Structural insight into the environment of the serine/threonine protein kinase domain of titin Dissertation zur Erlangung des akademischen Grades des Doktors der Naturwissenschaften an der Universitat¨ Konstanz Mathematisch-Naturwissenschaftliche Sektion Fachbereich Biologie vorgelegt von Simone Muller¨ Tag der mundlichen¨ Prufung:¨ 30.03.2006 1. Referent: Prof. Dr. Wolfram Welte 2. Referent: PD Dr. Matthias Wilmanns 3. Referent: Prof. Dr. Helmut Plattner Contents Abbreviations ix Summary xi Zusammenfassung xiii 1 Introduction 1 1.1 Striated muscle and the structure of the sarcomere . 1 1.2 Titin . 3 1.3 Structure and function of titin . 5 1.3.1 Z-disc and anchoring of titin . 5 1.3.2 I-band and elasticity of the sarcomere . 5 1.3.3 A-band and the thick filament . 6 1.3.4 M-line and anchoring function . 7 1.4 Modules in titin: Ig and FnIII domains . 7 1.5 Titin kinase . 9 1.6 Titin kinase signalling pathway . 11 1.7 Disease association of a titin kinase mutation . 12 1.8 Muscle-specific RING finger protein MURF . 13 1.9 NBR1 . 15 1.10 The protein p62 . 16 1.11 Aim of the work . 17 2 The A-band immunoglobulin domains A168 and A169 19 2.1 Introduction . 19 2.1.1 Immunoglobulin domains . 19 2.1.2 Interaction of titin A168-A169 and MURF . 20 2.2 Materials and Methods . 21 2.2.1 Purification of A168-A169 . 21 2.2.2 Preparation of Selenomethionine incorporated A168-A169 22 2.2.3 Purification of titin A168-A169-A170 . 22 2.2.4 Crystallisation of A168-A169-A170 and diffraction tests . 23 2.2.5 Crystallisation of A168-A169 . 24 2.2.6 A168-A169 X-ray data collection and processing . 24 2.2.7 Structure solution of A168-A169 . 27 2.2.8 Tilt and twist angle in module assembly . 28 2.3 Results . 29 i ii Table of Contents 2.3.1 Overall structure of titin A168-A169 . 29 2.3.2 Comparison of the Ig domains . 30 2.3.3 Comparison with other tandem Ig domains . 32 2.4 Discussion . 34 2.4.1 Insertion loop in A169 between strand A and A’ . 34 2.4.2 Continuous β-strand bridging the two domains . 37 2.4.3 Interdomain geometry . 37 2.4.4 Tight connection and rigid structure . 38 2.4.5 Relevance of the bulge in the function of A168-A169 . 39 3 The titin M-band immunoglobulin domain M1 41 3.1 Introduction . 41 3.1.1 MLCK and related kinases surrounded by FnIII and Ig domains . 41 3.1.2 Two examples of a subsequent Ig domain of a kinase - telokin and the twitchin Ig domain Ig26 . 41 3.1.3 Scope of the work . 42 3.2 Materials and Methods . 43 3.2.1 Preparation of titin M1 . 43 3.2.2 Crystallisation of titin M1 . 44 3.2.3 Data collection and processing . 44 3.2.4 Molecular replacement and refinement . 45 3.3 Results . 46 3.3.1 Purification and crystallisation of M1 . 46 3.3.2 Structure solution and refinement . 46 3.3.3 Overall structure of titin M1 . 47 3.3.4 Comparison with telokin and twitchin Ig26 . 49 3.4 Discussion . 50 4 NBR1 PB1 in complex with p62 PB1 53 4.1 Introduction . 53 4.1.1 Protein interaction via PB1 domains . 53 4.1.2 PB1 domains – Mode of interaction . 54 4.1.3 Specificity determination of PB1 domains . 56 4.1.4 Overview of PB1 domain structures . 56 4.2 Materials and Methods . 56 4.2.1 Preparation of NBR1 PB1 . 56 4.2.2 Crystallisation of NBR1 PB1 . 58 4.2.3 X-ray data collection and processing of NBR1 PB1 . 58 4.2.4 Structure solution of NBR1 PB1 by MAD . 59 4.2.5 Preparation of p62 PB1 and mutants . 60 4.2.6 Complex formation of NBR1 PB1 and p62 PB1 (DDAA) 61 4.2.7 Crystallisation of the PB1 complex . 62 4.2.8 Data collection and processing of the PB1 complex . 63 4.2.9 Structure solution of the PB1 complex . 64 4.3 Results . 65 4.3.1 Purification and crystallisation of NBR1 PB1 . 65 Table of Contents iii 4.3.2 Preparation of p62 PB1 and mutants . 65 4.3.3 Complex formation of NBR1 PB1 and p62 PB1 (DDAA) 66 4.3.4 Crystallisation and data collection of the PB1 complex . 66 4.3.5 Structure solution and refinement of NBR1 PB1 . 67 4.3.6 Overall structure of NBR1 PB1 . 67 4.3.7 Comparison with other PB1 domains . 69 4.3.8 Interaction surface of NBR1 PB1 domain . 70 4.3.9 Structure solution and refinement of the PB1 complex . 72 4.3.10 Cadmium chloride bound to p62 PB1 . 75 4.3.11 Overall structure of the heterodimer . 75 4.3.12 Heterodimeric PB1 domain interface . 76 4.3.13 Comparison with other heterodimeric PB1 complexes . 79 4.4 Discussion . 80 4.4.1 Cadmium bound to H66 in p62 PB1 . 80 4.4.2 The three classes of PB1 domains . 80 4.4.3 Model of the p62 PB1 homodimer . 80 4.4.4 Affinity of the NBR1/p62 PB1/PB1 heterodimer complex 81 4.4.5 p62 interactions . 81 4.4.6 Biological relevance of the NBR1/p62 heterodimer complex 81 5 Conclusions 83 A Titin kinase 87 A.1 Introduction . 87 A.2 Materials and Methods . 87 A.2.1 Cloning . 87 A.2.2 Expression of titin kinase . 88 A.2.3 Purification . 90 A.2.4 Western blot . 91 A.3 Results and Discussion . 91 A.3.1 Cloning . 91 A.3.2 Expression . 92 A.3.3 Purification . 93 A.3.4 Titin kinase truncations and mutations . 95 References 99 List of Figures 1.1 Striated muscle . 2 1.2 Titin . 4 1.3 Titin kinase . 10 1.4 Titin kinase downstream signalling pathway . 12 1.5 Domain architecture of MURF family members . 14 2.1 Titin kinase downstream signalling pathway . 21 2.2 Diffraction patterns of A168-A169-A170 . 25 2.3 Crystals of A168-A169 . 27 2.4 Overall structure of the tandem Ig domains . 29 2.5 Superimposition of the tandem Ig domains . 30 2.6 Schematic representation of the continuous β-strand interaction . 33 2.7 Tandem domain interface . 34 2.8 Structure-based sequence alignment of titin Ig-like domains . 35 2.9 Superimposition of A169 and 1Flt-D2 . 36 3.1 Schematic domain arrangement representation . 42 3.2 Titin kinase downstream signalling pathway . 43 3.3 Crystal of M1 . 44 3.4 Ramachandran plot for the M1 structure . 47 3.5 Structure-based sequence alignment on M1 . 48 3.6 Structure of M1 . 48 3.7 Electron density of M1 . 49 3.8 Superimposition of M1, telokin, and Ig26 . 50 3.9 Model of titin kinase with M1 . 52 4.1 Titin kinase downstream signalling pathway . 54 4.2 Model of PB1 interaction . 55 4.3 Crystals of the PB1 domain of NBR1 grown in ammonium sulphate 58 4.4 Crystals of the PB1 complex . 63 4.5 SEC of p62 PB1 (DA) and p62 PB1 (DDAA) . 66 4.6 SEC of the complex of the two PB1 domains . 67 4.7 ITC of NBR1 and p62 (DDAA) . 68 4.8 Structure of NBR1 PB1 . 70 4.9 Electron density of NBR1 PB1 . 72 4.10 Superimposition of PB1 domains . 73 4.11 Sequence alignment of PB1 domains . 74 v vi List of Figures 4.12 Anomalous difference fourier map . 75 4.13 Overall structure of the NBR1/p62 PB1/PB1 heterodimer . 76 4.14 Stereo view of the main PB1 interaction site . 77 4.15 Electrostatic potential of NBR1 PB1 and p62 PB1 . 78 4.16 Molecular interaction between p62 PB1/PB1 heterodimer . 78 A.1 Expression of 170TK and TKM1 . 93 A.2 Anion exchange chromatography of 170TK and TKM1 . 94 A.3 SDS-PAGE of 170TK and TKM1 . 95 List of Tables 1.1 Interaction partners of p62 . 17 2.1 X-ray data collection statistics of A168-A169 . 26 2.2 Selenium sites in A168-A169 . 27 2.3 Refinement statistics of A168-A169 . 28 2.4 Structural comparison of titin Ig domains . 31 3.1 X-ray data and structure refinement statistics of M1 . 45 3.2 Structural comparison of titin M1 . 49 4.1 PDB entries for PB1 domain structures . 57 4.2 X-ray data collection statistics of NBR1 PB1 . 59 4.3 Primers for cloning of p62 PB1 and mutants . 60 4.4 X-ray data collection statistics of the PB1 heterodimer complex . 64 4.5 Refinement statistics of NBR1 PB1 . 69 4.6 Structural comparison of PB1 domains . 71 4.7 Refinement statistics of the PB1 complex . ..