Browsing by Author "van Rooyen, Jason M"

Now showing 1 - 3 of 3
  • Thumbnail Image
    Item
    Open Access
    Crystal structure of the Large Type III Glutamine Synthetase from Bacteroides Fragilis
    (2010) van Rooyen, Jason M; Sewell, Bryan Trevor; Abratt, Valerie Rose
    Glutamine synthetases are one of the most ancient functioning enzymes in existence and these large oligomeric complexes are found in all extant forms of life where they play a critical role in nitrogen metabolism. Over the past five decades, extensive biochemical studies together with structural investigations have helped build a picture of the mechanism of functioning and regulation in the GSI and GSII families. The most divergent GSIII family, however, is poorly characterized and has only recently been recognized. Structural studies, using both cryo-EM and X-ray crystallography, were undertaken on the type III GS, GlnN, from the opportunistic human pathogen, Bacteroides fragilis, with a view to better understanding the GSIII family in the light of the known structure functionrelationships of the other GS enzymes, and to investigate the potential for the design of selective inhibitors against the divergent family. A low-resolution (16 Ã) reconstruction of GlnN was first determined by single particle cryo-EM and image processing. This structure revealed that GlnN was a double-ringed dodecamer with D6 symmetry and the arrangement of active sites within the hexameric rings closely matched the GSI structure. Following the design of a rapid purification protocol and improvements to the stability and solubility of GlnN, conditions were discovered for the production of diffraction quality inhibitor-bound crystals. A second better diffracting crystal form was also produced following proteolytic processing. The crystal structure of GlnN was solved to near atomic resolution (3.0 Ã) following phase extension of low-resolution SAD phases, taking into account the cryo-EM structure. The higher resolution of the crystal structure revealed that, surprisingly, the orientation of the hexameric rings in GlnN is inverted in comparison to other families. These results have raised interesting questions surrounding the mechanism and driving forces responsible for the evolution of quaternary structure in the GS enzymes and have suggested that the GSI and GSII structure arose following truncation of a large GSIII-like ancestor. Despite the differences in higher order assembly, the GlnN monomer displayed a high degree of similarity with the GSI and GSII structures in the core active site region, thus, suggesting a conservation of reaction mechanism. Structure-based multiple sequence alignment showed that the residues forming the nucleotide binding pocket are the least conserved in the GS superfamily, and several residue positions, which represent altered modes of ligand binding, were suggested as potential avenues for the design of selective inhibitors against GlnN.
  • Thumbnail Image
    Item
    Restricted
    Crystal structure of Type III glutamine synthetase: surprising reversal of the inter-ring interface
    (Elsevier, 2011) van Rooyen, Jason M; Abratt, Valerie R; Belrhali, Hassan; Sewell, Trevor
    Glutamine synthetases are ubiquitous, homo-oligomeric enzymes essential for nitrogen metabolism. Unlike types I and II, which are well described both structurally and functionally, the larger, type IIIs are poorly characterized despite their widespread occurrence. An understanding of the structural basis for this divergence and the implications for design of type-specific inhibitors has, therefore, been impossible. The first crystal structure of a GSIII enzyme, presented here, reveals a conservation of the GS catalytic fold but subtle differences in protein-ligand interactions suggest possible avenues for the design GSIII inhibitors. Despite these similarities, the divergence of the GSIII enzymes can be explained by differences in quaternary structure. Unexpectedly, the two hexameric rings of the GSIII dodecamer associate on the opposite surface relative to types I and II. The diversity of GS quaternary structures revealed here suggests a nonallosteric role for the evolution of the double-ringed architecture seen in all GS enzymes.
  • Thumbnail Image
    Item
    Restricted
    Three-dimensional structure of a type III glutamine synthetase by single-particle reconstruction
    (Elsevier, 2006) van Rooyen, Jason M; Abratt, Valerie R; Sewell, Trevor B
    GlnN, the type III glutamine synthetase (GSIII) from the medically important, anaerobic, opportunistic pathogen Bacteroides fragilis, has 82.8 kDa subunits that share only 9% sequence identity with the type I glutamine synthetases (GSI), the only family for which a structure is known. Active GlnN was found predominantly in a single peak that eluted from a calibrated gel-filtration chromatography column at a position equaivalent to 0.86(±0.08) MDa. Negative-stain electron microscopy enabled the identification of double-ringed particles and single hexameric rings (“pinwheels”) resulting from partial staining. A 2D average of these pinwheels showed marked similarity to the corresponding structures found in preparations of GSI, except that the arms of the subunits were 40% longer. Reconstructions from particles embedded in vitreous ice showed that GlnN has a double-ringed, dodecameric structure with a 6-fold dihedral space group (D6) symmetry and dimensions of 17.0 nm parallel with the 6-fold axis and 18.3 nm parallel with the 2-fold axes. The structures, combined with a sequence alignment based on structural principles, showed how many aspects of the structure of GSI, and most notably the α/β barrel fold active site were preserved. There was evidence for the presence of this structure in the reconstructed volume, thus, identifying the indentations between the pinwheel spokes as putative active sites and suggesting conservation of the overall molecular geometry found in GSI despite their low level of global homology. Furthermore, docking of GSI into the reconstruction left sufficient plausibly located unoccupied density to account for the additional residues in GSIII, thus validating the structure.