Insights into pneumococcal fratricide from the crystal structures of the modular killing factor LytC

Journal name:
Nature Structural & Molecular Biology
Volume:
17,
Pages:
576–581
Year published:
DOI:
doi:10.1038/nsmb.1817
Received
Accepted
Published online

Abstract

The first structure of a pneumococcal autolysin, that of the LytC lysozyme, has been solved in ternary complex with choline and a pneumococcal peptidoglycan (PG) fragment. The active site of the hydrolase module is not fully exposed but is oriented toward the choline-binding module, which accounts for its unique in vivo features in PG hydrolysis, its activation and its regulatory mechanisms. Because of the unusual hook-shaped conformation of the multimodular protein, it is only able to hydrolyze non–cross-linked PG chains, an assertion validated by additional experiments. These results explain the activation of LytC by choline-binding protein D (CbpD) in fratricide, a competence-programmed mechanism of predation of noncompetent sister cells. The results provide the first structural insights to our knowledge into the critical and central function that LytC plays in pneumococcal virulence and explain a long-standing puzzle of how murein hydrolases can be controlled to avoid self-lysis during bacterial growth and division.

At a glance

Figures

  1. 3D structure of LytC-choline-PG ternary complex.
    Figure 1: 3D structure of LytC–choline–PG ternary complex.

    (a) Stereoview representation of the general structure of LytC in complex with choline and a purified PG fragment. CBM is formed by the NI domain (repeats p1–p9) and the NII domain (repeats p10 and p11), colored in blue and green, respectively. Seven choline molecules (spheres) are bound to the choline-binding sites. The pneumococcal PG fragment (green sticks) is bound to the catalytic module (red). Loop Lc (residues 364–381) is labeled. (b) 3D structure of a choline-binding site in LytC. (c) 3D structure of GYMA sites indicates that they are formed by six aromatic residues from three consecutive repeats. In panels b and c, choline molecules and protein residues forming the cavity are depicted as capped sticks and are colored in magenta and white, respectively. (d) Details of PG recognition by LytC. The stereoview shows the interactions between LytC and a pneumococcal PG fragment. The residues forming the active site are drawn as capped sticks. Carbon atoms of the ligand are in green. Hydrogen bonds are shown as dashed lines.

  2. Localization of GFP-LytC, properties of Q1 chimeric protein, and differential behavior between LytC and Cpl-1.
    Figure 2: Localization of GFP-LytC, properties of Q1 chimeric protein, and differential behavior between LytC and Cpl-1.

    (a) Subcellular localization of LytC by fluorescence image of the GFP-LytC fusion protein added to the pneumococcal culture. (b) Schematic representation of the Q1 chimeric protein. The modular construction of the parental proteins is represented by different colors: red and orange for catalytic modules of LytC and Cpl-1, respectively; deep blue and green for repeats of the choline-binding motifs of LytC, and light blue for repeats of the choline-binding motifs of Cpl-1. The relevant enzymatic properties of the parental and chimeric proteins are presented. (c) Increase of in vitro LytC activity by the pretreatment of pneumococcal cell walls with small amounts of LytA added to cell walls before the action of LytC or Cpl-1.

  3. Proposed interaction between LytC and PG substrate attached to teichoic acid chains.
    Figure 3: Proposed interaction between LytC and PG substrate attached to teichoic acid chains.

    (a) Details of the active site of LytC with the bound PG fragment (green sticks). Choline molecule attached to the site formed by repeats p7 and p8 is about 9 Å away from the PG bound to the active site. Loop Lc (residues 364–381) is labeled. (b) Computational model of PG-TA moiety bound to LytC active site based on the crystal structure of the LytC–choline–PG ternary complex and the NMR structure of a TA chain from S. pneumoniae R6 strain15. The docked choline moieties are represented as spheres.

  4. Superimposition of LytC-choline-PG and Cpl-1-(2S5P)2 crystallographic complexes onto larger PG framework as deduced by its NMR structure.
    Figure 4: Superimposition of LytC–choline–PG and Cpl-1–(2S5P)2 crystallographic complexes onto larger PG framework as deduced by its NMR structure22.

    (a) Molecular surface of Cpl-1 catalytic module is colored in light blue and the CBM in light brown. PG model as determined by NMR is drawn with glycan chains as orange spheres and peptide-stems as green spheres. The (2S5P)2 PG analog bound to Cpl-1 (ref. 19) (PDB 2J8G) is drawn as black sticks. In the superimposed region NMR glycan chains are semitransparent for clarity. (b) LytC molecular surface is represented by modules colored as in panel a. Catalytic modules of Cpl-1 and LytC are in the same orientation, with the PG model superimposed as in panel a. PG fragment bound to LytC is drawn as black sticks. Left, CBM of LytC is not fully depicted (the black area) to appreciate steric encumbrance with peptide stems of PG that would exist in a crosslinked arrangement. The presence of the cross-linked peptide would abrogate binding.

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References

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Author information

Affiliations

  1. Grupo de Cristalografía Macromolecular y Biología Estructural, Instituto Química-Física Rocasolano, Consejo Superior de Investigaciones Científicas, Madrid, Spain.

    • Inmaculada Pérez-Dorado,
    • Reyes Sanles,
    • Martín Martínez-Ripoll &
    • Juan A Hermoso
  2. Departamento de Microbiología Molecular, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain.

    • Ana González,
    • María Morales,
    • José L García &
    • Pedro García
  3. Centro de Investigación Biomédica en Red de Enfermedades Respiratorias, Bunyola, Mallorca, Illes Balears, Spain.

    • Ana González,
    • María Morales &
    • Pedro García
  4. Mikrobielle Genetik, Universität Tübingen, Tübingen, Germany.

    • Waldemar Striker &
    • Waldemar Vollmer
  5. Department of Chemistry and Biochemistry, University of Notre Dame, South Bend, Indiana, USA.

    • Shahriar Mobashery
  6. Present addresses: Medical Research Council Laboratory of Molecular Biology, Cambridge, UK (I.P.-D.), Biological Chemistry and Drug Discovery, College of Life Science, University of Dundee, Dundee, UK (W.S.) and Center for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne, UK (W.V.).

    • Inmaculada Pérez-Dorado,
    • Waldemar Striker &
    • Waldemar Vollmer

Contributions

I.P.-D. performed crystallization and structural determination; A.G. and M.M. performed biochemical experiments; R.S. performed crystallization; W.S. and W.V. purified the PG; S.M. synthetized the PG ligands and wrote the manuscript; M.M.-R. wrote the manuscript; J.L.G., P.G. and J.A.H. conceived the study and wrote the manuscript.

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The authors declare no competing financial interests.

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