Structural studies on mammalian septins – New insights into filament formation Inaugural-Dissertation zur Erlangung des Doktorgrades des Fachbereiches Chemie der Universität Dortmund vorgelegt von Minhajuddin Sirajuddin aus Ranipet / Indien Dortmund 2007 ii Die vorliegende Arbeit wurde im Zeitraum October 2003 bis August 2007 in der Abteilung I (Structurelle Biologie) des Max-Planck-Instituts für Molekulare Physiologie in Dortmund unter der Anleitung von Prof. Dr. Alfred Wittinghofer angefertigt. 1. Gutachter: Prof. Dr. Alfred Wittinghofer 2. Gutachter: Prof. Dr. Herbert Waldmann iii iv Contents Contents 1 Introduction.................................................................................................... 1 1.1 Cytoskeleton ...................................................................................................1 1.1.1 Components of the cytoskeleton................................................................................ 1 1.1.2 Polymerization and dynamics of cytoskeletal systems.............................................. 4 1.2 An overview of the cell cycle .........................................................................5 1.3 Guanine nucleotide binding proteins ..............................................................7 1.3.1 Classification of GTPase superclass.......................................................................... 8 1.3.2 Characteristic features of the G domain .................................................................... 9 1.4 Septins identification ....................................................................................11 1.5 Domains and motifs......................................................................................13 1.6 Redundancy of the mammalian septin system..............................................14 1.7 Septins and disease .......................................................................................15 1.8 Septin orthologs between different organisms..............................................16 1.9 Septin filaments ............................................................................................18 1.10 Regulation of septin polymerization.............................................................21 1.10.1 Regulation by interacting partners......................................................................... 21 1.10.2 Regulation by covalent modifications ................................................................... 22 1.10.3 Role of nucleotide.................................................................................................. 23 1.11 Septin function..............................................................................................25 1.11.1 Septins and cytokinesis.......................................................................................... 25 1.11.2 Membrane organization and vesicle trafficking .................................................... 26 1.12 Objective of this work...................................................................................28 2 Materials and Methods................................................................................ 29 2.1 Materials .......................................................................................................29 2.2 Constructs .....................................................................................................30 2.3 Expression and purifcation of Septin 2.........................................................33 2.3.1 Growth and Harvest of bacteria............................................................................... 33 2.3.2 GSH-Sepharose affinity chromatography................................................................ 33 2.3.3 Concentrating proteins by ultra-filtration ................................................................ 34 2.3.4 Size Exclusion Chromatography ............................................................................. 34 v Contents 2.4 Purification of Human Septin Complex (HSC 2, 6, 7)................................. 34 2.4.1 Co-expression of SEPT2, SEPT6, and SEPT7 ........................................................34 2.4.2 Large scale co-expression of SEPT2, SEPT6, and SEPT7 ......................................35 2.4.3 Purification using Ni-NTA affinity chromatography...............................................35 2.4.4 Purification using amylose resin..............................................................................35 2.4.5 Size Exclusion Chromatography..............................................................................36 2.5 Crystallization and Crystallography............................................................. 36 2.5.1 Crystallization screens .............................................................................................36 2.5.2 Optimizing Crystals .................................................................................................37 2.5.3 Seeding ....................................................................................................................37 2.5.4 Crystal Soaking with Heavy atom compounds........................................................37 2.5.5 Cryo-Crystallography ..............................................................................................37 2.5.6 Data Collection ........................................................................................................38 2.5.7 Matthews Coefficient and Solvent content analysis ................................................38 2.5.8 Phase Determination – Importance of phases..........................................................39 2.5.9 Phase Determination using Heavy atoms derivatization..........................................40 2.5.10 Phase Determination using Molecular replacement (MR).....................................41 2.5.11 Phase improvement using density modification ....................................................41 2.5.12 Model building and Refinement ............................................................................42 2.6 Electron Microscopy (EM) and Single particle analysis.............................. 43 3 Results............................................................................................................44 3.1 Characterization of Septin 2......................................................................... 44 3.1.1 Purification ..............................................................................................................44 3.1.2 Nucleotide content...................................................................................................45 3.1.3 Monomer, dimer and oligomer equilibrium.............................................................45 3.2 Structure of Septin 2..................................................................................... 46 3.2.1 Crystallization of SEPT2-315..................................................................................46 3.2.2 Data collection and Data processing........................................................................47 3.2.3 Structure determination............................................................................................48 3.3 Overview of Septin structure and topology.................................................. 52 3.4 Dimerization of Septin2 ............................................................................... 53 3.4.1 G and NC - dimer ....................................................................................................53 3.4.2 Mutational analysis of the dimer interface...............................................................57 3.5 Crystallization of HSC ................................................................................. 58 3.5.1 Purification and initial characterization ...................................................................58 vi Contents 3.5.2 Crystallization.......................................................................................................... 60 3.5.3 Data collection and heavy atom derivatization........................................................ 61 3.6 Structure solution of HSC.............................................................................67 3.6.1 Phase determination................................................................................................. 67 3.6.2 Subunit assignment.................................................................................................. 68 3.6.3 Phase extension, Model building, and Refinement.................................................. 68 3.6.4 Trimer Model........................................................................................................... 71 3.7 Electron microscopy of human septin complex............................................72 3.8 Septin – Borg interaction studies..................................................................75 3.8.1 Purification of Borg3............................................................................................... 75 3.8.2 Septin and borg complexes...................................................................................... 77 4 Discussion...................................................................................................... 79 4.1 Dimerization of Septin G domain.................................................................79 4.2 Human septin complex is a linear hexamer..................................................82 4.3