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. 2021 Apr 23;12:2426. doi: 10.1038/s41467-021-22596-0

Fig. 1. Binary adduct formation and complex formation.

Fig. 1

a Schematic representation of covalently tethering FICD to BiP via TReNDs. First, the binary adduct is produced with TReNDs. Subsequently, a covalent ternary complex is formed with BiP in an AMPylation reaction. The ribose is displayed as pentagon, the triazole as circle, and the electrophilic moiety as encircled “+.” b Thiol-reactive nucleotide derivatives (TReNDs) that are used in this study. c Selection of residues (yellow) within FICD suitable for cysteine replacement (based on the structure of FICD E234G:ATP, PDB: 4U0723). d Reactivity of TReNDs toward FICD 102–458 E234G bearing cysteine substitutions resolved by Phos-TagTM SDS-PAGE. TReND-mix consists of TReND-1, TReND-2, and TReND-3 at equimolar concentrations. Representative gels are shown from three independent experiments. e Intact mass spectrometry indicating successful reaction of TReND-2 with FICD 102–458 E234G E404C. The mass deviation of unreacted FICD and FICDTReND-2 is −1 and +2 Da, respectively. Intact mass spectrometric data for all binary adducts of FICD 102–458 E234G E404C is shown in Supplementary Fig. 1. f SDS-PAGE displaying the formation of a covalently linked ternary complex of FICD 102–445 E234G E404CTReND-2 and BiP 19–654. A representative gel is shown from three independent experiments. g SDS-PAGE of the purified ternary complex (using BiP 28–549 T229A and FICD 102–445 T168A T183A E234G L258D E404C) submitted for crystallography. The covalent complex was purified at least two times with similar purity. h Intact mass spectrometry of the purified ternary complex that was used for crystallography. The mass deviation of the ternary complex is −1 Da. Source data are provided as a Source data file.