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

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. (Fig. 1A) (3, 8). Although these motifs are absent from the mammalian gp91 phox , they are present in non-phagocytic NADPH oxidases such as Nox5, Duox1, and Duox2 (9,10).Rboh is stimulated directly by Ca 2ϩ , likely through the N-terminal region (11). Recently, it has been reported that Ca 2ϩ activated the Arabidopsis NADPH oxidase, RHD2 (AtRbohC), to produce ROS at the growing tip of root hairs. Moreover, Ca 2ϩ binding to EF-hand motifs is required for activation of ROS production by RHD2 (12). Recent work has also demonstrated that calcium-dependent protein kinases activate potato RbohB (StRbohB) protein by phosphorylation of the N-terminal region (13), and that Ca 2ϩ binding and phosphorylation synergistically activate ROS production by AtRbohD (14). These results demonstrate that the extended N-terminal region plays a key role in the regulation of Rboh.OsRac1, a Rac GTPase located in the plasma membrane, functions as a positive regulator of NADPH oxidase activity and is involved in defense responses in rice (15,16). In fact, constitutively active and dominant-negative forms of OsRac1 were found to activate and suppress ROS production, respectively. Direct interaction between the constitutively active OsRac1 and the N-terminal region of OsRbohB was verified by yeast two-hybrid analysis, in vitro pulldown assays, NMR titration experiments, and in vivo fluorescence resonance energy trans- 81-45-508-7365; E-mail: shimizu@tsurumi.yokohama-cu.ac.jp. 2 The abbreviations used are: ROS, reactive oxygen species; GST, glutathione S-transferase; GMP-P(NH)P, gu...

Clathrin and Membrane Microdomains Cooperatively Regulate RbohD Dynamics and Activity in Arabidopsis

“…Additionally, we found that the number of bleaching steps ranged from one to two, indicating that GFP-RbohD at the plasma membrane exists in multiple oligomeric forms, including monomers and dimers. This is consistent with previous crystallographic studies (Oda et al, 2010), which reported that the active conformation of OsRbohB is a dimer and that monomers interact via their terminal cytoplasmic domains. The dispersion of the diffusion coefficient distribution and the different oligomeric states suggest that GFP-RbohD is highly mobile and heterogeneously distributed at the plasma membrane.…”

Plasma Membrane Microdomains Are Essential for Rac1-RbohB/H-Mediated Immunity in Rice

“…OsRbohB acts downstream of Os-Rac1 through a direct interaction of its N-terminal EF-hand motif with GTP-bound Os-Rac1 (Wong et al, 2007;Oda et al, 2010;Kosami et al, 2014), and, as shown in this study, Os-RbohH is also involved in Rac1-mediated immunity. Proteome analyses have identified Rbohs in DRMs of tobacco, Arabidopsis and rice (Morel et al, 2006;Kierszniowska et al, 2009;Fujiwara et al, 2009), and ROS production by Rbohs in pollen tube tips is dependent on PM microdomains in Picea meyeri (Liu et al, 2009).…”

“…Os-Rac1 interacts with the N-terminal EF-hand motif of Os-RbohB, leading to an increase in ROS production (Wong et al, 2007;Oda et al, 2010;Kosami et al, 2014). To understand whether PM microdomains are necessary for ROS production mediated by the Rac1-Rboh pathway, we measured ROS production in OsFAH1/2-KD using the L-012 reagent.…”

Reactive oxygen species and stress signaling in plants

“…В результате происходит образование активно-го димера, приводящее к усилению генерации АФК [20]. Позднее на примере одной из форм мембраносвязанной субъединицы NADPH оксидазы риса (OsRBOHB) было показано на-личие в Nтерминальной области EFрук, ука-зывающих на формирование димера с участием кальция [43]. Таким образом, повышение актив-ности NADPHоксидазы с участием кальция, повидимому, связано не только с активацией им протеинкиназы, но и с прямым взаимодействи-ем Ca 2+ с каталитической субъединицей.…”