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NORMAL GENOMIC

• Shaw N, Liu ZJ
• Role of the HIN domain in regulation of innate immune responses.
• Mol Cell Biol. 2014; 34: 2-15
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• The oligonucleotide/oligosaccharide binding (OB) fold is employed by proteins to bind nucleic acids during replication, transcription, and translation. Recently, a variation of the OB fold consisting of a tandem pair of OB folds named the HIN (hematopoietic expression, interferon-inducible nature, and nuclear localization) domain was shown to play essential roles in the regulation of innate immune responses originating from binding of nucleic acids in the cytoplasm or the nucleus of the cell. Although the two OB folds of the HIN domain are linked via a long linker region, conserved hydrophobic contacts between the two OB folds hold them together firmly, resulting in a single compact domain. This overall topology of the HIN domain seems to be highly conserved, and proteins containing the HIN domain have been grouped in the PYHIN family. Structures of the recently solved HIN domains reveal that these domains exhibit either absent in melanoma2 (Aim2) HIN-like or p202 HINa-like modes of DNA binding. These two modes of DNA binding seem to result in different responses and as a consequence confer distinct roles on the proteins. This review summarizes our current understanding of the structure and function of the HIN domains in context with the innate immune responses.

• Hassed SJ et al.
• RBPJ mutations identified in two families affected by Adams-Oliver syndrome.
• Am J Hum Genet. 2012; 91: 391-5
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• Through exome resequencing, we identified two unique mutations in recombination signal binding protein for immunoglobulin kappa J (RBPJ) in two independent families affected by Adams-Oliver syndrome (AOS), a rare multiple-malformation disorder consisting primarily of aplasia cutis congenita of the vertex scalp and transverse terminal limb defects. These identified mutations link RBPJ, the primary transcriptional regulator for the Notch pathway, with AOS, a human genetic disorder. Functional assays confirmed impaired DNA binding of mutated RBPJ, placing it among other notch-pathway proteins altered in human genetic syndromes.

• Jin T et al.
• Structures of the HIN domain:DNA complexes reveal ligand binding and activation mechanisms of the AIM2 inflammasome and IFI16 receptor.
• Immunity. 2012; 36: 561-71
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• Recognition of DNA by the innate immune system is central to antiviral and antibacterial defenses, as well as an important contributor to autoimmune diseases involving self DNA. AIM2 (absent in melanoma 2) and IFI16 (interferon-inducible protein 16) have been identified as DNA receptors that induce inflammasome formation and interferon production, respectively. Here we present the crystal structures of their HIN domains in complex with double-stranded (ds) DNA. Non-sequence-specific DNA recognition is accomplished through electrostatic attraction between the positively charged HIN domain residues and the dsDNA sugar-phosphate backbone. An intramolecular complex of the AIM2 Pyrin and HIN domains in an autoinhibited state is liberated by DNA binding, which may facilitate the assembly of inflammasomes along the DNA staircase. These findings provide mechanistic insights into dsDNA as the activation trigger and oligomerization platform for the assembly of large innate signaling complexes such as the inflammasomes.

• Luo S, Ye K
• Dimerization, but not phosphothreonine binding, is conserved between the forkhead-associated domains of Drosophila MU2 and human MDC1.
• FEBS Lett. 2012; 586: 344-9
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• Mutator 2 (MU2) in Drosophila melanogaster has been proposed to be the ortholog of human MDC1, a key mediator in DNA damage response. The forkhead-associated (FHA) domain of MDC1 is a dimerization module regulated by trans binding to phosphothreonine 4 from another molecule. Here we present the crystal structure of the MU2 FHA domain at 1.9A resolution, revealing its evolutionarily conserved role in dimerization. As compared to the MDC1 FHA domain, the MU2 FHA domain dimerizes using a different and more stable interface and contains a degenerate phosphothreonine-binding pocket. Our results suggest that the MU2 dimerization is constitutive and lacks phosphorylation-mediated regulation.

• VanderWielen BD, Yuan Z, Friedmann DR, Kovall RA
• Transcriptional repression in the Notch pathway: thermodynamic characterization of CSL-MINT (Msx2-interacting nuclear target protein) complexes.
• J Biol Chem. 2011; 286: 14892-902
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• The Notch pathway is a conserved cell-to-cell signaling mechanism that mediates cell fate decisions in metazoans. Canonical signaling results in changes in gene expression, which is regulated by the nuclear effector of the pathway CSL (CBF1/RBP-J, Su(H), Lag-1). CSL is a DNA binding protein that functions as either a repressor or an activator of transcription, depending upon whether it is complexed by transcriptional corepressor or coactivator proteins, respectively. In stark contrast to CSL-coactivator complexes, e.g. the transcriptionally active CSL-Notch-Mastermind ternary complex, the structure and function of CSL-corepressor complexes are poorly understood. The corepressor MINT (Msx2-interacting nuclear target protein) has been shown in vivo to antagonize Notch signaling and shown in vitro to biochemically interact with CSL; however, the molecular details of this interaction are only partially defined. Here, we provide a quantitative thermodynamic binding analysis of CSL-MINT complexes. Using isothermal titration calorimetry, we demonstrate that MINT forms a high affinity complex with CSL, and we also delineate the domains of MINT and CSL that are necessary and sufficient for complex formation. Moreover, we show in cultured cells that this region of MINT can inhibit Notch signaling in transcriptional reporter assays. Taken together, our results provide functional insights into how CSL is converted from a repressor to an activator of transcription.

• Kurth P, Preiss A, Kovall RA, Maier D
• Molecular analysis of the notch repressor-complex in Drosophila: characterization of potential hairless binding sites on suppressor of hairless.
• PLoS One. 2011; 6: 27986-27986
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• The Notch signalling pathway mediates cell-cell communication in a wide variety of organisms. The major components, as well as the basic mechanisms of Notch signal transduction, are remarkably well conserved amongst vertebrates and invertebrates. Notch signalling results in transcriptional activation of Notch target genes, which is mediated by an activator complex composed of the DNA binding protein CSL, the intracellular domain of the Notch receptor, and the transcriptional coactivator Mastermind. In the absence of active signalling, CSL represses transcription from Notch target genes by the recruitment of corepressors. The Notch activator complex is extremely well conserved and has been studied in great detail. However, Notch repressor complexes are far less understood. In Drosophila melanogaster, the CSL protein is termed Suppressor of Hairless [Su(H)]. Su(H) functions as a transcriptional repressor by binding Hairless, the major antagonist of Notch signalling in Drosophila, which in turn recruits two general corepressors--Groucho and C-terminal binding protein CtBP. Recently, we determined that the C-terminal domain (CTD) of Su(H) binds Hairless and identified a single site in Hairless, which is essential for contacting Su(H). Here we present additional biochemical and in vivo studies aimed at mapping the residues in Su(H) that contact Hairless. Focusing on surface exposed residues in the CTD, we identified two sites that affect Hairless binding in biochemical assays. Mutation of these sites neither affects binding to DNA nor to Notch. Subsequently, these Su(H) mutants were found to function normally in cellular and in vivo assays using transgenic flies. However, these experiments rely on Su(H) overexpression, which does not allow for detection of quantitative or subtle differences in activity. We discuss the implications of our results.

• Friedmann DR, Kovall RA
• Thermodynamic and structural insights into CSL-DNA complexes.
• Protein Sci. 2010; 19: 34-46
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• The Notch pathway is an intercellular signaling mechanism that plays important roles in cell fates decisions throughout the developing and adult organism. Extracellular complexation of Notch receptors with ligands ultimately results in changes in gene expression, which is regulated by the nuclear effector of the pathway, CSL (C-promoter binding factor 1 (CBF-1), suppressor of hairless (Su(H)), lin-12 and glp-1 (Lag-1)). CSL is a DNA binding protein that is involved in both repression and activation of transcription from genes that are responsive to Notch signaling. One well-characterized Notch target gene is hairy and enhancer of split-1 (HES-1), which is regulated by a promoter element consisting of two CSL binding sites oriented in a head-to-head arrangement. Although previous studies have identified in vivo and consensus binding sites for CSL, and crystal structures of these complexes have been determined, to date, a quantitative description of the energetics that underlie CSL-DNA binding is unknown. Here, we provide a thermodynamic and structural analysis of the interaction between CSL and the two individual sites that comprise the HES-1 promoter element. Our comprehensive studies that analyze binding as a function of temperature, salt, and pH reveal moderate, but distinct, differences in the affinities of CSL for the two HES-1 binding sites. Similarly, our structural results indicate that overall CSL binds both DNA sites in a similar manner; however, minor changes are observed in both the conformation of CSL and DNA. Taken together, our results provide a quantitative and biophysical basis for understanding how CSL interacts with DNA sites in vivo.

• Hoopes JT et al.
• Structural characterization of the E2 domain of APL-1, a Caenorhabditis elegans homolog of human amyloid precursor protein, and its heparin binding site.
• J Biol Chem. 2010; 285: 2165-73
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• The amyloid beta-peptide deposit found in the brain tissue of patients with Alzheimer disease is derived from a large heparin-binding protein precursor APP. The biological function of APP and its homologs is not precisely known. Here we report the x-ray structure of the E2 domain of APL-1, an APP homolog in Caenorhabditis elegans, and compare it to the human APP structure. We also describe the structure of APL-1 E2 in complex with sucrose octasulfate, a highly negatively charged disaccharide, which reveals an unexpected binding pocket between the two halves of E2. Based on the crystal structure, we are able to map, using site-directed mutagenesis, a surface groove on E2 to which heparin may bind. Our biochemical data also indicate that the affinity of E2 for heparin is influenced by pH: at pH 5, the binding appears to be much stronger than that at neutral pH. This property is likely caused by histidine residues in the vicinity of the mapped heparin binding site and could be important for the proposed adhesive function of APL-1.

• Del Bianco C et al.
• Notch and MAML-1 complexation do not detectably alter the DNA binding specificity of the transcription factor CSL.
• PLoS One. 2010; 5: 15034-15034
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• BACKGROUND: Canonical Notch signaling is initiated when ligand binding induces proteolytic release of the intracellular part of Notch (ICN) from the cell membrane. ICN then travels into the nucleus where it drives the assembly of a transcriptional activation complex containing the DNA-binding transcription factor CSL, ICN, and a specialized co-activator of the Mastermind family. A consensus DNA binding site motif for the CSL protein was previously defined using selection-based methods, but whether subsequent association of Notch and Mastermind-like proteins affects the DNA binding preferences of CSL has not previously been examined. PRINCIPAL FINDINGS: Here, we utilized protein-binding microarrays (PBMs) to compare the binding site preferences of isolated CSL with the preferred binding sites of CSL when bound to the CSL-binding domains of all four different human Notch receptors. Measurements were taken both in the absence and in the presence of Mastermind-like-1 (MAML1). Our data show no detectable difference in the DNA binding site preferences of CSL before and after loading of Notch and MAML1 proteins. CONCLUSIONS/SIGNIFICANCE: These findings support the conclusion that accrual of Notch and MAML1 promote transcriptional activation without dramatically altering the preferred sites of DNA binding, and illustrate the potential of PBMs to analyze the binding site preferences of multiprotein-DNA complexes.

• Gazave E et al.
• Origin and evolution of the Notch signalling pathway: an overview from eukaryotic genomes.
• BMC Evol Biol. 2009; 9: 249-249
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• BACKGROUND: Of the 20 or so signal transduction pathways that orchestrate cell-cell interactions in metazoans, seven are involved during development. One of these is the Notch signalling pathway which regulates cellular identity, proliferation, differentiation and apoptosis via the developmental processes of lateral inhibition and boundary induction. In light of this essential role played in metazoan development, we surveyed a wide range of eukaryotic genomes to determine the origin and evolution of the components and auxiliary factors that compose and modulate this pathway. RESULTS: We searched for 22 components of the Notch pathway in 35 different species that represent 8 major clades of eukaryotes, performed phylogenetic analyses and compared the domain compositions of the two fundamental molecules: the receptor Notch and its ligands Delta/Jagged. We confirm that a Notch pathway, with true receptors and ligands is specific to the Metazoa. This study also sheds light on the deep ancestry of a number of genes involved in this pathway, while other members are revealed to have a more recent origin. The origin of several components can be accounted for by the shuffling of pre-existing protein domains, or via lateral gene transfer. In addition, certain domains have appeared de novo more recently, and can be considered metazoan synapomorphies. CONCLUSION: The Notch signalling pathway emerged in Metazoa via a diversity of molecular mechanisms, incorporating both novel and ancient protein domains during eukaryote evolution. Thus, a functional Notch signalling pathway was probably present in Urmetazoa.

• Bertagna A, Toptygin D, Brand L, Barrick D
• The effects of conformational heterogeneity on the binding of the Notch intracellular domain to effector proteins: a case of biologically tuned disorder.
• Biochem Soc Trans. 2008; 36: 157-66
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• Cell-fate decisions in metazoans are frequently guided by the Notch signalling pathway. Notch signalling is orchestrated by a type-1 transmembrane protein, which, upon interacting with extracellular ligands, is proteolytically cleaved to liberate a large intracellular domain [NICD (Notch intracellular domain)]. NICD enters the nucleus where it binds the transcription factor CSL (CBF1/suppressor of Hairless/Lag-1) and activates transcription of Notch-responsive genes. In the present paper, the interaction between the Drosophila NICD and CSL will be examined. This interaction involves two separate binding regions on NICD: the N-terminal tip of NICD {the RAM [RBP-Jkappa (recombination signal-binding protein 1 for Jkappa)-associated molecule] region} and an ankyrin domain approximately 100 residues away. CD studies show that the RAM region of NICD lacks alpha-helical and beta-sheet secondary structure, and also lacks rigid tertiary structure. Fluorescence studies show that the tryptophan residues in RAM are highly solvated and are quenched by solvent. To assess the impact of this apparent disorder on the bivalent binding of NICD to CSL, we modelled the region between the RAM and ANK (ankyrin repeat)-binding regions using polymer statistics. A WLC (wormlike chain) model shows that the most probable sequence separation between the two binding regions is approximately 50 A (1 A=0.1 nm), matching the separation between these two sites in the complex. The WLC model predicts a substantial enhancement of ANK occupancy via effective concentration, and suggests that the linker length between the two binding regions is optimal for bivalent interaction.

• Stead MA, Rosbrook GO, Hadden JM, Trinh CH, Carr SB, Wright SC
• Structure of the wild-type human BCL6 POZ domain.
• Acta Crystallogr Sect F Struct Biol Cryst Commun. 2008; 64: 1101-4
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• BCL6 is a transcriptional repressor that is overexpressed in diffuse large B-cell lymphoma and follicular lymphoma. The N-terminal POZ domain of BCL6 interacts with transcriptional corepressors and targeting these associations is a promising therapeutic strategy. Previous structural studies of the BCL6 POZ domain have used a mutant form because of the low solubility of the wild-type recombinant protein. A method for the purification and crystallization of the wild-type BCL6 POZ domain is described and the crystal structure to 2.1 A resolution is reported. This will be relevant for the design of therapeutics that target BCL6 POZ-domain interaction interfaces.

• Krejci A, Bray S
• Notch activation stimulates transient and selective binding of Su(H)/CSL to target enhancers.
• Genes Dev. 2007; 21: 1322-7
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• The CSL [CBF1/Su(H)/Lag2] proteins [Su(H) in Drosophila] are implicated in repression and activation of Notch target loci. Prevailing models imply a static association of these DNA-binding transcription factors with their target enhancers. Our analysis of Su(H) binding and chromatin-associated features at 11 E(spl) Notch target genes before and after Notch revealed large differences in Su(H) occupancy at target loci that correlated with the presence of polymerase II and other marks of transcriptional activity. Unexpectedly, Su(H) occupancy was significantly and transiently increased following Notch activation, suggesting a more dynamic interaction with targets than hitherto proposed.

• Prevorovsky M, Puta F, Folk P
• Fungal CSL transcription factors.
• BMC Genomics. 2007; 8: 233-233
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• BACKGROUND: The CSL (CBF1/RBP-Jkappa/Suppressor of Hairless/LAG-1) transcription factor family members are well-known components of the transmembrane receptor Notch signaling pathway, which plays a critical role in metazoan development. They function as context-dependent activators or repressors of transcription of their responsive genes, the promoters of which harbor the GTG(G/A)GAA consensus elements. Recently, several studies described Notch-independent activities of the CSL proteins. RESULTS: We have identified putative CSL genes in several fungal species, showing that this family is not confined to metazoans. We have analyzed their sequence conservation and identified the presence of well-defined domains typical of genuine CSL proteins. Furthermore, we have shown that the candidate fungal protein sequences contain highly conserved regions known to be required for sequence-specific DNA binding in their metazoan counterparts. The phylogenetic analysis of the newly identified fungal CSL proteins revealed the existence of two distinct classes, both of which are present in all the species studied. CONCLUSION: Our findings support the evolutionary origin of the CSL transcription factor family in the last common ancestor of fungi and metazoans. We hypothesize that the ancestral CSL function involved DNA binding and Notch-independent regulation of transcription and that this function may still be shared, to a certain degree, by the present CSL family members from both fungi and metazoans.

• Masui T, Long Q, Beres TM, Magnuson MA, MacDonald RJ
• Early pancreatic development requires the vertebrate Suppressor of Hairless (RBPJ) in the PTF1 bHLH complex.
• Genes Dev. 2007; 21: 2629-43
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• PTF1a is an unusual basic helix-loop-helix (bHLH) transcription factor that is required for the development of the pancreas. We show that early in pancreatic development, active PTF1a requires interaction with RBPJ, the vertebrate Suppressor of Hairless, within a stable trimeric DNA-binding complex (PTF1). Later, as acinar cell development begins, RBPJ is swapped for RBPJL, the constitutively active, pancreas-restricted paralog of RBPJ. Moreover, the Rbpjl gene is a direct target of the PTF1 complex: At the onset of acinar cell development when the Rbpjl gene is first induced, a PTF1 complex containing RBPJ is bound to the Rbpjl promoter. As development proceeds, RBPJL gradually replaces RBPJ in the PTF1 complex bound to Rbpjl and appears on the binding sites for the complex in the promoters of other acinar-specific genes, including those for the secretory digestive enzymes. A single amino acid change in PTF1a that eliminates its ability to bind RBPJ (but does not affect its binding to RBPJL) causes pancreatic development to truncate at an immature stage, without the formation of acini or islets. These results indicate that the interaction between PTF1a and RBPJ is required for the early stage of pancreatic growth, morphogenesis, and lineage fate decisions. The defects in pancreatic development phenocopy those of Ptf1a-null embryos; thus, the first critical function of PTF1a is in the context of the PTF1 complex containing RBPJ. Action within an organ-specific transcription factor is a previously unknown function for RBPJ and is independent of its role in Notch signaling.

• Koelzer S, Klein T
• Regulation of expression of Vg and establishment of the dorsoventral compartment boundary in the wing imaginal disc by Suppressor of Hairless.
• Dev Biol. 2006; 289: 77-90
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• The transcription factor Suppressor of Hairless (Su(H)) belongs to the CSL transcription factor family, which are the main transcriptional effectors of the Notch-signaling pathway. Su(H) is the only family member in the Drosophila genome and should therefore be the main transcriptional effector of the Notch pathway in this species. Despite this fact, in many developmental situations, the phenotype caused by loss of function of Su(H) is too weak for a factor that is supposed to mediate most or all aspects of Notch signaling. One example is the Su(H) mutant phenotype during the development of the wing, which is weaker in comparison to other genes required for Notch signaling. Another example is the complete absence of a phenotype upon loss of Su(H) function during the formation of the dorsoventral (D/V) compartment boundary, although the Notch pathway is required for this process. Recent work has shown that Su(H)/CBF1 has a second function as a transcriptional repressor, in the absence of the activity of the Notch pathway. As a repressor, Su(H) acts in a complex together with Hairless (H), which acts as a bridge to recruit the co-repressors Groucho and CtBP, and acts in a Notch-independent manner to prevent the transcription of target genes. This raises the possibility that a de-repression of target genes can occur in the case of loss if function of Su(H). Here, we show that the weak phenotype of Su(H) mutants during wing development and the absence of a phenotype during formation of the D/V compartment boundary are caused by the concomitant loss of the Notch-independent repressor function. This loss of the repressor function of Su(H) results in a de-repression of expression of target genes to a different degree in each process. Loss of Su(H) function during wing development results in a transient de-repression of expression of the selector gene vestigial (vg). We show that this residual expression of vg is responsible for the weaker mutant phenotype of Su(H) in the wing. During the formation of the D/V compartment boundary, de-repression of target genes seems to be sufficiently strong, to compensate the loss of Su(H) activity. Thus, de-repression of its target genes obscures the involvement of Su(H) in this process. Furthermore, we provide evidence that Dx does not signal in a Su(H)-independent manner as has been suggested previously.

• Beres TM, Masui T, Swift GH, Shi L, Henke RM, MacDonald RJ
• PTF1 is an organ-specific and Notch-independent basic helix-loop-helix complex containing the mammalian Suppressor of Hairless (RBP-J) or its paralogue, RBP-L.
• Mol Cell Biol. 2006; 26: 117-30
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• PTF1 is a trimeric transcription factor essential to the development of the pancreas and to the maintenance of the differentiated state of the adult exocrine pancreas. It comprises a dimer of P48/PTF1a (a pancreas and neural restricted basic helix-loop-helix [bHLH] protein) and a class A bHLH protein, together with a third protein that we show can be either the mammalian Suppressor of Hairless (RBP-J) or its paralogue, RBP-L. In mature acinar cells, PTF1 exclusively contains the RBP-L isoform and is bound to the promoters of acinar specific genes. P48 interacts with the RBP subunit primarily through two short conserved tryptophan-containing motifs, similar to the motif of the Notch intracellular domain (NotchIC) that interacts with RBP-J. The transcriptional activities of the J and L forms of PTF1 are independent of Notch signaling, because P48 occupies the NotchIC docking site on RBP-J and RBP-L does not bind the NotchIC. Mutations that delete one or both of the RBP-interacting motifs of P48 eliminate RBP-binding and are associated with a human genetic disorder characterized by pancreatic and cerebellar agenesis, which indicates that the association of P48 and RBPs is required for proper embryonic development. The presence of related peptide motifs in other transcription factors indicates a broader Notch-independent function for RBPJ/SU(H).

• Cave JW, Loh F, Surpris JW, Xia L, Caudy MA
• A DNA transcription code for cell-specific gene activation by notch signaling.
• Curr Biol. 2005; 15: 94-104
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• BACKGROUND: Cell-specific gene regulation is often controlled by specific combinations of DNA binding sites in target enhancers or promoters. A key question is whether these sites are randomly arranged or if there is an organizational pattern or "architecture" within such regulatory modules. During Notch signaling in Drosophila proneural clusters, cell-specific activation of certain Notch target genes is known to require transcriptional synergy between the Notch intracellular domain (NICD) complexed with CSL proteins bound to "S" DNA sites and proneural bHLH activator proteins bound to nearby "A" DNA sites. Previous studies have implied that arbitrary combinations of S and A DNA binding sites (an "S+A" transcription code) can mediate the Notch-proneural transcriptional synergy. RESULTS: By contrast, we show that the Notch-proneural transcriptional synergy critically requires a particular DNA site architecture ("SPS"), which consists of a pair of specifically-oriented S binding sites. Native and synthetic promoter analysis shows that the SPS architecture in combination with proneural A sites creates a minimal DNA regulatory code, "SPS+A", that is both sufficient and critical for mediating the Notch-proneural synergy. Transgenic Drosophila analysis confirms the SPS orientation requirement during Notch signaling in proneural clusters. We also present evidence that CSL interacts directly with the proneural Daughterless protein, thus providing a molecular mechanism for this synergy. CONCLUSIONS: The SPS architecture functions to mediate or enable the Notch-proneural transcriptional synergy which drives Notch target gene activation in specific cells. Thus, SPS+A is an architectural DNA transcription code that programs a cell-specific pattern of gene expression.

• Beaster-Jones L, Okkema PG
• DNA binding and in vivo function of C.elegans PEB-1 require a conserved FLYWCH motif.
• J Mol Biol. 2004; 339: 695-706
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• Caenorhabditis elegans PEB-1 is a novel protein containing a DNA-binding domain in its N terminus, which includes a Cys/His-rich FLYWCH motif also found in Drosophila Mod(mdg4) proteins, and a large C-terminal domain of unknown function. PEB-1 is expressed in most pharyngeal cell types, but its molecular function remains unclear. Here we describe comparative and functional analyses of PEB-1. Characterization of the PEB-1 sequence from C.briggsae indicates highest conservation in the DNA-binding domain (including the FLYWCH motif) and the C terminus, suggesting two functional domains. The PEB-1 FLYWCH motif is essential for DNA-binding and in vivo function; however, it does not bind detectable metal. Likewise, the PEB-1 C terminus is necessary for full activity in vivo, although the DNA-binding domain alone is sufficient for partial function. Both the FLYWCH motif and the C-terminal domain are required for efficient nuclear localization, suggesting PEB-1 must bind DNA and other components to remain in the nucleus. Analysis of binding sites revealed a YDTGCCRW PEB-1 consensus-binding site, and matches to this consensus are widespread in the C.elegans genome.

• Hamaoka BY, Dann CE 3rd, Geisbrecht BV, Leahy DJ
• Crystal structure of Caenorhabditis elegans HER-1 and characterization of the interaction between HER-1 and TRA-2A.
• Proc Natl Acad Sci U S A. 2004; 101: 11673-8
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• HER-1 is a secreted protein that promotes male development in the nematode Caenorhabditis elegans. HER-1 inhibits the function of TRA-2A, a multipass integral membrane protein thought to serve as its receptor. We report here the 1.5-A crystal structure of HER-1. The structure was solved by the multiwavelength anomalous diffraction method by using selenomethionyl-substituted HER-1 produced in Chinese hamster ovary cells. The HER-1 structure consists of two all-helical domains and is not closely homologous to any known structure. Sites of amino acid substitutions known to impair HER-1 function were mapped on the HER-1 structure and classified according to the likely mechanism by which they affect HER-1 activity. A subset of these and other amino acid substitutions on the HER-1 surface were assayed for their ability to disrupt interactions between HER-1 and TRA-2A-expressing cells, and a localized region on the HER-1 surface important for mediating this interaction was identified.

• Zweifel ME, Leahy DJ, Hughson FM, Barrick D
• Structure and stability of the ankyrin domain of the Drosophila Notch receptor.
• Protein Sci. 2003; 12: 2622-32
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• The Notch receptor contains a conserved ankyrin repeat domain that is required for Notch-mediated signal transduction. The ankyrin domain of Drosophila Notch contains six ankyrin sequence repeats previously identified as closely matching the ankyrin repeat consensus sequence, and a putative seventh C-terminal sequence repeat that exhibits lower similarity to the consensus sequence. To better understand the role of the Notch ankyrin domain in Notch-mediated signaling and to examine how structure is distributed among the seven ankyrin sequence repeats, we have determined the crystal structure of this domain to 2.0 angstroms resolution. The seventh, C-terminal, ankyrin sequence repeat adopts a regular ankyrin fold, but the first, N-terminal ankyrin repeat, which contains a 15-residue insertion, appears to be largely disordered. The structure reveals a substantial interface between ankyrin polypeptides, showing a high degree of shape and charge complementarity, which may be related to homotypic interactions suggested from indirect studies. However, the Notch ankyrin domain remains largely monomeric in solution, demonstrating that this interface alone is not sufficient to promote tight association. Using the structure, we have classified reported mutations within the Notch ankyrin domain that are known to disrupt signaling into those that affect buried residues and those restricted to surface residues. We show that the buried substitutions greatly decrease protein stability, whereas the surface substitutions have only a marginal affect on stability. The surface substitutions are thus likely to interfere with Notch signaling by disrupting specific Notch-effector interactions and map the sites of these interactions.

• Pickles LM, Roe SM, Hemingway EJ, Stifani S, Pearl LH
• Crystal structure of the C-terminal WD40 repeat domain of the human Groucho/TLE1 transcriptional corepressor.
• Structure. 2002; 10: 751-61
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• Groucho (Gro)/TLE proteins are transcriptional corepressors that lack inherent DNA binding but interact with DNA-bound transcription factors and histones, and recruit histone deacetylases. Groucho-mediated repression is essential in embryonic development and involved in regulation of Wnt signaling in adult tissue. We have determined the 1.6 A crystal structure of a C-terminal fragment of human Groucho/TLE1, comprising part of the Ser/Pro-rich region and a seven-bladed beta propeller WD40 repeat domain, implicated in protein-protein interactions. The structure confirms the relationship to the yeast Tup1 corepressor, but reveals important structural differences specific to the metazoan system. Analysis of missense mutations in the C. elegans Groucho homolog UNC-37 identifies sites of interaction with repression effectors, and suggests an induced fit binding site for eh1 domains of Engrailed-type transcription factors.

• Oswald F et al.
• SHARP is a novel component of the Notch/RBP-Jkappa signalling pathway.
• EMBO J. 2002; 21: 5417-26
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• Notch proteins are the receptors for an evolutionarily highly conserved signalling pathway that regulates numerous cell fate decisions during development. Signal transduction involves the presenilin-dependent intracellular processing of Notch and nuclear translocation of the intracellular domain of Notch, Notch-IC. Notch-IC associates with the DNA-binding protein RBP-Jkappa/CBF-1 to activate transcription of Notch target genes. In the absence of Notch signalling, RBP-Jkappa/CBF-1 acts as a transcriptional repressor through the recruitment of histone deacetylase (HDAC) corepressor complexes. We identified SHARP as an RBP-Jkappa/CBF-1-interacting corepressor in a yeast two-hybrid screen. In cotransfection experiments, SHARP-mediated repression was sensitive to the HDAC inhibitor TSA and facilitated by SKIP, a highly conserved SMRT and RBP-Jkappa-interacting protein. SHARP repressed Hairy/Enhancer of split (HES)-1 promoter activity, inhibited Notch-1-mediated transactivation and rescued Notch-1-induced inhibition of primary neurogenesis in Xenopus laevis embryos. Based on our data, we propose a model in which SHARP is a novel component of the HDAC corepressor complex, recruited by RBP-Jkappa to repress transcription of target genes in the absence of activated Notch.

• Jeffries S, Robbins DJ, Capobianco AJ
• Characterization of a high-molecular-weight Notch complex in the nucleus of Notch(ic)-transformed RKE cells and in a human T-cell leukemia cell line.
• Mol Cell Biol. 2002; 22: 3927-41
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• Notch genes encode a family of transmembrane proteins that are involved in many cellular processes, such as differentiation, proliferation, and apoptosis. It is well established that all four Notch genes can act as oncogenes; however, the mechanism by which Notch proteins transform cells remains unknown. Previously, we reported that both nuclear localization and transcriptional activation are required for neoplastic transformation of RKE cells. Furthermore, we identified cyclin D1 as a direct transcriptional target of constitutively active Notch molecules. In an effort to understand the mechanism by which Notch functions in the nucleus, we sought to determine if Notch formed stable complexes using size exclusion chromatography. Herein, we report that the Notch intracellular domain (N(ic)) forms distinct high-molecular-weight complexes in the nuclei of transformed RKE cells. The largest complex is approximately 1.5 MDa and contains both endogenous CSL (for CBF1, Suppressor of Hairless, and Lag-1) and Mastermind-Like-1 (Maml). N(ic) molecules that do not have the high-affinity binding site for CSL (RAM) retain the ability to associate with CSL in a stable complex through interactions involving Maml. However, Maml does not directly bind to CSL. Furthermore, Maml can rescue Delta RAM transcriptional activity on a CSL-dependent promoter. These results indicate that deletion of the RAM domain does not equate to CSL-independent signaling. Moreover, in SUP-T1 cells, N(ic) exists exclusively in the largest N(ic)-containing complex. SUP-T1 cells are derived from a T-cell leukemia that harbors the t(7;9)(q34;q34.3) translocation and constitutively express N(ic). Taken together, our data indicate that complex formation is likely required for neoplastic transformation by Notch(ic).

• Lai EC
• Keeping a good pathway down: transcriptional repression of Notch pathway target genes by CSL proteins.
• EMBO Rep. 2002; 3: 840-5
• Display abstract

• CSL [CBF-1, Su(H), Lag-1]-type transcription factors are the primary effectors of the Notch pathway, a signal transduction cascade that is essential for the development of all metazoan organisms. Interestingly, CSL proteins were originally classified as transcriptional repressors in vertebrates, but as transcriptional activators in model invertebrate organisms. Resolution of this paradox came with the realization that repression and activation by CSL proteins occurs in both systems and that the switch involves recruitment of distinct co-repressor and co-activator complexes. Although CSL proteins appear to utilize a common co-activator complex of largely similar constitution, recent studies have demonstrated that vertebrate and Drosophila CSL interact with a variety of distinct co-repressor complexes. This review highlights differences in composition and similarities in function of different CSL co-repressor complexes, which actively repress Notch pathway target genes in the absence of Notch pathway activity.

• Wilson JR et al.
• Crystal structure and functional analysis of the histone methyltransferase SET7/9.
• Cell. 2002; 111: 105-15
• Display abstract

• Methylation of lysine residues in the N-terminal tails of histones is thought to represent an important component of the mechanism that regulates chromatin structure. The evolutionarily conserved SET domain occurs in most proteins known to possess histone lysine methyltransferase activity. We present here the crystal structure of a large fragment of human SET7/9 that contains a N-terminal beta-sheet domain as well as the conserved SET domain. Mutagenesis identifies two residues in the C terminus of the protein that appear essential for catalytic activity toward lysine-4 of histone H3. Furthermore, we show how the cofactor AdoMet binds to this domain and present biochemical data supporting the role of invariant residues in catalysis, binding of AdoMet, and interactions with the peptide substrate.

• Peng H, Zheng L, Lee WH, Rux JJ, Rauscher FJ 3rd
• A common DNA-binding site for SZF1 and the BRCA1-associated zinc finger protein, ZBRK1.
• Cancer Res. 2002; 62: 3773-81
• Display abstract

• More than 220 Kruppel-associated box-zinc finger protein (KRAB-ZFP) genes are encoded in the human genome. KRAB-ZFPs function as transcriptionalrepressors by binding DNA through their tandem zinc finger motifs.Gene silencing is mediated by the highly conserved KRAB domain, which recruits histone deacetylase complexes, histone methylases, and heterochromatin proteins. However, little is known of the biological programs regulated by KRAB-ZFPs, in large part because of the difficulty in identifying DNA-binding sites recognized by long arrays of zinc fingers. In an attempt to identify the natural target genes for a KRAB-ZFP, we chose SZF1, a hematopoietic progenitor-restricted, KRAB-ZFP that contains only four C(2)H(2) zinc finger motifs. Using recombinant SZF1 protein and a PCR-based binding site selection strategy, we identified a 15-bp consensus DNA sequence recognized by SZF1. Remarkably, this sequence is similar to the core DNA-binding site described recently for ZBRK1, a KRAB-ZFP that binds to BRCA1 and is involved in coordinating the cellular DNA damage response. The SZF1 and ZBRK1 proteins bind to both the experimentally derived SZF1 site and the canonical ZBRK1 site. The KRAB domain from SZF1 bound directly to the KAP-1 corepressor and displayed intrinsic silencing activity. Moreover, full-length SZF1 repressed a promoter containing ZBRK1 recognition sequences. Thus, SZF1 and ZBRK1 may regulate a common set of target genes in vivo.

• Kim SS, Zhang RG, Braunstein SE, Joachimiak A, Cvekl A, Hegde RS
• Structure of the retinal determination protein Dachshund reveals a DNA binding motif.
• Structure. 2002; 10: 787-95
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• The Dachshund proteins are essential components of a regulatory network controlling cell fate determination. They have been implicated in eye, limb, brain, and muscle development. These proteins cannot be assigned to any recognizable structural or functional class based on amino acid sequence analysis. The 1.65 A crystal structure of the most conserved domain of human DACHSHUND is reported here. The protein forms an alpha/beta structure containing a DNA binding motif similar to that found in the winged helix/forkhead subgroup of the helix-turn-helix family. This unexpected finding alters the previously proposed molecular models for the role of Dachshund in the eye determination pathway. Furthermore, it provides a rational framework for future mechanistic analyses of the Dachshund proteins in several developmental contexts.

• Barolo S, Stone T, Bang AG, Posakony JW
• Default repression and Notch signaling: Hairless acts as an adaptor to recruit the corepressors Groucho and dCtBP to Suppressor of Hairless.
• Genes Dev. 2002; 16: 1964-76
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• The DNA-binding transcription factor Suppressor of Hairless [Su(H)] functions as an activator during Notch (N) pathway signaling, but can act as a repressor in the absence of signaling. Hairless (H), a novel Drosophila protein, binds to Su(H) and has been proposed to antagonize N signaling by inhibiting DNA binding by Su(H). Here we show that, in vitro, H directly binds two corepressor proteins, Groucho (Gro) and dCtBP. Reduction of gro or dCtBP function enhances H mutant phenotypes and suppresses N phenotypes in the adult mechanosensory bristle. This activity of gro is surprising, because it is directed oppositely to its traditionally defined role as a neurogenic gene. We find that Su(H)-H complexes can bind to DNA with high efficiency in vitro. Furthermore, a H-VP16 fusion protein causes dominant-negative phenotypes in vivo, a result consistent with the proposal that H functions in transcriptional repression. Taken together, our findings indicate that "default repression" of N pathway target genes by an unusual adaptor/corepressor complex is essential for proper cell fate specification during Drosophila peripheral nervous system development.

• Oswald F et al.
• p300 acts as a transcriptional coactivator for mammalian Notch-1.
• Mol Cell Biol. 2001; 21: 7761-74
• Display abstract

• Notch-1 belongs to a family of transmembrane receptor proteins that direct the decisions as to various cell fates. After ligand binding, a proteolytic cleavage step occurs and the intracellular part of Notch-1, Notch-1-IC, translocates into the nucleus, where it targets the DNA binding protein RBP-J kappa/CBF1. RBP-J kappa mediates repression through recruitment of a histone deacetylase-containing complex. The Notch-1-IC/RBP-J kappa complex overcomes repression and activates the transcription of Notch target genes. We have identified a novel domain in Notch-1-IC, the EP domain, which is indispensable for full transcriptional activation. This transactivation domain is localized adjacent to the ankyrin repeats of Notch-1-IC. In cotransfection experiments, Notch-1-IC-mediated transcriptional activation was inhibited by E1A12S and p53, two proteins, which interfere with the function of the common coactivator p300. Protein-protein interaction assays demonstrated the association of Notch-1-IC and the CH3 region of p300. In addition, the interaction of mammalian Notch-1-IC with p300 was destabilized after deletion of the EP domain of Notch-1-IC. Based on physical interaction with Notch-1-IC and coactivator functions of p300, we propose a model for Notch-1-mediated gene regulation via p300.

• Lamar E et al.
• Nrarp is a novel intracellular component of the Notch signaling pathway.
• Genes Dev. 2001; 15: 1885-99
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• The Lin12/Notch receptors regulate cell fate during embryogenesis by activating the expression of downstream target genes. These receptors signal via their intracellular domain (ICD), which is released from the plasma membrane by proteolytic processing and associates in the nucleus with the CSL family of DNA-binding proteins to form a transcriptional activator. How the CSL/ICD complex activates transcription and how this complex is regulated during development remains poorly understood. Here we describe Nrarp as a new intracellular component of the Notch signaling pathway in Xenopus embryos. Nrarp is a member of the Delta-Notch synexpression group and encodes a small protein containing two ankyrin repeats. Nrarp expression is activated in Xenopus embryos by the CSL-dependent Notch pathway. Conversely, overexpression of Nrarp in embryos blocks Notch signaling and inhibits the activation of Notch target genes by ICD. We show that Nrarp forms a ternary complex with the ICD of XNotch1 and the CSL protein XSu(H) and that in embryos Nrarp promotes the loss of ICD. By down-regulating ICD levels, Nrarp could function as a negative feedback regulator of Notch signaling that attenuates ICD-mediated transcription.

• Mumm JS, Kopan R
• Notch signaling: from the outside in.
• Dev Biol. 2000; 228: 151-65

• Gouet P, Courcelle E, Stuart DI, Metoz F
• ESPript: analysis of multiple sequence alignments in PostScript.
• Bioinformatics. 1999; 15: 305-8
• Display abstract

• MOTIVATION: The program ESPript (Easy Sequencing in PostScript) allows the rapid visualization, via PostScript output, of sequences aligned with popular programs such as CLUSTAL-W or GCG PILEUP. It can read secondary structure files (such as that created by the program DSSP) to produce a synthesis of both sequence and structural information. RESULTS: ESPript can be run via a command file or a friendly html-based user interface. The program calculates an homology score by columns of residues and can sort this calculation by groups of sequences. It offers a palette of markers to highlight important regions in the alignment. ESPript can also paste information on residue conservation into coordinate files, for subsequent visualization with a graphics program. AVAILABILITY: ESPript can be accessed on its Web site at http://www.ipbs.fr/ESPript. Sources and helpfiles can be downloaded via anonymous ftp from ftp.ipbs.fr. A tar file is held in the directory pub/ESPript.

• Perrakis A, Morris R, Lamzin VS
• Automated protein model building combined with iterative structure refinement.
• Nat Struct Biol. 1999; 6: 458-63
• Display abstract

• In protein crystallography, much time and effort are often required to trace an initial model from an interpretable electron density map and to refine it until it best agrees with the crystallographic data. Here, we present a method to build and refine a protein model automatically and without user intervention, starting from diffraction data extending to resolution higher than 2.3 A and reasonable estimates of crystallographic phases. The method is based on an iterative procedure that describes the electron density map as a set of unconnected atoms and then searches for protein-like patterns. Automatic pattern recognition (model building) combined with refinement, allows a structural model to be obtained reliably within a few CPU hours. We demonstrate the power of the method with examples of a few recently solved structures.

• Lam LT, Bresnick EH
• Identity of the beta-globin locus control region binding protein HS2NF5 as the mammalian homolog of the notch-regulated transcription factor suppressor of hairless.
• J Biol Chem. 1998; 273: 24223-31
• Display abstract

• Previously, we characterized a DNA-binding protein, HS2NF5, that bound tightly to a conserved region within hypersensitive site 2 (HS2) of the human beta-globin locus control region (LCR) (Lam, L. T. , and Bresnick, E. H. (1996) J. Biol. Chem. 271, 32421-32429). The beta-globin LCR controls the chromatin structure, transcription, and replication of the beta-globin genes. We have now purified HS2NF5 to near-homogeneity from fetal bovine thymus. Two polypeptides of 56 and 61 kDa copurified with the DNA binding activity. The two proteins bound to the LCR recognition site with an affinity (3.1 nM) and specificity similar to mouse erythroleukemia cell HS2NF5. The amino acid sequences of tryptic peptides of purified HS2NF5 revealed it to be identical to the murine homolog of the suppressor of hairless transcription factor, also known as recombination signal binding protein Jkappa or C promoter binding factor 1 (CBF1). The CBF1 site within HS2 resides near sites for hematopoietic regulators such as GATA-1, NF-E2, and TAL1. An additional conserved, high affinity CBF1 site was localized within HS4 of the LCR. As CBF1 is a downstream target of the Notch signaling pathway, we propose that Notch may modulate LCR activity during hematopoiesis.

• Kimble J, Simpson P
• The LIN-12/Notch signaling pathway and its regulation.
• Annu Rev Cell Dev Biol. 1997; 13: 333-61
• Display abstract

• Notch, LIN-12, and GLP-1 are receptors that mediate a broad range of cell interactions during Drosophila and nematode development. Signaling by these receptors relies on a conserved pathway with three core components: DSL ligand, LNG receptor, and a CSL effector that links the receptor to its transcriptional response. Although key functional regions have been identified in each class of proteins, the mechanism for signal transduction is not yet understood. Diverse regulatory mechanisms influence signaling by the LIN-12/Notch pathway. Inductive signaling relies on the synthesis of ligand and receptor in distinct but neighboring cells. By contrast, lateral signaling leads to the transformation of equivalent cells that express both ligand and receptor into nonequivalent cells that express either ligand or receptor. This transformation appears to rely on regulatory feedback loops within the LIN-12/Notch pathway. In addition, the pathway can be regulated by intrinsic factors that are asymmetrically segregated during cell division or by extrinsic cues via other signaling pathways. Specificity in the pathway does not appear to reside in the particular ligand or receptor used for a given cell-cell interaction. The existence of multiple ligands and receptors may have evolved from the stringent demands placed upon the regulation of genes encoding them.

• Jones TA, Kjeldgaard M
• Electron-density map interpretation.
• Methods Enzymol. 1997; 277: 173-208

• el Hassan MA, Calladine CR
• Propeller-twisting of base-pairs and the conformational mobility of dinucleotide steps in DNA.
• J Mol Biol. 1996; 259: 95-103
• Display abstract

• When DNA is bent around a protein, it must distort. The distortion occurs by changes in the conformation of successive dinucleotide steps. Bending does not necessarily occur uniformly: some steps might remain particularly rigid, i.e. they might deform relatively little, while others might take more than their proportional share of deformation. We investigate here the deformational capacity of specific dinucleotide steps by examining a database of crystallized oligomers. Dividing the steps into ten types by sequence (AA( = TT), AC( = GT), AG( = CT), AT, CA( = TG), CG, GA( = TC), GC, GG( = CC) and TA), we find that some step types are practically rigid, while others have considerable internal mobility or conformational flexibility. Now in general base-pairs are not planar, but have Propeller-Twist. We find a clear empirical correlation between the level of Propeller-Twist in the base-pairs and the flexibility of the dinucleotide step which they constitute. Propeller-Twist in the base-pairs makes stacking into a dinucleotide step more awkward than in plane base-pairs. In particular, it provides a stereochemical "locking" effect which can make steps with highly Propeller-Twisted base-pairs rigid. Although the origins of Propeller-Twist are not yet clearly understood, this result provides a key to understanding the flexibility of DNA in bending around proteins.

• Chung CN, Hamaguchi Y, Honjo T, Kawaichi M
• Site-directed mutagenesis study on DNA binding regions of the mouse homologue of Suppressor of Hairless, RBP-J kappa.
• Nucleic Acids Res. 1994; 22: 2938-44
• Display abstract

• To map regions important for DNA binding of the mouse homologue of Suppressor of Hairless or RBP-J kappa protein, mutated mouse RBP-J kappa cDNAs were made by insertion of oligonucleotide linkers or base replacement. DNA binding assays using the mutated proteins expressed in COS cells showed that various mutations between 218 Arg and 227 Arg decreased the DNA binding activity drastically. The DNA binding activity was not affected by amino acid replacements within the integrase motif of the RBP-J kappa protein (230His-269His). Replacements between 291Arg and 323Tyr affected the DNA binding activity slightly but reproducibly. These results indicate that the region encompassing 218Arg-227Arg is critical for the DNA binding activity of RBP-J kappa. This region did not show any significant homology to motifs or domains of the previously described DNA binding proteins. Using a truncation mutant protein RBP-J kappa was shown to associate with DNA as a monomer.

• Graves BJ, Hatada MH, Hendrickson WA, Miller JK, Madison VS, Satow Y
• Structure of interleukin 1 alpha at 2.7-A resolution.
• Biochemistry. 1990; 29: 2679-84
• Display abstract

• The interleukin 1 (IL-1) family of proteins has a central role in modulating immune and inflammatory responses. Two major IL-1 proteins, designated alpha (IL-1 alpha) and beta (IL-1 beta), are produced by activated macrophages and other cell types. In an effort to understand the similarities and differences in the physicochemical and functional properties of these two proteins, a program was initiated to determine their structures. Crystals of IL-1 alpha were grown, and the three-dimensional structure at 2.7-A resolution was solved. The technique of multiple-wavelength anomalous dispersion (MAD) with the selenomethionine form of IL-1 alpha was utilized in combination with a single mercury derivative to provide the starting phases. Partial refinement of the IL-1 alpha model has been performed as well. The overall structure is composed of 14 beta-strands and a 3(10) helix. The core of this structure is a capped beta-barrell that possesses 3-fold symmetry and displays a topology similar to that observed for IL-1 beta [Priestle, J. P., et al. (1988) EMBO J. 7, 339-343] and soybean trypsin inhibitor (STI) [McLachlan, A. D. (1979) J. Mol. Biol. 133, 557-563]. In this paper, the overall structure of IL-1 alpha and the nature and fidelity of the internal 3-fold symmetry are discussed. Comparisons with IL-1 beta and STI are made within these contexts.

• Kabsch W, Sander C
• Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features.
• Biopolymers. 1983; 22: 2577-637

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