Structural and functional characterization of a phosphatase domain within yeast general transcription factor TFIIIC.
Taylor, N.M., Glatt, S., Hennrich, M.L., von Scheven, G., Grotsch, H., Fernandez-Tornero, C., Rybin, V., Gavin, A.C., Kolb, P. & Muller, C.W.
J Biol Chem. 2013 Apr 8.
Saccharomyces cerevisiae tau55, a subunit of the RNA polymerase III-specific general transcription factor TFIIIC, comprises an N-terminal histidine phosphatase domain (tau55-HPD) whose catalytic activity and cellular function is poorly understood. We solved the crystal structures of tau55-HPD and its closely related paralogue Huf and used in silico docking methods to identify phospho-serine and phospho-tyrosine containing peptides as possible substrates that were subsequently validated using in vitro phosphatase assays. A comparative phospho-proteomic study identified additional phosphopeptides as possible targets, which show the involvement of these two phosphatases in the regulation of a variety of cellular functions. Our results identify tau55-HPD and Huf as bona fide protein phosphatases, characterize their substrate specificities and provide a small set of regulated phosphosite targets in vivo.
Structural insights into Elongator function.
Glatt, S. & Muller, C.W.
Curr Opin Struct Biol. 2013 Mar 16. pii: S0959-440X(13)00038-9. doi:10.1016/j.sbi.2013.02.009.
The eukaryotic Elongator complex was initially identified in yeast as a RNA polymerase II (Pol II) associated transcription elongation factor, although there is accumulating evidence that its main cellular function is the specific modification of uridines at the wobble base position of tRNAs. Elongator complex is built up by six highly conserved subunits and was shown to be involved in a variety of different cellular activities. Here, we summarize structural and functional information on individual Elongator subunits or subcomplexes. On the basis of homology models of the Elp1, Elp2 and Elp3 subunits and the crystal structure of the Elp456 subcomplex, the role of each subunit in Elongator complex assembly and catalytic activity is discussed.
Bacterial expression, purification, and crystallization of tyrosine phosphorylated STAT proteins.
Baudin, F. & Muller, C.W.
Methods Mol Biol. 2013;967:301-17. doi: 10.1007/978-1-62703-242-1_21.
Signal Transducer and Activator of Transcription (STAT) proteins are latent cytoplasmic transcription -factors that become activated by phosphorylation at a C-terminal tyrosine residue. Upon activation STAT proteins translocate to the nucleus and bind to their specific target sites. Here, we describe the recombinant expression of tyrosine phosphorylated STAT proteins in bacteria. This method allows the production of large amounts of activated STAT proteins for structural and biochemical studies including the high-throughput screening of chemical libraries.
Elongator: transcriptional or translational regulator?
Glatt, S., Seraphin, B. & Muller, C.W.
Transcription. 2012 Aug 14;3(6).
The conserved multi-subunit Elongator complex was initially described as a RNA polymerase II (RNAPII) associated transcription elongation factor, but since has been shown to be involved a variety of different cellular activities. Here, we summarize recent developments in the field and discuss the resulting implications for the proposed multi-functionality of Elongator.
The Elongator subcomplex Elp456 is a hexameric RecA-like ATPase.
Glatt, S., Letoquart, J., Faux, C., Taylor, N.M., Seraphin, B. & Muller, C.W.
Nat Struct Mol Biol. 2012 Feb 19. doi: 10.1038/nsmb.2234.
Elongator was initially described as an RNA polymerase II-associated factor but has since been associated with a broad range of cellular activities. It has also attracted clinical attention because of its role in certain neurodegenerative diseases. Here we describe the crystal structure of the Saccharomyces cerevisiae subcomplex of Elongator proteins 4, 5 and 6 (Elp456). The subunits each show almost identical RecA folds that form a heterohexameric ring-like structure resembling hexameric RecA-like ATPases. This structural finding is supported by different complementary in vitro and in vivo approaches, including the specific binding of the hexameric Elp456 subcomplex to tRNAs in a manner regulated by ATP. Our results support a role of Elongator in tRNA modification, explain the importance of each of the Elp4, Elp5 and Elp6 subunits for complex integrity and suggest a model for the overall architecture of the holo-Elongator complex.
Analyzing RNA polymerase III by electron cryomicroscopy.
Fernandez-Martos, C., Bottcher, B., Rashid, U.J. & Muller, C.W.
RNA Biol. 2011 Sep 1;8(5).
Recent electron cryomicroscopy reconstructions have provided new insights into the overall organization of yeast RNA polymerase (Pol) III, responsible for the synthesis of small, non-translated RNAs. The structure of the free Pol III enzyme at 10 A resolution provides an accurate framework to better understand its overall architecture and the structural organization and functional role of two Pol III-specific subcomplexes. Cryo-EM structures of elongating Pol III bound to DNA/RNA scaffolds show the rearrangement of the Pol III-specific subcomplexes that enclose incoming DNA. In one reconstruction downstream DNA and newly transcribed RNA can be followed over considerably longer distances as in the crystal structure of elongating Pol II. The Pol III transcription machinery is increasingly recognized as a possible target for cancer therapy. The recent cryo-EM reconstructions contribute to the molecular understanding of Pol III transcription as a prerequisite for targeting its components.
Chromatin-modifying Complex Component Nurf55/p55 Associates with Histones H3 and H4 and Polycomb Repressive Complex 2 Subunit Su(z)12 through Partially Overlapping Binding Sites.
Nowak, A.J., Alfieri, C., Stirnimann, C.U., Rybin, V., Baudin, F., Ly-Hartig, N., Lindner, D. & Muller, C.W.
J Biol Chem. 2011 Jul 1;286(26):23388-96. Epub 2011 May 5.
Drosophila Nurf55 is a component of different chromatin-modifying complexes, including the PRC2 (Polycomb repressive complex 2). Based on the 1.75-A crystal structure of Nurf55 bound to histone H4 helix 1, we analyzed interactions of Nurf55 (Nurf55 or p55 in fly and RbAp48/46 in human) with the N-terminal tail of histone H3, the first helix of histone H4, and an N-terminal fragment of the PRC2 subunit Su(z)12 using isothermal calorimetry and pulldown experiments. Site-directed mutagenesis identified the binding site of histone H3 at the top of the Nurf55 WD40 propeller. Unmodified or K9me3- or K27me3-containing H3 peptides were bound with similar affinities, whereas the affinity for K4me3-containing H3 peptides was reduced. Helix 1 of histone H4 and Su(z)12 bound to the edge of the beta-propeller using overlapping binding sites. Our results show similarities in the recognition of histone H4 and Su(z)12 and identify Nurf55 as a versatile interactor that simultaneously contacts multiple partners.
Recognizing and remodeling the nucleosome.
Glatt, S., Alfieri, C. & Muller, C.W.
Curr Opin Struct Biol. 2011 Jun;21(3):335-41. Epub 2011 Mar 4.
The X-ray structure of the nucleosome core particle (NCP) has been a major milestone in the structural biology of chromatin. Since, our understanding how NCPs interact with multiple partners has been extending from single chromatin-binding domains recognizing post-translational modifications (PTMs) in histone tails towards the recognition of higher-order chromatin structure by multi-subunit chromatin remodeling complexes. The current review summarizes recent progress in the structural biology of nucleosome-recognition from chromatin-binding domains to multi-protein remodeling complexes.
Insights into the function of the CRM1 cofactor RanBP3 from the structure of its Ran-binding domain.
Langer, K., Dian, C., Rybin, V., Muller, C.W. & Petosa, C.
PLoS One. 2011 Feb 25;6(2):e17011.
Proteins bearing a leucine-rich nuclear export signal (NES) are exported from the nucleus by the transport factor CRM1, which forms a cooperative ternary complex with the NES-bearing cargo and with the small GTPase Ran. CRM1-mediated export is regulated by RanBP3, a Ran-interacting nuclear protein. Unlike the related proteins RanBP1 and RanBP2, which promote disassembly of the export complex in the cytosol, RanBP3 acts as a CRM1 cofactor, enhancing NES export by stabilizing the export complex in the nucleus. RanBP3 also alters the cargo selectivity of CRM1, promoting recognition of the NES of HIV-1 Rev and of other cargos while deterring recognition of the import adaptor protein Snurportin1. Here we report the crystal structure of the Ran-binding domain (RBD) from RanBP3 and compare it to RBD structures from RanBP1 and RanBP2 in complex with Ran and CRM1. Differences among these structures suggest why RanBP3 binds Ran with unusually low affinity, how RanBP3 modulates the cargo selectivity of CRM1, and why RanBP3 promotes assembly rather than disassembly of the export complex. The comparison of RBD structures thus provides an insight into the functional diversity of Ran-binding proteins.
Mass Spectrometry Reveals Stable Modules in holo and apo RNA Polymerases I and III.
Lane, L.A., Fernandez-Tornero, C., Zhou, M., Morgner, N., Ptchelkine, D., Steuerwald, U., Politis, A., Lindner, D., Gvozdenovic, J., Gavin, A.C., Muller, C.W. & Robinson, C.V.
Structure. 2011 Jan 12;19(1):90-100.
RNA polymerases are essential enzymes which transcribe DNA into RNA. Here, we obtain mass spectra of the cellular forms of apo and holo eukaryotic RNA polymerase I and III, defining their composition under different solution conditions. By recombinant expression of subunits within the initiation heterotrimer of Pol III, we derive an interaction network and couple this data with ion mobility data to define topological restraints. Our data agree with available structural information and homology modeling and are generally consistent with yeast two hybrid data. Unexpectedly, elongation complexes of both Pol I and III destabilize the assemblies compared with their apo counterparts. Increasing the pH and ionic strength of apo and holo forms of Pol I and Pol III leads to formation of at least ten stable subcomplexes for both enzymes. Uniquely for Pol III many subcomplexes contain only one of the two largest catalytic subunits. We speculate that these stable subcomplexes represent putative intermediates in assembly pathways.
Human importin alpha and RNA do not compete for binding to influenza A virus nucleoprotein.
Boulo, S., Akarsu, H., Lotteau, V., Muller, C.W., Ruigrok, R.W. & Baudin, F.
Virology. 2011 Jan 5;409(1):84-90. Epub 2010 Oct 25.
Influenza virus has a segmented genome composed of eight negative stranded RNA segments. Each segment is covered with NP forming ribonucleoproteins (vRNPs) and carries a copy of the heterotrimeric polymerase complex. As a rare phenomenon among the RNA viruses, the viral replication occurs in the nucleus and therefore implies interactions between host and viral factors, such as between importin alpha and nucleoprotein. In the present study we report that through binding with the human nuclear receptor importin alpha5 (Impalpha5), the viral NP is no longer oligomeric but maintained as a monomer inside the complex. In this regard, Impalpha5 acts as a chaperone until NP is delivered in the nucleus for viral RNA encapsidation. Moreover, we show that the association of NP with the host transporter does not impair the binding of NP to RNA. The complex human Impalpha5-NP binds RNA with the same affinity as wt NP alone, whereas engineered monomeric NP through point mutations binds RNA with a strongly reduced affinity.
A systematic screen for protein-lipid interactions in Saccharomyces cerevisiae.
Gallego, O., Betts, M.J., Gvozdenovic-Jeremic, J., Maeda, K., Matetzki, C., Aguilar-Gurrieri, C., Beltran-Alvarez, P., Bonn, S., Fernandez-Tornero, C., Jensen, L.J., Kuhn, M., Trott, J., Rybin, V., Muller, C.W., Bork, P., Kaksonen, M., Russell, R.B. & Gavin, A.C.
Mol Syst Biol. 2010 Nov 30;6:430. doi: 10.1038/msb.2010.87.
Protein-metabolite networks are central to biological systems, but are incompletely understood. Here, we report a screen to catalog protein-lipid interactions in yeast. We used arrays of 56 metabolites to measure lipid-binding fingerprints of 172 proteins, including 91 with predicted lipid-binding domains. We identified 530 protein-lipid associations, the majority of which are novel. To show the data set's biological value, we studied further several novel interactions with sphingolipids, a class of conserved bioactive lipids with an elusive mode of action. Integration of live-cell imaging suggests new cellular targets for these molecules, including several with pleckstrin homology (PH) domains. Validated interactions with Slm1, a regulator of actin polarization, show that PH domains can have unexpected lipid-binding specificities and can act as coincidence sensors for both phosphatidylinositol phosphates and phosphorylated sphingolipids.
Full repression of RNA polymerase III transcription requires interaction between two domains of its negative regulator Maf1.
Gajda, A., Towpik, J., Steuerwald, U., Muller, C.W., Lefebvre, O. & Boguta, M.
J Biol Chem. 2010 Nov 12;285(46):35719-27. Epub 2010 Sep 3.
Maf1, first identified in yeast Saccharomyces cerevisiae, is a general negative regulator of RNA polymerase III (Pol III). Transcription regulation by Maf1 is important under stress conditions and during the switch between fermentation and respiration. Maf1 is composed of two domains conserved during evolution. We report here that these two domains of human Maf1 are resistant to mild proteolysis and interact together as shown by pull-down and size-exclusion chromatography and that the comparable domains of yeast Maf1 interact in a two-hybrid assay. Additionally, in yeast, a mutation in the N-terminal domain is compensated by mutations in the C-terminal domain. Integrity of both domains and their direct interaction are necessary for Maf1 dephosphorylation and subsequent inhibition of Pol III transcription on a nonfermentable carbon source. These data relate Pol III transcription inhibition to Maf1 structural changes.
Solving the NES problem.
Mattaj, I.W. & Muller, C.W.
Nat Struct Mol Biol. 2010 Nov;17(11):1288-9. PubMed
Conformational flexibility of RNA polymerase III during transcriptional elongation.
Fernandez-Tornero, C., Bottcher, B., Rashid, U.J., Steuerwald, U., Florchinger, B., Devos, D.P., Lindner, D. & Muller, C.W.
EMBO J. 2010 Nov 17;29(22):3762-72. Epub 2010 Oct 22
RNA polymerase (Pol) III is responsible for the transcription of genes encoding small RNAs, including tRNA, 5S rRNA and U6 RNA. Here, we report the electron cryomicroscopy structures of yeast Pol III at 9.9 A resolution and its elongation complex at 16.5 A resolution. Particle sub-classification reveals prominent EM densities for the two Pol III-specific subcomplexes, C31/C82/C34 and C37/C53, that can be interpreted using homology models. While the winged-helix-containing C31/C82/C34 subcomplex initiates transcription from one side of the DNA-binding cleft, the C37/C53 subcomplex accesses the transcription bubble from the opposite side of this cleft. The transcribing Pol III enzyme structure not only shows the complete incoming DNA duplex, but also reveals the exit path of newly synthesized RNA. During transcriptional elongation, the Pol III-specific subcomplexes tightly enclose the incoming DNA duplex, which likely increases processivity and provides structural insights into the conformational switch between Pol III-mediated initiation and elongation.
Structural basis of TBX5-DNA recognition: the T-box domain in its DNA-bound and -unbound form.
Stirnimann, C.U., Ptchelkine, D., Grimm, C. & Muller, C.W.
J Mol Biol. 2010 Jul 2;400(1):71-81. Epub 2010 May 5.
TBX5, a member of the T-box transcription factor family, plays an important role in heart and limb development. More than 60 single point or deletion mutations of human TBX5 are associated with Holt-Oram syndrome that manifests itself as heart and limb malformations in 1 out of 100,000 live births. The majority of these mutations are located in the TBX5 T-box domain. We solved the crystal structures of the human TBX5 T-box domain in its DNA-unbound form and in complex with a natural DNA target site allowing for the first time the comparison between unbound and DNA-bound forms. Our analysis identifies a 3(10)-helix at the C-terminus of the T-box domain as an inducible recognition element, critically required for the interaction with DNA, as it only forms upon DNA binding and is unstructured in the DNA-unbound form. Using circular dichroism, we characterized the thermal stability of six TBX5 mutants containing single point mutations in the T-box domain (M74V, G80R, W121G, G169R, T223M, and R237W) and compared them with wild-type protein. Mutants G80R and W121G show drastically reduced thermal stability, while the other mutants only show a marginal stability decrease. For all TBX5 mutants, binding affinities to specific and nonspecific DNA sequences were determined using isothermal titration calorimetry. All TBX5 mutants show reduced binding affinities to a specific DNA target site, although to various degrees. Interestingly, all tested TBX5 mutants differ in their ability to bind unspecific DNA, indicating that both sequence-specific and unspecific binding might contribute to the misregulation of target gene expression.
WD40 proteins propel cellular networks.
Stirnimann, C.U., Petsalaki, E., Russell, R.B. & Muller, C.W.
Trends Biochem Sci. 2010 Oct;35(10):565-74. Epub 2010 May 5. Review
Recent findings indicate that WD40 domains play central roles in biological processes by acting as hubs in cellular networks; however, they have been studied less intensely than other common domains, such as the kinase, PDZ or SH3 domains. As suggested by various interactome studies, they are among the most promiscuous interactors. Structural studies suggest that this property stems from their ability, as scaffolds, to interact with diverse proteins, peptides or nucleic acids using multiple surfaces or modes of interaction. A general scaffolding role is supported by the fact that no WD40 domain has been found with intrinsic enzymatic activity despite often being part of large molecular machines. We discuss the WD40 domain distributions in protein networks and structures of WD40-containing assemblies to demonstrate their versatility in mediating critical cellular functions.
A novel key element in post-meiotic male genome reprogramming
Rousseaux, S., Petosa, C., Muller, C.W. & Khochbin, S.
Med Sci (Paris). 2010 Feb;26(2):130-2. PubMed
Cooperative binding of two acetylation marks on a histone tail by a single bromodomain.
Moriniere, J., Rousseaux, S., Steuerwald, U., Soler-Lopez, M., Curtet, S., Vitte, A.L., Govin, J., Gaucher, J., Sadoul, K., Hart, D.J., Krijgsveld, J., Khochbin, S., Muller, C.W. & Petosa, C.
Nature. 2009 Oct 1;461(7264):664-8.
A key step in many chromatin-related processes is the recognition of histone post-translational modifications by effector modules such as bromodomains and chromo-like domains of the Royal family. Whereas effector-mediated recognition of single post-translational modifications is well characterized, how the cell achieves combinatorial readout of histones bearing multiple modifications is poorly understood. One mechanism involves multivalent binding by linked effector modules. For example, the tandem bromodomains of human TATA-binding protein-associated factor-1 (TAF1) bind better to a diacetylated histone H4 tail than to monoacetylated tails, a cooperative effect attributed to each bromodomain engaging one acetyl-lysine mark. Here we report a distinct mechanism of combinatorial readout for the mouse TAF1 homologue Brdt, a testis-specific member of the BET protein family. Brdt associates with hyperacetylated histone H4 (ref. 7) and is implicated in the marked chromatin remodelling that follows histone hyperacetylation during spermiogenesis, the stage of spermatogenesis in which post-meiotic germ cells mature into fully differentiated sperm. Notably, we find that a single bromodomain (BD1) of Brdt is responsible for selectively recognizing histone H4 tails bearing two or more acetylation marks. The crystal structure of BD1 bound to a diacetylated H4 tail shows how two acetyl-lysine residues cooperate to interact with one binding pocket. Structure-based mutagenesis that reduces the selectivity of BD1 towards diacetylated tails destabilizes the association of Brdt with acetylated chromatin in vivo. Structural analysis suggests that other chromatin-associated proteins may be capable of a similar mode of ligand recognition, including yeast Bdf1, human TAF1 and human CBP/p300 (also known as CREBBP and EP300, respectively). Our findings describe a new mechanism for the combinatorial readout of histone modifications in which a single effector module engages two marks on a histone tail as a composite binding epitope.
Molecular recognition of histone lysine methylation by the Polycomb group repressor dSfmbt.
Grimm, C., Matos, R., Ly-Hartig, N., Steuerwald, U., Lindner, D., Rybin, V., Muller, J. & Muller, C.W.
EMBO J. 2009 Jul 8;28(13):1965-77. Epub 2009 Jun 4.
Polycomb group (PcG) proteins repress transcription by modifying chromatin structure in target genes. dSfmbt is a subunit of the Drosophila melanogaster PcG protein complex PhoRC and contains four malignant brain tumour (MBT) repeats involved in the recognition of various mono- and dimethylated histone peptides. Here, we present the crystal structure of the four-MBT-repeat domain of dSfmbt in complex with a mono-methylated histone H4 peptide. Only a single histone peptide binds to the four-MBT-repeat domain. Mutational analyses show high-affinity binding with low peptide sequence selectivity through combinatorial interaction of the methyl-lysine with an aromatic cage and positively charged flanking residues with the surrounding negatively charged surface of the fourth MBT repeat. dSfmbt directly interacts with the PcG protein Scm, a related MBT-repeat protein with similar methyl-lysine binding activity. dSfmbt and Scm co-occupy Polycomb response elements of target genes in Drosophila and they strongly synergize in the repression of these target genes, suggesting that the combined action of these two MBT proteins is crucial for Polycomb silencing.
Structural basis for LEAFY floral switch function and similarity with helix-turn-helix proteins.
Hames, C., Ptchelkine, D., Grimm, C., Thevenon, E., Moyroud, E., Gerard, F., Martiel, J.L., Benlloch, R., Parcy, F. & Muller, C.W.
EMBO J. 2008 Oct 8;27(19):2628-37. Epub 2008 Sep 11.
The LEAFY (LFY) protein is a key regulator of flower development in angiosperms. Its gradually increased expression governs the sharp floral transition, and LFY subsequently controls the patterning of flower meristems by inducing the expression of floral homeotic genes. Despite a wealth of genetic data, how LFY functions at the molecular level is poorly understood. Here, we report crystal structures for the DNA-binding domain of Arabidopsis thaliana LFY bound to two target promoter elements. LFY adopts a novel seven-helix fold that binds DNA as a cooperative dimer, forming base-specific contacts in both the major and minor grooves. Cooperativity is mediated by two basic residues and plausibly accounts for LFY's effectiveness in triggering sharp developmental transitions. Our structure reveals an unexpected similarity between LFY and helix-turn-helix proteins, including homeodomain proteins known to regulate morphogenesis in higher eukaryotes. The appearance of flowering plants has been linked to the molecular evolution of LFY. Our study provides a unique framework to elucidate the molecular mechanisms underlying floral development and the evolutionary history of flowering plants.
Structure of the Drosophila nucleosome core particle highlights evolutionary constraints on the H2A-H2B histone dimer.
Clapier, C.R., Chakravarthy, S., Petosa, C., Fernandez-Tornero, C., Luger, K. & Muller, C.W.
Proteins. 2008 Apr;71(1):1-7.
We determined the 2.45 A crystal structure of the nucleosome core particle from Drosophila melanogaster and compared it to that of Xenopus laevis bound to the identical 147 base-pair DNA fragment derived from human alpha-satellite DNA. Differences between the two structures primarily reflect 16 amino acid substitutions between species, 15 of which are in histones H2A and H2B. Four of these involve histone tail residues, resulting in subtly altered protein-DNA interactions that exemplify the structural plasticity of these tails. Of the 12 substitutions occurring within the histone core regions, five involve small, solvent-exposed residues not involved in intraparticle interactions. The remaining seven involve buried hydrophobic residues, and appear to have coevolved so as to preserve the volume of side chains within the H2A hydrophobic core and H2A-H2B dimer interface. Thus, apart from variations in the histone tails, amino acid substitutions that differentiate Drosophila from Xenopus histones occur in mutually compensatory combinations. This highlights the tight evolutionary constraints exerted on histones since the vertebrate and invertebrate lineages diverged.
Structural and functional analyses of methyl-lysine binding by the malignant brain tumour repeat protein Sex comb on midleg.
Grimm, C., de Ayala Alonso, A.G., Rybin, V., Steuerwald, U., Ly-Hartig, N., Fischle, W., Muller, J. & Muller, C.W.
EMBO Rep. 2007 Nov;8(11):1031-7. Epub 2007 Oct 12.
Sex comb on midleg (Scm) is a member of the Polycomb group of proteins involved in the maintenance of repression of Hox and other developmental control genes in Drosophila. The two malignant brain tumour (MBT) repeats of Scm form a domain that preferentially binds to monomethylated lysine residues either as a free amino acid or in the context of peptides, while unmodified or di- or trimethylated lysine residues are bound with significantly lower affinity. The crystal structure of a monomethyl-lysine-containing histone tail peptide bound to the MBT repeat domain shows that the methyl-lysine side chain occupies a binding pocket in the second MBT repeat formed by three conserved aromatic residues and one aspartate. Insertion of the monomethylated side chain into this pocket seems to be the main contributor to the binding affinity. Functional analyses in Drosophila show that the MBT domain of Scm and its methyl-lysine-binding activity are required for repression of Hox genes.
Insights into transcription initiation and termination from the electron microscopy structure of yeast RNA polymerase III.
Fernandez-Tornero, C., Bottcher, B., Riva, M., Carles, C., Steuerwald, U., Ruigrok, R.W., Sentenac, A., Muller, C.W. & Schoehn, G.
Mol Cell. 2007 Mar 23;25(6):813-23.
RNA polymerase III (RNAPIII) synthesizes tRNA, 5S RNA, U6 snRNA, and other small RNAs. The structure of yeast RNAPIII, determined at 17 A resolution by cryo-electron microscopy and single-particle analysis, reveals a hand-like shape typical of RNA polymerases. Compared to RNAPII, RNAPIII is characterized by a bulkier stalk and by prominent features extending from the DNA binding cleft. We attribute the latter primarily to five RNAPIII-specific subunits, present as two distinct subcomplexes (C82/C34/C31 and C53/C37). Antibody labeling experiments localize the C82/C34/C31 subcomplex to the clamp side of the DNA binding cleft, consistent with its known role in transcription initiation. The C53/C37 subcomplex appears to be situated across the cleft, near the presumed location of downstream DNA, accounting for its role in transcription termination. Our structure rationalizes available mutagenesis and biochemical data and provides insights into RNAPIII-mediated transcription.
Structure of the tau60/Delta tau91 subcomplex of yeast transcription factor IIIC: insights into preinitiation complex assembly.
Mylona, A., Fernandez-Tornero, C., Legrand, P., Haupt, M., Sentenac, A., Acker, J. & Muller, C.W.
Mol Cell. 2006 Oct 20;24(2):221-32.
Yeast RNA polymerase III is recruited upon binding of subcomplexes tauA and tauB of transcription factor IIIC (TFIIIC) to the A and B blocks of tRNA gene promoters. The tauB subcomplex consists of subunits tau60, tau91, and tau138. We determined the 3.2 A crystal structure of tau60 bound to a large C-terminal fragment of tau91 (Deltatau91). Deltatau91 protein contains a seven-bladed propeller preceded by an N-terminal extension, whereas tau60 contains a structurally homologous propeller followed by a C-terminal domain with a novel alpha/beta fold. The two propeller domains do not have any detectable DNA binding activity and mediate heterodimer formation that may serve as scaffold for tau138 assembly. We show that the C-terminal tau60 domain interacts with the TATA binding protein (TBP). Recombinant tauB recruits TBP and stimulates TFIIIB-directed transcription on a TATA box containing tRNA gene, implying a combined contribution of tauA and tauB to preinitiation complex formation.
HDAC6-p97/VCP controlled polyubiquitin chain turnover.
Boyault, C., Gilquin, B., Zhang, Y., Rybin, V., Garman, E., Meyer-Klaucke, W., Matthias, P., Muller, C.W. & Khochbin, S.
EMBO J. 2006 Jul 26;25(14):3357-66. Epub 2006 Jun 29.
HDAC6 is a unique cytoplasmic deacetylase capable of interacting with ubiquitin. Using a combination of biophysical, biochemical and biological approaches, we have characterized the ubiquitin-binding domain of HDAC6, named ZnF-UBP, and investigated its biological functions. These studies show that the three Zn ion-containing HDAC6 ZnF-UBP domain presents the highest known affinity for ubiquitin monomers and mediates the ability of HDAC6 to negatively control the cellular polyubiquitin chain turnover. We further show that HDAC6-interacting chaperone, p97/VCP, dissociates the HDAC6-ubiquitin complexes and counteracts the ability of HDAC6 to promote the accumulation of polyubiquitinated proteins. We propose that a finely tuned balance of HDAC6 and p97/VCP concentrations determines the fate of ubiquitinated misfolded proteins: p97/VCP would promote protein degradation and ubiquitin turnover, whereas HDAC6 would favour the accumulation of ubiquitinated protein aggregates and inclusion body formation.
Karyopherin flexibility in nucleocytoplasmic transport.
Conti, E., Muller, C.W. & Stewart, M.
Curr Opin Struct Biol. 2006 Apr;16(2):237-44. Epub 2006 Mar 29.
Recent structural work on nuclear transport factors of the importin-beta superfamily of karyopherins has shown that these proteins are superhelices of HEAT repeats that are able to assume different conformations in different functional states. The inherent flexibility of these helicoids facilitates the accommodation of different binding partners by an induced-fit type of mechanism. Moreover, the energy stored by distorting these molecules may partially balance binding energies to enable assembly and disassembly of their complexes with relatively small energy changes. Flexibility appears to be an intrinsic feature of such superhelices and might be functionally important not only for karyopherins and nuclear transport, but also for HEAT repeat proteins from other biological systems.
Expression, purification, crystallization and preliminary X-ray analysis of a C-terminal fragment of the Epstein-Barr virus ZEBRA protein.
Morand, P., Budayova-Spano, M., Perrissin, M., Müller, C.W. & Petosa, C.
Acta Crystallograph Sect F Struct Biol Cryst Commun. 2006 Mar 1;62(Pt3):210-4. Epub 2006 Feb 10.
A C-terminal fragment of the Epstein-Barr virus immediate-early transcription factor ZEBRA has been expressed as a recombinant protein in Escherichia coli and purified to homogeneity. The fragment behaves as a dimer in solution, consistent with the presence of a basic region leucine-zipper (bZIP) domain. Crystals of the fragment in complex with a DNA duplex were grown by the hanging-drop vapour-diffusion technique using polyethylene glycol 4000 and magnesium acetate as crystallization agents. Crystals diffract to better than 2.5 A resolution using synchrotron radiation (lambda = 0.976 A). Crystals belong to space group C2, with unit-cell parameters a = 94.2, b = 26.5, c = 98.1 A, beta = 103.9 degrees.
Structural basis of lytic cycle activation by the Epstein-Barr virus ZEBRA protein.
Petosa, C., Morand, P., Baudin, F., Moulin, M., Artero, J.B. & Muller, C.W.
Mol Cell. 2006 Feb 17;21(4):565-72.
Epstein-Barr virus (EBV) causes infectious mononucleosis and is linked to several human malignancies. EBV has a biphasic infection cycle consisting of a latent and a lytic, replicative phase. The switch from latent to lytic infection is triggered by the EBV immediate-early transcription factor ZEBRA (BZLF1, Zta, Z, EB1). We present the crystal structure of ZEBRA's DNA binding domain bound to an EBV lytic gene promoter element. ZEBRA exhibits a variant of the basic-region leucine zipper (bZIP) fold in which a C-terminal moiety stabilizes the coiled coil involved in dimer formation. The structure provides insights into ZEBRA's broad target site specificity, preferential activation of specific EBV promoters in their methylated state, ability to dimerize despite lacking a leucine zipper motif, and failure to heterodimerize with cellular bZIP proteins. The structure will allow for the design of new therapeutic agents that block activation of the EBV lytic cycle.
Expression, proteolytic analysis, reconstitution, and crystallization of the tau60/tau91 subcomplex of yeast TFIIIC.
Mylona, A., Acker, J., Fernandez-Tornero, C., Sentenac, A. & Muller, C.W.
Protein Expr Purif. 2006 Feb;45(2):255-61. Epub 2005 Jul 26.
The transcription factor IIIC (TFIIIC) is a multisubunit DNA-binding factor required for promoter recognition and TFIIIB assembly on tRNA genes transcribed by RNA polymerase III. Yeast TFIIIC consists of six subunits, organized in the two globular subcomplexes tauA and tauB, which recognize two internal tDNA promoter elements, the A and the B block, respectively. As a first step toward a detailed structural analysis of TFIIIC, we report here the expression, proteolytic analysis, reconstitution, and crystallization of the complex between yeast TFIIIC subunits tau91 and tau60. Proteolysis provided an insight into the domain structure of tau60 and tau91. Both the proteins form a stable complex that does not require an N-terminal, protease-sensitive extension of tau91. Crystals diffracting beyond 3.2 A were obtained from a complex formed by full-length tau60 and the N-terminally truncated form of tau91 lacking this extension.
The histone fold subunits of Drosophila CHRAC facilitate nucleosome sliding through dynamic DNA interactions.
Hartlepp, K.F., Fernandez-Tornero, C., Eberharter, A., Grune, T., Müller, C.W. & Becker, P.B.
Mol Cell Biol 2005 Nov;25(22):9886-96.
The chromatin accessibility complex (CHRAC) is an abundant, evolutionarily conserved nucleosome remodeling machinery able to catalyze histone octamer sliding on DNA. CHRAC differs from the related ACF complex by the presence of two subunits with molecular masses of 14 and 16 kDa, whose structure and function were not known. We determined the structure of Drosophila melanogaster CHRAC14-CHRAC16 by X-ray crystallography at 2.4-A resolution and found that they dimerize via a variant histone fold in a typical handshake structure. In further analogy to histones, CHRAC14-16 contain unstructured N- and C-terminal tail domains that protrude from the handshake structure. A dimer of CHRAC14-16 can associate with the N terminus of ACF1, thereby completing CHRAC. Low-affinity interactions of CHRAC14-16 with DNA significantly improve the efficiency of nucleosome mobilization by limiting amounts of ACF. Deletion of the negatively charged C terminus of CHRAC16 enhances DNA binding 25-fold but leads to inhibition of nucleosome sliding, in striking analogy to the effect of the DNA chaperone HMGB1 on nucleosome sliding. The presence of a surface compatible with DNA interaction and the geometry of an H2A-H2B heterodimer may provide a transient acceptor site for DNA dislocated from the histone surface and therefore facilitate the nucleosome remodeling process.
Interaction of influenza virus proteins with nucleosomes.
Garcia-Robles, I., Akarsu, H., Müller, C.W., Ruigrok, R.W. & Baudin, F.
Virology 2005 Feb 5;332(1):329-36.
During influenza virus infection, transcription and replication of the viral RNA take place in the cell nucleus. Directly after entry in the nucleus the viral ribonucleoproteins (RNPs, the viral subunits containing vRNA, nucleoprotein and the viral polymerase) are tightly associated with the nuclear matrix. Here, we have analysed the binding of RNPs, M1 and NS2/NEP proteins to purified nucleosomes, reconstituted histone octamers and purified single histones. RNPs and M1 both bind to the chromatin components but at two different sites, RNP to the histone tails and M1 to the globular domain of the histone octamer. NS2/NEP did not bind to nucleosomes at all. The possible consequences of these findings for nuclear release of newly made RNPs and for other processes during the infection cycle are discussed.
Architecture of CRM1/Exportin1 suggests how cooperativity is achieved during formation of a nuclear export complex.
Petosa, C., Schoehn, G., Askjaer, P., Bauer, U., Moulin, M., Steuerwald, U., Soler-Lopez, M., Baudin, F., Mattaj, I.W. & Muller, C.W.
Mol Cell 2004 Dec 3;16(5):761-75.
CRM1/Exportin1 mediates the nuclear export of proteins bearing a leucine-rich nuclear export signal (NES) by forming a cooperative ternary complex with the NES-bearing substrate and the small GTPase Ran. We present a structural model of human CRM1 based on a combination of X-ray crystallography, homology modeling, and electron microscopy. The architecture of CRM1 resembles that of the import receptor transportin1, with 19 HEAT repeats and a large loop implicated in Ran binding. Residues critical for NES recognition are identified adjacent to the cysteine residue targeted by leptomycin B (LMB), a specific CRM1 inhibitor. We present evidence that a conformational change of the Ran binding loop accounts for the cooperativity of Ran- and substrate binding and for the selective enhancement of CRM1-mediated export by the cofactor RanBP3. Our findings indicate that a single architectural and mechanistic framework can explain the divergent effects of RanGTP on substrate binding by many import and export receptors.
The histone octamer is invisible when NF-kappaB binds to the nucleosome.
Angelov, D., Lenouvel, F., Hans, F., Muller, C.W., Bouvet, P., Bednar, J., Moudrianakis, E.N., Cadet, J. & Dimitrov, S.
J Biol Chem 2004 Oct 8;279(41):42374-82. Epub 2004 Jul 21.
The transcription factor NF-kappaB is involved in the transcriptional control of more than 150 genes, but the way it acts at the level of nucleosomal templates is not known. Here we report on a study examining the interaction of NF-kappaB p50 with its DNA recognition sequence in a positioned nucleosome. We demonstrate that NF-kappaB p50 was able to bind to the nucleosome with an apparent association constant close to that for free DNA. In agreement with this, the affinity of NF-kappaB p50 binding does not depend on the localization of its recognition sequence relative to the nucleosome dyad axis. In addition, the binding of NF-kappaB p50 does not induce eviction of histones and does not perturb the overall structure of the nucleosome. The NF-kappaB p50-nucleosome complex exhibits, however, local structural alterations within the NF-kappaB p50 recognition site. Importantly, these alterations were very similar to those found in the NF-kappaB p50-DNA complex. Our data suggest that NF-kappaB p50 can accommodate the distorted, bent DNA within the nucleosome. This peculiar property of NF-kappaB p50 might have evolved to meet the requirements for its function as a central switch for stress responses.
Structure of an activated Dictyostelium STAT in its DNA-unbound form.
Soler-Lopez, M., Petosa, C., Fukuzawa, M., Ravelli, R.B.G., Williams, J.G. & Müller, C.W.
Mol Cell 2004 Mar 26;13(6):791-804.
Dd-STATa is a STAT protein which transcriptionally regulates cellular differentiation in Dictyostelium discoideum, the only non-metazoan known to employ SH2 domain signaling. The 2.7 A crystal structure of a tyrosine phosphorylated Dd-STATa homodimer reveals a four-domain architecture similar to that of mammalian STATs 1 and 3, but with an inverted orientation for the coiled-coil domain. Dimerization is mediated by SH2 domain:phosphopeptide interactions and by a direct interaction between SH2 domains. The unliganded Dd-STATa dimer adopts a fully extended conformation remarkably different from that of the DNA-bound mammalian STATs, implying a large conformational change upon target site recognition. Buried hydrophilic residues predicted to destabilize the coiled-coil domain suggest how hydrophobic residues may become exposed and mediate nuclear export. Functional and evolutionary implications for metazoan STAT proteins are discussed.
Crystal structure of the M1 protein-binding domain of the influenza A virus nuclear export protein (NEP/NS2).
Akarsu, H., Burmeister, W.P., Petosa, C., Petit, I., Muller, C.W., Ruigrok, R.W. & Baudin, F.
EMBO J 2003 Sep 15;22(18):4646-55.
During influenza virus infection, viral ribonucleoproteins (vRNPs) are replicated in the nucleus and must be exported to the cytoplasm before assembling into mature viral particles. Nuclear export is mediated by the cellular protein Crm1 and putatively by the viral protein NEP/NS2. Proteolytic cleavage of NEP defines an N-terminal domain which mediates RanGTP-dependent binding to Crm1 and a C-terminal domain which binds to the viral matrix protein M1. The 2.6 A crystal structure of the C-terminal domain reveals an amphipathic helical hairpin which dimerizes as a four-helix bundle. The NEP-M1 interaction involves two critical epitopes: an exposed tryptophan (Trp78) surrounded by a cluster of glutamate residues on NEP, and the basic nuclear localization signal (NLS) of M1. Implications for vRNP export are discussed.
Crystal structure and functional analysis of a nucleosome recognition module of the remodeling factor ISWI.
Grune, T., Brzeski, J., Eberharter, A., Clapier, C.R., Corona, D.F., Becker, P.B. & Müller, C.W.
Mol Cell 2003 Aug;12(2):449-60.
Energy-dependent nucleosome remodeling emerges as a key process endowing chromatin with dynamic properties. However, the principles by which remodeling ATPases interact with their nucleosome substrate to alter histone-DNA interactions are only poorly understood. We have identified a substrate recognition domain in the C-terminal half of the remodeling ATPase ISWI and determined its structure by X-ray crystallography. The structure comprises three domains, a four-helix domain with a novel fold and two alpha-helical domains related to the modules of c-Myb, SANT and SLIDE, which are linked by a long helix. An integrated structural and functional analysis of these domains provides insight into how ISWI interacts with the nucleosomal substrate.
Solution study of the NF-kappaB p50-DNA complex by UV laser protein-DNA cross-linking.
Angelov, D., Charra, M., Muller, C.W., Cadet, J. & Dimitrov, S.
Photochem Photobiol 2003 Jun;77(6):592-6.
In this study, we describe a new approach for studying protein-DNA interactions in solution. The approach is based on mapping the UV laser-induced protein-DNA cross-links between the amino acids of the protein and the DNA bases that are in direct contact. The approach was applied for studying the solution structure of the human necrosis factor (NF)-kappaB p50 homodimer bound to a 37 base pair DNA. Several points of contact identical to those observed in the NF-kappaB-DNA crystal structure were found between the two biomolecules. Evidence is provided for the occurrence of two new contact points, one for each DNA strand. These new points of contact are located symmetrically a base apart from the extremity of the binding sequence.
Structure of the GCM domain-DNA complex: a DNA-binding domain with a novel fold and mode of target site recognition.
Cohen, S.X., Moulin, M., Hashemolhosseini, S., Kilian, K., Wegner, M. & Müller, C.W.
EMBO J 2003 Apr 15;22(8):1835-45.
Glia cell missing (GCM) transcription factors form a small family of transcriptional regulators in metazoans. The prototypical Drosophila GCM protein directs the differentiation of neuron precursor cells into glia cells, whereas mammalian GCM proteins are involved in placenta and parathyroid development. GCM proteins share a highly conserved 150 amino acid residue region responsible for DNA binding, known as the GCM domain. Here we present the crystal structure of the GCM domain from murine GCMa bound to its octameric DNA target site at 2.85 A resolution. The GCM domain exhibits a novel fold consisting of two domains tethered together by one of two structural Zn ions. We observe the novel use of a beta-sheet in DNA recognition, whereby a five- stranded beta-sheet protrudes into the major groove perpendicular to the DNA axis. The structure combined with mutational analysis of the target site and of DNA-contacting residues provides insight into DNA recognition by this new type of Zn-containing DNA-binding domain.
X-ray crystal structure of STAT proteins and structure-activity relationships.
Muller, C.W., Soler-Lopez, M., Gewinner, C., & Groner, B.
In "Signal Transducers and Activators of Transcription (STATs): Activation and Biology." P.B. Sehgal, D.E. Levy, & T. Hirano (Eds). Kluwer Academic Publishers, Dordrecht, The Netherlands.
The GCM domain is a Zn-coordinating DNA-binding domain.
Cohen, S.X., Moulin, M., Schilling, O., Meyer-Klaucke, W., Schreiber, J., Wegner, M. & Muller, C.W.
FEBS Lett 2002 Sep 25;528(1-3):95-100.
Glial cells missing (GCM) proteins form a small family of transcriptional regulators involved in different developmental processes. They contain a DNA-binding domain that is highly conserved from flies to mice and humans and consists of approximately 150 residues. The GCM domain of the mouse GCM homolog a was expressed in bacteria. Extended X-ray absorption fine structure and particle-induced X-ray emission analysis techniques showed the presence of two Zn atoms with four-fold coordination and cysteine/histidine residues as ligands. Zn atoms can be removed from the GCM domain by the Zn chelator phenanthroline only under denaturating conditions. This suggests that the Zn ions are buried in the interior of the GCM domain and that their removal abolishes DNA-binding because it impairs the structure of the GCM domain. Our results define the GCM domain as a new type of Zn-coordinating, sequence-specific DNA-binding domain.
Structure of the DNA-bound T-box domain of human TBX3, a transcription factor responsible for ulnar-mammary syndrome.
Coll, M., Seidman, J.G. & Muller, C.W.
Structure (Camb) 2002 Mar;10(3):343-56.
T-box genes encode transcription factors involved in morphogenesis and organogenesis of vertebrates and invertebrates. Mutations in human T-box genes TBX3, TBX5, and TBX1 cause severe genetic disorders known as Ulnar-Mammary syndrome (UMS), Holt-Oram syndrome (HOS), and DiGeorge syndrome, respectively. The crystal structure of the T-box domain of the first human T-box transcription factor, TBX3, in complex with DNA at 1.7 A resolution explains structural consequences of T-box domain point mutations observed in UMS and HOS patients. Comparison with the structure of the T-box domain from Xenopus laevis (Xbra) bound to DNA shows differences in several secondary structure elements and in the quaternary structure of the two complexes. TBX3 independently recognizes the two binding sites present in the palindromic DNA duplex, whereas in Xbra, binding to the palindrome is stabilized through interactions between the two monomers. The different quaternary structures suggest different DNA binding modes for T-box transcription factors.
Crystal structure of the ankyrin repeat domain of Bcl-3: a unique member of the IkappaB protein family.
Michel, F., Soler-Lopez, M., Petosa, C., Cramer, P., Siebenlist, U. & Muller, C.W.
EMBO J 2001 Nov 15;20(22):6180-90.
IkappaB proteins associate with the transcription factor NF-kappaB via their ankyrin repeat domain. Bcl-3 is an unusual IkappaB protein because it is primarily nucleoplasmic and can lead to enhanced NF-kappaB-dependent transcription, unlike the prototypical IkappaB protein IkappaBalpha, which inhibits NF-kappaB activity by retaining it in the cytoplasm. Here we report the 1.9 A crystal structure of the ankyrin repeat domain of human Bcl-3 and compare it with that of IkappaBalpha bound to NF-kappaB. The two structures are highly similar over the central ankyrin repeats but differ in the N-terminal repeat and at the C-terminus, where Bcl-3 contains a seventh repeat in place of the acidic PEST region of IkappaBalpha. Differences between the two structures suggest why Bcl-3 differs from IkappaBalpha in selectivity towards various NF-kappaB species, why Bcl-3 but not IkappaBalpha can associate with its NF-kappaB partner bound to DNA, and why two molecules of Bcl-3 but only one of IkappaBalpha can bind to its NF-kappaB partner. Comparison of the two structures thus provides an insight into the functional diversity of IkappaB proteins.
Comparison of ARM and HEAT protein repeats.
Andrade, M.A., Petosa, C., O'Donoghue, S.I., Muller, C.W. & Bork, P.
J Mol Biol 2001 May 25;309(1):1-18.
ARM and HEAT motifs are tandemly repeated sequences of approximately 50 amino acid residues that occur in a wide variety of eukaryotic proteins. An exhaustive search of sequence databases detected new family members and revealed that at least 1 in 500 eukaryotic protein sequences contain such repeats. It also rendered the similarity between ARM and HEAT repeats, believed to be evolutionarily related, readily apparent. All the proteins identified in the database searches could be clustered by sequence similarity into four groups: canonical ARM-repeat proteins and three groups of the more divergent HEAT-repeat proteins. This allowed us to build improved sequence profiles for the automatic detection of repeat motifs. Inspection of these profiles indicated that the individual repeat motifs of all four classes share a common set of seven highly conserved hydrophobic residues, which in proteins of known three-dimensional structure are buried within or between repeats. However, the motifs differ at several specific residue positions, suggesting important structural or functional differences among the classes. Our results illustrate that ARM and HEAT-repeat proteins, while having a common phylogenetic origin, have since diverged significantly. We discuss evolutionary scenarios that could account for the great diversity of repeats observed.
Transcription factors: global and detailed views.
Curr Opin Struct Biol 2001 Feb;11(1):26-32.
During the past year, much progress has been made in understanding the structural basis of transcriptional regulation. Low-resolution electron microscopy structures of general transcription factor complexes have shed light on their global organization. These results are complemented by the structural and biochemical analysis of individual general transcription factors. High-resolution crystal structures of sequence-specific transcription factors still yield unexpected results. Detailed analysis of DNA recognition by different family members of the same class of sequence-specific transcription factors shows considerable variations in the way they interact with DNA.
DNA recognition by NF kappa B and STAT transcription factors.
Ernst Schering Res Found Workshop 2001;(34):143-66. PubMed
Nuclear import factors importin alpha and importin beta undergo mutually induced conformational changes upon association.
Cingolani, G., Lashuel, H.A., Gerace, L. & Muller, C.W.
FEBS Lett 2000 Nov 10;484(3):291-8
A heterodimer of importin alpha and importin beta accomplishes the nuclear import of proteins carrying classical nuclear localization signals (NLS). The interaction between the two import factors is mediated by the IBB domain of importin alpha and involves an extended recognition surface as shown by X-ray crystallography. Using a combination of biochemical and biophysical techniques we have investigated the formation of the importin beta:IBB domain complex in solution. Our data suggest that upon binding to the IBB domain, importin beta adopts a compact, proteolytically resistant conformation, while simultaneously the IBB domain folds into an alpha helix. We suggest a model to describe how these dual mutually induced conformational changes may orchestrate the nuclear import of NLS cargo in vivo.
Structure of the specificity domain of the Dorsal homologue Gambif1 bound to DNA.
Cramer, P., Varrot, A., Barillas-Mury, C., Kafatos, F.C. & Muller, C.W.
Structure Fold Des 1999 Jul 15;7(7):841-52
BACKGROUND: NF-kappa B/Rel transcription factors play important roles in immunity and development in mammals and insects. Their activity is regulated by their cellular localization, homo- and heterodimerization and association with other factors on their target gene promoters. Gambif1 from Anopheles gambiae is a member of the Rel family and a close homologue of the morphogen Dorsal, which establishes dorsoventral polarity in the Drosophila embryo. RESULTS: We present the crystal structure of the N-terminal specificity domain of Gambif1 bound to DNA. This first structure of an insect Rel protein-DNA complex shows that Gambif1 binds a GGG half-site element using a stack of three arginine sidechains. Differences in affinity to Dorsal binding sites in target gene promoters are predicted to arise from base changes in these GGG elements. An arginine that is conserved in class II Rel proteins (members of which contain a transcription activation domain) contacts the outermost guanines of the DNA site. This previously unseen specific contact contributes strongly to the DNA-binding affinity and might be responsible for differences in specificity between Rel proteins of class I and II. CONCLUSIONS: The Gambif1-DNA complex structure illustrates how differences in Dorsal affinity to binding sites in developmental gene promoters are achieved. Comparison with other Rel- DNA complex structures leads to a general model for DNA recognition by Rel proteins.
Structure of importin-beta bound to the IBB domain of importin-alpha
Cingolani, G., Petosa, C., Weis, K. & Müller, C.W.
Nature 1999 May 20;399(6733):221-9
Cytosolic proteins bearing a classical nuclear localization signal enter the nucleus bound to a heterodimer of importin-alpha and importin- beta (also called karyopherin-alpha and -beta). The formation of this heterodimer involves the importin-beta-binding (IBB) domain of importin- alpha, a highly basic amino-terminal region of roughly 40 amino-acid residues. Here we report the crystal structure of human importin-beta bound to the IBB domain of importin-alpha, determined at 2.5 A and 2.3 A resolution in two crystal forms. Importin-beta consists of 19 tandemly repeated HEAT motifs and wraps intimately around the IBB domain. The association involves two separate regions of importin-beta, recognizing structurally distinct parts of the IBB domain: an amino- terminal extended moiety and a carboxy-terminal helix. The structure indicates that significant conformational changes occur when importin- beta binds or releases the IBB domain domain and suggests how dissociation of the importin-alpha/beta heterodimer may be achieved upon nuclear entry.
Different TBX5 interactions in heart and limb defined by Holt-Oram syndrome mutations.
Basson, C.T., Huang, T., Lin, R.C., Bachinsky, D.R., Weremowicz, S., Vaglio, A., Bruzzone, R., Quadrelli, R., Lerone, M., Romeo, G., Silengo, M., Pereira, A., Krieger, J., Mesquita, S.F., Kamisago, M., Morton, C.C., Pierpont, M.E., Muller, C.W., Seidman, J.G. & Seidman, C.E.
Proc Natl Acad Sci U S A 1999 Mar 16;96(6):2919-24
To better understand the role of TBX5, a T-box containing transcription factor in forelimb and heart development, we have studied the clinical features of Holt-Oram syndrome caused by 10 different TBX5 mutations. Defects predicted to create null alleles caused substantial abnormalities both in limb and heart. In contrast, missense mutations produced distinct phenotypes: Gly80Arg caused significant cardiac malformations but only minor skeletal abnormalities; and Arg237Gln and Arg237Trp caused extensive upper limb malformations but less significant cardiac abnormalities. Amino acids altered by missense mutations were located on the three-dimensional structure of a related T-box transcription factor, Xbra, bound to DNA. Residue 80 is highly conserved within T-box sequences that interact with the major groove of target DNA; residue 237 is located in the T-box domain that selectively binds to the minor groove of DNA. These structural data, taken together with the predominant cardiac or skeletal phenotype produced by each missense mutation, suggest that organ-specific gene activation by TBX5 is predicated on biophysical interactions with different target DNA sequences.
A firm hand on NFkappaB: structures of the IkappaBalpha-NFkappaB complex.
Cramer, P. & Muller, C.W.
This is a review article.
Structure Fold Des 1999 Jan 15;7(1):R1-6
Two crystal structures of an IkappaB-NFkappaB complex have recently been determined. The structures show in detail how IkappaB controls the subcellular localization and activity of the eukaryotic transcription factor NFkappaB.
Expression of a tyrosine phosphorylated, DNA binding Stat3beta dimer in bacteria.
Becker, S., Corthals, G.L., Aebersold, R., Groner, B. & Muller, C.W.
FEBS Lett 1998 Dec 11;441(1):141-7.
The signal transducer and activator of transcription (STAT) proteins deliver signals from the cell membrane to the nucleus. An N-terminally truncated fragment of murine Stat3beta, Stat3betatc (127-722), was produced in bacteria. STAT proteins must be specifically phosphorylated at a single tyrosine residue for dimerization and DNA binding. Therefore, Stat3betatc was coexpressed with the catalytic domain of the Elk receptor tyrosine kinase. Stat3betatc was quantitatively phosphorylated by this kinase domain. Gel filtration chromatography revealed a Stat3betatc dimer. Y705 was identified as the major phosphorylated residue of Stat3betatc. This corresponds to the tyrosine residue which is phosphorylated by the Janus kinase in vivo. The phosphorylated Stat3betatc specifically bound to DNA binding sites. The described protocol allows the production of large amounts of activated protein for biochemical and pharmaceutical studies.
Three-dimensional structure of the Stat3beta homodimer bound to DNA.
Becker, S., Groner, B. & Muller, C.W.
Nature 1998 Jul 9;394(6689):145-51.
STAT proteins are a family of eukaryotic transcription factors that mediate the response to a large number of cytokines and growth factors. Upon activation by cell-surface receptors or their associated kinases, STAT proteins dimerize, translocate to the nucleus and bind to specific promoter sequences on their target genes. Here we report the first crystal structure of a STAT protein bound to its DNA recognition site at 2.25 A resolution. The structure provides insight into the various steps by which STAT proteins deliver a response signal directly from the cell membrane to their target genes in the nucleus.
Structure of the human NF-kappaB p52 homodimer-DNA complex at 2.1 A resolution.
Cramer, P., Larson, C.J., Verdine, G.L. & Muller, C.W.
EMBO J 1997 Dec 1;16(23):7078-90.
The crystal structure of human NF-kappaB p52 in its specific complex with the natural kappaB DNA binding site MHC H-2 has been solved at 2.1 A resolution. Whereas the overall structure resembles that of the NF-kappaB p50-DNA complex, pronounced differences are observed within the 'insert region'. This sequence segment differs in length between different Rel proteins. Compared with NF-kappaB p50, the compact alpha-helical insert region element is rotated away from the core of the N-terminal domain, opening up a mainly polar cleft. The insert region presents potential interaction surfaces to other proteins. The high resolution of the structure reveals many water molecules which mediate interactions in the protein-DNA interface. Additional complexity in Rel protein-DNA interaction comes from an extended interfacial water cavity that connects residues at the edge of the dimer interface to the central DNA bases. The observed water network might acount for differences in binding specificity between NF-kappaB p52 and NF-kappaB p50 homodimers.
Crystallographic structure of the T domain-DNA complex of the Brachyury transcription factor.
Muller, C.W. & Herrmann, B.G.
Nature 1997 Oct 23;389(6653):884-8.
The mouse Brachyury (T) gene is the prototype of a growing family of so-called T-box genes which encode transcriptional regulators and have been identified in a variety of invertebrates and vertebrates, including humans. Mutations in Brachyury and other T-box genes result in drastic embryonic phenotypes, indicating that T-box gene products are essential in tissue specification, morphogenesis and organogenesis. The T-box encodes a DNA-binding domain of about 180 amino-acid residues, the T domain. Here we report the X-ray structure of the T domain from Xenopus laevis in complex with a 24-nucleotide palindromic DNA duplex. We show that the protein is bound as a dimer, interacting with the major and the minor grooves of the DNA. A new type of specific DNA contact is seen, in which a carboxy-terminal helix is deeply embedded into an enlarged minor groove without bending the DNA. Hydrophobic interactions and an unusual main-chain carbonyl contact to a guanine account for sequence-specific recognition in the minor groove by this helix. Thus the structure of this T domain complex with DNA reveals a new way in which a protein can recognize DNA.
Structure of the NF-kappa B p50 homodimer bound to DNA.
Muller, C.W., Rey, F.A., Sodeoka, M., Verdine, G.L. & Harrison, S.C.
Nature 1995 Jan 26;373(6512):311-7.
The structure of a large fragment of the p50 subunit of the human transcription factor NF-kappa B, bound as a homodimer to DNA, reveals that the Rel-homology region has two beta-barrel domains that grip DNA in the major groove. Both domains contact the DNA backbone. The amino-terminal specificity domain contains a recognition loop that interacts with DNA bases; the carboxy-terminal dimerization domain bears the site of I-kappa B interaction. The folds of these domains are related to immunoglobulin-like modules. The amino-terminal domain also resembles the core domain of p53.