Sensitivity-enhanced solid-state NMR detection of expansin's target in plant cell walls

Journal Article · Thu Aug 29 00:00:00 EDT 2013 · Proceedings of the National Academy of Sciences of the United States of America

DOI:https://doi.org/10.1073/pnas.1316290110· OSTI ID:1127457

Wang, Tuo; Park, Yong Bum; Caporini, Marc A.; Rosay, Melanie; Zhong, Linghao; Cosgrove, Daniel J.; Hong, Mei

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 ¹³C spin diffusion from trace concentrations of ¹³C, ¹⁵N-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 ¹³C-¹³C 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 ¹³C 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.

• Research Organization: Ames Lab., Ames, IA (United States)
• Sponsoring Organization: USDOE Office of Science (SC)
• DOE Contract Number: AC02-07CH11358
• OSTI ID: 1127457
• Report Number(s): IS-T-8152
• Journal Information: Proceedings of the National Academy of Sciences of the United States of America, Vol. 110, Issue 41; ISSN 0027-8424
• Publisher: National Academy of Sciences, Washington, DC (United States)
• Country of Publication: United States
• Language: English