title: Crystallization, neutron data collection, initial structure refinement and analysis of a xyloglucan heptamer bound to an engineered carbohydrate-binding module from xylanase.
abstract: 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.
authors.affiliation: Department of Immunotechnology, Lund University, Medicon Village, Building 406, 223 81 Lund, Sweden.
authors.forename: Mats
authors.initials: M
authors.lastname: Ohlin
authors.affiliation: Department of Immunotechnology, Lund University, Medicon Village, Building 406, 223 81 Lund, Sweden.
authors.forename: Laura
authors.initials: L
authors.lastname: von Schantz
authors.affiliation: Jülich Centre for Neutron Science (JCNS) at Heinz Maier-Leibnitz Zentrum (MLZ), Forschungszentrum Jülich GmbH, Lichtenbergstrasse 1, 85747 Garching, Germany.
authors.forename: Tobias E
authors.initials: TE
authors.lastname: Schrader
authors.affiliation: Heinz Maier-Leibnitz Zentrum (MLZ), Technische Universität München, Lichtenbergstrasse 1, 85748 Garching, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Ostermann
authors.affiliation: Department of Chemistry, Lund University, PO Box 124, 221 00 Lund, Sweden.
authors.forename: Derek T
authors.initials: DT
authors.lastname: Logan
authors.affiliation: Scientific Activities Division, European Spallation Source, Tunavägen 24, 221 00 Lund, Sweden.
authors.forename: S Zoë
authors.initials: SZ
authors.lastname: Fisher
mesh_terms: D020816:Amino Acid Motifs; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D003001:Cloning, Molecular; D003460:Crystallization; D003461:Crystallography; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005936:Glucans / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D033363:Neutron Diffraction; D011485:Protein Binding; D015202:Protein Engineering; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics; D045245:Rhodothermus / Q000737:chemistry* / Q000201:enzymology; D014990:Xylans / Q000737:chemistry*; D047071:beta-Glucans / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D005936:Glucans; D011993:Recombinant Fusion Proteins; D014990:Xylans; D047071:beta-Glucans; C029353:xyloglucan; D043364:Endo-1,4-beta Xylanases
keywords: carbohydrate-binding module; hydrogen bond; neutron crystallography