Structure of the putative 32 kDa myrosinase-binding protein from Arabidopsis (At3g16450.1) determined by SAIL-NMR

Authors


M. Kainosho, Graduate School of Science, Institute for Advanced Research, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan
Fax: +81 52 747 6433
Tel: +81 52 747 6474
E-mail: kainosho@nagoya-u.jp

J. L. Markley, Center for Eukaryotic Structural Genomics, Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, WI 53706 1344, USA
Fax: +1 608 262 3759
Tel: +1 608 263 9349
E-mail: markley@nmrfam.wisc.edu

Abstract

The product of gene At3g16450.1 from Arabidopsis thaliana is a 32 kDa, 299-residue protein classified as resembling a myrosinase-binding protein (MyroBP). MyroBPs are found in plants as part of a complex with the glucosinolate-degrading enzyme myrosinase, and are suspected to play a role in myrosinase-dependent defense against pathogens. Many MyroBPs and MyroBP-related proteins are composed of repeated homologous sequences with unknown structure. We report here the three-dimensional structure of the At3g16450.1 protein from Arabidopsis, which consists of two tandem repeats. Because the size of the protein is larger than that amenable to high-throughput analysis by uniform 13C/15N labeling methods, we used stereo-array isotope labeling (SAIL) technology to prepare an optimally 2H/13C/15N-labeled sample. NMR data sets collected using the SAIL protein enabled us to assign 1H, 13C and 15N chemical shifts to 95.5% of all atoms, even at a low concentration (0.2 mm) of protein product. We collected additional NOESY data and determined the three-dimensional structure using the cyana software package. The structure, the first for a MyroBP family member, revealed that the At3g16450.1 protein consists of two independent but similar lectin-fold domains, each composed of three β-sheets.

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