Masaru Tanokura

Kinesin-1 walks along microtubules by alternating ATP hydrolysis and movement of its two motor domains (“head”). The detached head preferentially binds to the forward tubulin-binding site after ATP binds to the microtubule-bound head, but the mechanism preventing premature microtubule binding while the partner head awaits ATP remains unknown. Here, we examined the role of the neck linker, the segment connecting two heads, in this mechanism. Structural analyses of the nucleotide-free head revealed a bulge just ahead of the neck linker’s base, creating an asymmetric constraint on its mobility. While the neck linker can stretch freely backward, it must navigate around this bulge to extend forward. We hypothesized that increased neck linker tension suppresses premature binding of the tethered head, which was supported by molecular dynamics simulations and single-molecule fluorescence assays. These findings demonstrate a tension-dependent allosteric mechanism that coordinates the movement of two heads, where neck linker tension modulates the allosteric conformational changes rather than directly affecting the nucleotide state.

DNA-binding with one-finger (Dof) proteins are a family of plant-specific transcription factors distinguished by the highly conserved Dof DNA-binding domain. Various members play crucial roles in diverse plant biological processes. However, it remains unclear how the Dof domain recognizes a restricted set of promoters for gene regulation by binding to just four nucleotides, AAAG/CTTT. Here we present the crystal structure of the Dof domain of CYCLING DOF FACTOR 1 (CDF1), a well-characterized Dof protein acting as a transcriptional repressor by binding to the CONSTANS promoter to regulate photoperiodic flowering, in complex with DNA containing two cis elements. The data reveal that the Dof domain exhibits a unique zinc ribbon fold that includes a three-stranded antiparallel β-sheet and a carboxy-terminal loop, enabling DNA recognition accompanied by directional expansion of the major groove. These features facilitate binding to contiguous target cis elements in a proper arrangement to effectively regulate gene expression.

The smoke-derived butenolides, karrikins (KARs), regulate many aspects of plant growth and development. However, KARs and a plant hormone, strigolactones (SLs), have high resemblance in signal perception and transduction, making it hard to delineate KARs response due to the shortage of chemical-genetic tools. Here, we identify a triazole urea KK181N1 as an inhibitor of the KARs receptor KAI2. KK181N1 selectively depress the KAR-induced phenotypes in Arabidopsis. We further elucidate the antagonistic, KAI2 binding mechanism of KK181N1, showing that KK181N1 binds to the catalytic pockets of KAI2 in a non-covalent binding manner. Our experiments also demonstrate the binding affinity of triazole urea compounds are regulated by the structured water molecule networks. By fine-tuning this network, we successfully develop a more potent derivative of KK181N1. We anticipate that these chemicals will be applicable to the elucidation of KARs biology, especially for discriminating the molecular and physiological aspects of KARs and SL signaling.

SUMMARY: Brassinosteroids (BRs) are plant steroid hormones that regulate plant development and environmental responses. BIL1/BZR1, a master transcription factor that regulates approximately 3000 genes in the BR signaling pathway, is transported to the nucleus from the cytosol in response to BR signaling; however, the molecular mechanism underlying this process is unknown. Here, we identify a novel BR signaling factor, BIL7, that enhances plant growth and positively regulates the nuclear accumulation of BIL1/BZR1 in Arabidopsis thaliana . BIL7 ‐overexpressing plants were resistant to the BR biosynthesis inhibitor Brz and taller than wild‐type (WT) plants were due to increased cell division. BIL7 is mainly localized to the plasma membrane, but during the early stages of cell growth, it was also localized to the nucleus. BIL7 was directly phosphorylated by the kinase BIN2, and nuclear localization of BIL7 was enhanced by the BIN2 inhibitor bikinin. BIL7 was found to bind to BIL1/BZR1, and nuclear accumulation of BIL1/BZR1 was strongly enhanced by BIL7 overexpression. Finally, double overexpression of BIL1/BZR1 and BIL7 led to greatly elongated hypocotyls in the presence of Brz. These findings suggest that BIL7 mediates nuclear accumulation of BIL1/BZR1, which activates inflorescence elongation in plants via BR signaling.

RNA silencing is a post-transcriptional gene-silencing mechanism mediated by microRNAs (miRNAs). However, the regulatory mechanism of RNA silencing during viral infection is unclear. TAR RNA-binding protein (TRBP) is an enhancer of RNA silencing that induces miRNA maturation by interacting with the ribonuclease Dicer. TRBP interacts with a virus sensor protein, laboratory of genetics and physiology 2 (LGP2), in the early stage of viral infection of human cells. Next, it induces apoptosis by inhibiting the maturation of miRNAs, thereby upregulating the expression of apoptosis regulatory genes. In this study, we show that TRBP undergoes a functional conversion in the late stage of viral infection. Viral infection resulted in the activation of caspases that proteolytically processed TRBP into two fragments. The N-terminal fragment did not interact with Dicer but interacted with type I interferon (IFN) signaling modulators, such as protein kinase R (PKR) and LGP2, and induced ER stress. The end results were irreversible apoptosis and suppression of IFN signaling. Our results demonstrate that the processing of TRBP enhances apoptosis, reducing IFN signaling during viral infection.

Chloroplast development adapts to the environment for performing suitable photosynthesis. Brassinosteroids (BRs), plant steroid hormones, have crucial effects on not only plant growth but also chloroplast development. However, the detailed molecular mechanisms of BR signaling in chloroplast development remain unclear. Here, we identify a regulator of chloroplast development, BPG4, involved in light and BR signaling. BPG4 interacts with GOLDEN2-LIKE (GLK) transcription factors that promote the expression of photosynthesis-associated nuclear genes (PhANGs), and suppresses their activities, thereby causing a decrease in the amounts of chlorophylls and the size of light-harvesting complexes. BPG4 expression is induced by BR deficiency and light, and is regulated by the circadian rhythm. BPG4 deficiency causes increased reactive oxygen species (ROS) generation and damage to photosynthetic activity under excessive high-light conditions. Our findings suggest that BPG4 acts as a chloroplast homeostasis factor by fine-tuning the expression of PhANGs, optimizing chloroplast development, and avoiding ROS generation.

NADPH oxidases/RBOHs catalyze apoplastic ROS production and act as key signaling nodes, integrating multiple signal transduction pathways regulating plant development and stress responses. Although RBOHs have been suggested to be activated by Ca ²⁺ binding and phosphorylation by various protein kinases, a mechanism linking Ca ²⁺ binding and phosphorylation in the activity regulation remained elusive. Chitin-triggered ROS production required cytosolic Ca ²⁺ elevation and Ca ²⁺ binding to MpRBOHB in a liverwort Marchantia polymorpha . Heterologous expression analysis of truncated variants revealed that a segment of the N-terminal cytosolic region highly conserved among land plant RBOHs encompassing the two EF-hand motifs is essential for the activation of MpRBOHB. Within the conserved regulatory domain, we have identified two Ser residues whose phosphorylation is critical for the activation in planta . Isothermal titration calorimetry analyses revealed that phosphorylation of the two Ser residues increased the Ca ²⁺ binding affinity of MpRBOHB, while Ca ²⁺ binding is indispensable for the activation, even if the two Ser residues are phosphorylated. Our findings shed light on a mechanism through which phosphorylation potentiates the Ca ²⁺ -dependent activation of MpRBOHB, emphasizing the pivotal role of Ca ²⁺ binding in mediating the Ca ²⁺ and phosphorylation-driven activation of MpRBOHB, which is likely to represent a fundamental mechanism conserved among land plant RBOHs.

Lenalidomide, an immunomodulatory drug (IMiD), is commonly used as a first-line therapy in many haematological cancers, such as multiple myeloma (MM) and 5q myelodysplastic syndromes (5q MDS), and it functions as a molecular glue for the protein degradation of neosubstrates by CRL4^CRBN. Proteolysis-targeting chimeras (PROTACs) using IMiDs with a target protein binder also induce the degradation of target proteins. The targeted protein degradation (TPD) of neosubstrates is crucial for IMiD therapy. However, current IMiDs and IMiD-based PROTACs also break down neosubstrates involved in embryonic development and disease progression. Here, we show that 6-position modifications of lenalidomide are essential for controlling neosubstrate selectivity; 6-fluoro lenalidomide induced the selective degradation of IKZF1, IKZF3, and CK1α, which are involved in anti-haematological cancer activity, and showed stronger anti-proliferative effects on MM and 5q MDS cell lines than lenalidomide. PROTACs using these lenalidomide derivatives for BET proteins induce the selective degradation of BET proteins with the same neosubstrate selectivity. PROTACs also exert anti-proliferative effects in all examined cell lines. Thus, 6-position-modified lenalidomide is a key molecule for selective TPD using thalidomide derivatives and PROTACs.

Plants respond to severe temperature changes by inducing the expression of numerous genes whose products enhance stress tolerance and responses. Dehydration-responsive element (DRE)–binding protein 1/C-repeat binding factor (DREB1/CBF) transcription factors act as master switches in cold-inducible gene expression. Since DREB1 genes are rapidly and strongly induced by cold stress, the elucidation of the molecular mechanisms of DREB1 expression is vital for the recognition of the initial responses to cold stress in plants. A previous study indicated that the circadian clock–related MYB-like transcription factors REVEILLE4/LHY-CCA1-Like1 (RVE4/LCL1) and RVE8/LCL5 directly activate DREB1 expression under cold stress conditions. These RVEs function in the regulation of circadian clock–related gene expression under normal temperature conditions. They also activate the expression of HSF-independent heat-inducible genes under high-temperature conditions. Thus, there are thought to be specific regulatory mechanisms whereby the target genes of these transcription factors are switched when temperature changes are sensed. We revealed that NIGHT LIGHT–INDUCIBLE AND CLOCK-REGULATED (LNK) proteins act as coactivators of RVEs in cold and heat stress responses in addition to regulating circadian-regulated genes at normal temperatures. We found that among the four Arabidopsis LNKs, LNK1 and LNK2 function under normal and high-temperature conditions, and LNK3 and LNK4 function under cold conditions. Thus, these LNK proteins play important roles in inducing specific genes under different temperature conditions. Furthermore, LNK3 and LNK4 are specifically phosphorylated under cold conditions, suggesting that phosphorylation is involved in their activation.