Extended Data Fig. 6: Structural comparison of RTCB catalytic centers.
(a) Detailed view of the interaction of the CTD of PYROXD1 with the catalytic center of RTCB. Bound magnesium ions are depicted as green spheres. (b) Structural superposition of the PYROXD1 CTD onto the catalytic cleft of DNA-bound PhRtcB (PDB: 7LFQ)28. Steric clashes are indicated by a dotted circle. (c) Structural superposition of the PYROXD1-RTCB complex and GMP-bound RTCB (PDB: 7P3B)9. (d) Structural superposition of the PYROXD1 CTD with the catalytic center of GMP-bound human RTCB (PDB: 7P3B)9. Clashes are indicated by a dotted circle. (e) Structural comparison of the position of the CTD of PYROXD1 in the catalytic center of guanylylated human RTCB (PDB: 8ODO)10. Clashes are indicated by a dotted circle. (f) Fraction of guanylylated RTCB in pull-down samples, as determined by LC-MS. Guanylylation levels are indicated for three independent replicates. (g) Representative mass spectra of the input, unbound (supernatant) and bound (elution) fractions. Mass and intensity of each peak are indicated. Red asterisk indicates a mass peak corresponding to GSH-modified RTCB. (h) In vitro ligation assay to determine the effects of guanylylation on the oxidative inactivation of RTCB. The ligation assay was performed in a three-step procedure, where RTCB was incubated with GTP and Archease (see labels on the left), followed by incubation with increasing concentrations of H2O2 in the presence or absence of PYROXD1. In the last steps all samples were supplemented with GTP and Archease to allow for multiple-turnover ligation of an RNA substrate. This experiment was repeated three times with similar results. (i) Co-precipitation of PYROXD1 by immobilized StrepII-GFP-RTCB in the presence of GTP, GMP or Archease. Strep-Tactin beads were washed to remove unbound PYROXD1, and bound proteins were analyzed by SDS-PAGE and Coomassie blue staining. This experiment was repeated three times with similar results.
