Structural basis for chitin recognition by defense proteins: GlcNAc residues are bound in a multivalent fashion by extended binding sites in hevein domains

Background: Many plants respond to pathogenic attack by producing defense p roteins that are capable of reversible binding to chitin, a polysaccharide present in the cell wall of fungi and the exoskeleton of insects. Most of t hese chitin-binding proteins include a common structural motif of 30 to 43 residues organized around a conserved four-disulfide core, known as the 'he vein domain' or 'chitin-binding' motif. Although a number of structural and thermodynamic studies on hevein-type domains have been reported, these stu dies do not clarify how chitin recognition is achieved. Results: The specific interaction of hevein with several (GlcNAc)(n) oligom ers has been studied using nuclear magnetic resonance (NMR), analytical ult racentrifugation and isothermal titration microcalorimetry (ITC). The data demonstrate that hevein binds (GlcNAc)(2-4) in 1:1 stoichiometry with milli molar; affinity. In contrast, for (GlcNAc)(5), a significant increase in bi nding affinity is observed. Analytical ultracentrifugation studies on the h evein-(GlcNAc)(5,8) interaction allowed detection of protein-carbohydrate c omplexes with a ratio of 2:1 in solution. NMR structural studies on the hev ein-(GlcNAc)(5) complex showed the existence of an extended binding site wi th at least five GlcNAc units directly involved in protein-sugar contacts. Conclusions: The first detailed structural model for the hevein-chitin comp lex is presented on the basis of the analysis of NMR data. The resulting mo del, in combination with ITC and analytical ultracentrifugation data, concl usively shows that recognition of chitin by hevein domains is a dynamic pro cess, which is not exclusively restricted to the binding of the nonreducing end of the polymer as previously thought. This allows chitin to bind with high affinity to a variable number of protein molecules, depending on the p olysaccharide chain length. The biological process is multivalent.