In Vivo Function of Tryptophans in the Arabidopsis UV-B Photoreceptor UVR8

Arabidopsis thaliana UV RESISTANCE LOCUS8 (UVR8) is a photoreceptor specifically for UV-B light that initiates photomorphogenic responses in plants. UV-B exposure causes rapid conversion of UVR8 from dimer to monomer, accumulation in the nucleus, and interaction with CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1), which functions with UVR8 in UV-B responses. Studies in yeast and with purified UVR8 implicate several tryptophan amino acids in UV-B photoreception. However, their roles in UV-B responses in plants, and the functional significance of all 14 UVR8 tryptophans, are not known. Here we report the functions of the UVR8 tryptophans in vivo. Three tryptophans in the β-propeller core are important in maintaining structural stability and function of UVR8. However, mutation of three other core tryptophans and four at the dimeric interface has no apparent effect on function in vivo. Mutation of three tryptophans implicated in UV-B photoreception, W233, W285, and W337, impairs photomorphogenic responses to different extents. W285 is essential for UVR8 function in plants, whereas W233 is important but not essential for function, and W337 has a lesser role. Ala mutants of these tryptophans appear monomeric and constitutively bind COP1 in plants, but their responses indicate that monomer formation and COP1 binding are not sufficient for UVR8 function.     

The C‐terminal 17 amino acids of the photoreceptor UVR8 is involved in the fine‐tuning of UV‐B signaling

Plant UV-B responses are mediated by the photoreceptor UV RESISTANCE LOCUS 8(UVR8). In response to UV-B irradiation, UVR8 homodimers dissociate into monomers that bind to the E3 ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENIC1(COP1). The interaction of the C27 domain in the C-terminal tail of UVR8 with the WD40 domain of COP1 is critical for UV-B signaling. However, the function of the last 17 amino acids(C17) of the C-terminus of UVR8, which are adjacent to C27, is unknown, although they are largely conserved in land plants. In this study, we established that Arabidopsis thaliana UVR8 C17 binds to full-length UVR8, but not to COP1, and reduces COP1 binding to the remaining portion of UVR8, including C27. We hypothesized that overexpression of C17 in a wild-type background would have a dominant negative effect on UVR8 activity;however, C17 overexpression caused strong silencing of endogenous UVR8, precluding a detailed analysis. We therefore generated YFP-UVR8~(N423) transgenic lines, in which C17 was deleted, to examine C17 function indirectly. YFP-UVR8~(N423) was more active than YFP-UVR8,suggesting that C17 inhibits UV-B signaling by attenuating binding between C27 and COP1. Our study reveals an inhibitory role for UVR8 C17 in fine-tuning UVR8–COP1 interactions during UV-B signaling.     

Organization of protein complexes under photomorphogenic UV-B in Arabidopsis

Low-fluence and long-wavelength UV-B light promotes photomorphogenic development in Arabidopsis. CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) is a positive regulator in this pathway while it is a negative regulator of the traditional photomorphogenesis triggered by far-red and visible light. We have recently reported the mechanism by which the switch of COP1 function is accomplished in distinct light contexts. In response to photomorphogenic UV-B, the photoactivated UV RESISTANCE LOCUS 8 (UVR8) associates with the COP1- SUPRESSOR OF PHYA (SPA) core complexes, resulting in the physical and functional disassociation of COP1-SPA from the CULLIN4-DAMAGED DNA BINDING PROTEIN 1 (CUL4-DDB1) E3 scaffold. These UV-B dependent UVR8-COP1-SPA complexes promote the stability and activity of ELONGATED HYPOCOTYL 5 (HY5), and eventually cause COP1 to switch from repressing to promoting photomorphogenesis. In addition, it is possible that CUL4-DDB1 might simultaneously recruit alternative DDB1 BINDING WD40 (DWD) proteins to repress this UV-B-specific signaling. Further investigation is required, however, to verify this hypothesis. Overall, the coordinated organization of various protein complexes facilitates an efficient and balanced UV-B signaling.     

光受体UVR8对UV-B响应机制研究进展

来自太阳光谱中的UV-B辐射被认为是一种重要的环境信号,可以被植物感受并诱导植物调整自身生长和发育状态以适应环境。人们对植物中光敏色素、隐花色素和蓝光受体向光素的研究已非常深入,但对植物响应UV-B的机制仅在最近才取得一些突破性进展。这些研究发现,植物中存在着UV-B受体UVR8(UV Resistance Locus 8)。目前认为,UVR8二聚体感应UV-B后瞬间解聚为单体,并与E3泛素连接酶COP1(constitutively photomorphogenic 1)相互作用,从而激活UV-B响应基因的表达。该文从UVR8的发现、UVR8的结构和感受UV-B机制、UVR8二聚体重新形成以及UV-B信号传导与可见光信号传导途径间的差异等方面综述了关于UV-B受体UVR8的最新研究成果。     

Photoactivated UVR8-COP1 Module Determines Photomorphogenic UV-B Signaling Output in Arabidopsis

In Arabidopsis, ultraviolet (UV)-B-induced photomorphogenesis is initiated by a unique photoreceptor UV RESISTANCE LOCUS 8 (UVR8) which utilizes its tryptophan residues as internal chromophore to sense UV-B. As a result of UV-B light perception, the UVR8 homodimer shaped by its arginine residues undergoes a conformational switch of monomerization. Then UVR8 associates with the CONSTITUTIVELY PHOTOMORPHOGENIC 1-SUPPRESSOR OF PHYA (COP1-SPA) core complex(es) that is released from the CULLIN 4-DAMAGED DNA BINDING PROTEIN 1 (CUL4-DDB1) E3 apparatus. This association, in turn, causes COP1 to convert from a repressor to a promoter of photomorphogenesis. It is not fully understood, however, regarding the biological significance of light-absorbing and dimer-stabilizing residues for UVR8 activity in photomorphogenic UV-B signaling. Here, we take advantage of transgenic UVR8 variants to demonstrate that two light-absorbing tryptophans, W233 and W285, and two dimer-stabilizing arginines, R286 and R338, play pivotal roles in UV-B-induced photomorphogenesis. Mutation of each residue results in alterations in UV-B light perception, UVR8 monomerization and UVR8-COP1 association in response to photomorphogenic UV-B. We also identify and functionally characterize two constitutively active UVR8 variants, UVR8^W285A and UVR8^R338A, whose photobiological activities are enhanced by the repression of CUL4, a negative regulator in this pathway. Based on our molecular and biochemical evidence, we propose that the UVR8-COP1 affinity in plants critically determines the photomorphogenic UV-B signal transduction coupling with UVR8-mediated UV-B light perception.     

The Carboxy-Terminal Domain of Erb1 Is a Seven-Bladed ?-Propeller that Binds RNA

Erb1 (Eukaryotic Ribosome Biogenesis 1) protein is essential for the maturation of the ribosomal 60S subunit. Functional studies in yeast and mammalian cells showed that altogether with Nop7 and Ytm1 it forms a stable subcomplex called PeBoW that is crucial for a correct rRNA processing. The exact function of the protein within the process remains unknown. The N-terminal region of the protein includes a well conserved region shown to be involved in PeBoW complex formation whereas the carboxy-terminal half was predicted to contain seven WD40 repeats. This first structural report on Erb1 from yeast describes the architecture of a seven-bladed β-propeller domain that revealed a characteristic extra motif formed by two α-helices and a β-strand that insert within the second WD repeat. We performed analysis of molecular surface and crystal packing, together with multiple sequence alignment and comparison of the structure with other β-propellers, in order to identify areas that are more likely to mediate protein-protein interactions. The abundance of many positively charged residues on the surface of the domain led us to investigate whether the propeller of Erb1 might be involved in RNA binding. Three independent assays confirmed that the protein interacted in vitro with polyuridilic acid (polyU), thus suggesting a possible role of the domain in rRNA rearrangement during ribosome biogenesis.     

Identification of a structural motif that confers specific interaction with the WD40 repeat domain of Arabidopsis COP1

Arabidopsis COP1 is a photomorphogenesis repressor capable of directly interacting with the photomorphogenesis-promoting factor HY5. This interaction between HY5 and COP1 results in targeted deg radation of HY5 by the 26S proteasome. Here we characterized the WD40 repeat domain-mediated interactions of COP1 with HY5 and two new proteins. Mutational analysis of those interactive partners revealed a conserved motif responsible for the interaction with the WD40 domain. This novel motif, with the core sequence V-P-E/D-φ-G (φ = hydrophobic residue) in conjunction with an upstream stretch of 4-5 negatively charged residues, interacts with a defined surface area of the ss-propeller assembly of the COP1 WD40 repeat domain through both hydrophobic and ionic interactions. Several residues in the COP1 WD40 domain that are critical for the interaction with this motif have been revealed. The fact that point mutations either in the COP1 WD40 domain or in the HY5 motif that abolish the interaction between COP1 and HY5 in yeast result in a dramatic reduction of HY5 degradation in transgenic plants validates the biological significance of this defined interaction.     

Structure of the WD40 domain of SCAP from fission yeast reveals the molecular basis for SREBP recognition

The sterol regulatory element-binding protein (SREBP) and SREBP cleavage-activating protein (SCAP) are central players in the SREBP pathway, which control the cellular lipid homeostasis. SCAP binds to SREBP through their carboxyl (C) domains and escorts SREBP from the endoplasmic reticulum to the Golgi upon sterol depletion. A conserved pathway, with the homologues of SREBP and SCAP being Sre1 and Scp1, was identified in fission yeast Schizosaccharomyces pombe. Here we report the in vitro reconstitution of the complex between the C domains of Sre1 and Scp1 as well as the crystal structure of the WD40 domain of Scp1 at 2.1 Å resolution. The structure reveals an eight-bladed β-propeller that exhibits several distinctive features from a canonical WD40 repeat domain. Structural and biochemical characterization led to the identification of two Scp1 elements that are involved in Sre1 recognition, an Arg/Lys-enriched surface patch on the top face of the WD40 propeller and a 30-residue C-terminal tail. The structural and biochemical findings were corroborated by in vivo examinations. These studies serve as a framework for the mechanistic understanding and further functional characterization of the SREBP and SCAP proteins in fission yeast and higher organisms.     

WDR26 Functions as a Scaffolding Protein to Promote Gβγ-mediated Phospholipase C β2 (PLCβ2) Activation in Leukocytes

We have recently identified WDR26 as a novel WD40 repeat protein that binds Gβγ and promotes Gβγ signaling during leukocyte migration. Here, we have determined the mechanism by which WDR26 enhances Gβγ-mediated phospholipase C β2 (PLCβ2) activation in leukocytes. We show that WDR26 not only directly bound Gβγ but also PLCβ2. The binding sites of WDR26 and PLCβ2 on Gβ₁γ₂ were overlapping but not identical. WDR26 used the same domains for binding Gβγ and PLCβ but still formed a signaling complex with Gβγ and PLCβ2 probably due to the fact that WDR26 formed a higher order oligomer through its Lis homology and C-terminal to LisH (LisH-CTLH) and WD40 domains. Additional studies indicated that the formation of higher order oligomers was required for WDR26 to promote PLCβ2 interaction with and activation by Gβγ. Moreover, WDR26 was required for PLCβ2 translocation from the cytosol to the membrane in polarized leukocytes, and the translocation of PLCβ2 was sufficient to cause partial activation of PLCβ2. Collectively, our data indicate that WDR26 functions as a scaffolding protein to promote PLCβ2 membrane translocation and interaction with Gβγ, thereby enhancing PLCβ2 activation in leukocytes. These findings have identified a novel mechanism of regulating Gβγ signaling through a scaffolding protein.     

Olfactomedin-1 Has a V-shaped Disulfide-linked Tetrameric Structure

Olfactomedin-1 (Olfm1; also known as noelin and pancortin) is a member of the olfactomedin domain-containing superfamily and a highly expressed neuronal glycoprotein important for nervous system development. It binds a number of secreted proteins and cell surface-bound receptors to induce cell signaling processes. Using a combined approach of x-ray crystallography, solution scattering, analytical ultracentrifugation, and electron microscopy we determined that full-length Olfm1 forms disulfide-linked tetramers with a distinctive V-shaped architecture. The base of the “V” is formed by two disulfide-linked dimeric N-terminal domains. Each of the two V legs consists of a parallel dimeric disulfide-linked coiled coil with a C-terminal β-propeller dimer at the tips. This agrees with our crystal structure of a C-terminal coiled-coil segment and β-propeller combination (Olfm1^coil-Olf) that reveals a disulfide-linked dimeric arrangement with the β-propeller top faces in an outward exposed orientation. Similar to its family member myocilin, Olfm1 is stabilized by calcium. The dimer-of-dimers architecture suggests a role for Olfm1 in clustering receptors to regulate signaling and sheds light on the conformation of several other olfactomedin domain family members.     

Structural Characterization of Bardet-Biedl Syndrome 9 Protein (BBS9)

The Bardet-Biedl syndrome protein complex (BBSome) is an octameric complex that transports membrane proteins into the primary cilium signaling organelle in eukaryotes and is implicated in human disease. Here we have analyzed the 99-kDa human BBS9 protein, one of the eight BBSome components. The protein is composed of four structured domains, including a β-stranded N-terminal domain. The 1.8 Å crystal structure of the 46-kDa N-terminal domain reveals a seven-bladed β-propeller. A structure-based homology search suggests that it functions in protein-protein interactions. We show that the Bardet-Biedl syndrome-causing G141R mutation in BBS9 likely results in misfolding of the β-propeller. Although the C-terminal half of BBS9 dimerizes in solution, the N-terminal domain only does so in the crystal lattice. This C-terminal dimerization interface might be important for the assembly of the BBSome.     

A novel interaction between Glycogen Synthase Kinase-3α (GSK-3α) and the scaffold protein Receptor for Activated C-Kinase 1 (RACK1) regulates the circadian clock

Glycogen synthase kinase-3α (GSK-3α) and GSK-3β are intracellular kinases with largely redundant functions. However, the deletion of each GSK-3 isoform in the mouse has distinct consequences, suggesting that these related enzymes also have non-overlapping isoform-specific functions. A yeast two-hybrid screen for GSK-3α interacting partners revealed an interaction with the Receptor for Activated C-Kinase 1 (RACK1). We confirm this interaction in mammalian cells, and provide evidence that RACK1 does not interact with GSK-3β. Structure-function analyses revealed that WD repeats 5-6 are required to interact with GSK-3α. Furthermore, this interaction is independent of GSK-3α activity. Finally, our data show that the GSK-3α-RACK1 interaction is necessary for regulating the circadian clock in mammalian cells. In summary, our data provides a mechanistic link between GSK-3 and RACK-1 in the regulation of the circadian clock, and demonstrates that this effect is specific to the GSK-3α isoform.     

A Direct Interaction with NEDD1 Regulates γ-Tubulin Recruitment to the Centrosome

The centrosome is the primary microtubule organizing centre of the cell. γ-tubulin is a core component of the centrosome and is required for microtubule nucleation and centrosome function. The recruitment of γ-tubulin to centrosomes is mediated by its interaction with NEDD1, a WD40-repeat containing protein. Here we demonstrate that NEDD1 is likely to be oligomeric in vivo and binds directly to γ-tubulin through a small region of just 62 residues at the carboxyl-terminus of the protein. This carboxyl-terminal domain that binds γ-tubulin has a helical structure and is a stable tetramer in solution. Mutation of residues in NEDD1 that disrupt binding to γ-tubulin result in a mis-localization of γ-tubulin away from the centrosome. Hence, this study defines the binding site on NEDD1 that is required for its interaction with γ-tubulin, and shows that this interaction is required for the correct localization of γ-tubulin.     

Inhibition of αIIbβ3 Ligand Binding by an αIIb Peptide that Clasps the Hybrid Domain to the βI Domain of β3

Agonist-stimulated platelet activation triggers conformational changes of integrin αIIbβ3, allowing fibrinogen binding and platelet aggregation. We have previously shown that an octapeptide, ^p1YMESRADR⁸, corresponding to amino acids 313–320 of the β-ribbon extending from the β-propeller domain of αIIb, acts as a potent inhibitor of platelet aggregation. Here we have performed in silico modelling analysis of the interaction of this peptide with αIIbβ3 in its bent and closed (not swing-out) conformation and show that the peptide is able to act as a substitute for the β-ribbon by forming a clasp restraining the β3 hybrid and βI domains in a closed conformation. The involvement of species-specific residues of the β3 hybrid domain (E356 and K384) and the β1 domain (E297) as well as an intrapeptide bond (^pE315-^pR317) were confirmed as important for this interaction by mutagenesis studies of αIIbβ3 expressed in CHO cells and native or substituted peptide inhibitory studies on platelet functions. Furthermore, NMR data corroborate the above results. Our findings provide insight into the important functional role of the αIIb β-ribbon in preventing integrin αIIbβ3 head piece opening, and highlight a potential new therapeutic approach to prevent integrin ligand binding.     

The regulation of integrin function by divalent cations

Integrins are a family of α/β heterodimeric adhesion metalloprotein receptors and their functions are highly dependent on and regulated by different divalent cations. Recently advanced studies have revolutionized our perception of integrin metal ion-binding sites and their specific functions. Ligand binding to integrins is bridged by a divalent cation bound at the MIDAS motif on top of either α I domain in I domain-containing integrins or β I domain in α I domain-less integrins. The MIDAS motif in β I domain is flanked by ADMIDAS and SyMBS, the other two crucial metal ion binding sites playing pivotal roles in the regulation of integrin affinity and bidirectional signaling across the plasma membrane. The β-propeller domain of α subunit contains three or four β-hairpin loop-like Ca²⁺-binding motifs that have essential roles in integrin biogenesis. The function of another Ca²⁺-binding motif located at the genu of α subunit remains elusive. Here, we provide an overview of the integrin metal ion-binding sites and discuss their roles in the regulation of integrin functions.     

The Interaction of Integrin αIIbβ3 with Fibrin Occurs through Multiple Binding Sites in the αIIb β-Propeller Domain

The currently available antithrombotic agents target the interaction of platelet integrin α_IIbβ₃ (GPIIb-IIIa) with fibrinogen during platelet aggregation. Platelets also bind fibrin formed early during thrombus growth. It was proposed that inhibition of platelet-fibrin interactions may be a necessary and important property of α_IIbβ₃ antagonists; however, the mechanisms by which α_IIbβ₃ binds fibrin are uncertain. We have previously identified the γ370–381 sequence (P3) in the γC domain of fibrinogen as the fibrin-specific binding site for α_IIbβ₃ involved in platelet adhesion and platelet-mediated fibrin clot retraction. In the present study, we have demonstrated that P3 can bind to several discontinuous segments within the α_IIb β-propeller domain of α_IIbβ₃ enriched with negatively charged and aromatic residues. By screening peptide libraries spanning the sequence of the α_IIb β-propeller, several sequences were identified as candidate contact sites for P3. Synthetic peptides duplicating these segments inhibited platelet adhesion and clot retraction but not platelet aggregation, supporting the role of these regions in fibrin recognition. Mutant α_IIbβ₃ receptors in which residues identified as critical for P3 binding were substituted for homologous residues in the I-less integrin α_Mβ₂ exhibited reduced cell adhesion and clot retraction. These residues are different from those that are involved in the coordination of the fibrinogen γ404–411 sequence and from auxiliary sites implicated in binding of soluble fibrinogen. These results map the binding of fibrin to multiple sites in the α_IIb β-propeller and further indicate that recognition specificity of α_IIbβ₃ for fibrin differs from that for soluble fibrinogen.