期刊
CHEMISTRY & BIOLOGY
卷 7, 期 7, 页码 529-543出版社
CELL PRESS
DOI: 10.1016/S1074-5521(00)00136-8
关键词
analytical ultracentrifugation; chitin recognition; hevein domains; microcalorimetry; NMR; protein-carbohydrate interactions
Background: Many plants respond to pathogenic attack by producing defense proteins that are capable of reversible binding to chitin, a polysaccharide present in the cell wall of fungi and the exoskeleton of insects. Most of these chitin-binding proteins include a common structural motif of 30 to 43 residues organized around a conserved four-disulfide core, known as the 'hevein domain' or 'chitin-binding' motif. Although a number of structural and thermodynamic studies on hevein-type domains have been reported, these studies do not clarify how chitin recognition is achieved. Results: The specific interaction of hevein with several (GlcNAc)(n) oligomers has been studied using nuclear magnetic resonance (NMR), analytical ultracentrifugation and isothermal titration microcalorimetry (ITC). The data demonstrate that hevein binds (GlcNAc)(2-4) in 1:1 stoichiometry with millimolar; affinity. In contrast, for (GlcNAc)(5), a significant increase in binding affinity is observed. Analytical ultracentrifugation studies on the hevein-(GlcNAc)(5,8) interaction allowed detection of protein-carbohydrate complexes with a ratio of 2:1 in solution. NMR structural studies on the hevein-(GlcNAc)(5) complex showed the existence of an extended binding site with at least five GlcNAc units directly involved in protein-sugar contacts. Conclusions: The first detailed structural model for the hevein-chitin complex is presented on the basis of the analysis of NMR data. The resulting model, in combination with ITC and analytical ultracentrifugation data, conclusively shows that recognition of chitin by hevein domains is a dynamic process, 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 polysaccharide chain length. The biological process is multivalent.
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