Dissertation zur Erlangung des Doktorgrades der Fakultät für Chemie und Pharmazie der Ludwig-Maximilians-Universität München Structural and Functional Studies on Myosin Type V Dependent Transport in Vertebrates Hana Velvarská aus Brandeis a.d. Elbe, Tschechische Republik 2012 Erklärung Diese Dissertation wurde im Sinne von § 7 der Promotionsordnung vom 28. November 2011 von Herrn Prof. Dr. Klaus Förstemann betreut. Eidesstattliche Versicherung Diese Dissertation wurde eigenständig, ohne unerlaubte Hilfe erarbeitet. München, ……………………………………… Hana Velvarská Dissertation eingereicht am 24. September 2012 1. Gutachter: Prof. Dr. Klaus Förstemann 2. Gutachter: Prof. Dr. Mario Halic Mündliche Prüfung am 19. November 2012 TABLE OF CONTENTS SUMMARY ................................................................................................................................................. 1 1. INTRODUCTION ............................................................................................................................ 3 1.1 Intracellular transport ............................................................................................................. 3 1.2 Microtubules ................................................................................................................................ 4 1.3 Dynein and kinesin proteins ................................................................................................. 4 1.4 EB1 ................................................................................................................................................... 5 1.5 Actin ................................................................................................................................................. 6 1.6 Unconventional myosins ........................................................................................................ 6 1.7 Type V Myosin ............................................................................................................................. 7 1.8 Type V myosin in human ........................................................................................................ 8 1.8.1 Myo5a ................................................................................................................................................... 9 1.8.2 Myo5b ................................................................................................................................................. 10 1.8.3 Myo5c .................................................................................................................................................. 11 1.8.4 Autoinhibition ................................................................................................................................. 12 1.8.5 Regulation by Ca2+ ......................................................................................................................... 14 1.8.6 Regulation by cargo attachement ........................................................................................... 16 1.8.7 Phosphorylation ............................................................................................................................. 16 1.8.8 Griscelli syndrome ........................................................................................................................ 17 1.9 Melanophilin ............................................................................................................................. 18 1.10 Rab GTPases ............................................................................................................................ 20 1.10.1 Rab27a ............................................................................................................................................. 21 1.10.2 Rab11a ............................................................................................................................................. 22 1.11 Type V myosin in yeast and plants ................................................................................ 22 1.11.1 Myo2p .............................................................................................................................................. 23 1.11.2 Myo4p .............................................................................................................................................. 24 1.11.3 Myosins in plant ........................................................................................................................... 25 1.12 Myo6 ........................................................................................................................................... 25 1.13 Objectives ................................................................................................................................. 27 2. RESULTS ........................................................................................................................................ 28 2.1 Structural studies on Melanophilin ................................................................................. 28 2.1.1 Melanophilin-Rab27a complex ................................................................................................ 28 2.1.2 Melanophilin-Myo5a complex .................................................................................................. 29 2.1.3 Melanophilin-EB1 complex ....................................................................................................... 32 2.2 Structural studies on Myo6 CBD ...................................................................................... 34 2.2.1 Myo6 CBD crystallization ........................................................................................................... 35 2.2.2 Myo6 interaction with Miranda ............................................................................................... 37 2.2.3 Myo6 Initial NMR experiments ................................................................................................ 38 2.3 Structural studies on Myo5a .............................................................................................. 40 2.3.1 Defining the limits of Myo5a GD and crystallization attempts .................................. 40 2.3.2 Shape analysis by Small Angle X-ray Scattering ............................................................... 41 2.3.3 Shape analysis by SAXS of Myo5a, Myo5b, and Myo5c GDs ........................................ 42 2.3.4 Crystallization of the Myo5a GD .............................................................................................. 44 2.3.5 Structure determination of the Myo5a GD ......................................................................... 46 2.3.6 Structure determination of the Myo5b GD ......................................................................... 48 2.3.7 Model of Myo5c GD ....................................................................................................................... 50 2.3.8 Myo5a GD-mutations found to cause Griscelli-syndrome ........................................... 51 2.3.9 Comparison of the human MyoV GD with the yeast type V Myosins ...................... 53 2.3.10 Comparison of surface properties of the Myo5a, Myo5b, and Myo5c GD ........... 54 2.3.11 Investigation of structurally similar proteins ................................................................. 57 2.3.12 Residues required for Myo5a autoinhibition are conserved in Myo5b and Myo5c ...... 58 2.3.13 Not only Myo5a, but also Myo5b and Myo5c GD mediates autoinhibition ........ 59 2.3.14 Binding of Rab11a does not relieve the autoinhibition .............................................. 60 2.3.15 Docking of Myo5a GD crystal structure into EM density of autoinhibited Myo5a ........ 62 2.3.16 The loop I and II of the GD are required for autoinhibition ..................................... 64 2.3.17 A phospho-mimicking mutation in Myo5a loop I abolishes autoinhibition ...... 66 3. DISCUSSION ................................................................................................................................. 67 3.1 Myo5 regulation by autoinhibition ................................................................................. 68 3.2 Myo5 regulation by cargo binding and phosphorylation ...................................... 69 3.3 Regulation by Ca2+ concentration .................................................................................... 71 3.4 The Myo5a autoinhibition model ..................................................................................... 72 4. MATERIALS AND METHODS ................................................................................................ 75 4.1 Chemicals ................................................................................................................................... 75 4.2 DNA Oligonucleotides for cloning .................................................................................... 75 4.3 Plasmids ...................................................................................................................................... 78 4.4 Competent bacterial strains ............................................................................................... 80 4.5 Insect cells lines ....................................................................................................................... 80 4.6 Antibodies .................................................................................................................................