Structural and biochemical characterisation of the C-type lectin receptor DNGR-1 and its binding to F-actin Pavel Hanč University College London and Cancer Research UK London Research Institute PhD Supervisor: Caetano Reis e Sousa, DPhil, FMedSci A thesis submitted for the degree of Doctor of Philosophy University College London September 2015 Declaration I, Pavel Hanč, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. 2 Abstract DNGR-1 is a C-type lectin receptor that has been implicated in the regulation of endocytic trafficking and cross-presentation of dead cell-associated antigens. Dendritic cells deficient in DNGR-1 are impaired in priming effector T-cell responses against cytopathic viruses and other dead cell-associated antigens. The ligand for DNGR-1 is the polymerized form of actin (F-actin) revealed in dead cells upon loss of membrane integrity. In this study we set out to determine biophysical, biochemical, and structural properties of DNGR-1 and its interaction with F-actin. First, we describe a conformational change that occurs in the neck region of the receptor in a pH- and ionic strength-dependent manner. Notably, the conformational change happens between conditions corresponding to the extracellular environment and the environment present in the vesicles of the endosomal pathway respectively, suggesting a possible role in the spatial regulation of the DNGR-1 function. Second, in collaboration with Keichii Namba and Takashi Fujii (RIKEN Quantitative Biology Center, Osaka, Japan) we used electron cryomicroscopy to solve the structure of DNGR-1 bound to F-actin at 7.7 Å resolution. Interestingly, DNGR-1 binds into the groove between actin protofilaments, making contacts with three actin subunits that are helically arranged in the F-actin structure. We identify the residues directly involved in the interaction, confirm their contribution to the binding and demonstrate the importance of avidity of the multivalent interaction between DNGR-1 and F-actin. Additionally, in collaboration with David Sancho and Salvador Iborra (Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain) we formally demonstrate that ligand recognition is prerequisite for the biological function of DNGR-1 in dendritic cells. Third, by using heterodimeric DNGR-1 proteins in which one half of the dimer is incapable of binding to ligand, we demonstrate that DNGR-1 can bind with both ligand-binding domains to a single actin filament, suggesting an exceptional flexibility of the neck region, and demonstrating an absence of rigid dimerization interface between the ligand-binding domains. In summary, we provide a comprehensive description of the structural and biophysical properties of DNGR-1, offering novel insights into its function and shedding light into innate immune mechanisms involved in recognition of cell death. 3 Acknowledgement First and foremost, I would like to thank my supervisor, Caetano Reis e Sousa, for giving me the chance to carry out this work in his lab, for giving me his guidance when needed and at the same time allowing me to develop my independence and learn from my mistakes, for showing me how to be a better scientist in myriad ways both small and large, and for always being supportive and encouraging, even when the data were not. Many thanks go out to my thesis committee, Facundo Batista and Erik Sahai, for all the helpful advice I’ve received from them over the years and their supportive attitude during our meetings. None of my work would have been possible without our numerous collaborators. Namely, I’d like to thank Keiichi Namba and Takashi Fujii for lending their Cryo-EM expertise, David Sancho and Salvador Iborra for helpful discussions and indispensable assistance with antigen-presentation assays, Steve Martin for his help with circular dichroism measurements, Thomas Surrey and Christian Duellberg for their assistance with TIRF-based experiments, Svend Kjær for numerous size exclusion chromatography purifications as well as many helpful discussions about anything “protein-related,” and last but not least Michael Way for all his advice regarding the properties and behaviour of actin. I’m truly grateful for the great time I’ve had in the Immunobiology lab during the last four years, for the friendly, helpful and supportive, yet stimulating and highly productive environment, and for all the things that I’ve had a chance to learn. In one way or another I’m indebted to all members of the lab, past and present, but in particular to Oliver Schultz for showing me the ropes when I joined the lab and for many helpful discussions over the years, to the other members of the “DNGR-1 team,” Jatta Huotari and formerly Susan Ahrens for all their help, to Santiago Zelenay for insightful comments and plentiful DNGR-1 (and otherwise) centred lunch-time conversations, to Janneke, Kat, Oliver, Naren and Jan for making Friday evenings worth having, and to everyone else for making the lab a place where one can not only work but also have fun! Outside of the lab, I’d like to thank my family and friends for their continual support during what was an exciting, but not always an easy time. And finally, special 4 thanks to Kristyna for having endless patience with me, for listening to my moaning and complaining when things went wrong without ever telling me to be quiet, for always being there, and last but not least, for proof-reading this thesis and finding all the little details that I have no eye for. 5 Table of Contents Abstract ............................................................................................................... 3 Acknowledgement .............................................................................................. 4 Table of Contents ................................................................................................ 6 Table of figures ................................................................................................. 10 List of tables ...................................................................................................... 13 Abbreviations .................................................................................................... 14 Chapter 1. Introduction .................................................................................. 17 1.1 Brief overview of the conceptual development of immunology ....... 17 1.2 Innate and adaptive immune system and their roles in homeostasis maintenance .................................................................................................. 18 1.2.1 Innate immune system ...................................................................... 19 1.2.2 Adaptive immune system .................................................................. 20 1.3 Dendritic cells ........................................................................................ 22 1.3.1 Function of dendritic cells ................................................................. 23 1.3.2 DC subsets and their roles within immune system ........................... 25 1.4 PAMP and DAMP recognition .............................................................. 26 1.4.1 PRRs and PAMP recognition ............................................................ 27 1.4.2 DAMP recognition by innate immune receptors ................................ 29 1.5 C-type lectin receptors ......................................................................... 32 1.5.1 C-type lectin terminology .................................................................. 32 1.5.2 Structural features of C-type lectins .................................................. 32 1.5.3 C-type lectins in the immune system ................................................ 34 1.5.4 Signalling by C-type lectins ............................................................... 35 1.5.5 HemITAM containing C-type lectins .................................................. 38 1.6 DNGR-1 ................................................................................................... 39 1.6.1 Discovery of DNGR-1 ....................................................................... 39 1.6.2 Genomic arrangement and isoforms of DNGR-1 .............................. 40 1.6.3 Structure of hDNGR-1 CTLD ............................................................ 41 1.6.4 DNGR-1 is involved in cross-presentation of dead cell-associated antigens ....................................................................................................... 42 1.6.5 DNGR-1 controls endocytic handling of dead cell-associated antigens ....................................................................................................... 44 1.6.6 Identification of DNGR-1 ligand ........................................................ 45 1.6.7 Cellular distribution of DNGR-1 ......................................................... 46 1.6.8 Antigen targeting to DNGR-1 promotes immune response .............. 46 1.7 Summary and objectives ...................................................................... 48 Chapter 2. Materials & Methods .................................................................... 49 2.1 Reagents ................................................................................................ 49 2.1.1 Buffers ............................................................................................... 49 2.1.2 Proteins ............................................................................................