title: The Target of β-Expansin EXPB1 in Maize Cell Walls from Binding and Solid-State NMR Studies.
abstract: The wall-loosening actions of β-expansins are known primarily from studies of EXPB1 extracted from maize (Zea mays) pollen. EXPB1 selectively loosens cell walls (CWs) of grasses, but its specific binding target is unknown. We characterized EXPB1 binding to sequentially extracted maize CWs, finding that the protein primarily binds glucuronoarabinoxylan (GAX), the major matrix polysaccharide in grass CWs. This binding is strongly reduced by salts, indicating that it is predominantly electrostatic in nature. For direct molecular evidence of EXPB1 binding, we conducted solid-state nuclear magnetic resonance experiments using paramagnetic relaxation enhancement (PRE), which is sensitive to distances between unpaired electrons and nuclei. By mixing13C-enriched maize CWs with EXPB1 functionalized with a Mn2+tag, we measured Mn2+-induced PRE Strong1H and13C PREs were observed for the carboxyls of GAX, followed by more moderate PREs for carboxyl groups in homogalacturonan and rhamnogalacturonan-I, indicating that EXPB1 preferentially binds GAX In contrast, no PRE was observed for cellulose, indicating very weak interaction of EXPB1 with cellulose. Dynamics experiments show that EXPB1 changes GAX mobility in a complex manner: the rigid fraction of GAX became more rigid upon EXPB1 binding while the dynamic fraction became more mobile. Combining these data with previous results, we propose that EXPB1 loosens grass CWs by disrupting noncovalent junctions between highly substituted GAX and GAX of low substitution, which binds cellulose. This study provides molecular evidence of β-expansin's target in grass CWs and demonstrates a new strategy for investigating ligand binding for proteins that are difficult to express heterologously.
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 (T.W., M.H.); and.|Department of Biology, Pennsylvania State University, University Park, Pennsylvania 16802 (Y.C., A.T., D.J.C.).
authors.forename: Tuo
authors.initials: T
authors.lastname: Wang
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 (T.W., M.H.); and.|Department of Biology, Pennsylvania State University, University Park, Pennsylvania 16802 (Y.C., A.T., D.J.C.).
authors.forename: Yuning
authors.identifier: 0000-0002-8730-6218
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 (T.W., M.H.); and.|Department of Biology, Pennsylvania State University, University Park, Pennsylvania 16802 (Y.C., A.T., D.J.C.).
authors.forename: Akira
authors.identifier: 0000-0001-9012-3562
authors.initials: A
authors.lastname: Tabuchi
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 (T.W., M.H.); and meihong@mit.edu dcosgrove@psu.edu.|Department of Biology, Pennsylvania State University, University Park, Pennsylvania 16802 (Y.C., A.T., D.J.C.) meihong@mit.edu dcosgrove@psu.edu.
authors.forename: Daniel J
authors.identifier: 0000-0002-4020-5786
authors.initials: DJ
authors.lastname: Cosgrove
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 (T.W., M.H.); and meihong@mit.edu dcosgrove@psu.edu.|Department of Biology, Pennsylvania State University, University Park, Pennsylvania 16802 (Y.C., A.T., D.J.C.) meihong@mit.edu dcosgrove@psu.edu.
authors.forename: Mei
authors.initials: M
authors.lastname: Hong
mesh_terms: D002122:Calcium Chloride / Q000494:pharmacology; D002247:Carbon Isotopes; D002473:Cell Wall / Q000378:metabolism*; D005109:Extracellular Matrix / Q000187:drug effects / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008345:Manganese / Q000378:metabolism; D008958:Models, Molecular; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D019346:Sodium Acetate / Q000494:pharmacology; D003313:Zea mays / Q000166:cytology* / Q000378:metabolism*
chemical_list: D002247:Carbon Isotopes; D010940:Plant Proteins; D011134:Polysaccharides; C095825:expansin protein, plant; D008345:Manganese; D019346:Sodium Acetate; D002122:Calcium Chloride