Sensitivity-enhanced solid-state NMR detection of expansin's target in plant cell walls.
Structure determination of protein binding to noncrystalline macromolecular assemblies such as plant cell walls (CWs) poses a significant structural biology challenge. CWs are loosened during growth by expansin proteins, which weaken the noncovalent network formed by cellulose, hemicellulose, and pectins, but the CW target of expansins has remained elusive because of the minute amount of the protein required for activity and the complex nature of the CW. Using solid-state NMR spectroscopy, combined with sensitivity-enhancing dynamic nuclear polarization (DNP) and differential isotopic labeling of expansin and polysaccharides, we have now determined the functional binding target of expansin in the Arabidopsis thaliana CW. By transferring the electron polarization of a biradical dopant to the nuclei, DNP allowed selective detection of (13)C spin diffusion from trace concentrations of (13)C, (15)N-labeled expansin in the CW to nearby polysaccharides. From the spin diffusion data of wild-type and mutant expansins, we conclude that to loosen the CW, expansin binds highly specific cellulose domains enriched in xyloglucan, whereas more abundant binding to pectins is unrelated to activity. Molecular dynamics simulations indicate short (13)C-(13)C distances of 4-6 Å between a hydrophobic surface of the cellulose microfibril and an aromatic motif on the expansin surface, consistent with the observed NMR signals. DNP-enhanced 2D (13)C correlation spectra further reveal that the expansin-bound cellulose has altered conformation and is enriched in xyloglucan, thus providing unique insight into the mechanism of CW loosening. DNP-enhanced NMR provides a powerful, generalizable approach for investigating protein binding to complex macromolecular targets.
Department of Chemistry and Ames Laboratory, Iowa State University, Ames, IA 50011.
Tuo
T
Wang
Yong Bum
YB
Park
Marc A
MA
Caporini
Melanie
M
Rosay
Linghao
L
Zhong
Daniel J
DJ
Cosgrove
Mei
M
Hong
D017360:Arabidopsis / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002247:Carbon Isotopes / Q000032:analysis; D002473:Cell Wall / Q000737:chemistry* / Q000378:metabolism; D002482:Cellulose / Q000032:analysis* / Q000378:metabolism; D005936:Glucans / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D020894:Microfibrils / Q000378:metabolism*; D056004:Molecular Dynamics Simulation; D009587:Nitrogen Isotopes / Q000032:analysis; D010940:Plant Proteins / Q000378:metabolism*; D014990:Xylans / Q000378:metabolism*
D002247:Carbon Isotopes; D005936:Glucans; D009587:Nitrogen Isotopes; D010940:Plant Proteins; D014990:Xylans; C095825:expansin protein, plant; C029353:xyloglucan; D002482:Cellulose
CBM; carbohydrate-binding module