Structural and functional study of the interaction between CXCL12 chemokine and its receptors : CXCR4 and ACKR3/CXCR7 Pasquale Cutolo To cite this version: Pasquale Cutolo. Structural and functional study of the interaction between CXCL12 chemokine and its receptors : CXCR4 and ACKR3/CXCR7. Immunotherapy. Université Paris Saclay (COmUE), 2016. English. NNT : 2016SACLS550. tel-01674198v2 HAL Id: tel-01674198 https://tel.archives-ouvertes.fr/tel-01674198v2 Submitted on 2 Jan 2018 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. 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NNT 2016SACLS550 Thèse de doctorat De L’Université Paris-Saclay Ecole Doctorale n° 569 INNOVATION THÉRAPEUTIQUE : DU FONDAMENTAL À L’APPLIQUÉ Spécialité de doctorat : IMMUNOLOGIE ET BIOTHÉRAPIES Présentée par Pasquale CUTOLO Etude de l’interaction structurelle et fonctionnelle entre la chimiokine CXCL12 et ses récepteurs : CXCR4 et ACKR3/CXCR7 Thèse présentée et soutenue à Châtenay-Malabry, le 16 septembre 2016 Composition du Jury : Françoise BACHELERIE DR, Inserm UMR-S 996, Clamart Directrice de thèse Esther KELLENBERGER Prof., Faculté de Pharmacie de Strasbourg Rapportrice Eric REITER DR, INRA, Tour Rapporteur Philippe DETERRE DR, Inserm UMR-S 945, Paris Examinateur Thierry DURROUX DR, CNRS – IGF, Montpellier Examinateur Sandrine ONGERI Prof. de l’Université Paris-Saclay Examinatrice INDEX Abbreviations .................................................................................................................. 4 Introduction .................................................................................................................... 7 Chapter I - Chemokines structure and interactions ................................................................................................ 7 Preamble on Chemokines .................................................................................................................................................... 7 I.1 Primary structure and classification of Chemokines (CKs) ......................................................................... 9 I.2 Topology and Folding of CKs ....................................................................................................................................12 I.3 Oligomerization of CKs ...............................................................................................................................................14 I.4 Interaction with Glycosaminoglycans (GAGs)..................................................................................................18 I.5 Interaction of CKs with receptors ..........................................................................................................................24 Chapter II - G-protein Coupled Receptors (GPCRs) structural basis ............................................................ 38 Preamble on GPCRs ...........................................................................................................................................................38 II.1 Current Chemokine Receptors (CKRs) Crystal Structures ........................................................................40 II.2 Oligomerization of GPCRs ........................................................................................................................................41 II.3 Atypical Chemokine Receptors (ACKRs)............................................................................................................47 II.4 CXCR4 receptor-dependent signaling pathways ...........................................................................................53 Chapter III - Nanobodies (NBs) as new innovative tools .................................................................................... 59 Preamble on NBs: the smallest natural antigen-binding fragment .................................................................59 III.1 Antibodies for biomedical applications ...........................................................................................................63 III.2 NBs in structural biology ........................................................................................................................................64 III.3 CXCR4- and CXCR7/ACKR3- targeting NBs ...................................................................................................67 Objectives ...................................................................................................................... 68 Results ........................................................................................................................... 69 Paper n°1 ............................................................................................................................................................................ 72 Paper n°2 ......................................................................................................................................................................... 118 Paper n°3 ......................................................................................................................................................................... 158 1 Discussion and perspectives ......................................................................................... 196 I - Mechanism of the interaction and stoichiometry of CXCL12 with CXCR4 and CXCR7/ACKR3 receptors .......................................................................................................................................................................... 197 I.1 Modeling the three-dimensional structure of GPCRs ................................................................................. 197 I.2 Modeling the binding of ligands to GPCRs ...................................................................................................... 199 I.3 Conclusions .................................................................................................................................................................... 201 II – Nanobodies ............................................................................................................................................................. 203 II.1 Characterization of NBs against CXCR4 ........................................................................................................ 203 II.2 NBs applications........................................................................................................................................................ 203 II.3 Conclusions .................................................................................................................................................................. 204 References .................................................................................................................... 207 Annexes ....................................................................................................................... 238 Curriculum Vitae .......................................................................................................................................................... 243 Acknowledgement ....................................................................................................................................................... 238 2 “Eppur si muove” "And yet it moves" or "Albeit it does move", one of the most famous Galileo Galilei’s citation for Earth movement, is the good sentence to introduce my thesis. The Italian scientist spoke about the movement of our planet and its “migration” around the Sun, people are moving around the world during their life, but deeply inside this movement is also the base of our biology, from the embryogenesis to the adult life. How can our cells “migrate” and decide where and when “be or not to be”? The answer is in small molecules called chemokines and their fascinating and highly intricate networks with chemokine receptors. 3 Abbreviations 7-TM Seven transmembrane TM Transmembrane BRET Bioluminescent resonance energy transfer C-terminus Carboxyl-terminal extremity CDRs Complementary determining regions CFP Cyan fluorescent protein CKs Chemokines CKRs Chemokines Receptors CRS1 Chemokine recognition site 1 CRS2 Chemokine recognition site 2 CS Chondroitin sulfate CX3R, CX3C chemokine receptors CXCR CXC chemokine receptor Cyt Cytoplasmic region DS Dermatan sulfate ECL Extracellular loop ECM Extracellular matrix ELR Glu-Leu-Arg sequences Fig Figure FRET Förster Resonance Energy Transfer GAGs Glycosaminoglycans GPCRs G-protein coupled receptors HA Hyaluronic acid HCAbs Heavy-chain antibodies HIV Human Immunodeficiency Virus HS Heparan sulfate HTRF Homogeneous time-resolved FRET ICL Intracellular loop KS Keratin sulfate mAbs Monoclonal antibodies MOR mu opioid receptor N-terminus Amino-terminal extremity NB Nanobody 4 NMR Nuclear magnetic resonance PDB Protein DATA Bank sTyr Sulfotyrosine Tab Table TM Transmembrane region TNF Tumor necrosis factor VHH Camelid heavy-chain antibody YFP Yellow fluorescent protein - Table