Identifying critical unrecognized sugar-protein interactions in GH10 xylanases from Geobacillus stearothermophilus using STD NMR

¹H solution NMR spectroscopy is used synergistically with 3D crystallographic structures to map experimentally significant hydrophobic interactions upon substrate binding in solution under thermodynamic equilibrium. Using saturation transfer difference spectroscopy (STD NMR), a comparison is made between wild-type xylanase XT6 and its acid/base catalytic mutant E159Q - a non-active, single-heteroatom alteration that has been previously utilized to measure binding thermodynamics across a series of xylooligosaccharide-xylanase complexes [Zolotnitsky et al. (2004) Proc Natl Acad Sci USA 101, 11275-11280). In this study, performing STD NMR of one substrate screens binding interactions to two proteins, avoiding many disadvantages inherent to the technique and clearly revealing subtle changes in binding induced upon mutation of the catalytic Glu. To visualize and compare the binding epitopes of xylobiose-xylanase complexes, a 'SASSY' plot (saturation difference transfer spectroscopy) is used. Two extraordinarily strong, but previously unrecognized, non-covalent interactions with H₂-5 of xylobiose were observed in the wild-type enzyme but not in the E159Q mutant. Based on the crystal structure, these interactions were assigned to tryptophan residues at the -1 subsite. The mutant selectively binds only the β-xylobiose anomer. The ¹H solution NMR spectrum of a xylotriose-E159Q complex displays non-uniform broadening of the NMR signals. Differential broadening provides a unique subsite assignment tool based on structural knowledge of face-to-face stacking with a conserved tyrosine residue at the +1 subsite. The results obtained herein by substrate-observed NMR spectroscopy are discussed further in terms of methodological contributions and mechanistic understanding of substrate-binding adjustments upon a charge change in the E159Q construct. Saturation transfer difference ¹H NMR spectroscopy epitope maps are used to compare a wild type xylanase with a non-catalytic mutant. Critical hydrophobic interactions at a highly conserved Trp pocket are exposed in the wild-type enzyme. Mutation induced anomeric specificity is revealed and interpreted in terms of looser binding. A novel subsite assignment tool based on aromatic shielding is introduced.