hype!terminal  (SourceForge net)

Journal: bioRxiv

Article Title: A Ca 2+ -regulated deAMPylation switch in human and bacterial FIC proteins

doi: 10.1101/323253

Figure Lengend Snippet: A: Structure of the EfFIC monomer showing the FIC motif (pink), the C-terminal α-helix bearing the inhibitory glutamate (orange) and the β-hairpin predicted to bind protein substrates (cyan). The ADP moiety of ATPγS is shown in sticks. B: The inhibitory glutamate from EfFIC WT (orange) is structurally equivalent to the glutamate found in the C-terminus of NmFIC ( , PDB 2G03) and in the N-terminus of Bacteroides BtFIC (PDB 3CUC), Clostridium CdFIC ( , PDB 4X2E) and of human FICD/HYPE ( , PDB 4U04). Superpositions are done on the structurally highly conserved FIC motif. C: Interactions of the inhibitory glutamate with the active site of ATPγS-bound EfFIC WT . Hydrogen bonds are depicted by dotted lines. D: The positions of the α- and β-phosphates of ATPγS bound to EfFIC WT (yellow) diverge from those of ATPγS bound to NmFIC WT (cyan, , PDB 3S6A) and of ATP bound to CdFIC (blue, , S31A/E35A mutant, PDB 4X2D) in a non-canonical conformations. Note that only the ADP moiety of ATPγS is visible in the EfFIC WT and NmFIC WT crystal structures. E: The α- and β-phosphates of ATPγS bound to EfFIC WT (yellow) superpose well to those of ATP bound to NmFIC (purple, , E186G mutant, PDB 3ZLM) and of CDP-choline bound to AnkX (green, , H229A mutant, PDB 4BET) in a PTM-competent conformation. F: AutoAMPylation of EfFIC WT and EfFIC E190G . The level of AMPylated proteins (indicated as AMP* EfFIC WT ) was measured by autoradiography using radioactive [α- P]-ATP in the presence of 100 μM Mg 2+ . The reaction was carried out for one hour for EfFIC WT and five minutes for EfFIC E190G . The total amount of EfFIC WT measured by Coomassie staining in the same sample is shown.

Article Snippet: The codon-optimized gene encoding human FICD/HYPE (residues 45-459) carrying an N-terminal 6-His tag followed by SUMO tag was from GeneArt Gene Synthesis and cloned into a pET151/D-TOPO vector (ThermoFisher Scientific).

Techniques: Mutagenesis, Autoradiography, Staining

Journal: bioRxiv

Article Title: A Ca 2+ -regulated deAMPylation switch in human and bacterial FIC proteins

doi: 10.1101/323253

Figure Lengend Snippet: A: FICD WT does not AMPylate the BiP chaperone. Reactions were carried out for one hour in presence of 1mM Mg 2+ or 1mM Ca 2+ . The level of AMPylated proteins (indicated as AMP-FAM FICD WT and AMP-FAM BIP) was measured by fluorescence. The total amount of proteins measured by Coomassie staining in the same sample is shown. B: Ca 2+ inhibits deAMPylation of BiP by FICD/HYPE WT . BiP was first AMPylated for one hour by the hyperactive FICD E234G mutant in the presence of 100 μM Mg 2+ , then AMP-FAM BIP was purified to remove Mg 2+ , PPi and ATP-FAM. DeAMPylation of AMP-FAM BIP was then triggered by addition of wild-type or mutant FICD as indicated in the presence of 1mM Mg 2+ or 1mM Ca 2+ . AMP-FAM BiP levels were measured by fluorescence. The total amount of BiP measured by Coomassie staining in each sample is shown. C: The net deAMPylation efficiency of FICD WT is tuned by the Mg 2+ /Ca 2+ ratio. DeAMPylation was carried out as in using a fixed Mg 2+ concentration (200µM) and 0, 1, 2 or 3mM Ca 2+ . AMPylation levels were measured by fluorescence, normalized to the fluorescence intensity of FICD and expressed as the percentage of maximal deAMPylation level obtained in the absence of Ca 2+ . All data have p-values <0.05 with respect to the control in the absence of Ca 2+ . The total amount of BiP measured by Coomassie staining in each sample is shown.

Article Snippet: The codon-optimized gene encoding human FICD/HYPE (residues 45-459) carrying an N-terminal 6-His tag followed by SUMO tag was from GeneArt Gene Synthesis and cloned into a pET151/D-TOPO vector (ThermoFisher Scientific).

Techniques: Fluorescence, Staining, Mutagenesis, Purification, Concentration Assay