Université Grenoble 1 — Joseph Fourier Sciences et Géographie N attribué par la bibliothèque Thèse de Tim GRÜNE Doctorat: Chimie et Sciences du Vivant — Biologie Discipline: Aspect Moleculaires et Cellulaires de la Biologie Structural studies on ISWI, an ATPdependent nucleosome remodelling factor Thèse dirigée par: Christoph W. MÜLLER Laboratoire Européenne de Biologie Moleculaire, Grenoble Soutenance publique le 3 Octobre 2003 Jury: Christoph W. MÜLLER EMBL Grenoble Directeur de thèse Elena CONTI EMBL Heidelberg Rapportrice Félix REY CNRS GifsurYvette Rapporteur Hans GEISELMANN UJF Grenoble Président du Jury Saadi KHOCHBIN UJF Grenoble membre du Jury 2 3 Abstract The Imitation Switch protein, or ISWI, from D. melanogaster is an essential enzyme that uses the energy from ATP hydrolysis in order to rearrange nucleosomes in chromatin. It plays an important role in gene expression because access to DNA and especially to promoter sites is altered by nucleosome positioning. ISWI can thus act both as an enhancer and repressor of transcription. In all eukaryotes one can find a large number of complexes involved in chromatin remodelling. Despite the diversity and variety of functioning, all these complexes contain an ATPase with a homologous socalled SNF2 domain that is conserved through all eukaryotes. Four groups of remodelling ATPases can be distinguished, SNF2, SNF2L, CHD1, and INO80 of which only the first three have been further characterised according to conserved domains they contain besides the SNF2 domain. For more than 15 years these complexes have been known and a large pool of data is available to characterise a process that, together with covalent histone modifications, alters the chromatin structure and has important influence on processes like transcription, DNA repair, and replication. But to date detailed information that might shed light on the mechanism of how remodelling is carried out has been missing and only coarse hypotheses have been proposed. The work summarised in this document began in January 2000 with the aim to find structural information about ISWI. The starting point was a clone and a protocol that allowed to produce the enzyme recombinantly, but the path was certainly not straight. A manifold of attempts was undertaken, many of which are not described here. Acf1 and ISWI build one of the complexes that can be found in vivo. The possibility of ISWI being stabilised by Acf1 was considered, but neither could Acf1 expression be detected in bacteria nor could the complex be produced in insect cells in sufficient amounts for structural studies. Binding to substrates like the Nterminal tail of histone H4, ATP and nonhydrolisable analogues of ATP, or cruciform DNA was investigated but did not produce useful results. Given that ATPases often undergo large conformational changes, it was not unexpected that crystallisation trials with the fulllength protein (Mr = 120 kDa) failed. One of the first experiments was therefore to find subdomains of the protein by restricted proteolysis. It showed that the enzyme consists of two flexibly linked main parts, the Nterminus that makes two thirds of the protein, and the Cterminus, about one third. These two parts were subcloned, but only the Cterminal part proved to be stable and gave crystals. Many Nterminal clones could be purified but were difficult to concentrate, even worse than the fulllength protein. Crystals of the Cterminal fragment of ISWI were obtained in July 2001. Since then, most of the effort went into improving the crystals‘ data collection, but the structure could only be solved with a new crystal form that suddenly appeared in May 2002. The introduction of this thesis provides a short overview on chromatin remodelling complexes but emphasises the ISWI family. Chromatin remodelling covers a wide network of intertwined actions that include histone tail modifications, transcription, silencing and the condensation and decondensation of chromatin. No attempt was made to fully cover the literature. The in vitro assays used to characterise the functioning and interaction with substrate are explained in a general way and only the results specific to ISWI are described in further detail. The part following the introduction is dedicated to the techniques that were used to gain insight into the struc ture of ISWI. These obviously include protein crystallisation and crystallography (with emphasis on the phase problem), but also a short section about circular dichroism. The main result of this work is the crystal structure of ISWI [691:991] to 1.9 Å resolution. The elongated structure consists of three domains which are described separately in detail. The very Nterminal domain presents a new fold and has been named the “Hand” domain. It is in direct contact with the following SANT domain. The last domain, SLIDE, is separated from the rest of the molecule by a straight 50 Ålong spacer helix. The fragment has an asymmetric charge distribution of acidic and basic residues between the Nterminal domains and the Cterminal domain. Most of the molecule has a negative charged distribution on the surface, but especially the SLIDE domain contains some positive patches. The following analysis showed that this patch is probably the main area where the Cterminal part of ISWI contacts the DNA of the nucleosome by a classical helixturnhelix motif. The water, glucose, and glycerol molecules, that were found in the structure, are described in detail because they are important for crystal contacts. The description includes a beautiful composition with a water molecule sitting right on one of the symmetry axes that forms a pentagonal water ring with two more water molecules from the asymmetric unit and their symmetry mates. Most of the water molecules of the structure concentrate at the Cterminus of the protein where they build part of the interface between two protein molecules. Functional interpretation of the structure was based on the homology of both the SANT and the SLIDE domain with DNA binding proteins like the oncogene product cMyb and the homeodomain protein Pax6. The SLIDE domain is only distantly related to SANT domains which had been proposed to be DNA binding. However, we now found strong evidence that in fact the more remotely related SLIDE domain contacts the DNA directly while the helix of the SANT domain that, according to the structural homology, should be in contact with the DNA is too negatively charged to bind DNA. The thesis finishes with a suggestion of how the Cterminal fragment of ISWI might bind the nucleosome and hence act as a substrate recognition module for fulllength ISWI. Note for reading Like during this introduction, I sometimes refer to the names of the domains in the structure of the Cterminal fragment of ISWI. In order to better understand the text I recommend the reader to first look at figure 13.1 in order to get an overview of the model and the domain names and their borders. Furthermore, 4 I sometimes use the term ISWIC. With this term I refer to any of the Cterminal clones of ISWI I prepared during this work where a distinction is not necessary. These are mainly the ones that crystallised: ISWI [691:991], ISWI [701:991] and ISWI [713:991]. 5 Résumé1 La protéine Imitation Switch de Drosophila melanogaster est une enzyme essentielle qui utilise l’énergie de l’hydrolyse de l’adénosine triphosphate pour réarranger des nucléosomes dans la chromatine. Elle joue un rôle important dans l’expression des gènes parce que l’accès à l’ADN et aux sites promoteurs est modifié par le po sitionnement des nucléosomes. ISWI peut donc agir comme activateur mais aussi comme répresseur de la trans cription. Dans tous les Eucaryotes, on peut trouver un grand nombre de complexes multiprotéiques impliqués dans le réarrangement de la chromatine. En dépit de leur diversité et de leur varieté de fonctionnement, tous ces com plexes contiennent une ATPase présentant un domaine homologue très conservé nommé SNF2. Une caractérisation plus approfondie de ces ATPases impliquées dans le remodelage de la chromatine a permis de distinguer quatre groups : SNF2, SNF2L, CHD1 et INO80. En quinze ans, depuis la découverte de ces complexes, un grand nombre d’informations ont été collectées pour caractériser le processus de remodelage de la chromatine qui, avec les mo difications covalentes des histones, affecte la structure de la chromatine et a donc une influence importante sur des processus comme la réplication, la réparation de l’ADN et la transcription. Mais jusqu’ici le manque d’informa tions détaillées qui pourraient éclairer le mécanisme de remodelage du nucléosome a seulement conduit à proposer des hypothèses. Le travail récapitulé dans ce document a commencé en janvier 2000 avec le but de l’obtention d’informa tions structurales sur ISWI. Le point de départ était un clone et un protocole permettant de produire l’enzyme ISWI recombinante chez E.coli. Mais le chemin conduisant à des résultats a été ramifié : une multitude d’expé riences différentes a été effectuées, dont beaucoup ne sont pas décrites ici. Par exemple, ISWI établit avec Acf1 un complexe qui peut être trouvé in vivo. La possibilité de stabiliser ISWI par association à Acf1 a été conside rée. Malheureusement aucune expression d’Acf1 n’a été détectée dans les bactéries et les quantités de complexe produites en cellules d’insecte étaient insuffisantes pour des études structurles. L’association d’ISWI avec des sub strats dont l’extrémité Nterminale de l’histone H4, l’ATP, des analogues nonhydrolysables de l’ATP ou de l’ADN cruciforme a été étudiée mais n’a pas produit de résultats utiles. Sachant que les ATPases subissent de grands changements de conformation durant leur activité enzymatique, il n’est pas surprenant que les essais de cristallisation avec la protéine entière (Mr = 120 kDa) aient échoué. L’une des premières expériences a donc été une protéolyse restreinte de la protéine entière afin d’identifier des sous domaines.