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Crystallization, neutron data collection, initial structure refinement and analysis of a xyloglucan heptamer bound to an engineered carbohydrate‐binding module from xylanase
Author(s) -
Ohlin Mats,
von Schantz Laura,
Schrader Tobias E.,
Ostermann Andreas,
Logan Derek T.,
Fisher S. Zoë
Publication year - 2015
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 37
ISSN - 2053-230X
DOI - 10.1107/s2053230x15011383
Subject(s) - xyloglucan , carbohydrate , neutron diffraction , xylanase , carbohydrate binding module , crystallography , chemistry , protein data bank (rcsb pdb) , protein crystallization , crystallization , biochemistry , protein structure , protein data bank , enzyme , glycoside hydrolase , crystal structure , organic chemistry
Carbohydrate‐binding modules (CBMs) are discrete parts of carbohydrate‐hydrolyzing enzymes that bind specific types of carbohydrates. Ultra high‐resolution X‐ray crystallographic studies of CBMs have helped to decipher the basis for specificity in carbohydrate–protein interactions. However, additional studies are needed to better understand which structural determinants confer which carbohydrate‐binding properties. To address these issues, neutron crystallographic studies were initiated on one experimentally engineered CBM derived from a xylanase, X‐2 L110F, a protein that is able to bind several different plant carbohydrates such as xylan, β‐glucan and xyloglucan. This protein evolved from a CBM present in xylanase Xyn10A of Rhodothermus marinus . The protein was complexed with a branched xyloglucan heptasaccharide. Large single crystals of hydrogenous protein (∼1.6 mm 3 ) were grown at room temperature and subjected to H/D exchange. Both neutron and X‐ray diffraction data sets were collected to 1.6 Å resolution. Joint neutron and X‐ray refinement using phenix.refine showed significant density for residues involved in carbohydrate binding and revealed the details of a hydrogen‐bonded water network around the binding site. This is the first report of a neutron structure of a CBM and will add to the understanding of protein–carbohydrate binding interactions.