Structure calculation of proteins from solution and solid-state NMR data : Application to monomers and symmetric aggregates Inaugural-Dissertation to obtain the academic degree Doctor rerum naturalium (Dr. rer. nat.) submitted to the Department of Biology, Chemistry and Pharmacy of the Freie Universitat¨ Berlin by Benjamin Bardiaux from Nantes, France January, 2009 1st Reviewer: Prof. Dr. Michael Nilges (Institut Pasteur, Paris) 2nd Reviewer: Prof. Dr. Hartmut Oschkinat (FMP, Berlin) date of defence: 8 Sept. 2009 This work was carried out from October 2004 to September 2008 under the joint supervision of Prof. Dr. Michael Nilges (Institut Pasteur, Paris), Dr. Ther´ ese` E. Malliavin (Institut Pasteur, Paris) and Prof. Dr. Hartmut Oschkinat (Leibnizinstitut fur¨ Molekulare Pharmakologie, Berlin) at the Unite´ de Bioinformatique Structurale of the Institut Pasteur in Paris. iii Acknowledgements I would like to thank Michael Nilges for giving me the opportunity to work in an excellent scientific environment and entrusting me with all ARIA related projects during the last four years. I am deeply grateful to Ther´ ese` Malliavin for introducing me to the world of NMR and structure calculations. Her everyday support, inventiveness and cheerfulness was of great help during my thesis. I wish to thank Hartmut Oschkinat for his effective co-supervision of my thesis from Berlin. I would also like to express my gratitude to the persons across Europe without whom this work would not have been possible : Michael Habeck and Wolfgang Rieping who initiated me to the mysteries of the ARIA program; Anja Bockmann¨ for her constant enthusiasm for structure calculations; Antoine Loquet, Carole Gardiennet, Manuel Etzkorn and Marc Baldus for fruitful and pleasant discussions; Richard Lavery for welcoming me in his lab and Alexey Mazur for his contribution to the work on ICMD; Ernest Laue, Tim Stevens, Wim Vranken and all the CCPN developer team; Michael Sattler and Bernd Simon for providing data on the B2 dimer; Christian Wasmer and Beat Meier for discussions on HET-s fibrils; Michele Fossi for sharing his experi- ence on SOLARIA; Inaki˜ Guijarro and all ARIA users and contributors. My visits to Berlin were always a great pleasure, thanks to the availability and kindness of Hartmut Oschkinat, Frank Eisenmenger and Victoria Highman. Particular thanks to Johanna Becker and Barth-Jan van Rossum for their close collaboration on CA150.WW2 fibrils and SH3 projects. My everyday work at the Institut Pasteur has been greatly facilitated by Tru Huyn and Renee´ Communal. I would like to thank both of them for their crucial help with computer and office matters. I am thankful to all the BIS people for the pleasant atmosphere in the lab and more particularly Olivier, Giacommo, Laura and Elodie. Special thanks to Aymeric and David who were always present for work or fun. I would like to thank the French “Ministere` de la Recherche“ for financial support through the “Action Concerte´ Incitative” ICMD RMN and the ANR “Regul lipids+”. This work was also supported by the EU projects SPINE and Extend-NMR. Je remercie egalement´ mes parents, Marie et Jean-Claude, qui m’ont toujours soutenu du- rant mes etudes,´ ainsi que mon frere` et Catherine. Un grand merci a` Annie et toute la famille de Morgane. Of course, I could not have done this thesis without the support of Morgane, my beloved little fay, who is always beside me. iv CONTENTS Abbreviations viii 1 General Introduction 1 1.1 Concepts in NMR spectroscopy . 1 1.1.1 Chemical shift . 2 1.1.2 Scalar and dipolar couplings . 2 1.1.3 Magical angle spinning solid-state NMR spectroscopy . 3 1.2 NMR data for protein structure determination . 3 1.2.1 Nuclear Overhauser Effects . 4 1.2.2 J couplings . 5 1.2.3 Chemical shifts . 6 1.2.4 Residual dipolar couplings . 6 1.2.5 Structural restraints from solid-state MAS NMR . 7 1.3 Structure calculation algorithms . 8 1.3.1 Molecular dynamics simulated annealing . 8 1.3.2 NMR data energy target functions . 9 1.3.3 Torsion angle dynamics . 11 1.3.4 Internal Coordinate Molecular Dynamics approach . 12 1.3.5 Inferential Structure Determination . 12 1.4 Automated NOE assignment and structure calculation . 13 1.4.1 Current state of the field . 13 1.4.2 ARIA . 14 1.5 Validation of NMR structures . 15 1.5.1 Experimental data validation . 15 1.5.2 Geometric quality evaluation . 16 1.6 NMR structure determination of symmetric oligomers . 17 1.7 Objectives of the thesis . 19 2 Programming contribution to the ARIA software 21 2.1 Description of the ARIA program . 21 2.1.1 Detailed protocol steps . 21 2.1.2 Bookkeeping in ARIA . 27 2.1.3 General ARIA implementation . 27 2.2 Network anchoring in ARIA . 28 2.3 Symmetric homo–dimer calculation . 29 2.4 Adaptation to solid-state MAS NMR data . 31 2.5 Graphical analysis of NMR structural quality . 32 2.5.1 Interactive analysis of peaks assignments . 32 2.5.2 Per-residue structural quality . 34 v 3 Influence of automated NOE assignment and NMR structure calculation protocols on struc- tural quality 35 3.1 Effect of network anchoring on the structure of the HRDC domain . 36 3.1.1 Calculation schemes . 36 3.1.2 Results . 36 3.2 Comparison of different torsion angle approaches for NMR structure determination. 40 3.2.1 Calculation schemes . 40 3.2.2 Analysis of the ICMD structures with floating restraints . 44 3.2.3 Ambiguous and floating form of restraints . 46 3.2.4 Comparison between ICMD and RECOORD . 47 3.3 Conclusion . 52 4 Automated structure determination of symmetric homo-dimers with ARIA. 55 4.1 Symmetric homo-dimers from solution NMR . 55 4.1.1 Calculation schemes . 55 4.1.2 Efficiency of the chain assignment . 57 4.1.3 Application of the network anchoring with ambiguous data . 60 4.1.4 Impact of the spin diffusion correction with different levels of ambiguity . 62 4.2 Structure determination of the Crh homo-dimer from solid-state NMR . 64 4.2.1 Experimental context . 64 4.2.2 Automatic assignments of highly ambiguous restraints. 65 4.2.3 Calculation of the Crh 3D structure from the ARIA unambiguous restraints . 67 4.2.4 Comparison with other protein structures obtained from ssNMR data . 69 4.3 Conclusion . 69 5 Analysis of the conformational landscape of Crh from solution and solid-state NMR data 71 5.1 Introduction . 71 5.2 Materials and Methods . 73 5.2.1 Input files of the conformers calculations . 73 5.2.2 ARIA-CNS calculation . 73 5.2.3 Structure analysis . 74 5.2.4 Clustering algorithms . 75 5.2.5 Molecular dynamics simulations . 75 5.2.6 Ensemble crystallographic refinement . 75 5.3 Results . 76 5.3.1 Filtering of the liquid-state NMR restraints . 76 5.3.2 Presentation of data inputs and dimeric calculations . 77 5.3.3 Convergence of the calculation and fit to the NMR restraints . 78 5.3.4 Conformers quality and accuracy . 82 5.3.5 Relative monomer orientation in the dimer . 83 5.3.6 Dimer and tetramer architecture . 86 5.4 Discussion . 89 vi 6 A general method for NMR structure calculation of symmetric aggregates 91 6.1 Introduction . 91 6.2 Material and Methods . 92 6.2.1 Symmetric systems and data sets used . 92 6.2.2 Structure calculation with strict symmetry . 93 6.3 Results and Discussion . 95 6.3.1 Phospholamban homo-pentamer . 95 6.3.2 Modelling of amyloid fibrils from solid-state NMR data . 99 6.3.3 Refinement of SH3 domain microcrystalline structure with crystal symmetries . 104 6.4 Conclusion . 105 7 General conclusions and Perspectives 107 7.1 Concluding remarks . 107 7.2 Perspectives . 109 7.3 Publications . 110 References 111 Summary 123 Zusammenfassung 124 Resum´ e´ 125 C.V. 127 Appendix 129 vii ABBREVIATIONS 2D two-dimensional 3D three-dimensional ADR ambiguous distance restraint ARIA ambiguous restraints for iterative assignments CPU Central Processing Unit Crh.