Structural and Functional Insights into Peptidoglycan Access for the Lytic Amidase LytA of Streptococcus pneumoniae

A muropeptide fragment comprising a pentasaccharide motif fits snugly within the Y-shaped substrate-binding crevice of LytA^AMI. (A) Surface of LytA^AMI, with the Y-shaped substrate-binding crevice colored in green and the three contiguous subregions α, β, and γ indicated. The catalytic Zn²⁺ ion is displayed as a pink sphere, and the locations of key substrate-interacting residues are marked. (B) A molecular model of a PG fragment consisting of five sugars (MurNAc-GlcNAc-MurNAc-GlcNAc-MurNAc, with a stem peptide linked to the central MurNAc) was placed within the substrate-binding crevices of LytA^AMI. The position of the substrate fragment M5P within the active site was first obtained by superposing the crystal structure of LytA^AMI on PGRP-Iα in complex with M5P (Protein Data Bank [PDB] accession number 2APH). Sugar chains and peptide stems were thereafter added to both ends of M5P in order to create the M(GM5P) GM fragment. The surface is colored according to electrostatic potentials; purple represents acidic and red basic residues. (C) The PG motif M(GM5P) GM fits snugly into the binding crevice of AmiE, taking the same conformation as in the molecular model of LytA. (D) The surface representation of the crystal structure of AmpD in complex with the reaction product reveals significant differences in the structural shapes and the electrostatic potentials of the substrate-binding crevice.

A motif corresponding to the tetrasaccharide muropeptide substrate (di-GM5P) is cleaved by LytA. (A) Incubation of the GM5P substrate with LytA or inactive LytA-H133A does not result in any product peak, as demonstrated by the HPAEC-PAD analysis. The chromatogram for GM5P incubated in buffer alone is provided for comparison. The masses of the substrate peak were determined using MALDI-TOF MS (right panels). Calculated masses and a cartoon of the corresponding substrate are shown (lower panels). (B) Incubation of the tetra-saccharide substrate di-GM5P (peak 1) with either wt-LytA or Lyt^AMI resulted in the production of two new peaks (2 and 3), as determined by HPAEC-PAD. In contrast, no peaks appeared following incubation with inactive LytA-H133A. The profile of di-GM5P incubated with buffer alone is also provided as a reference. The masses of the substrate and of each product peak were determined using MALDI-TOF MS (right panels). Calculated masses of the substrate (di-GM5P) and products (GMGM5P and 5P) and corresponding cartoons are shown (lower panels).

The C-terminal domain of phage LytA contains six choline-binding sites. (A) Two perpendicular views of the crystal structure of p-LytA^CBD, comprising residues 173 to 318. The numbering of the β-strands, starting with number 8, reflects their positions in the full-length LytA (numbers 1 to 7 in the amidase domain). The β-hairpin formed by β8 and β9 constitute the last-step, solenoid-shaped choline-binding domain, which faces the amidase domain on its N-terminal side. (B) The classical choline-binding site, localized between residues Trp178 and Tyr185, was not occupied by a choline molecule in the crystal, most likely due to a crystal packing artifact. However, a clear electron density for choline was present in the second nonclassical choline-binding site formed by residues Trp186, Tyr194, and Tyr214. The 1σ electron density in the 2F_o-F_c omit map is displayed in blue. (C) The dimer of p-LytA^CBD, generated along the crystallographic 2-fold axis, indicates the possibility for simultaneous binding of 12 choline molecules. Orange boxes represent consensus choline binding sites that were not occupied by cholines in the crystal structure.