Fig. 6: Specific recognition of cUMP by PycTIR proteins.
From: Structural and functional characterization of cyclic pyrimidine-regulated anti-phage system
a, b Enlarged view of the ligand-binding pocket of (a) cUMP-bound PsPycTIRCNBD and (b) cAMP-bound EcCRP. The hydrogen-bonding interactions between them are shown in black dashed lines. The specificity-determining residues (N143 for uracil and S128 for adenine) are labeled with an asterisk (*). c Sequence alignment of CRP proteins with PycTIR proteins. The conserved residues interacting with cyclic mononucleotides are indicated by triangles (▼). The specificity-determining residue of PycTIR proteins and CRP proteins is indicated by an asterisk (*). d NAD+ cleavage activity of PsPycTIR under different cUMP concentrations (0, 0.15625, 0.3125, 0.625, 1.25, 2.5, 5, 10 μM). Source data are provided as a Source Data file. e NAD+ cleavage activity of PsPycTIR in the absence or presence of different cyclic mononucleotides at a concentration of 10 μM. Source data are provided as a Source Data file. f NAD+ cleavage activity of wild-type and single mutant R138A, N143A, N143S and E114R of PsPycTIR in the absence or presence of 10 μM cUMP. Source data are provided as a Source Data file. The data in (d–f) were shown as mean ± standard deviation for n = 3 independent replicates. g The growth curves of E. coli cells overexpressing PsPycTIRWT (red), E. coli cells overexpressing R138A mutant of PsPycTIR (blue), and E. coli cells expressing N143A mutant of PsPycTIR (green). All of them were supplemented with 5 mM cUMP to medium. E. coli cells with empty vector serve as a negative control (black). Source data are provided as a Source Data file. h The growth curves of E. coli cells overexpressing wild-type PsPysar system containing PsPycC and PsPycTIR (red), E. coli cells overexpressing PsPysar system containing PsPycC and PsPycTIRN143A (green), E. coli cells overexpressing PsPysar system containing PsPycC and PsPycTIRE114R (cyan), and E. coli cells carrying empty vector (black) with T2 phage infection at an MOI of 0.01. The E. coli cells harboring PsPycsar system or empty vector without T2 phage infection serve as negative controls. Source data are provided as a Source Data file. The experiments in (g) and (h) were performed for n = 3 biological replicates and each of them is shown.
