Extended Data Fig. 4: Analysis of Cap2 mutants and epitope-tagged CD-NTase and evidence that Cap2 conjugates the CD- NTase C-terminus to a target.
From: An E1–E2 fusion protein primes antiviral immune signalling in bacteria
(a) Efficiency of plating of the indicated phage when infecting E. coli expressing CBASS with the indicated genotype. Data plotted as in Fig. 1b. (b) Efficiency of plating of the indicated phage when infecting E. coli expressing CBASS with the indicated genotype. Data plotted as in Fig. 1b. (c) Western blot analysis of cell lysates from E. coli expressing CBASS with the indicated genotypes demonstrating that the mutations do not affect expression levels. (d) Western blot analysis of cell lysates from E. coli expressing CBASS with the indicated genotypes demonstrating that the mutations do not affect protein expression levels. (e) SDS-PAGE analysis of E. cloacae Cap2 activity assay. The indicated genotypes of His6-Cap2 and the CD-NTase were expressed from a single plasmid and the formation of a CD-NTase–His6-Cap2 conjugate was used as an indicator of Cap2 activity. (-): no CD-NTase; (+): wild-type CD-NTase; (∆C): CD-NTase lacking its C-terminal 19 residues; C.D. Cap2: C548A/C109A. Blue asterisk indicates a putative intermediate with CD-NTase thioester-linked to the Cap2 E1 catalytic cysteine (C548). The formation of a CD-NTase–Cap2 conjugate in the absence of a functional E1 catalytic cysteine (C548A) indicates that in vitro, this residue is dispensable for catalysis and the nearby E2 catalytic cysteine (C109) can function in instead. (f) Cap2 E1 active site (yellow) in Cap2–CD-NTase cryoEM structure with the residues mutated in (a) indicated and the E1 active-site cysteine residue (C548 for Cap2) shown as a sphere and labeled. The CD-NTase C-terminus (orange) conjugated to AMP (black). (g) Left: SDS-PAGE analysis of Ni2+-purified E. cloacae His6-Cap2, expressed either alone or with full-length CD-NTase. Right: Protease treatment (TEV or Cap3) of the CD-NTase–His6-Cap2 conjugate. (h) Schematic of the inferred CD-NTase–His6-Cap2 conjugate formed upon coexpression of E. cloacae His6-Cap2 and CD-NTase, with cleavage sites for Cap3 and TEV protease indicated. (i) SDS-PAGE analysis with detection by coomassie (left) or αHA western blot (right) of αHA immunoprecipitated E. cloacae Cap2 coexpressed with HA-CD-NTase. C.D. Cap2: C548A/C109A. Red asterisk indicates band used for tryptic mass spectrometry analysis in (f-g). (j) Peptides detected in tryptic mass spectrometry of the marked band in (e), showing conjugation of CD-NTase to the N-terminus of a second HA-CD-NTase molecule. See Supplementary Table 7 for mass spectrometry data. (k) Collision-induced dissociation mass spectrum of the peptide indicated in (f), with b1 peak indicated (mass of 350.1533 is that of Met+(H+)+(Phe-Ala)). (l) SDS-PAGE analysis of Ni2+-purified E. cloacae His6-Cap2 with CD-NTase with the indicated genotype.
