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Structural and biochemical characterisation of the C-type lectin receptor DNGR 1 and its binding to F-actin

Hanc, P; (2015) Structural and biochemical characterisation of the C-type lectin receptor DNGR 1 and its binding to F-actin. Doctoral thesis , UCL (University College London). Green open access

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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.

Type: Thesis (Doctoral)
Title: Structural and biochemical characterisation of the C-type lectin receptor DNGR 1 and its binding to F-actin
Event: UCL
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Third party copyright material has been removed from ethesis.
UCL classification: UCL
UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences
URI: https://discovery.ucl.ac.uk/id/eprint/1472936
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