Structural and functional characterization of the N-terminal acetyltransferase NatC Inaugural-Dissertation to obtain the academic degree Doctor rerum naturalium (Dr. rer. nat.) submitted to the Department of Biology, Chemistry, Pharmacy of Freie Universität Berlin by Stephan Grunwald Berlin October 01, 2019 Die vorliegende Arbeit wurde von April 2014 bis Oktober 2019 am Max-Delbrück-Centrum für Molekulare Medizin unter der Anleitung von PROF. DR. OLIVER DAUMKE angefertigt. Erster Gutachter: PROF. DR. OLIVER DAUMKE Zweite Gutachterin: PROF. DR. ANNETTE SCHÜRMANN Disputation am 26. November 2019 iii iv Erklärung Ich versichere, dass ich die von mir vorgelegte Dissertation selbstständig angefertigt, die benutzten Quellen und Hilfsmittel vollständig angegeben und die Stellen der Arbeit – einschließlich Tabellen, Karten und Abbildungen – die anderen Werken im Wortlaut oder dem Sinn nach entnommen sind, in jedem Einzelfall als Entlehnung kenntlich gemacht habe; und dass diese Dissertation keiner anderen Fakultät oder Universität zur Prüfung vorgelegen hat. Berlin, 9. November 2020 Stephan Grunwald v vi Acknowledgement I would like to thank Prof. Oliver Daumke for giving me the opportunity to do the research for this project in his laboratory and for the supervision of this thesis. I would also like to thank Prof. Dr. Annette Schürmann from the German Institute of Human Nutrition (DifE) in Potsdam-Rehbruecke for being my second supervisor. From the Daumke laboratory I would like to especially thank Dr. Manuel Hessenberger, Dr. Stephen Marino and Dr. Tobias Bock-Bierbaum, who gave me helpful advice. I also like to thank the rest of the lab members for helpful discussions. I deeply thank my wife Theresa Grunwald, who always had some helpful suggestions and kept me alive while writing this thesis. vii Abbreviations AcCoA / Acetyl-CoA (Acetyl-) coenzyme A ARFRP1 ADP-ribosylation factor-related protein 1 (a trans-Golgi-associated small GTPase) Cryo-EM Cryogenic electron microscopy EPR Electropositive Region ES Ribosomal RNA expansion segments Gag Major capsid protein of the Saccharomyces cerevisiae virus L-A GNAT GCN5-related N-acetyltransferase superfamily HAT Histone acetyl transferase HRV 3C Human rhinovirus 3C protease / recognition site IMAC Immobilized metal affinity chromatography KAT Lysine acetyl transferase MALDI-TOF MS Matrix-assisted laser desorption/ionization – time-of-flight – mass spectrometry MetAP Methionine aminopeptidase NAA/Naa Nα-acetyltransferase gene/protein, respectively. NAT N-terminal acetyltransferase (enzyme that the catalyzes the Nt-acetylation) Nt-acetylation N-terminal acetylation (also known as Nα-acetylation) pI Isoelectric point PE Ribosome nascent polypetide exit tunnel PTM Post-translational modification SEC Size-exclusion chromatography yArl3 ADP-ribosylation factor-like protein 3 from S. cerevisieae (the yeast ortholog of human ARFRP1) X-ray X-radiation, a form of high-energy electromagnetic radiation. The above list represents only frequently occurring abbreviations that are beneficial for text comprehension. Further abbreviations are found in the materials and methods sections or defined upon first use. viii Table of contents 1 Introduction ..............................................................................................................................1 1.1 The ‘missing’ genetic information in eukaryotic genomes ................................................................ 1 1.2 Post-translational modifications — a major source of protein diversity? .......................................... 2 1.3 Protein acetylation ............................................................................................................................. 3 1.4 Evolutionary development of N-terminal acetylation ........................................................................ 4 1.5 The eukaryotic NAT machinery ........................................................................................................ 5 1.6 Co-translational Nt-acetylation at the ribosome ................................................................................ 9 1.7 Diverse functions of N-terminal acetylation .................................................................................... 10 1.8 The structure of NAT enzymes ........................................................................................................ 11 1.9 General catalytic mechanism ........................................................................................................... 16 1.10 Objectives of this work ................................................................................................................ 17 2 Materials ..................................................................................................................................18 2.1 Instruments ...................................................................................................................................... 18 2.2 Consumables and Kits ..................................................................................................................... 18 2.3 Chemicals ........................................................................................................................................ 19 2.4 Antibodies ....................................................................................................................................... 20 2.5 Enzymes .......................................................................................................................................... 20 2.6 Bacterial strains ............................................................................................................................... 20 2.7 Plasmids and cDNA clones ............................................................................................................. 20 2.8 Peptides ........................................................................................................................................... 21 2.9 DNA oligonucleotides ..................................................................................................................... 22 2.10 Growth media .............................................................................................................................. 24 2.11 Software ....................................................................................................................................... 25 3 Methods ...................................................................................................................................26 3.1 Cloning of the NatC complex .......................................................................................................... 26 3.1.1 PCR reactions .......................................................................................................................... 26 3.1.2 Enzymatic assembly of DNA using restriction digestion and ligation .................................... 27 3.1.3 Cloning of NatC constructs ..................................................................................................... 27 3.2 Expression and purification of the NatC complex ........................................................................... 28 3.2.1 Preparation of chemically competent E. coli cells ................................................................... 28 3.2.2 Transformation of chemically competent E. coli BL21(DE3) ................................................. 28 3.2.3 Small-scale IPTG induction test .............................................................................................. 29 3.2.4 Expression of the native NatC complex .................................................................................. 29 3.2.5 Expression of the selenomethionine-labeled NatC complex ................................................... 30 3.2.6 Purification of the NatC complex ............................................................................................ 31 3.3 MALDI-TOF mass spectrometry .................................................................................................... 32 3.4 Purification of yeast 80S ribosomes ................................................................................................ 33 3.5 NatC-ribosome sedimentation assay ................................................................................................ 34 3.5.1 Silver staining .......................................................................................................................... 34 ix 3.5.2 Western Blot, Ponceau S- and immunostaining ....................................................................... 34 3.6 Acetyltransferase assay .................................................................................................................... 35 3.6.1 Assay adaptations for different peptide substrates ................................................................... 37 3.6.2 Determination of peptide concentrations.................................................................................. 38 3.6.3 Pathlength calculations for the acetyltransferase assay ............................................................ 38 3.7 Crystallization of NatC .................................................................................................................... 39 3.8 Data collection ................................................................................................................................