The SERK3 elongated allele defines a role for BIR ectodomains in brassinosteroid signalling

Abstract

The leucine-rich repeat receptor kinase (LRR-RK) BRASSINOSTEROID INSENSITIVE 1 (BRI1) requires a shape-complementary SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) co-receptor for brassinosteroid sensing and receptor activation. Interface mutations that weaken the interaction between receptor and co-receptor in vitro reduce brassinosteroid signalling responses. The SERK3 elongated (elg) allele maps to the complex interface and shows enhanced brassinosteroid signalling, but surprisingly no tighter binding to the BRI1 ectodomain in vitro. Here, we report that rather than promoting the interaction with BRI1, the elg mutation disrupts the ability of the co-receptor to interact with the ectodomains of BRI1-ASSOCIATED-KINASE1 INTERACTING KINASE (BIR) receptor pseudokinases, negative regulators of LRR-RK signalling. A conserved lateral surface patch in BIR LRR domains is required for targeting SERK co-receptors and the elg allele maps to the core of the complex interface in a 1.25 Å BIR3-SERK1 structure. Collectively, our structural, quantitative biochemical and genetic analyses suggest that brassinosteroid signalling complex formation is negatively regulated by BIR receptor ectodomains.

Specifying the role of BAK1‐interacting receptor‐like kinase 3 in brassinosteroid signaling

“…Our computational modeling demonstrates that the cytoplasmic domains of BRI1, BAK1 and BIR3 are indeed likely to play a role in the interactions of the whole proteins, providing an explanation for the differential interactions observed for the ectodomains and whole proteins. In line with the interaction of the ectodomains (Hohmann et al ), our analysis shows that BRI1 and BIR3 compete for the overlapping interaction surfaces, but that the interaction between BIR3 and BAK1 is the more favorable one (Figure ). We further demonstrate that BRI1, BAK1 and BIR3‐containing nanoclusters are possible based on our computational simulations.…”

“…Among the known inhibitory mechanisms (Wang et al ; Jaillais et al ; Lin et al ), the BIR3‐related one was of particular interest for us, because it implicated the interaction of BIR3 with both BRI1 and BAK1 (Imkampe et al ). Here, the ectodomains have been shown to play a deciding role in the interaction of BAK1 and BIR3 (Hohmann et al ). However, this does not preclude a role of the cytoplasmic domains in the interactions of the whole proteins, since the experimentally observed interaction of BIR3 and BRI1 (Imkampe et al ) and this study does not appear to be mediated by the ectodomains of these proteins (Hohmann et al ).…”

“…Among the native functions of BIR3 are the stabilization of BAK1 and to keep the BRI1‐BAK1 hetero‐oligomer inactive in the absence of BR or at low BR concentrations, in concert with the other known inhibitory mechanisms (Wang et al ; Jaillais et al ; Lin et al ). Therefore, the loss of BIR3 function would result in a BR hypersensitive phenotype as described for the elg mutant that perturbs interaction of BIRs and BAK1 (Hohmann et al ). Thus, a fine‐tuning of the BIR3 protein amount or interaction capability, for example by post‐translational modification, would provide an elegant possibility for modulating the BRI1‐BAK1 hetero‐oligomer for its quantitative response to BR.…”

“…This suggests that the complex binding energy of the BRI1 BR -BAK1 complex is more negative than the complex binding energy of the BIR3-BAK1 complex, the most stable cytoplasmic complex present in the proposed nanoclusters. Indeed, experiments have demonstrated that BRI1 BL can compete with BIR3 for BAK1 complex formation (Hohmann et al 2018). From a kinetic perspective, the switch of interaction surfaces has to happen rapidly as BR-induced alterations in cell Analysis of BIR3 inhibition of brassinosteroid signaling 7 www.jipb.net XXX 2019 | Volume XXXX | Issue XXXX | XXX-XX physiology such as hyperpolarization of the plasma membrane, cell wall expansion ) and dephosphorylation of BRI1-EMS suppressor (BES1) (He et al 2002) are observed within a few minutes after BR application.…”

“…After application of BR, BIR3 is released from BAK1 and BRI1, allowing downstream signaling (Imkampe et al 2017). This has been substantiated by the interaction study of the extracellular domains (Hohmann et al 2018), where the ectodomains of BAK1 and BIR3 bind with a stoichiometry of 1:1 and the BL-bound ectodomain of BRI1 (BRI1 BL ) can compete with BIR3 for the interaction with BAK1. Interestingly, the ectodomains of BRI1 and BIR3 do not appear to interact in this essay (Hohmann et al 2018), suggesting that transmembrane and cytoplasmic domains also play a role in the experimentally observed interactions of the whole proteins (Imkampe et al 2017).…”