Structural insights into oligomerization and mitochondrial remodeling of dynamin 1-like protein Dissertation zur Erlangung des akademischen Grades des Doktors der Naturwissenschaften (Dr. rer. nat.) eingereicht im Fachbereich Biologie, Chemie, Pharmazie der Freien Universität Berlin vorgelegt von Dipl.-Ing. Chris Fröhlich aus der Lutherstadt Eisleben Berlin 2013 ii Die vorliegende Arbeit wurde im Zeitraum April 2009 bis März 2013 am Max-Delbrück-Centrum für Molekulare Medizin Berlin-Buch unter Anleitung von PROF. DR. OLIVER DAUMKE angefertigt. 1. Gutachter: Prof. Dr. Udo Heinemann 2. Gutachter: Prof. Dr. Oliver Daumke Tag der Disputation: 29.07.2013 iii … to boldly go where no man has gone before. iv Contents ___________________________________________________________________________ Contents Contents ................................................................................................................................................... v List of Figures ........................................................................................................................................... ix List of Tables ............................................................................................................................................ xi 1. Introduction ..................................................................................................................................... 1 1.1. Mitochondria ........................................................................................................................... 1 1.1.1. The mitochondrial compartment .................................................................................... 2 1.1.2. The mitochondrial network ............................................................................................. 5 1.1.3. Mitochondria associated diseases .................................................................................. 6 1.2. G proteins ................................................................................................................................ 8 1.3. The dynamin superfamily of G proteins ................................................................................ 10 1.3.1. Dynamin ........................................................................................................................ 12 1.3.1.1. Dynamins are key players in clathrin-mediated endocytosis ................................ 12 1.3.1.2. The G domain and the bundle signaling element ................................................. 14 1.3.1.3. G domain dimerization is crucial for dynamin function ........................................ 15 1.3.1.4. Dynamin's PH domain mediates lipid binding ....................................................... 17 1.3.1.5. The stalk is the central assembly hub for dynamin oligomerization ..................... 17 1.3.1.6. Regulatory functions of the stalk .......................................................................... 20 1.3.1.7. Functional models of dynamin's mechano-chemical action ................................. 20 1.3.2. Myxovirus resistance (Mx) proteins .............................................................................. 22 1.3.2.1. Mx proteins mediate antiviral host response ....................................................... 22 1.3.2.2. Different functions - similar structures: MxA and dynamin 1 ............................... 23 1.3.2.3. The MxA stalk mediates oligomerization and regulatory function ....................... 24 1.3.2.4. The MxA stalk mediates assembly in rings rather than helices ............................ 25 1.3.3. Bacterial dynamin-like proteins (BDLPs) ....................................................................... 26 1.3.4. Guanylate-binding proteins (GBPs) ............................................................................... 28 1.3.5. Eps15 homology-domain containing proteins (EHDs) ................................................... 29 1.3.6. Mitochondrial fusion dynamins..................................................................................... 31 1.3.6.1. Mitochondrial outer membrane fusion dynamins ................................................ 31 1.3.6.2. Mitochondrial inner membrane fusion dynamins ................................................ 32 1.3.7. The mitochondrial fission dynamin 1-like protein (DNM1L) ......................................... 32 1.3.7.1. DNM1L is a key player in mitochondrial fission .................................................... 32 v Contents ___________________________________________________________________________ 1.3.7.2. Recruitment of DNM1L to mitochondria scission sites involves certain adaptor proteins and the endoplasmic reticulum (ER) ........................................................................... 33 1.3.7.3. Two-start versus one-start helix ............................................................................ 34 1.4. Scope of this work ................................................................................................................. 36 2. Materials and Methods ................................................................................................................. 38 2.1. Materials ................................................................................................................................ 38 2.1.1. Chemicals ....................................................................................................................... 38 2.1.2. Antibodies...................................................................................................................... 38 2.1.3. Enzymes ......................................................................................................................... 38 2.1.4. Kits ................................................................................................................................. 39 2.1.5. Microorganisms and cell lines ....................................................................................... 39 2.1.6. Vectors ........................................................................................................................... 40 2.1.7. cDNA clone .................................................................................................................... 40 2.1.8. Primers .......................................................................................................................... 40 2.1.8.1. Cloning Primers ..................................................................................................... 40 2.1.8.2. Quick change mutagenesis primers ...................................................................... 40 2.1.9. Media and antibiotics .................................................................................................... 42 2.1.10. Buffers ........................................................................................................................... 42 2.2. Molecular biology methods................................................................................................... 43 2.2.1. Polymerase chain reaction ............................................................................................ 43 2.2.2. DNA digestion ................................................................................................................ 43 2.2.3. Agarose gel electrophoresis .......................................................................................... 43 2.2.4. DNA purification ............................................................................................................ 43 2.2.5. Ligation .......................................................................................................................... 44 2.2.6. Preparation of chemically competent E. coli ................................................................. 44 2.2.7. Transformation of chemically competent E. coli ........................................................... 44 2.2.8. Isolation of plasmid DNA ............................................................................................... 44 2.2.9. DNA sequencing ............................................................................................................ 44 2.2.10. Site specific mutagenesis............................................................................................... 44 2.2.11. Sequence alignments .................................................................................................... 45 2.2.12. Bacterial storage ............................................................................................................ 45 2.2.13. Construct design ............................................................................................................ 45 2.3. Biochemical methods ............................................................................................................ 45 2.3.1. SDS PAGE ....................................................................................................................... 45 2.3.2. Protein over-expression test in E. coli ........................................................................... 45 vi Contents ___________________________________________________________________________ 2.3.3. Protein solubility test .................................................................................................... 46 2.3.4. Large scale protein over-expression in E. coli ..............................................................