Figure 1. Substrate recognition by the catalytic module of LytB.

(A) Schematic representation of the modular nature of LytB is shown. The 18 repeats (R1–R18) composing the choline-binding module of LytB are labeled. The position of the catalytic residue E585 is indicated by a triangle.

(B) Apo structure of the complete catalytic module of LytB in its closed conformation. The three domains building the catalytic module are colored differently and labeled. The catalytic E585 residue is represented as capped sticks and labeled. The calcium ion found attached to the SH3b domain is represented as a red sphere and coordinating residues as capped sticks.

(C) Detailed view of the differences in the catalytic loop between the closed (salmon) and the open (gray) conformations in the apo state. Some relevant residues are represented as capped sticks and labeled. Polar contacts are represented as dotted lines.

(D) Three-dimensional structure of the LytB_cat:NAG₄ complex in its open conformation, with NAG₄ depicted in capped sticks colored by atom type (green for the carbons). Sites occupied by the ligand are labeled.

(E) Detailed view of substrate recognition by LytB as observed in the LytB_cat-E585Q:(NAG-NAM)₂ complex. Substrate spanning from site −3 to +2 is depicted as capped sticks colored by atom type (green for carbon). Relevant active-site residues are given in capped sticks (colored white for carbons) and labeled. Hydrogen-bond interactions are represented as dotted lines.

(F) LytB_cat:PG fragment complex model in its closed conformation. Peptide stems and glycan chains are colored by atom type with yellow and dark green for carbon atoms, respectively.

(G) Phase-contrast microscopy images of WT, lytB-GH73-Y₆₃₅A, lytB-SH3b-K₄₂₆E, lytB-GH73-2Mut (Y₆₀₆A/D₆₀₇K), lytB-GH73-3Mut (Y₆₅₄A/S₆₅₆A/D₆₅₇K), lytB-GH73-E₅₈₅A, lytB-GH73-E₅₈₅Q, lytB-WW-5Mut (Y₄₇₇A/E₄₇₉K/Y₄₈₆A/Y₄₈₈A/Y₅₁₁A), and ΔlytB cells. Scale bar, 2 μm.

(H) Percentage of cells with a chaining phenotype (minimum four cells per chain), and n indicates the number of cells scored from three independent experiments. The error bar and the data points overlapping the histogram (mean of three experiments) represent the SEM and the mean of each experiment, respectively. Statistical comparison was done with one-way ANOVA with Tukey’s multiple comparison test. ****p < 0.0001 and ns, not significant, p > 0.05.