Computational protocol: Structural and biochemical characterization of a trapped coenzyme A adduct of Caenorhabditis elegans glucosamine-6-phosphate N-acetyltransferase 1

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Protocol publication

[…] Protein (17 mg ml−1) was pre-incubated on ice for 1 min with a final concentration of 5 mM of both substrates (acetyl-CoA and GlcN-6-P) as well as 5 mM of the product N-acetyl-d-glucosamine 6-phosphate. The sitting-drop vapour-diffusion method was used to produce crystals by mixing 0.5 µl of the protein/ligand solution with an equal volume of mother liquor [0.1 M Tris–HCl pH 8.5, 0.2 M sodium acetate trihydrate and 30%(v/v) PEG 3350 in the presence of 11 mM BaCl2] at 293 K. Bar-shaped crystals (space group P212121) grew within 2 d and bipyramidal-shaped crystals (P61) grew within 12 d. The crystals were cryoprotected using 10% ethylene glycol in the mother liquor (P212121) or 10% glycerol in the mother liquor (P61) and were cooled in a nitrogen-gas stream at 100 K. A single-wavelength SAD experiment was carried out using an in-house generator (Rigaku MicroMax-007 rotating-anode generator equipped with an R-AXIS IV++ detector) using a native hexagonal crystal and a crystal soaked for 20 min in mother liquor containing 20 mM HgCl2. The soaked crystal was cryoprotected with mother liquor containing 5% PEG 400. Data were processed with the HKL suite (Otwinowski & Minor, 1997). Phasing, phase extension and solvent flattening were performed with SOLVE (Terwilliger & Berendzen, 1999), exploiting the anomalous signal of three mercury sites. The resulting 2.0 Å resolution electron-density map was partially autotraced using ARP/wARP (Perrakis et al., 1999), followed by refinement with CNS (Brünger et al., 1998) interspersed with model building with O (Jones et al., 1991). This model was used to solve the subsequent structures CeGNA1–Cys–CoA and CeGNA1–CoA–GlcNAc-6P to 1.55 and 1.75 Å resolution, respectively, using data collected on BM14, ESRF, Grenoble. Ligands were included when unambiguously defined by unbiased |F o| − |F c|, ϕcalc maps. Ligand topologies and coordinates were generated with PRODRG (Schüttelkopf & van Aalten, 2004). The full polypeptide chain was built for both structures, with the exception of Met1. However, poor electron density was observed in the following regions. In the P61 structure chain A residues 16–17, 143–151 and chain B residues 17–20 are disordered. In addition, the adenosine moiety of the CoA molecule had poor electron density. Consequently, these regions have higher B factors. The side chains of Leu18, Ser41, Ser45, Ser76, Ser99, Pro143 and Glu144 in subunit A and of Ser41, Ser76 and Leu122 in subunit B did not appear to have dual conformations. In the P212121 structure residues 16–20 are slightly disordered in molecule A as well as the β-mercaptoethylamine (bME) moiety of the CoA molecule in subunit B. Two ethylene glycol molecules are bound to the backbone N and O atoms of Val89 in subunits A and B of the P212121 structure. All residues in the P61 data set occupy allowed regions of the Ramachandran plot. One amino acid (0.3%) in the P212121 data set was built into a disallowed region. This residue (Leu18 in chain A) is part of the partially disordered region 16–20. All figures were produced with PyMOL (DeLano, 2004). […]

Pipeline specifications

Software tools ARP/wARP, PRODRG, PyMOL
Applications Drug design, Protein structure analysis
Organisms Caenorhabditis elegans
Chemicals Bismuth, Phosphates