeta)(2) dimers, in solution, the hexamer being destabilized by high ionic strength and, to a lower extent, by the reaction product NADP(+). Hexamerization seems to decrease the catalytic efficiency of the alphabeta protomer only 3-fold by increasing the K(m) values measured for l-Gln and 2-OG. However, it cannot be ruled out that the (alphabeta)(6) hexamer acts as a scaffold for the assembly of multienzymatic complexes of nitrogen metabolism or that it provides a means to regulate the activity of the enzyme through an as yet unknown ligand.Journal of Biological Chemistry 04/2008; 283(13):8237-49. · 4.77 Impact Factor
