title: Cooperative binding of LysM domains determines the carbohydrate affinity of a bacterial endopeptidase protein.
abstract: Cellulose, chitin and peptidoglycan are major long-chain carbohydrates in living organisms, and constitute a substantial fraction of the biomass. Characterization of the biochemical basis of dynamic changes and degradation of these β,1-4-linked carbohydrates is therefore important for both functional studies of biological polymers and biotechnology. Here, we investigated the functional role of multiplicity of the carbohydrate-binding lysin motif (LysM) domain that is found in proteins involved in bacterial peptidoglycan synthesis and remodelling. The Bacillus subtilis peptidoglycan-hydrolysing NlpC/P60 D,L-endopeptidase, cell wall-lytic enzyme associated with cell separation, possesses four LysM domains. The contribution of each LysM domain was determined by direct carbohydrate-binding studies in aqueous solution with microscale thermophoresis. We found that bacterial LysM domains have affinity for N-acetylglucosamine (GlcNac) polymers in the lower-micromolar range. Moreover, we demonstrated that a single LysM domain is able to bind carbohydrate ligands, and that LysM domains act additively to increase the binding affinity. Our study reveals that affinity for GlcNAc polymers correlates with the chain length of the carbohydrate, and suggests that binding of long carbohydrates is mediated by LysM domain cooperativity. We also show that bacterial LysM domains, in contrast to plant LysM domains, do not discriminate between GlcNAc polymers, and recognize both peptidoglycan fragments and chitin polymers with similar affinity. Finally, an Ala replacement study suggested that the carbohydrate-binding site in LysM-containing proteins is conserved across phyla.
authors.affiliation: Department of Molecular Biology and Genetics, Centre for Carbohydrate Recognition and Signalling, Aarhus University, Denmark.
authors.forename: Jaslyn E M M
authors.initials: JE
authors.lastname: Wong
authors.forename: Husam M A B
authors.initials: HM
authors.lastname: Alsarraf
authors.forename: Jørn Døvling
authors.initials: JD
authors.lastname: Kaspersen
authors.forename: Jan Skov
authors.initials: JS
authors.lastname: Pedersen
authors.forename: Jens
authors.initials: J
authors.lastname: Stougaard
authors.forename: Søren
authors.initials: S
authors.lastname: Thirup
authors.forename: Mickaël
authors.initials: M
authors.lastname: Blaise
mesh_terms: D001412:Bacillus subtilis / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002686:Chitin / Q000378:metabolism; D009088:Mucoproteins / Q000378:metabolism; D010457:Peptidoglycan / Q000378:metabolism; D017434:Protein Structure, Tertiary
chemical_list: D001426:Bacterial Proteins; D009088:Mucoproteins; D010457:Peptidoglycan; C010393:lysin, gastropoda; D002686:Chitin
keywords: NlpC/P60 endopeptidase; autolysin; chitin; lysin motif (LysM) domain; peptidoglycan