Structure of the N-terminal regulatory domain of a plant NADPH oxidase and its functional implications.
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.
Graduate School of Nanobioscience, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan.
Takashi
T
Oda
Hiroshi
H
Hashimoto
Naoyuki
N
Kuwabara
Satoko
S
Akashi
Kokoro
K
Hayashi
Chojiro
C
Kojima
Hann Ling
HL
Wong
Tsutomu
T
Kawasaki
Ko
K
Shimamoto
Mamoru
M
Sato
Toshiyuki
T
Shimizu
D020816:Amino Acid Motifs / Q000502:physiology; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D019255:NADPH Oxidases / Q000737:chemistry* / Q000378:metabolism; D012275:Oryza / Q000201:enzymology*; D010940:Plant Proteins / Q000737:chemistry* / Q000378:metabolism; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D017382:Reactive Oxygen Species / Q000737:chemistry / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000737:chemistry / Q000378:metabolism
D010940:Plant Proteins; D017382:Reactive Oxygen Species; D019255:NADPH Oxidases; D020830:rac1 GTP-Binding Protein; D002118:Calcium