Identifying Critical Unrecognized Sugar - Protein Interactions in GH10 Xylanases from Geobacillus stearothermophilus Using STD NMR.
Related Articles Identifying Critical Unrecognized Sugar - Protein Interactions in GH10 Xylanases from Geobacillus stearothermophilus Using STD NMR.
FEBS J. 2013 Jul 17;
Authors: Balazs YS, Lisitsin E, Carmiel O, Shoham G, Shoham Y, Schmidt A
Abstract
(1) H solution NMR spectroscopy is used synergistically with three-dimensional 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 previously utilized to measure binding thermodynamics across a series of xylooligosaccharide - xylanase complexes (Zolonitsky, et*al., PNAS (2004) 101: 11275). In this study, STD NMR of one substrate screens binding interactions to two different 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 introduced. Two extraordinarily strong, yet previously unrecognized, non-covalent interactions with H2 -5 of xylobiose are observed in the wild-type enzyme and lacking in the E159Q mutant. Based on the crystal structure, these interactions are assigned to tryptophan residues at the -1 subsite. The mutant selectively binds only the ?-xylobiose anomer. The (1) H solution NMR spectrum of a xylotriose - E159Q complex, displays non-uniform broadening of the NMR signals. This 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 further discussed both in terms of methodological contributions and mechanistic understanding of substrate binding adjustments upon a charge change in the E159Q construct. This article is protected by copyright. All rights reserved.
PMID: 23863045 [PubMed - as supplied by publisher]
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