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Resolving the Ligand-Binding to Pattern Recognition Receptor for Advanced Glycation End Products (RAGE)

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Resolving the Ligand-Binding to Pattern Recognition Receptor for Advanced Glycation End Products (RAGE) Resolving the ligand-binding to pattern recognition Receptor for Advanced Glycation End products (RAGE) INAUGULRALDISSERTATION Zur Erlangung des Doktorgrades Der Fakultät Chemie und Pharmazie Der Albert-Ludwigs-Universität Freiburg in Breisgau Vorgelegt von Roya Tadayon Geboren 27.08.1986 in Teheran, Iran 2016 i Vorsitzender des Promotionsausschusses: Prof. Dr. Stefan Weber Referent: Prof. Dr. Oliver Einsle Korreferent: PD. Dr. Günter Fritz Datum der mündlichen Prüfung: 30 .01.2017 ii This thesis is dedicated to my beloved mother Farideh for all her love and encouragement from the first day of my life. iii SUMMARY The Receptor for Advanced Glycation Endproducts (RAGE) is a pattern recognition receptor and key in the innate immune response. It is a type 2 membrane protein with an ectodomain consisting of three immunoglobulin-like domains, V, C1, and C2 domain. RAGE activation triggers the initiation and perpetuation of the inflammatory response. Hyper-activation of RAGE is associated with chronic inflammatory disorders, diabetic complications, tumor outgrowth and neurodegenerative disorders. Wide varieties of structurally diverse ligands bind to RAGE and trigger intracellular signal cascades. The cellular response evoked upon RAGE-ligand interaction is dependent on the nature of the ligand, its concentration, and affinity towards the receptor. In order to understand the molecular basis of receptor activation, I was studying the interaction of this unique receptor with several of its ligands. Key ligands of RAGE are Danger-Associated Molecular Pattern molecules (DAMPs) like e.g. S100A9, S100A12 and S100A8/A9. Using isothermal calorimetry, I have characterized binding of S100A9 to RAGE-VC1 tandem domain. Only the Ca2+- and Zn2+-bound form of S100A9 interacts with VC1. Analysis of the binding data suggests that the interaction at a Kd of 4 µM is largely entropy driven. Further I have characterized the interaction of S100 proteins with RAGE applying surface plasmon resonance and microscale thermophoresis. The X-ray structure of S100A9 in complex with Ca2+- and Zn2+ revealed drastic metal ion-induced conformational changes exposing hydrophobic pocket required for high-affinity RAGE binding. Blocking the interaction of S100A9 with RAGE represents a promising pharmaceutical approach in the therapy of chronic inflammatory diseases. Therefore, I characterized the binding of new compounds which block S100A9-receptor interaction. I have analyzed a series of compounds from the Quinoline-3- carboxamides family (Q-compounds) by ITC and X-ray crystallography. Strikingly, all different compounds bound to the hydrophobic pocket of S100A9. The structural data presented here give first insights into the molecular mechanism of inhibition and provide the basis for the development of more potent and specific drugs in the future. 1 ZUSAMMENFASSUNG Der Rezeptor RAGE (Receptor for Advanced Glycation Endproducts) ist ein sog. Pattern-Recognition Rezeptor und ein Schlüsselmolekül in der angeborenen Immunantwort. RAGE ist ein Typ-2 Membranprotein, wobei der extrazelluläre Teil von RAGE aus den drei Immunglobulin (Ig)-artigen Domänen V, C1 und C2 besteht. Die Aktivierung von RAGE initiiert die physiologische Entzündungsreaktion und hält diese aufrecht. Eine Überaktivierung von RAGE findet sich bei chronischen Entzündungserkrankungen, Diabetesfolgeerkrankungen, wachsenden Tumoren und neurodegenerativen Erkrankungen. RAGE bindet eine Vielzahl verschiedener Liganden, die jeweils eine intrazelluläre Signalkaskade auslösen. Die daraus resultierende zelluläre Antwort ist abhängig von der Art des Liganden, dessen Konzentration und Affinität zum Rezeptor. In der vorliegenden Arbeit untersuchte ich die Wechselwirkungen des Rezeptors RAGE mit verschiedenen Liganden. Wichtige RAGE-Liganden sind die sog. Danger-Associated-Molecular-Pattern molecules (DAMPs), wie zum Beispiel S100A9, S100A12 und S100A8/A9. Mithilfe von isothermaler Kalorimetrie ‚(ITC) untersuchte ich die Bindung von S100A9 an die Tandem-Ig-Domänen V und C1 von RAGE. S100A9 bindet an diese VC1 Domänen nur in der Gegenwart von Ca2+ und Zn2+ wobei die Wechselwirkung vor allem durch Entropiegewinn zustande kommt. Eine Dissoziationskonstante (Kd) on 4 µM wurde bestimmt. Weiterhin untersuchte ich die Wechselwirkung von anderen S100 Proteinen mit RAGE mittels Oberflächenplasmon-Resonanz (SPR) und Mikro- Thermophorese (MST). Die Kristallstruktur von S100A9 im Ca2+- und Zn2+- gebundenen Zustand zeigte, dass sich die Konformation des Proteins nach Metallionenbindung drastisch ändert und eine tiefe hydrophobe Tasche ausgebildet wird. Die Bindung von S100A9 an RAGE erfolgt über diese hydrophobe Bindetasche. Eine mögliche Therapie von chronischen Entzündungserkrankungen wäre die Bindung von S100A9 an RAGE zu unterbinden. In meiner Arbeit untersuchte eine Reihe Moleküle, welche die genau diese Wechselwirkung von S100A9 mit RAGE blockieren. Mehrere Moleküle aus der Familie der Chinolin-3-Carboxamide wurden auf ihre Bindung an S100A9 hin mittels ITC und anhand der Röntgenkristallographie charakterisiert. Überraschenderweise zeigte sich, dass alle Moleküle in der hydrophoben Bindetasche von S100A9 binden und diese effektiv blockieren. Die in dieser Arbeit vorgestellten Ergebnisse geben so einen erstmals Einblick in den molekularen Wirkmechanismen der Wirkstoffe aus der Chinolin-3-Carboxamid 2 Familie und bilden die Basis für die Entwicklung weiterer potenter und spezifischer Wirkstoffe. 3 Table of contents 1. INTRODUCTION ---------------------------------------------- 8 1.1 Receptor for advanced glycation end products -------------------------------------- 8 1.1.1 RAGE’s ligands and signaling cascades --------------------------------------- 11 1.1.2 Effect of glycosylation on RAGE ligand binding ------------------------------ 13 1.2 S100 Protein family ----------------------------------------------------------------------- 14 1.2.1 Biological function --------------------------------------------------------------------- 14 1.2.2 S100 proteins and metal Ion binding -------------------------------------------- 15 1.2.3 S100A9/A9 and S100A8/A9 history ---------------------------------------------- 17 1.3 The interaction of the RAGE with S100 Proteins---------------------------------- 18 1.4 Quinoline-3-carboxamides (Q compounds) ---------------------------------------- 19 1.5 Aim of this thesis --------------------------------------------------------------------------- 21 2 MATERIAL AND METHODS ----------------------------- 23 2.1 MATERIAL ---------------------------------------------------------------------------------- 23 2.1.1 Chemicals ------------------------------------------------------------------------------- 23 2.1.2 Q-Compounds ------------------------------------------------------------------------- 26 2.1.3 Buffers, Media, Gels and antibiotics --------------------------------------------- 33 2.1.3.1 Medias ------------------------------------------------------------------------------ 33 2.1.3.2 Buffers, solutions, and antibiotics ------------------------------------------- 34 2.1.3.3 Buffers and gels for SDS-PAGE --------------------------------------------- 34 2.1.3.4 Purification buffers for S100A9 ---------------------------------------------- 35 2.1.3.5 Purification buffers for VC1243 ------------------------------------------------ 36 2.1.3.6 Purification buffers for sRAGE ----------------------------------------------- 36 2.1.3.7 ITC, MST and SPR buffers --------------------------------------------------- 37 4 2.1.4 Kits and SPR sensor chips --------------------------------------------------------- 38 2.1.5 Enzymes--------------------------------------------------------------------------------- 38 2.1.6 Chromatography Columns ---------------------------------------------------------- 38 2.1.7 Proteins and sequences ------------------------------------------------------------ 39 2.1.7.1 S100A9 ----------------------------------------------------------------------------- 39 2.1.7.2 S100A8/A9 ------------------------------------------------------------------------ 39 2.1.7.3 S100A12 --------------------------------------------------------------------------- 40 2.1.7.4 S100B ------------------------------------------------------------------------------ 40 2.1.7.5 VC1243 ------------------------------------------------------------------------------ 40 2.1.7.6 sRAGE ----------------------------------------------------------------------------- 41 2.1.8 Bacterial strains ----------------------------------------------------------------------- 42 2.1.9 Crystallization screens --------------------------------------------------------------- 42 2.2 METHODS ---------------------------------------------------------------------------------- 43 2.2.1 Microbiological methods ------------------------------------------------------------ 43 2.2.1.1 Preparation of chemically competent E.coli cells ----------------------- 43 2.2.1.2 Transformation of competent cells ------------------------------------------ 44 2.2.1.3 Recombinant protein expression -------------------------------------------- 44 2.2.1.4 His6-VC1243 protein expression in E.coli Origami B (DE3) ----------- 45 2.2.1.5 sRAGE expression in E.coli Rosettagami B (DE3) --------------------- 45 2.2.1.6 S100 A9-C3S expression in E.coli BL21 (DE3) ------------------------- 46 2.2.1.7 Expression of recombinant glycosylated VC1 --------------------------- 47 2.2.2 Biochemical and biophysical methods ------------------------------------------ 47 2.2.2.1 Purification
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