Interactions defining the specificity between fungal xylanases and the xylanase-inhibiting protein XIP-I from wheat

Ruth Flatman, W. Russell McLauchlan, Nathalie Juge, Caroline Furniss, Jean Guy Berrin, Richard K. Hughes, Paloma Manzanares, John E. Ladbury, Ronan O'Brien, Gary Williamson

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We previously reported on the xylanase-inhibiting protein I (XIP-I) from wheat [McLauchlan, Garcia-Conesa, Williamson, Roza, Ravestein and Maat (1999), Biochem, J. 338, 441-446]. In the present study, we show that XIP-I inhibits family-10 and -11 fungal xylanases. Ihe K1 values for fungal xylanases ranged from 3.4 to 610 nM, but bacterial family-10 and -11 xylanases were not inhibited. Unlike many glycosidase inhibitors, XIP-I was not a slow-binding inhibitor of the Aspergillus niger xylanase. Isothermal titration calorimetry of the XIP-I-A. niger xylanase complex showed the formation of a stoichiometric (1:1) complex with a heat capacity change of -1.38 kJ·mol-1·K-1, leading to a predicted buried surface area of approx. 2200 ± 500 Å2 at the complex interface. For this complex with A. niger xylanase (Ki = 320 nM at pH 5.5), titration curves indicated that an observable interaction occurred at pH 4-7, and this was consistent with the pH profile of inhibition of activity. In contrast, the stronger complex between A. nidulans xylanase and XIP-I (Ki = 9 nM) led to an observable interaction across the entire pH range tested (3-9). Using surface plasmon resonance, we show that the differences in the binding affinity of XIP-I for A. niger and A. nidulans xylanase are due to a 200-fold lower dissociation rate koff for the latter, with only a small difference in association rate kon.

Original languageEnglish
Pages (from-to)773-781
Number of pages9
JournalBiochemical Journal
Issue number3
Publication statusPublished - 1 Aug 2002
Externally publishedYes


  • Plant inhibitor
  • Protein-protein interaction

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