Structure of the N-terminal Regulatory Domain of a Plant NADPH Oxidase and Its Functional Implications

Takashi Oda,Hiroshi Hashimoto,Naoyuki Kuwabara,Satoko Akashi,Kokoro Hayashi,Chojiro Kojima,Hann Ling Wong,Tsutomu Kawasaki,Ko Shimamoto,Mamoru Sato,Toshiyuki Shimizu

doi:10.1074/jbc.m109.058909

Takashi Oda, Hiroshi Hashimoto + Show 9 more

Open Access

https://doi.org/10.1074/jbc.m109.058909

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Abstract

Plant NADPH oxidases (Rboh, for respiratory burst oxidase homolog) produce reactive oxygen species that are key regulators of various cellular events including plant innate immunity. Rbohs possess a highly conserved cytoplasmic N-terminal region containing two EF-hand motifs that regulate Rboh activity. Rice (Oryza sativa) RbohB (OsRbohB) is regulated by the direct binding of a small GTPase (Rac1) to this regulatory region as well as by Ca(2+) binding to the EF-hands. Here, we present the atomic structure of the N-terminal region of OsRbohB. The structure reveals that OsRbohB forms a unique dimer stabilized by swapping the EF-hand motifs. We identified two additional EF-hand-like motifs that were not predicted from sequence data so far. These EF-hand-like motifs together with the swapped EF-hands form a structure similar to that found in calcineurin B. We observed conformational changes mediated by Ca(2+) binding to only one EF-hand. Structure-based in vitro pulldown assays and NMR titration experiments defined the OsRac1 binding interface within the coiled-coil region created by swapping the EF-hands. In addition, we demonstrate a direct intramolecular interaction between the N and C terminus, and that the complete N-terminal cytoplasmic region is required for this interaction. The structural features and intramolecular interactions characterized here might be common elements shared by Rbohs that contribute to the regulation of reactive oxygen species production.

Highlights

reactive oxygen species (ROS) are generated by plant NADPH oxidases and genes termed rboh that encode homologs of the mammalian NADPH oxidase catalytic subunit gp91phox, have been isolated from many plant species including rice, Arabidopsis, tobacco, and potato [2,3,4,5,6,7]
Unlike the mammalian gp91phox, all plant Rboh proteins carry a cytosolic N-terminal region with two EF-hands that bind Ca2ϩ (Fig. 1A) [3, 8]. These motifs are absent from the mammalian gp91phox, they are present in non-phagocytic NADPH oxidases such as Nox5, Duox1, and Duox2 [9, 10]
It has been reported that Ca2ϩ activated the Arabidopsis NADPH oxidase, RHD2 (AtRbohC), to produce ROS at the growing tip of root hairs

Summary

Introduction

Unlike the mammalian gp91phox, all plant Rboh proteins carry a cytosolic N-terminal region with two EF-hands that bind Ca2ϩ (Fig. 1A) [3, 8]. We present the first crystal structure of the N-terminal region of OsRbohB, revealing that two molecules form a dimer with swapped EF-hands and that OsRbohB contains two additional EF-hand-like motifs so far not predicted from sequence analysis.

Results

Conclusion