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Peptidoglycan binding: Calcium-free killing

PSI-SGKB [doi:10.1038/fa_psisgkb.2010.22]
Featured Article - June 2010
Short description: NMR spectroscopy reveals the molecular basis for Ca2+-independent recognition by bactericidal Reg family lectins.

Human REG3A (also known as HIP/PAP) is directly bactericidal for Gram-positive bacteria. It is highly expressed in the small intestine and a member of the mammalian Reg (regenerating) family proteins. These proteins contain a C-type lectin-like domain (CTLD) and bind to bacterial cell wall peptidoglycan. Despite having a CTLD, Reg proteins lack the conserved sequences required for Ca2+-dependent carbohydrate binding that are found in other lectins. Lora Hooper and colleagues now identify a motif in Reg proteins that is essential for binding of the peptidoglycan carbohydrate backbone, and propose a model to explain how the bacterial surface is specifically recognised even in the presence of soluble peptidoglycan fragments.

The CTLD of REG3A has a typical structure with two loop regions. In other lectins, a tripeptide EPN motif in Loop 2 contacts 3- and 4-OH groups of mannose or N-acetylglucosamine (GlcNAc) polysaccharides. Although this Loop 2 EPN binding is Ca2+ dependent, REG3A binds mannose and GlcNAc without calcium, and its Loop 2 lacks the EPN motif.

Using solution nuclear magnetic resonance (NMR) spectroscopy, the authors discovered that REG3A contains a Loop 1 EPN sequence, rather than Loop 2. Soluble peptidoglycan was titrated into labelled REG3A, and chemical shift perturbations of backbone amide resonances were measured. These indicate changes in the environment of specific residues, allowing binding sites to be mapped.

Primary sequence alignments showed that the Loop 1 EPN motif is conserved among Ca2+-independent mannose and GlcNAc-binding lectins, and absent in their Ca2+-dependent counterparts. Mutational analysis confirmed that it is critical for peptidoglycan binding activity by REG3A, and for successful bacterial killing.

To investigate the binding preferences of the REG3A EPN motif, the authors used analogs of the peptidoglycan carbohydrate backbone and fractions derived from native peptidoglycan and determined that binding affinity is dictated by polysaccharide chain length. This explains how shorter peptidoglycan fragments, which are constantly shed by intestinal bacteria, do not out-compete REG3A for binding to the bacterial surface. The peptide moiety did not form a significant part of the REG3A–peptidoglycan interaction.

Mucin–binding lectins, which have high affinity for extended rather than monovalent ligands, have been previously shown to 'bind and slide' from sugar to sugar in a manner that relates chain length to binding affinity. The authors propose that REG3A and other mammalian Loop 1 EPN-containing Reg proteins use a similar dynamic recognition process. This enables high-affinity binding to the bacterial cell wall and emphasizes that the clustered in vivo presentation of carbohydrate epitopes is critical for recognition by lectins.

Emma Leah

References

  1. R. E. Lehotzky et al. Molecular basis for peptidoglycan recognition by a bactericidal lectin.
    Proc. Natl Acad. Sci. USA 107, 7722-7727 (2010). doi:10.1073/pnas.0909449107

Structural Biology Knowledgebase ISSN: 1758-1338
Funded by a grant from the National Institute of General Medical Sciences of the National Institutes of Health