Cooperative binding of LysM domains determines the carbohydrate affinity of a bacterial endopeptidase protein

<jats:p>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 (<jats:styled-content style="fixed-case">L</jats:styled-content>ys<jats:styled-content style="fixed-case">M</jats:styled-content>) domain that is found in proteins involved in bacterial peptidoglycan synthesis and remodelling. The <jats:italic><jats:styled-content style="fixed-case">B</jats:styled-content>acillus subtilis</jats:italic> peptidoglycan‐hydrolysing <jats:styled-content style="fixed-case">N</jats:styled-content>lp<jats:styled-content style="fixed-case">C</jats:styled-content>/<jats:styled-content style="fixed-case">P</jats:styled-content>60 <jats:sc>d</jats:sc>,<jats:sc>l</jats:sc>‐endopeptidase, cell wall‐lytic enzyme associated with cell separation, possesses four <jats:styled-content style="fixed-case">L</jats:styled-content>ys<jats:styled-content style="fixed-case">M</jats:styled-content> domains. The contribution of each <jats:styled-content style="fixed-case">L</jats:styled-content>ys<jats:styled-content style="fixed-case">M</jats:styled-content> domain was determined by direct carbohydrate‐binding studies in aqueous solution with microscale thermophoresis. We found that bacterial <jats:styled-content style="fixed-case">L</jats:styled-content>ys<jats:styled-content style="fixed-case">M</jats:styled-content> domains have affinity for N‐acetylglucosamine (GlcNac) polymers in the lower‐micromolar range. Moreover, we demonstrated that a single <jats:styled-content style="fixed-case">L</jats:styled-content>ys<jats:styled-content style="fixed-case">M</jats:styled-content> domain is able to bind carbohydrate ligands, and that <jats:styled-content style="fixed-case">L</jats:styled-content>ys<jats:styled-content style="fixed-case">M</jats:styled-content> domains act additively to increase the binding affinity. Our study reveals that affinity for <jats:styled-content style="fixed-case">G</jats:styled-content>lc<jats:styled-content style="fixed-case">NA</jats:styled-content>c polymers correlates with the chain length of the carbohydrate, and suggests that binding of long carbohydrates is mediated by <jats:styled-content style="fixed-case">L</jats:styled-content>ys<jats:styled-content style="fixed-case">M</jats:styled-content> domain cooperativity. We also show that bacterial <jats:styled-content style="fixed-case">L</jats:styled-content>ys<jats:styled-content style="fixed-case">M</jats:styled-content> domains, in contrast to plant <jats:styled-content style="fixed-case">L</jats:styled-content>ys<jats:styled-content style="fixed-case">M</jats:styled-content> domains, do not discriminate between <jats:styled-content style="fixed-case">G</jats:styled-content>lc<jats:styled-content style="fixed-case">NA</jats:styled-content>c polymers, and recognize both peptidoglycan fragments and chitin polymers with similar affinity. Finally, an Ala replacement study suggested that the carbohydrate‐binding site in <jats:styled-content style="fixed-case">L</jats:styled-content>ys<jats:styled-content style="fixed-case">M</jats:styled-content>‐containing proteins is conserved across phyla.</jats:p>