University of Dundee DOCTOR of PHILOSOPHY Structural Studies Of
Total Page：16
File Type：pdf, Size：1020Kb
University of Dundee DOCTOR OF PHILOSOPHY Structural studies of paired PHD finger-bromodomain chromatin-binding modules targeting epigenetic readers with chemical probes Amato, Anastasia Award date: 2018 Link to publication General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 09. Oct. 2021 Structural studies of paired PHD finger- bromodomain chromatin-binding modules: targeting epigenetic readers with chemical probes Anastasia Amato Ph.D. Thesis January 2018 Supervisor: Professor Alessio Ciulli A mia madre e mio padre. To my mother and my father Acknowledgments Firstly, I would like to thank my supervisor Prof. Alessio Ciulli, who gave me the possibility to start this adventure four years ago. Thanks for all the guidance you provided along this journey and also for the opportunity you gave me to follow my own ideas. Thanks for believing in me! Thanks to the School of Life Science of the University of Dundee that funded this research. Thanks to all the past and current members of the Ciulli group. Everybody has contributed in some way to this experience, not only from the scientific point of view! A special thanks to Dr. Borto, who guided my first steps into biology and was always there to support me and to…stand me! Yes, I know it was not always easy, but it was great and you handle it very well A special thanks to Dr. Lucas who greatly contributed to rescue me from a lost world. Thanks for our fruitful conversations, scientific and not! Thanks to Teresa, sharing this experience made it a bit lighter and without you everything would have been more boring…since from the beginning. Thanks to Ester, the only one with the superpower to convince me that there is still life outside of the lab! Thanks to Wei-Wei, your friendship was a valuable discovery on the way…a bit less your ‘banani’ sound! Thanks to Gugui and Mattia, you were the light that suddenly appeared in Dundee Thanks to Tonia, our friendship is gonna last over the distance! Thanks to Nicola, our weekends in the lab were more enjoyable than a night in Dukes’! Thanks to Andrea, because our ‘discussions’ were always….productive. Thanks to my big star, always by my side. Thanks to my mum who is the bravest woman I have ever met and she’s my power! To Marina and Rossella, sisters I’ve never had! To the Joy group, because no matter where I go…our friendship lasts since ages. Declaration I declare that this study was carried out by myself under the supervision of Professor Alessio Ciulli. This work is original and where performed in collaboration with other people was explicitly stated in the text. This thesis has not been previously submitted for higher degree. All the references cited have been previously consulted. Anastasia Amato September 2017 Publication Part of the work presented in this thesis has led to the following publication: Chapter 1 Bortoluzzi, A., Amato , A., Lucas, X., Blank, M., Ciulli, A., Structural basis of molecular recognition of helical histone H3 tail by PHD finger domains. Biochem J, 2017. 474(10): p. 1633-1651. Summary The use of chemical probes is a powerful tool that can help to address important biological questions. The study presented in this thesis aims to target reader domains of chromatin- associated proteins with small molecules, in order to provide information on their ligandability, useful to develop - potent chemical probes. This thesis work is divided in three parts. In the first and second part it is shown how structural information obtained by the use of synthetic peptides to study the binding mode of reader domains with their natural binding partner can be combined with fragment screening to gauge future optimization of small molecules. The first section described the disclosure of the binding mode of the H3 histone tail by the PHD zinc finger of BAZ2A. A crystal structure of the complex of BAZ2A with the H3 10-mer peptide identified a helical conformation of H3 upon binding with the PHD. This information coupled with further structural and biophysical analysis led to the identification of a subfamily of PHD characterized by an acidic patch on the helical turn, which is responsible of inducing helicity on H3 tail upon binding. The second part of the work investigated the ligandability of the PHD zinc finger domains of BAZ2A and BAZ2B. Using a combination of biophysical techniques and X-ray crystallography it was probed that it is possible to target these reader domains. Despite the similarities of the two PHDs, comparison of the fragment-bound crystal structures of the two proteins highlighted some differences in the binding mode. The last part of the project describes the several attempts performed in trying to elucidate the histone binding partner of the PHD-BrD tandem of the chromatin-related proteins BAZ1B and TRIM66, both involved in diseases. List of Abbreviations Alpha - amplified luminescent proximity homogeneous assay ASU - asymmetric unit BAZ1A - Bromodomain Adjacent to Zinc finger containing protein 1A BAZ1B - Bromodomain Adjacent to Zinc finger containing protein 1B BAZ2A - Bromodomain Adjacent to Zinc finger containing protein 2A BAZ2B - Bromodomain Adjacent to Zinc finger containing protein 2B BrD - bromodomain BET - Bromodomain and Extra Terminal domain containing protein BHC80 - synonym PHF21A BLI - biolayer interferometry BPTF - Bromodomain and PHD finger-containing transcription factor BRCT - BRCA1 C terminus BRD3 - Bromodomain-containing protein 3 BRD4 - Bromodomain-containing protein 4 BRDT - Bromodomain testis-specific protein CHD5 - chromodomain-helicase-DNA-binding protein 5 CpG – cytosine preceding guanine CPMG - Carr-Purcel-Meiboom-Gill CSP - chemical shift perturbation Da – Dalton DCM - dichloromethane DDT - DNA binding homeobox and Different Transcription factors domain DMF - dimethylformamide DMSO - dimethylsulfoxide DNMT – DNA methyltransferase DPF – double PHD fingers DSF - differential scanning fluorimetry DTT - dithiothreitol E. coli - Escherichia coli ECL – enhanced chemiluminescence EZH2 – enhancer of zeste homolog 2 FBLD – fragment-based ligand discovery Fmoc - Fluorenylmethyloxycarbonyl chloride HAT - histone acetyl-transferases HATU - (1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate HBTU-(2-91H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophospahate) HDAC - histone deacetylases HEPES - 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HPLC - high-performance liquid chromatography HSQC - Heteronuclear Single Quantum Coherence HTS - high-throughput screening JARID1A – Jumonji/ARID domain-containing protein 1A IC50 - half maximal inhibitory concentration IPTG - isopropyl-β-D-thiogalactopyranoside ITC - isothermal titration calorimetry KA - association constant Kac - acetyl lysine KD - dissociation constant Kex – exchange constant KDMs – lysine demethylase Kme –methyl lysine KMT – kysine methyltransferase LB - lysogeny broth LC/MS - liquid chromatography–mass spectrometry LE - ligand efficiency LH domain - Leucine-rich helical domain MBD - Methyl-CpG-Binding Domain MBT – malignant brain tumor MeCP2 – methyl-CpG binding protein 2 MORF – mortality factor 4-like protein 1 MOZ – Monocytic leukemia zinc finger protein (MYST3) MW - molecular weight MWCO – molecular weight cut-off MYST3 - MOZ NMP - N-methylpyrrolidinone NMM – N-methylmorpholine NOE - Nuclear Overhauser Effect NoRC - Nucleolar remodelling complex OD600 – Optical density at 600 nm PDB - Protein Data Bank PHD - plant homeodomain PHF21A – PHD finger protein 21A PTM - Post-Translational Modification Pygo2 – Pygopus homolog 2 RDMs – arginine demethylases RFU - relative fluorescence units RING – really interesting new gene RMSD - root mean square deviation RMT – arginine methyltransferase S/B - signal to background ratio SAM – S-adenosyl methionine cofactor SET - Su(var)3-9, Enhancer-of-zeste and Trithorax SGC - Structural Genomics Consortium Sp100C – Nuclear autoantigen Sp-100 SPR – Surface Plasmon Resonance STD - Saturation Transfer Difference SUMO – small ubiquitin-like modifier TAF1L - TBP-associated factor RNA polymerase 1-like TCEP - tris(2-carboxyethyl)phosphine TEV - Tobacco Etch Virus protease TFA - trifluoroacetic acid TIF1 – transcriptional intermediary factor 1 TPS - triisopropylsilane Tm - melting temperature TRIM24 – tripartite motif family 24 TRIM28 – tripartite motif family 28 TRIM33 – tripartite motif family 33 TRIM66 – tripartite motif family 66 Tris - trisaminomethane TROSY – transverse relaxation-optimized spectroscopy v/v - volume to volume ratio WAC – (WSTF/Acf1/cbp146) WaterLOGSY - Water-ligand observed via gradient spectroscopy WBS- William-Beuren syndrom WSTF – William syndrome transcription factor Z' - Z-factor Table of contents Acknowledgments Declaration Publication Summary List of abbreviations Table of contents