Engineering molecular recognition of endoxylanase enzymes and their inhibitors through phage display

J Mol Recognit. Mar-Apr 2007;20(2):103-12. doi: 10.1002/jmr.818.

Abstract

Specific binding of interacting proteins generally depends on a limited set of amino acid residues located at the contact interface. We have applied a phage-display-based screening method to simultaneously evaluate the role of multiple residues of endo-beta-1,4-xylanase enzymes in conferring binding specificity towards two different endoxylanase inhibitors. Seven residues of the two beta-strand 'thumb' region of Trichoderma longibrachiatum endo-beta-1,4-xylanase XynII were targeted for randomization. The generated combinatorial library representing 62,208 site-directed variants was displayed on the surface of filamentous phage and selected against xylanase inhibitor protein (XIP) and Triticum aestivum xylanase inhibitor (TAXI). DNA sequence analysis of phagemid panning isolates provided information on the occurrence of particular amino acids at distinct positions. In particular, residues at positions 124 (Asn) and 131 (Thr) were found to be critical for specific inhibitor binding. These residue predictions derived from the combinatorial exploration of the thumb region and accompanying sequence analyses were experimentally confirmed by testing the inhibitor sensitivity of a limited set of recombinantly expressed XynII mutants. In addition, we successfully altered the inhibition susceptibility of the bacterial Bacillus subtilis endoxylanase XynA from XIP-insensitive to XIP-sensitive.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Endo-1,4-beta Xylanases / antagonists & inhibitors*
  • Endo-1,4-beta Xylanases / chemistry*
  • Endo-1,4-beta Xylanases / genetics
  • Endo-1,4-beta Xylanases / metabolism
  • Enzyme Inhibitors / chemical synthesis*
  • Models, Biological
  • Models, Molecular
  • Molecular Sequence Data
  • Mutant Proteins / analysis
  • Peptide Library*
  • Protein Binding / drug effects
  • Protein Engineering / methods*
  • Trichoderma / enzymology
  • Triticum / enzymology

Substances

  • Enzyme Inhibitors
  • Mutant Proteins
  • Peptide Library
  • Endo-1,4-beta Xylanases