Journal of Biological Chemistry
Volume 289, Issue 41, 10 October 2014, Pages 28569-28578
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Plant Biology
The Crystal Structure of the Plant Small GTPase OsRac1 Reveals Its Mode of Binding to NADPH Oxidase*

https://doi.org/10.1074/jbc.M114.603282Get rights and content
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Rac/Rop proteins are Rho-type small GTPases that act as molecular switches in plants. Recent studies have identified these proteins as key components in many major plant signaling pathways, such as innate immunity, pollen tube growth, and root hair formation. In rice, the Rac/Rop protein OsRac1 plays an important role in regulating the production of reactive oxygen species (ROS) by the NADPH oxidase OsRbohB during innate immunity. However, the molecular mechanism by which OsRac1 regulates OsRbohB remains unknown. Here, we report the crystal structure of OsRac1 complexed with the non-hydrolyzable GTP analog guanosine 5′-(β,γ-imido)triphosphate at 1.9 Å resolution; this represents the first active-form structure of a plant small GTPase. To elucidate the ROS production in rice cells, structural information was used to design OsRac1 mutants that displayed reduced binding to OsRbohB. Only mutations in the OsRac1 Switch I region showed attenuated interactions with OsRbohB in vitro. In particular, Tyr39 and Asp45 substitutions suppressed ROS production in rice cells, indicating that these residues are critical for interaction with and activation of OsRbohB. Structural comparison of active-form OsRac1 with AtRop9 in its GDP-bound inactive form showed a large conformational difference in the vicinity of these residues. Our results provide new insights into the molecular mechanism of the immune response through OsRac1 and the various cellular responses associated with plant Rac/Rop proteins.

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*

This work was supported in part by Grants-in-aid for science research (to I. O., Y. K., C. K., and K. S.), the Targeted Proteins Research Program (to C. K. and K. S.), a Ministry of Agriculture, Forestry and Fisheries Genomics for Agricultural Innovation grant (to K. S.), Grants-in-aid for JSPS Fellows (to K. K. and M. N.), and the Platform for Drug Discovery, Informatics, and Structural Life Science from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

The atomic coordinates and structure factors (code 4U5X) have been deposited in the Protein Data Bank (http://wwpdb.org/).

This article contains supplemental Figs. S1–S6.

This work is dedicated to Ko Shimamoto, who died September 28, 2013.

1

These authors contributed equally to this work.

4

The abbreviations used are:

    ROS

    reactive oxygen species

    GEF

    guanine nucleotide exchange factor

    CA

    constitutively activated

    DN

    dominant-negative

    GMPPNP

    guanosine 5′-(β,γ-imido)triphosphate

    BisTris

    2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol

    PDB

    Protein Data Bank

    HSQC

    heteronuclear single quantum coherence

    GTPγS

    guanosine 5′-O-(thiotriphosphate).