Fig. 4: ThsB1 interacts with Mhp to recruit ThsB2.
From: Bacterial TIR-based immune systems sense phage capsids to initiate defense
a, SDS–PAGE analysis of purified His6-ThsB1, ThsB2-His6 or Mhp, and His6-ThsB1 incubated with ThsB2-His6 (left). Native PAGE analysis of ThsB1, ThsB2 and Mhp with and without NAD+. Each lane is labelled with the molar ratio ThsB:Mhp (right). The strong bands that appear ~100 kDa in the presence of ThsB1–THsB2–Mhp and without NAD+ probably represent nonspecific aggregates or the ThsB1–Mhp complex that should form in the absence of NAD+. Free ThsB1 is expected but may not separate clearly from the other higher-order structures. b, SEC analysis of the Mhp–ThsB1–ThsB2 complex. Fractions corresponding to peaks marked as a,b and c were collected for SDS–PAGE analysis. c, SDS–PAGE analysis of purified His6-ThsB1, ThsB2-His6 or Mhp, and SEC fractions a, b and c. Molecular weight (MW) in kDa of protein markers are shown. d, Coomassie Blue-stained SDS–PAGE of proteins isolated from staphylococci expressing hexahystidyl- (H) tagged versions of either ThsB1 or ThsB2, uninfected or infected with Φ80α-vir, after cobalt resin affinity chromatography. Protein molecular weight (kDa) markers are shown. e, Coomassie Blue-stained SDS–PAGE of proteins isolated from staphylococci expressing hexahystidyl- (H) or 3× Flag- (F) tagged versions of ThsB1 and ThsB2 in the absence and presence of wild type, V273A or W84K Mhp expressed from a plasmid, after cobalt resin affinity chromatography.
