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

Vorsitzender des Promotionsausschusses: Prof. Dr. Stefan Weber Referent: Prof. Dr. Oliver Einsle Korreferent: PD. Dr. Günter Fritz Datum der mündlichen Prüfung: 30 .01.2017

This thesis is dedicated to my beloved mother Farideh for all her love and encouragement from the first day of my life.

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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.

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.

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 of His6-VC1243 and His6-sRAGE ------47

2.2.2.2 Purification of S100A9-C3S------49

2.2.2.3 Purification of recombinant glycosylated VC1 ------51

2.2.2.4 Isothermal titration calorimetry (ITC) ------51

2.2.2.4.1 ITC titrations of S100A9 with Q-compounds ------53

2.2.2.5 Micro scale thermophoresis (MST) ------53

2.2.2.5.1 MST of VC1243 and S100A9------55

2.2.2.6 Surface plasmon resonance (SPR) ------55

2.2.2.6.1 SPR for VC1243 and S100 proteins ------56

2.2.2.7 Protein X-ray crystallography------58

2.2.2.7.1 Concept of protein X-ray crystallography ------59

2.2.2.7.2 Crystallization ------59

2.2.2.7.3 The vapor diffusion technique ------60

2.2.2.7.4 Crystallization of S100A9-Ca2+-Zn2+ ------61

2.2.2.7.5 Soaking of compounds in to S100A9 ------62

2.2.2.7.6 Crystallization of S100A9 and of a VC1243-S100A9 complex --- 62

2.2.2.7.7 Data collection, and X-ray diffraction data analysis ------63

3 RESULTS ------69

3.1 Expression and purification of His6-VC1243 ------69

3.1.1 Expression of His6-VC1243 out of Origami B (DE3)------69

3.1.2 Purification of His6-VC1 243 ------70

3.2 Expression and purification of sRAGE ------74

3.2.1 Expression of sRAGE in E.coli Rosettagami B (DE3) ------74

3.2.2 Purification of sRAGE ------75

3.3 Expression and purification of S100A9 ------80

3.3.1 Expression of S100A9-C3S out of E.coli BL21 (DE3) ------80 6

3.3.2 Purification of S100A9-C3S ------81

3.4 Co-crystallization of S100A9 with VC1243 ------84

3.4.1 Optimizations of crystallization of VC1-S100A9 ------86

3.5 Monitoring interactions of S100A9 with VC1 domain using MST ------88

3.6 Monitoring binding of inhibitors and Zn2+ to S100A9 using ITC ------89

3.6.1 Calorimetric titrations of Zn2+ ions into S100A9 homodimer ------89

3.6.2 Calorimetric titrations of Q-compounds into S100A9 homodimer ------92

3.7 SPR measurements for S100 proteins to immobilized VC1 ------95

3.7.1 Binding of S100A9, S100A8/A9, S100A12 to immobilized VC1 ------95

3.8 Crystallization and structure of S100A9-Ca2+-Zn2+ ------100

3.8.1 Structure of S100A9 in complex Q-compounds ------104

4 DISCUSSION ------110

4.1 Conformational changes upon Zn2+ binding ------110

4.2 Analysis of RAGE–S100 interactions ------111

4.3 Effect of N-glycosylation on ligand binding affinity of RAGE ------112

4.4 Analysis of S100A9-inhibitors interactions ------113

4.5 Characterization of Q-compounds binding sites on S100A9. ------114

5 References ------116

6 Supplements ------124

6.1 List of figures ------124

6.2 List of tables------128

6.3 Abbreviations ------129

6.4 Acknowledgments ------130

1. INTRODUCTION

1.1 Receptor for advanced glycation end products

Receptor of advanced glycation end products (RAGE) is a transmembrane multi- ligand pattern recognition receptor (Kierdorf & Fritz 2013) with a molecular mass of 35 kDa. It was first described in 1992 (Neeper et al. 1992) and is implicated in various human diseases like cancer (Leclerc & Vetter 2015), muscular dystrophy (Macaione et al. 2007), inflammatory disorders (Foell et al. 2003), autoimmune diabetes (Yatime & Andersen 2013) and neurodegeneration (Zimmer et al. 2005; Kierdorf & Fritz 2013). RAGE is a protein unique to mammals(Degani et al. 2015) and the encoding gene localizes in the major histocompatibility complex class III region on chromosome 6. There are different RAGE splice variants that have been named RAGE, RAGE_v1 to RAGE_v19. RAGE is expressed at low levels in many cell types in humans such as leukocytes, dendritic cells, endothelial cells, smooth muscle cells, neurons and microglia and is highly abundant in the lung (Ott et al. 2014).The following Figure (Figure 1) shows the canonical sequence of human RAGE and its domain organization.

MAAGTAVGAWVLVLSLWGAVVGAQNITARIGEPLVLKCKGAPKKPPQRLEWKLNTGRTE AWKVLSPQGGGPWDSVARVLPNGSLFLPAVGIQDEGIFRCQAMNRNGKETKSNYRVRVYQ IPGKPEIVDSASELTAGVPNKVGTCVSEGSYPAGTLSWHLDGKPLVPNEKGVSVKEQTRRHP ETGLFTLQSELMVTPARGGDPRPTFSCSFSPGLPRHRALRTAPIQPRVWEPVPLEEVQLVVE PEGGAVAPGGTVTLTCEVPAQPSPQIHWMKDGVPLPLPPSPVLILPEIPQDQGTYSCVATHS SHGPQESRAVSISIIEPGEEGPTAGSVGGSGLGTLALALGILGGLGTAALLIGVILWQRRQRRG

Figure 1: Primary structure of RAGE and domain organization.

The amino acid residues highlighted in red belong to V domain, those highlighted in gray belong to C1 domain, and in yellow mark C2 domain. The bold letters indicate N- glycosylation sites.

RAGE is a member of the type I cell surface receptor family. The ectodomain is composed of three immunoglobulin (Ig)-like domains, the N-terminal V-type Ig domain (V-domain), and two C-type domains termed C1 and C2. V- and C1-domain form a structural and functional unit (VC1-domain) that represents the binding site for almost all RAGE ligands (Figure 2). The ectodomain is anchored in the cytoplasmic membrane by a single transmembrane helix that is followed by a short cytoplasmic C-terminal tail that is indispensable for intracellular signaling (Yan et al. 2008).The 3D-structure of the RAGE extracellular portion (V–C1–C2), of VC1 and V, have been determined, providing first insights into the putative molecular mechanism of RAGE activation (Xie et al. 2008; Xie et al. 2013; Ding & Keller 2005) (Figure 3). Endogenous soluble RAGE (sRAGE) isoforms are produced through two mechanisms: splicing of the transmembrane region (esRAGE) or cleaving off the extracellular domain (ectodomain or ECD) at the cell surface by ectodomain shedding (Galichet et al. 2008; Raucci et al. 2008). Shedding is required for some physiological functions but can be deregulated in various pathological disorders where altered levels of metalloproteinases are found (Hayashida et al. 2010). RAGE exhibits the most unusual feature, that it binds various ligands of different structure, size, or chemical nature. This characteristic designates RAGE as a pattern recognition receptor (PRR) among other well-known pattern recognition receptors of the innate immune system like e.g. the Toll-like receptors or scavenger receptors. All ligands of RAGE reported so far are characterized by a negative surface charge that mediates most likely high-affinity binding to the positively charged VC1 domain (Deane et al. 2003). A subpopulation of RAGE is modified by carboxylated glycans, which are reported to influence the interaction with HMGB1, S100A8/S100A9, and S100A12. Activation of RAGE initiates the inflammatory response that is usually terminated after a certain period leading to resolution of the inflammation and tissue repair. However, the perpetuation of RAGE signaling causes chronic inflammation and is associated with a number of different disorders (Clynes et al. 2007).

Figure 2: Structure of RAGE and ligand-binding sites.

The receptor RAGE is composed of an N-terminal ectodomain, one transmembrane helix, and a short cytoplasmic C-terminal tail. The ectodomain is composed of three immunoglobulin-like domains, V, C1, and C2. A schematic drawing of the structure is shown on the right-hand side; a molecular model is shown on the left-hand side. The V-domain is positively charged (blue) and serves as ligand binding site for the different ligands like S100 proteins, advanced glycation end products (AGEs), amyloids β (Aβ), high mobility group box protein 1 (HMGB1), or lysophosphatidic acid (LPA) (Kierdorf & Fritz 2013).

Figure 3: Structure of the Tandem VC1 Domains of RAGE .

Stereo ribbon diagram of VC1 with the V domain in green and the C1 domain in magenta. The two Ig domains adopt a fixed orientation that is stabilized by hydrogen bonds and hydrophobic contacts (Koch et al. 2010).

1.1.1 RAGE’s ligands and signaling cascades

RAGE ligands include advanced glycation end products (AGE), S100 proteins, amyloid-β and high mobility group box 1 protein (HMGB1) (Fritz 2011). The large group of Advanced Glycation End products (AGEs) derives from unspecific reactions between sugars and proteins or lipids, followed often by oxidation and cross-linking reactions. These molecules were first identified in the 1980’s and once termed the receptor binding these molecules. AGEs occur at elevated levels in Diabetes patients and are the main causative for diabetic complications such as accelerated atherosclerosis, kidney failure, or chronic inflammation. The second large group of RAGE ligands is formed by the so-called Danger Associated Molecular Pattern molecules (DAMPs) such as high mobility group box 1 (HMGB1) or the S100 proteins (Sparvero et al. 2009; Leclerc et al. 2009). All ligands reported so far are characterized by a negative surface charge that mediates most likely high-affinity binding to the positively charged VC1 domain of RAGE (Deane et al. 2003; Koch et al. 2010)

Activation of RAGE initiates intercellular signaling cascades activating finally the proinflammatory master regulator NF-κB (Y.-J. Chen et al. 2015). Several studies suggested that RAGE multimerization is essential for the induction of various signaling cascades (Yatime & Andersen 2013; Koch et al. 2010). Many components of these signal transduction pathways have been identified in the past years, which include mitogen-activated protein (MAP) kinases, phosphatidylinositol 3-kinase, Janus kinase and signal transducers and activators of transcription (Jak/STAT) (Hofmann et al. 1999), and the Rho GTPases Rac-1 and Cdc42 (Hudson et al. 2008) (Figure 4). Despite the detailed knowledge available on the signal pathway, the initiation of this cascade is still poorly understood at a molecular level.

The nature and the strength of signaling by RAGE are dependent on the affinity between receptor and ligands on the lifetime of the formed receptor-ligand complexes. In the inflammatory response elevated RAGE expression cause a positive feedback resulting in sustained NF-κB activation. Thereby, a chronic pathophysiological state is established and the resulting inflammation is not resolved (Fritz 2011).

Several members of S100 protein family bind and activate RAGE. RAGE induces a multimerization of the receptor (Fritz 2011). This multimerization of RAGE might trigger the initiation of the signaling cascade. However, there is so far there no molecular or detailed structural information on a signal-competent RAGE-ligand complex available. Moreover, there is a lack of information on the structural properties of S100 proteins required for high-affinity RAGE interaction.

Figure 4: Activation of RAGE and signaling pathway.

Ligand binding induces receptor multimerization. Diaphanous-1 (Dia1) is binding to the cytoplasmic part of RAGE mediating the signal and initiating an intracellular signal cascade leading to the activation of NF-ĸB. NF-ĸB induces the expression of pro-inflammatory molecules and of RAGE itself (Kierdorf & Fritz 2013).

To date, it is still unclear, exactly how the different ligands activate the intracellularly signaling cascades and what are the common properties of these ligands? At the first glance, RAGE ligands do not share any similarities. However, common to all is a negative charge and a tendency to oligomerize (Fritz 2011).

Several studies have demonstrated that blocking RAGE signaling impairs the development of numerous pathologic states and therefore defines RAGE as an important therapeutic target.

1.1.2 Effect of glycosylation on RAGE ligand binding

RAGE ectodomain contains two putative N-glycosylation motifs N–X–S/T that have been noticed in the deduced amino acid sequence the first site is located in the amino terminus adjacent to the V-region-like domain, and the second site is located within the V domain. Both sites are modified by complex N-glycans(Osawa et al.

2007). Some studies reported that N-glycosylation of RAGE modulated ligand binding e.g. RAGE proteins that lacked glycosylation in the V domain showed 1000-fold higher affinity to glycolaldehyde-derived AGEs (Osawa et al. 2007; Yonekura et al. 2003) or the binding affinity of amphoterin (HMGB1) to RAGE was decreased (Srikrishna et al. 2002). These findings suggest that glycosylation on the V domain affects specificity or affinity to ligands. However, there is no evidence that carboxylated glycans present on RAGE have effects on binding of S100 proteins. One part of the present study was designed to examine whether the glycosylation state of RAGE might affect the affinity to different S100 proteins.

1.2 S100 Protein family

1.2.1 Biological function

S100 proteins are small and acidic proteins and constitute a family of 27 different members in human. S100 proteins occur only in vertebrates. Owing to their solubility in 100% saturated ammonium sulfate solutions the proteins were called S100 and were first identified by B.W. Moore in 1965 (Moore 1965).They operate a wide range of activities and have been studied meanwhile since more than five decades. They are involved in regulation of homeostasis, proliferation, differentiation, inflammation, apoptosis, migration, phosphorylation through interactions with a variety of target proteins. Several S100 proteins serve as damage-associated molecular pattern recognition factors (DAMPS) in the adaptive and innate immune systems (Donato et al. 2013; Donato 2001; Chen et al. 2014) and are known to be recruited to sites of inflammation.

The S100 proteins take action as intracellular regulators and as extracellular signaling proteins. Several S100 proteins act in a cytokine-like manner (Heizmann et al. 2002) by binding to cell surface receptors like RAGE (Leclerc et al. 2009), toll-like receptor 4 (TLR4) (Zhang et al. 2012) , G-protein-coupled receptors, scavenger receptors, or heparan sulfate proteoglycans and N-glycans (Donato et al. 2013) 14

Many of those are closely associated with human diseases, such as cancer (Chen et al. 2014; Salama et al. 2008), autoimmune Diabetes (Chen et al. 2004), or inflammatory disorders (Goyette & Geczy 2011). S100A9, S100A8, and S100A12 belong to a subgroup of S100 proteins which are called calgranulins and have been associated with acute/chronic inflammatory disorders (Foell et al. 2003; Goyette & Geczy 2011). Moreover, S100 proteins serve as markers in clinical diagnosis and represent possible targets for therapeutic intervention.

1.2.2 S100 proteins and metal Ion binding

Each S100 monomer consists of two helix-loop-helix motifs. The N-terminal or ‘pseudo EF-hand’ domain contains two helices (HI and HII) connected by one loop which represents the calcium binding site. The C-terminal domain consists like the N- terminal domain of two helices (HIII and HIV) forming a canonical EF-hand (Heizmann et al. 2002; Santamaria-Kisiel et al. 2006)(Figure 5). The first characterization the of metal ion binding properties of S100 proteins was performed around 30 years ago (Baudier et al. 1986; Physique 1982) who showed that S100B binds Ca2+ and Zn2+ with high affinity.

Besides Ca2+, many other S100 proteins bind Zn2+ and Cu2+. Ion binding is resulting in conformational rearrangements that lead to the exposure of hydrophobic patch that represents the target interactions site (Fritz & Heizmann 2006; Donato et al. 2013). Most S00 proteins form homodimers and in the dimer one helix of the C-terminal EF- hand and one helix of N-terminal EF-hand (HI and HIV) from both molecules interact and form a tight four-helix bundle. In this helix bundle any movements of the helices are restricted and allow only movements of the two other two helices (HII and HIII) (Sastry et al. 1998; Kilby et al. 1996) (Figure 6). Conformational changes in S100 proteins upon calcium binding were characterized by X-ray crystallography and NMR spectroscopy studies (Heizmann 2009; Donato 2001; Heizmann et al. 2002)

A B

Figure 5: Structural organization of EF-Hands.

A: The canonical EF-hand loaded with calcium ion. B: A pair of EF-hands from the N- terminal domain contains two EF hand with two calcium ions (Gifford et al. 2007). Helices are shown in green and Ca2+ as yellow spheres.

Figure 6: Conformational changes of S100 protein upon ca2+ binding and target binding.

A: Schematic drawing of a S100 protein dimer with subunits in yellow and blue color. Ca2+ binding induces conformational change that is required for binding to the target. B: Conformational rearrangements that occur upon calcium binding in S100B and expose the hydrophobic residues for target binding (Bresnick et al. 2015).

1.2.3 S100A9/A9 and S100A8/A9 history

S100A9 protein (also known as myeloid-related protein 14 , MRP 14, or calgranulin B) with 14 kDa molecular mass and is composed of 114 amino acids (Ryckman et al. 2003). Expression of S100A9 in macrophages was reported for the first time in 1987 by Odink et al. (Odink et al. 1987). S100A9 is involved in Ca2+- dependent regulation of several intracellular activities such as Ca2+ hemostasis, phosphorylation and differentiation (Itou et al. 2002). S100A9 is also classified as Damage-associated molecular patterns proteins (DAMPs) and is released by damaged or death cells triggering an inflammatory response via pattern recognition receptors (PRR) activation like TLR4 (Tsai et al. 2014). The crystal structure of S100A9/A9 was first described 2002 by Itou et al. at 2.1Å resolution (Itou et al. 2002). This study showed that each EF-hand binds to 1 calcium ion and calcium binding causes a large conformational change (Figure 8).

Figure 7: The cartoon representation of S100A9 dimer.

The two subunits are shown in different colors (red and blue) with yellow calcium ions close to loop regions (Itou et al. 2002).

S100A9 also exists as a heterodimer with S100A8 (Markowitz & Carson 2013). It was reported that S100A8/A9 has an important role during different steps of the 17

tumorigenic process such as cell growth, cell apoptosis, tumor microenvironment, cell differentiation and cell cycle (Chen et al. 2014) as well as in inflammatory disorders (Narumi et al. 2015) via activation of RAGE. Under inflammatory conditions S100A8/A9 is secreted by neutrophils and acts as a cytokine. Moreover, tumor and non-tumor cells in cancer tissues express S100A8/A9 to modulate tumor growth and metastasis. In different studies, it was shown that various signaling pathways in different pathological states were stimulated by S100A8/A9 via RAGE (Ang et al. 2010; Brautigam et al. 2016; Jones et al. 2010; Basso et al. 2013).

1.3 The interaction of the RAGE with S100 Proteins

Several S100 proteins are reported as RAGE’s ligands with the affinities in the range of nM to µM rage (Fritz 2011). Activation of RAGE by S100 proteins is largely dependent on the concentration of S100 proteins (Rani et al. 2014).

A number of studies have demonstrated that V and C1 domains together act as S100 binding site (Xie et al. 2008; Dattilo et al. 2007). However, most S100 proteins bind to V domain while C1 domain stabilizes the V domain (Penumutchu et al. 2014; Ding & Keller 2005).

Despite the fact that several members of the S100 protein family such as S100A12 (Xie et al. 2007), S10013 (Rani et al. 2014) and S100P (Penumutchu et al. 2014) have been shown to interact with RAGE , the structure and mechanism of S100A9 interacting with RAGE is still unclear (Wu et al. 2015). There are several studies showing that S100A9/A9 and S100A8/A9 increase the inflammatory response, and promote tumor growth via the interaction with RAGE. The prominent role in disease development makes these proteins an interesting target for therapeutic intervention (Wu et al. 2015; B. Chen et al. 2015; Narumi et al. 2015). It was shown that blocking the interaction of S100A9 with RAGE could inhibit its pro-inflammatory and tumorigenic activity in vitro and in vivo (Wu et al. 2015). In 2009, Björk et al characterized S100A9 binding to RAGE by surface plasmon resonance (SPR) The interaction of S100A9 with RAGE was strictly dependent on the presence of Ca2+ and

Zn2+ (Figure 8). The authors also showed that S100A9 serves as a ligand for quinoline-3-carboxamides, which have been identified previously as drugs in the treatment of autoimmune disease (Björk et al. 2009).

Figure 8: SPR sensograms of S100A9-RAGE and S100A8/A9-RAGE interaction.

SPR sensorgrams showing association and dissociation phase of S100A9 (upper) and S100A8/9 (lower) interaction with immobilized RAGE (Björk et al. 2009).

1.4 Quinoline-3-carboxamides (Q compounds)

Quinoline-3-carboxamides (Q-compounds) were first described in the 1980s.Among those linomide was the first lead molecule which was found to stimulate the immune response and inhibit the growth and metastasis of cancer cells in preclinical models. However, the following clinical trials were stopped because of unacceptable toxicity of linomide. Over the next years several Q-compounds were developed and tested (Isaacs 2010).

In 1995 Active Biotech Company in Sweden produced the second generation of Q- compounds. Among the various substances developed several of Q-compounds

have reached phase II clinical trials. For example, successful trials involved tasquinimod and laquinimod (Figure 9 and Figure 10), although its mechanisms have not been fully elucidated. Laquinimod (Figure 10) is currently in phase III clinical trial for relapsing-remitting multiple sclerosis (Andersen et al. 1996).

Noteworthy, a complete treatment of autoimmune diseases such as multiple sclerosis (MS), rheumatoid arthritis, and systemic lupus erythematosus is still not possible. Q- compounds might serve as a part of the therapy of these disorders.

Figure 9: Chemical structure of Q-compound ABR 215050.

(Tasquinimod) (Isaacs 2010).

Figure 10: Chemical structure of Quinoline-3-carboxamide ABR 21215062.

(Laquinimod) (Isaacs 2010).

I studied the binding of Q compounds to S100A9 that are able to block S100A9- RAGE interaction as shown in Figure 11.

Figure 11: Scheme of blockage of RAGE-S100A9 interaction by inhibitors.

Q-compounds bind to S100A9 and thereby block the binding of S100A9 to RAGE abrogating pro-inflammatory signaling.

1.5 Aim of this thesis

The calgranulin subgroup of the S100 protein family encompasses S100A9 homodimer, S100A8/A9 heterodimer and S100A12 homodimer. They are highly expressed in certain types of leukocytes and act extracellularly as proinflammatory molecules via the innate immune receptors RAGE and TLR-4. The interaction is dependent on calcium and zinc ions, however the molecular mechanism of RAGE activation by S100 has remained unknown so far. The aim of the thesis was to characterize the interaction between S100 proteins and RAGE to provide the thermodynamic and kinetic properties. With these parameters one can address

several key questions of RAGE activation by S100 proteins: (i) what is the affinity of different ligands to the receptor? (ii) What are the molecular properties of an active RAGE-ligand complex, i.e. what is the stoichiometry of RAGE and ligands in a complex, and what is the lifetime of RAGE-ligand complexes? (iii) What is the effect of zinc ions on the interaction and do post-translational modifications such as receptor glycosylation affect RAGE-S100 interaction? These questions should be addressed applying different methods suited to characterize the molecular interactions such as isothermal calorimetry, surface plasmon resonance and microscale thermophoresis.

S100A9 has been identified as a target of anti-inflammatory drugs derived from Q- compounds (Björk et al.). Recently, the first structure of S100A9 blocked by a Q- compound has been described (Giesler, 2015) revealing a deep pocket in S100A9 as receptor interaction site. However, it remained unknown whether well-established drugs of this compound family bind to the same pocket. Meanwhile, also a series of structurally different compounds have been developed which block RAGE-S100A9 interaction with high specificity. I aimed to describe the molecular basis of inhibition and the binding properties of these well-established drugs as well as of the new compounds by isothermal calorimetry and X-ray crystallography.

2 MATERIAL AND METHODS

2.1 MATERIAL

2.1.1 Chemicals

All standard chemicals used in this work were of analytical grade (p.a.) and obtained from the following companies: AppliChem (Darmstadt, Germany), Carl Roth (Karlsruhe, Germany), Fluka (Steinheim, Germany), Merck (Darmstadt, Germany), Riedel-de Haën (Seelze, Germany), Roche (Basel, Switzerland), Serva (Heidelberg, Germany) and Sigma-Aldrich (Deisenhofen, Germany) (Table 1).

Table 1: Name of chemicals and companies.

Name Company 2-Propanol AppliChem Acetic acid AppliChem Sulphuric acid AppliChem Acrylamide 4K solution 30% (37.5:1) AppliChem Acrylamide 2x crystallized Carl Roth Bisacrylamide 2x crystallized Carl Roth Agar AppliChem Ammonium acetate Carl Roth Ammonium chloride Carl Roth Ammonium sulfate Carl Roth Ammonium peroxidisulfate Merck BIS-TRIS Carl Roth Bis-Tris propane Sigma-Aldrich Boric acid Sigma-Aldrich

Bromophenol blue AppliChem BSA Sigma-Aldrich Calcium chloride dehydrated Merck CHES Carl Roth Citric acid monohydrate Merck Cobalt (II) chloride hexahydrate Sigma-Aldrich Coomassie Violet R 150 Merck Coomassie Brilliant Blau G-250 AppliChem Copper (II) chloride Merck D(+)-Glucose Riedel-de Haën DABCO Carl Roth Dimethylsulfoxide Merck Dithiothreitol AppliChem Ethanol, absolute Sigma-Aldrich Ferric chloride Sigma-Aldrich Formaldehyde - Solution 37 % AppliChem Glycerol AppliChem Glycine AppliChem Guanidine hydrochloride BioChemica AppliChem HEPES AppliChem Hydrochloric acid 37% Sigma-Aldrich Imidazole Sigma-Aldrich Isopropyl β-D-1-thiogalactopyranoside AppliChem Lactose AppliChem Lithium sulfate Fluka Methanol AppliChem Magnesiumacetate tetrahydrate Carl Roth Magnesium chloride hexahydrate Merck Magnesium sulfate hydrate Sigma-Aldrich Malic acid Sigma-Aldrich Manganese(II) chloride dihydrate Merck MES hydrate Sigma-Aldrich Mowiol 4-88 Carl Roth

Nickel(II) chloride hexahydrate AppliChem Paraformaldehyde Carl Roth PEG1000 Fluka PEG1500 Fluka PEG200 Fluka PEG2000 Fluka PEG3350 Sigma-Aldrich PEG400 Sigma-Aldrich PEG4000 Serva PEG5000 MME Fluka PEG550MME Fluka PEG600 Fluka PEG750MME Fluka PEG8000 Sigma-Aldrich PIPES Fluka Phenylmethylsulfonyl fluoride AppliChem Potassium chloride Merck Di-Potassium hydrogen phosphate Riedel-de Haën Potassium hydroxide Riedel-de Haën Sodium dodecyl sulfate Carl Roth Silver nitrate AppliChem Sodium acetate trihydrate Merck Sodium azid Merck Sodium cacodylate trihydrate Sigma Sodium carbonate Riedel-de Haën Sodium chloride Sigma-Aldrich Sodium citrate Serva Sodium thiosulfate-5-hydrate Riedel-de Haën Sodium hydrogen phosphate Sigma-Aldrich Sodium hydroxide Carl Roth Sodium molybdate dihydrate Sigma-Aldrich Sodium selenite pentahydrate Fluka Sodium sulfate Merck

Tetramethylethylenediamine AppliChem Tricine Sigma-Aldrich Triethylenglycol Sigma-Aldrich Tris Carl Roth Trizma Base Sigma-Aldrich Tween 20 Fluka Yeast Extract Carl Roth Zinc acetate dihydrate Fluka Zinc chloride Merck Zinc sulfate monohydrate Sigma-Aldrich

2.1.2 Q-Compounds

Quinoline-3-carboxamide compounds were provided by Active Biotech Company (Lund, Sweden) and stock solutions were prepared in 100% DMSO which were subsequently diluted in buffer for ITC measurements, co-crystallization and crystal soaking experiments. All stock solutions were stored at -20 °C. The following figures (Figure 12 to Figure 24) depict different Q-compounds which were used in this study.

Figure 12: Chemical structure of Quinoline-3-carboxamide ABR 215050.

Figure 13: Chemical structure of Quinoline-3-carboxamide ABR 239071.

Figure 14: Chemical structure of Quinoline-3-carboxamide ABR 239981.

Figure 15: Chemical structure of Quinoline-3-carboxamide ABR 239105.

Figure 16: Chemical structure of Quinoline-3-carboxamide ABR 239709.

Figure 17: Chemical structure of Quinoline-3-carboxamide ABR 240007.

Figure 18: Chemical structure of Quinoline-3-carboxamide ABR 249974.

Figure 19: Chemical structure of Quinoline-3-carboxamide ABR 240011.

Figure 20: Chemical structure of Quinoline-3-carboxamide ABR 239979.

Figure 21: Chemical structure of Quinoline-3-carboxamide ABR240010

Figure 22: Chemical structure of Quinoline-3-carboxamide ABR239397

Figure 23: Chemical structure of Quinoline-3-carboxamide ABR239338

Figure 24: Chemical structure of Quinoline-3-carboxamide ABR239508

2.1.3 Buffers, Media, Gels, and antibiotics

2.1.3.1 Medias

SOB per liter DYT-Media LB-Media 20 g/L Trypton 16 g/L Tryptone 10 g/L Tryptone 5 g/L Yeast extracts 10 g/L Yeast extract 5 g/L Yeast extract 10 mM NaCl 5 g/L NaCl 10 g/L NaCl 2,5 mM KCl 50 mM Na2HPO4 pH 7.4 Autoclaved Autoclaved 10 mM MgCl2 10mM MgSO4 Adjust pH 7.0;

SOC LB-Agar ZY 33

like SOB plus Glucose LB-Media + 15 g/L agar 10 g/L Tryptone 20 mM Glucose Autoclaved 5 g/l Yeast Extract Autoclaved

2.1.3.2 Buffers, solutions, and antibiotics

Metal Mix (TMM) [x1000] TB (Transformation Tetracyclin (Fluka) 50 mM FeCl3 Buffer) 100 ml 15 mg/mL in ethanol 20 mM CaCl2 10 mM Pipes Sterilize by filtering 10 mM MnCl2 15 mM CaCl2 through 0.2 µm 10 mM ZnSO4 250 mM KCl 2 mM CoCl2 add ca 80 ml H2O 2 mM CuCl2 Adjust pH to 6.7 with 5N 2 mM NiCl2 KOH 2 mM Na2MoO4 Add 55 mM MnCl2 2 mM Na2SeO3 Add 100 ml with H2O 2 mM H3BO3

Ampicillin Glucose [30%] IPTG 100 mg/mL in H2O 50 mg/mL in H2O 1 M in H2O Sterilize by filtering through Sterilize by filtering Sterilize by filtering 0.2 µm through 0.2 µm through 0.2 µm

Kanamycin [50 mg/ml] 50 mg/ml in ddH2O Sterilize by filtering through 0.2 µm

2.1.3.3 Buffers and gels for SDS-PAGE

SDS-PAGE running buffer Separating SDS gel Stacking SDS gel [5%] 25 mM Tris-HCl [15%] 5% polyacrylamide 190 mM Glycin 8% polyacrylamide [30%] [30%] 0.1% SDS 250 mM Tris-HCl pH 8,8 125 mM Tris-HCl pH 6,0 34

pH 8.3 0.1% SDS [10%] 0.1% SDS [10%] 0.1% APS [10%] 0.1% APS [10%] 1:2000 TEMED 1:1000 TEMED

Coomassie- quick Running Buffer SDS Schaegger separating stain+destain gel[1x] gel (10%) 5% Acetic acid 25 mM Tris/HCl pH 8.3 6 mL AB-3 stock solution 10% EtOH 190 mM Glycine 10 mL Schaegger PAGE 0.016% Coomassie brilliant 0.1% SDS gel buffer(10x) blue G-250 or 3 g Glycerol Coomassie Violet R 150 Add 30 mL H2O 150 µL APS 10% 15 µL TEMED

Schaegger stacking gel (4%) Laemmli [4x] AB-3 stock solution 1 mL AB-3 stock solution 250 mM Tris-HCl pH 6,8 48 g acrylamide 3 mL Schaegger PAGE gel 40% Glycerin 1.5 g bisacrylamide buffer (10x) 5% SDS Add 100 mL H2O Add 12 mL H2O 0.005% Bromophenol blue 90 µL APS 10% 9 µL TEMED

Schaegger PAGE anode Schaegger PAGE Schaegger PAGE gel buffer (10x) cathode buffer(10x) buffer (10x) 1 M Tris pH 8.9 1 M Tris pH 8.25 3 M Tris pH 8.45 0.225 M HCl 1 M Tricine 1 M HCl 1% SDS 0.3% SDS

2.1.3.4 Purification buffers for S100A9

100A9 SEC buffer S100A9 dialysis buffer S100A9 lysis buffer 30 mM Tris/HCl pH 8.0 30 mM Tris/HCl pH 8.0 30 mM Tris/HCl pH 8.0 150 mM NaCl 1 mM EDTA 1 mM EDTA 0.2 µm sterile filtrated 0.2 µm sterile filtrated 0.2 µm sterile filtrated 1 tablet protease inhibitor cocktail Spatula tip DNAseI 0.5 mM PMSF 0.5 mM MgCl2

S100A9 IEX buffer 2 S100A9 denaturing S100A9 IEX buffer 1 30 mM Tris/HCl pH 8.0 buffer 30 mM Tris/HCl pH 8.0 35

0.5 mM EDTA 6 M GdHCl 0.5 mM EDTA 1 mM NaCl 50 mMTris/HCl 0.2 µm sterile filtrated 0.2 µm sterile filtrated 150 mM NaCl 1 mM EDTA PMSF [0.2 M] pH 8,0 solved in isopropanol 0.2 µm sterile filtrated

2.1.3.5 Purification buffers for VC1243

VC1243 SEC buffer VC1243 dialysis buffer VC1243 digestion buffer 100 mM Na acetate 20 mM HEPES 20 mM HEPES 150 mM NaCl 400 mM NaCl 300 mM NaCl pH 5,2 pH 6,5 pH 8,0 0.2 µm sterile filtrated 0.2 µm sterile filtrated 0.2 µm sterile filtrated

VC1243 lysis buffer VC1243 IMAC VC1243 IMAC elution 20 mM HEPES pH 7.4 equilibration buffer buffer 300 mM NaCl 20 mM HEPES 20 mM HEPES 0.2 µm sterile filtrated 300 mM NaCl 300 mM NaCl 30 mM Imidazole 500 mM Imidazole pH 7,4 pH 7,4 0.2 µm sterile filtrated 0.2 µm sterile filtrated

2.1.3.6 Purification buffers for sRAGE

sRAGE SEC buffer sRAGE dialysis buffer sRAGE digestion 10 mM HEPES 20 mM Tris/HCl pH 8.0 buffer 300 mM NaCl 300 mM NaCl 20 mM Tris/HCl pH 8.0 36

pH 7,5 pH 8 300 mM NaCl 0.2 µm sterile filtrated 0.2 µm sterile filtrated pH 8 1 mM CaCl2 0.2 µm sterile filtrated sRAGE lysis buffer sRAGE IMAC sRAGE IMAC elution 50 mM NaK phosphate pH 7.4 equilibration buffer buffer 0.2 µm sterile filtrated 50 mM NaK phosphate 50 mM NaK phosphate 300 mM NaCl 300 mM NaCl 30 mM Imidazole 500 mM Imidazole pH 7,4 pH 7,4 0.2 µm sterile filtrated 0.2 µm sterile filtrated

2.1.3.7 ITC, MST and SPR buffers

ITC buffer MST buffer Regeneration solution 50 mM Tris pH 7, 5 50 mM HEPES pH 7, 5 for NTA sensor chip 100 mM NaCl 100 mM NaCl 350 mM EDTA in Water 2 mM CaCl2 2 mM CaCl2 pH~8.3 5 mM MgCl2 5 mM MgCl2 Add 20 µM ZnCl2 for diluting Add 20 µM ZnCl2 for S100A9 diluting S100A9

Nickel solution for NTA Wash solution for NTA SPR His VC1243 sensor chip sensor chip Immobilization 0.5 mM NiCl2 350 mM EDTA in Water solution 20 mM NaAcetate 150 mM NaCl Tween 0.005% pH~ 5.2 SPR Runinng buffer for S100 proteins 20 mM Tris 150 mM NaCl 2 mMCaCl2 37

0.005% Tween and 1mg/ml BSA pH~7.6

2.1.4 Kits and SPR sensor chips

Name company

Monolith NT.115 protein labeling kit Blue-NHS (Amine Reactive) NanoTemper Monolith NT115TM series capillaries Hydrophilic NanoTemper Zyppy™ Plasmid Miniprep Kit Zymmo SPR NTA sensor chip BIACORE/GE 2.1.5 Enzymes

Name company DNAseI I Sigma Thrombin from bovine plasma Serva

2.1.6 Chromatography Columns

Name company Source 15Q resin GE Healthcare Superdex 75 16/60 GE Healthcare Superdex 75 26/60 GE Healthcare Ni-Sepharose High Performance GE Healthcare

2.1.7 Proteins and sequences

2.1.7.1 S100A9

Recombinant human S100A9 protein with a C3S mutation and expressed in Escherichia coli BL21 (DE3). Cysteine residues on the protein surface are prone to oxidation and can form artificial disulfide bonds. Therefore, the cysteine at position 3 (primary amino acid sequence) was mutated into serine. It is known that this mutation does not have any influence on function or structure of S100A9.

S100A9-C3S in composed of114 amino acids and has molecular mass of 13242 Da (NCBI: NM_002965, pdb: P06702).

MTSKMSQLERNIETIINTFHQYSVKLGHPDTLNQGEFKELVRKDLQNFLKKENKNEK VIEHIMEDLDTNADKQLSFEEFIMLMARLTWASHEKMHEGDEGPGHHHKPGLGEGT P

2.1.7.2 S100A8/A9

Recombinant human S100A8 protein with mutation C42S was expressed purified as an S100A8/A9 heterodimer and kindly provided by Sven Zapf and Dr. Stephan Giesler (working group Fritz).

S100A8-C42S contains 93 amino acids and has a molecular mass of10575 Da (NCBI: NM_002964, pdb: P05109).

MLTELEKALNSIIDVYHKYSLIKGNFHAVYRDDLKKLLETESPQYIRKKGADVWFKEL DI NTDGAVNFQE FLILVIKMGV AAHKKSHEES HKE

2.1.7.3 S100A12

Recombinant human S100A12 was kindly provided by PD Dr. Günter Fritz.

S100A12 in composed of 92 amino acids and has a molecular mass of 10835 Da (NCBI: NM_005621, pdb: P80511).

MTKLEEHLEGIVNIFHQYSVRKGHFDTLSKGELKQLLTKELANTIKNIKDKAVIDEIFQ GLDANQDEQVD FQEFISLVAI ALKAAHYHTH KE

2.1.7.4 S100B

S100B is composed of 92 amino acids and has molecular mass of 10713Da (NCBI: NM_006272, pdb: P04271).

MSELEKAMVALIDVFHQYSGREGDKHKLKKSELKELINNELSHFLEEIKEQEVVDKV METLDNDGDGECDFQEFMAFVAMVTTACHEFFEHE

2.1.7.5 VC1243

The VC1243 construct contains the cDNA encoding residues 23-243 of human RAGE. It comprises Ig domains V and C1 (Dattilo et al. 2007) and a short linker region which 40

normally connects VC1 to the C2 domain. The cDNA was cloned into a pET-15b vector which contains an N-terminal His-tag and a thrombin cleavage site (Dattilo et al. 2007).

Protein sequence of VC1243 after thrombin cleavage is composed of 221 amino acids and has molecular mass of 24456Da (pdb: Q15109)

GSHMAQNITARIGEPLVLKCKGAPKKPPQRLEWKLNTGRTEAWKVLSPQGGGPWD SVARV LPNGSLFLPA VGIQDEGIFR CQAMNRNGKE TKSNYRVRVY QIPGKPEIVD SASELTAGVPNKVGTCVSEGSYPAGTLSWHLDGKPLVPNEKGVSVKEQTRRHPET GLFTLQSELMVTPARGGDPRPTFSCSFSPGLPRHRALRTAPIQRVWEPVPLEEVQL VVE

The residues highlighted in red indicate V domain, the residues in gray C1 domain and in yellow C2 domain.

2.1.7.6 sRAGE

Recombinant sRAGE protein encompasses residues 23-326 of human RAGE and has a molecular mass of 35050.9 kDa.

Sequence of sRAGE with N-terminal His-tag:

MGSSHHHHHHSSGLVPRGSHMAQNITARIGEPLVLKCKGAPKKPPQRLEWKLNTG RTEAWKVLSPQGGGPWDSVARVLPNGSLFLPAVGIQDEGIFRCQAMNRNGKETKS NYRVRVYQIPGKPEIVDSASELTAGVPNKVGTCVSEGSYPAGTLSWHLDGKPLVPN EKGVSVKEQTRRHPETGLFTLQSELMVTPARGGDPRPTFSCSFSPGLPRHRALRT APIQPRVWEPVPLEEVQLVVEPEGGAVAPGGTVTLTCEVPAQPSPQIHWMKDGVP LPLPPSPVLILPEIGPQDQGTYSCVATHSSHGPQESRAVSISIIEPGEEG

Sequence after His-tag removal by thrombin cleavage:

GSHMAQNITARIGEPLVLKCKGAPKKPPQRLEWKLNTGRTEAWKVLSPQGGGPWD SVARVLPNGSLFLPAVGIQDEGIFRCQAMNRNGKETKSNYRVRVYQIPGKPEIVDSA 41

SELTAGVPNKVGTCVSEGSYPAGTLSWHLDGKPLVPNEKGVSVKEQTRRHPETGL FTLQSELMVTPARGGDPRPTFSCSFSPGLPRHRALRTAPIQPRVWEPVPLEEVQLV VEPEGGAVAPGGTVTLTCEVPAQPSPQIHWMKDGVPLPLPPSPVLILPEIGPQDQG TYSCVATHSSHGPQESRAVSISIIEPGEEG

2.1.8 Bacterial strains

S100 proteins were expressed in E.coli BL21 (DE3). For expression of recombinant human His6-VC243 E. coli Origami B (DE3) was used. E. coli Rosettagami B (DE3) strain (Novagen, Darmstadt, Germany) was chosen for expression of sRAGE.

2.1.9 Crystallization screens

Initial crystallization screens were performed on 96well INTELLI-PLATES® with commercially available screens. All crystallization experiments were performed at 20°C. Initial screens were performed by Anita Arnold and Dr. Stephan Giesler (Workgroup Dr. Fritz) using a Phoenix dispenser (Art Robbins Instruments) in the laboratory of Prof. Dr. Einsle.

Five screens listed in Table 2 were used to identify initial crystallization conditions for VC1 in complex with S100A9. To enable the complex formation the molar ratio of RAGE and S100A9 was always 1:1. The final protein concentrations for the initial screenings were 2.7 mg/ml for VC1 and 2.9 mg/ml for S100A9. The protein solutions for co-crystallization in all screens were supplied with a final concentration of 10 mM

CaCl2 and two-fold molar amount of ZnCl2 per homodimeric S100A9. In order to increase protein stability and solubility the protein solutions containing VC1 with S100 proteins were supplemented with 150 mM NaCl. Moreover, to improve the crystallization conditions individual screening with small variations of pH, salt or PEG concentration were prepared.

Table 2: Name and source of Initial screens.

Index Screen Hampton Research PEGs Grid I and II Workgroup Prof.Dr. Einsle Morpheus Screen MRC Laboratory of Molecular Biology Footprint I-IV Screen Workgroup Prof.Dr. Einsle MemGold Molecular Dimension

2.2 METHODS

2.2.1 Microbiological methods

2.2.1.1 Preparation of chemically competent E.coli cells

Chemically competent cells of a desired E. coli strain were prepared according to the method developed by Inoue, Nojima, & Okayama, 1990. For the preparation of the competent cells each strain was streaked on LB agar plates from glycerol stocks. The plates were supplemented with the respective antibiotics for the specific strain. 4 ml LB medium was inoculated with a single colony and incubated it overnight shaking at 37°C. The preculture was used to inoculate the main culture. The main culture was grown in 250 ml SOB media with one respective antibiotic at the temperature 18°C. At an optical density of 0.6 the flask was put on ice for 10 minutes shaking gently to cool the culture. Cells were harvested by Centrifugation for 10 min at 2500 g and 4 °C. All following steps were performed on ice. The pellet was gently resuspended in 20 ml ice-cold TB buffer and incubated on ice for 10 min. The cells were again 43

centrifuged (as above), and the pellet was resuspended in 10 ml of TB buffer. 7% DMSO was added to the cell suspension and the cells were incubated on the ice for further 10 minutes. The cell suspension was aliquoted in 200 to 500 µl per eppi and each aliquot was immediately frozen in liquid nitrogen. Competent cells were stored at -80 °C.

2.2.1.2 Transformation of competent cells

For the transformation of competent expression strains E.coli Origami B (DE3) and E.coli Rosettagami B (DE3) the RAGE constructs were used. A standard transformation was performed as described below. The competent cells were thawed on ice for 5-10 min and gently mixed. Between 50 ng and 200 ng of plasmid DNA in a volume of 1-5 µl was added to the cells. The solution was inverted 4-5 times and incubated for 30 min on ice to allow the attachment of plasmid DNA at the bacteria cells. Afterward a heat shock was performed for 90 sec at 42°C to trigger the plasmid DNA uptake into the bacterial cells. Then incubation on ice for 5 min followed immediately. SOB media supplied with 20 mM glucose was added and mixed gently. The cells were incubated at 37°C for 45 to 60 min with 250 rpm rotation and streaked on LB agar plates supplied with the selective antibiotic

2.2.1.3 Recombinant protein expression

E.coli strain BL21 (DE3) was used for protein expression of the S100A9-C3S protein and E.coli Origami B (DE3) and E.coli Rosettagami B (DE3) were used for expression of human RAGE constructs

2.2.1.4 His6-VC1243 protein expression in E.coli Origami B (DE3)

The glycerol stock E.coli Origami B (DE3) transformed with pET-15b His6-VC1243 vector construct were streaked on LB agar plates supplied with 100 µg/ml ampicillin and 0.2% glucose and grown overnight at 37°C. A preculture with DYT, 100 µg/ml ampicillin, and0.2% glucose was prepared and a single colony of E.coli Origami B

(DE3) transformed with pET-15b His6-VC1243 vector was added. After overnight growth at 37°C in shaking culture (150 rpm), the preculture was centrifuged for 5 min at 5000 rpm at 4°C and the pellet was re-suspended in the same media conditions. The main culture containing DYT supplied with 100 µg/ml ampicillin, 0.2% glucose, and 0.2 x TMM was prepared and inoculated 1:100 with the resuspended preculture. The TMM was added to improve the bacterial growth. The bacterial culture was grown at 37°C until the OD600 of 0.8 was reached. Then the expression of His6-

VC1243 was started by adding 0.5 mM IPTG and the culture was further grown overnight at 23°C. Next the main culture was centrifuged at 8000 x g for 15 min at 4°C. The pellet was immediately frozen in liquid nitrogen and stored at -80°C.

2.2.1.5 sRAGE expression in E.coli Rosettagami B (DE3)

The glycerol stock E.coli Rosettagami B (DE3) transformed with pET-15b His6- sRAGE vector construct were streaked on LB agar plates supplied with 100 µg/ml ampicillin and 0.2% glucose and grown overnight at 37°C. A preculture with DYT, 100 µg/ml ampicillin, and 0.2% glucose was prepared and a single colony of E.coli

Rosettagami B (DE3) transformed with pET-15b His6-sRAGE vectors was added. After overnight growth at 37°C in shaking culture (150 rpm), the preculture was centrifuged for 5 min at 5000 rpm at 4°C and the pellet was resuspended in the same media conditions. Expression was performed in shaking cultures in DYT medium supplemented with appropriate antibiotics, 100 µg/ml ampicillin, 0.2% glucose, and 0.2x TMM to improve the bacterial growth. For inoculation of expression cultures, the overnight culture was diluted 1:100 in the same medium. The culture was grown at an OD600 nm of 0.8 was reached and expression was induced by addition of IPTG (Applichem) to a final concentration 0.5 mM. The culture was further grown overnight at 23°C. Cells from expression cultures were harvested by centrifugation at 8000 x g for 15 min at 4°C. The pellet was immediately frozen in liquid nitrogen and stored at - 80°C.

2.2.1.6 S100 A9-C3S expression in E.coli BL21 (DE3)

E.coli BL21 (DE3) transformed with pGEMEX-2-S100A9-C3S was streaked out on LB agar plates from glycerol stocks.LB plates were supplied with 100 µg/ml ampicillin and 0.2% glucose and incubated at 37°C overnight. A single colony was used to inoculate a 50mL DYT preculture supplemented with 100 µg/ml ampicillin, and 0.2% glucose. After overnight growth at 37°C in shaking culture (150 rpm), the preculture was centrifuged for 5 min at 5000 rpm at RT and the pellet was resuspended in the fresh media. The main culture containing DYT supplemented with 100 µg/ml ampicillin, 0.2% glucose and 0.2x TMM (trace metal mix) was prepared and inoculated 1:200 with the resuspended preculture. The bacterial suspension was grown at 37°C until an OD600 of 0.8 was reached. Then the expression of S100A9 was started by adding 0.5 mM IPTG and the culture was further grown for 4-5 h while expressing S100A9. The expression culture was centrifuged at 8000 x g for 15 min at 4°C. The pellet was immediately frozen in liquid nitrogen and stored at -80°C until further use.

2.2.1.7 Expression of recombinant glycosylated VC1

VC1 of human RAGE were kindly produced in the methylotrophic yeast Pichia pastoris by Dr. Genny Degani at the university of Milano. This chapter is taken from her recent publication (Degani et al. 2015). This microorganism (Pichia pastoris) has an efficient apparatus of protein glycosylation, folding, and secretion that may be beneficial for the expression of naturally secreted glycoproteins. In addition, glycosylation, together with the formation of disulfide bonds, confers stability to proteins and also improves their solubility.

Recombinant plasmids for integrative recombination in P. pastoris were obtained by cloning XhoI-digested PCR fragments into the XhoI site of pHIL-S1 (Invitrogen), to generate in-frame fusions with the secretion signal of P. pastoris PHO1, encoding an extracellular acid phosphatase. PCR was carried out using as a template pET-15b-

VC1243. To induce the expression of the recombinant proteins, the positive clones were grown overnight at 28°C in 10 mL of glycerol-containing medium (BMGY or MGY) under strong agitation and at a ratio between volume of culture and capacity of the flask of 1:10. Then, appropriate amount of cells were collected and transferred to methanol-containing medium (BMMY or MMY) in order to obtain an initial OD600 of 1.

Growth was monitored by increase of OD600. Fresh methanol was added daily to 0.5% (v/v) final concentration. To monitor protein expression, supernatants from 1 mL-aliquots of culture, withdrawn at intervals after induction, were obtained by centrifugation, flash-frozen and stored at 20° C until analysis by SDS–PAGE.

2.2.2 Biochemical and biophysical methods

2.2.2.1 Purification of His6-VC1243 and His6-sRAGE

The cell pellet was resuspended in lysis buffer (20 mM HEPES, 300 mM NaCl, pH 7.4) with approximately 2 to 3 ml buffer per gram wet weight cells. One tablet of complete protease inhibitor cocktail (EDTA-free Roche Applied Science), a little bit

DNase I, 0.5 mM MgCl2 and 0.5 mM PMSF were added to the buffer. Cell breakage was performed by Microfluidizer treatment. The pressure was set to 1000 bar and treatment was repeated two to three times. The crude extract was ultra-centrifuged at 100 000 x g for 60 min at 4°C to remove insoluble components. The supernatant was finally filtered through 0.22 µm and diluted 4 to 5 fold in the equilibration buffer. Before loading the supernatant to column one more tablet of complete protease inhibitor cocktail was dissolved in the supernatant. The first Ni-Sepharose chromatography was performed at room temperature (RT) with the ÄKTA™ Purifier System (GE Healthcare). The supernatant was loaded on Ni-Sepharose column equilibrated with 20 mM HEPES, 300 mM NaCl and 30 mM Imidazole, pH 7.4. The column was washed with the same buffer until OD280 reached approximately baseline level. Subsequently, bound protein was eluted with 20 mM HEPES, 300 mM NaCl, 500 mM Imidazole, pH 7.4 and protein was collected in 3 ml fractions. Protein purity in the fractions was checked by SDS-PAGE. Fractions containing most pure protein were combined. The high imidazole content was reduced by dialysis. A dialysis tube (Spectrum Laboratories) with a molecular weight cut-off (MWCO) of 6000-8000 Da was used. 5% glycerol was added to the combined fractions to stabilized protein. Afterwards, the protein solution was dialyzed overnight against dialysis buffer (20 mM NaAcetate pH 5.2, 150 mM NaCl) at room temperature with gentle stirring. For SPR experiments His6-VC1243 was prepared. For other experiments the N-terminal His6- tag was removed by thrombin cleavage. Thrombin cleaves specifically between added thrombin recognition site Leu, Val, Arg, Gly, and Ser. Prior cleavage 2 mM

CaCl2 was and cleavage was started by adding 2 to 3 units thrombin. After three hours the digestion process was checked by SDS-PAGE. Aggregates and precipitated protein was removal by centrifugation at 20,000 g for 10 min. 5% glycerol was added and protein was concentrated by ultrafiltration 5000 rpm at 4°C with a 10 kDa MWCO filtration device (PALL Corporation) until a volume suited for size exclusion chromatography was reached. The protein solution was stored on ice until it was applied on size exclusion chromatography. During concentration, the flow through and the supernatant were analyzed by UV-Vis measurement. In size exclusion chromatography molecules are separated based on their size. The

HiloadTM 26/600 superdex 75 (column volume 318 ml, GE Healthcare) was used. The matrix of the column consists of dextran and highly crosslinked agarose. The selectivity of dextran and the high stability of agarose allows a well-defined separation of molecules with a size between 3000 and 70 000 Da. The used buffers are listed in chapter 2.1.3.5 and 2.1.3.6 and the chromatography was performed with an ÄKTA™ Purifier System (GE Healthcare). The column was equilibrated with 2 CVs of SEC buffer (10 mM Na acetate pH 5.2, 150 mM NaCl), and the protein solution was 0.22 µm filtered before application via the injection valve to the column. This step was necessary since during concentration process a small amount of protein was precipitated or aggregates had formed. Proteins were eluted with 1.2 CVs of SEC buffer with a flow of 2.5 ml/min. The fractions were analyzed by SDS-

PAGE and the fractions containing the His6-VC1243 or sRAGE, respectively, were combined. Next the proteins were concentrated at 5000 rpm at 4°C by a 10 kDa MW cut-off filtration tube (PALL Corporation) until the desired protein concentration was reached. The flow through and the supernatant were analyzed during the process by

UV-Vis measurement. Finally, the protein solutions of His6-VC1243 and sRAGE were aliquoted, frozen in liquid nitrogen and stored at -80°C.

2.2.2.2 Purification of S100A9-C3S

The harvesting and lysis of S100A9 expressing E.coli BL21 (DE3) cells was performed on ice or at 4°C. The used buffers are listed in 0. Per 1 g of wet weight cells 2 ml of ice-cold lysis buffer was added and the cells were resuspended using a homogenizer. Cell breakage was performed by Microfluidizer treatment. The pressure was set to 1000 bar and treatment was repeated two to three times. Next the suspension was ultra-centrifuged at 100 000 x g for 1 h at 4°C for separate soluble cytoplasmic proteins from cell debris, membranes and inclusion bodies which contained the expressed S100A9 protein. The pellet was resuspended in ice-cold denaturing buffer to solubilize the inclusion bodies of S100A9. Then the solution was ultra-centrifuged at 20 000 x g for 40 min at 4°C to remove insoluble components. The supernatant, containing the S100A9 protein, was filled into a dialysis tubes (Spectrum Laboratories) with a molecular weight cut-off (MWCO) of 6000-8000 Da and dialyzed for 2 h at 4°C in dialysis buffer. After 2 h the dialysis buffer was

exchanged for another 16 h dialysis. Next the dialysate was ultra-centrifuged at 100000 x g for 1 h at 4°C to remove precipitated protein. The supernatant was finally filtered through 0.22 µm. Ion exchange chromatography is well suited for separation of hydrophilic or charged molecules. Because S100A9 is a negatively charged protein the anion exchange chromatography SOURCETM 15Q with a column volume (CV) of 10 ml from GE healthcare was used. The matrix consists of polystyrene/divinyl benzene beads of 15 µm diameter. The beads are positively charged due to the quaternary ammonium cation and can therefore bind the negatively charged S100A9.

All used buffers are mentioned in chapter 2.1.3.4 and the chromatography was performed at room temperature (RT) with the ÄKTA™ Purifier System (GE Healthcare). The first step was to equilibrate the column with 1.5 column volumes (CVs) IEX buffer 1 (30 mM Tris/HCl pH 8.0, 0.5 mM EDTA) and the protein solution was applied to a slow flow of 2.5 ml/min to the column to ensure binding of complete

S100A9 loading. Next, the column was washed with IEX buffer 1 until the OD280 nm reached the baseline level. Bound proteins were eluted in a linear gradient of IEX buffer 2 (30 mM Tris/HCl pH 8.0, 0.5 mM EDTA, 1 M NaCl) to 60%. The negative charged chloride ions of IEX buffer 2 displace the bound proteins from the beads. The collected fractions were analyzed by SDS-PAGE. Fractions containing S100A9 were combined and the protein was concentrated by ultrafiltration using a 3 kDa MW cut-off centrifugal filter (PALL Corporation) with 5000 rpm at 4°C until a volume suited for application to size exclusion chromatography was reached. The protein concentration of the flow through and the supernatant was analyzed during and after the concentration process by UV-Vis measurement. The protein solution was stored on ice until it was applied to size exclusion chromatography. The HiloadTM 26/600 superdex 75 (column volume 318 ml, GE Healthcare) was used. The concentrated protein solution of S10A9 from IEX was applied to the column via the injection valve. Protein was eluted with 1.2 CVs of SEC buffer at a flow of 2.5 ml/min. The eluted fractions were analyzed by SDS-PAGE. Fractions containing S100A9 were combined and concentrated by ultrafiltration using a 3 kDa MW cut-off centrifugal filter (PALL Corporation) at 5000 rpm at 4°C. Finally the protein solution of S100A9 was aliquoted, froze in liquid nitrogen and stored at -80°C.

2.2.2.3 Purification of recombinant glycosylated VC1

This purification was kindly performed by Dr. Genny Degani at the university of Milano. This chapter is taken from her recent publication (Degani et al. 2015). For protein small-scale purification, 80 mL-culture of induced P. pastoris cells was centrifuged at 4000 g for 10 min and the supernatant was filtered through cellulose nitrate filters (1.2 lm pore size, Sartorius) to remove residual cells and debris. After dialysis (Spectra/Por membranes, cut-off 6000–8000) at 4°C for 16 h against 10 mM Na-acetate pH 5.0, the sample was applied to a cation-exchange RESOURCE S column (1.6 15 cm; GE Healthcare) connected to an ÄKTA-FPLC system (GE Healthcare) and equilibrated with 10 mM Na-acetate pH 5.0. The column was washed with the same buffer until the A280 nm reached the baseline. The protein was then eluted with 10 mM Na-acetate pH 5.0 containing 1 M NaCl. The fractions containing the recombinant protein, as judged by SDS–PAGE, were combined and applied to a Superdex 75 (10/30) gel filtration column (GE Healthcare) equilibrated with 10 mM Na-acetate, pH 5.0, 1 M NaCl. Protein in the fractions was precipitated by trichloroacetic acid and analyzed by SDS–PAGE. To estimate the native molecular mass of VC1, the column was calibrated with standard proteins under the same conditions (BSA dimer, 132 kDa; BSA monomer, 66 kDa; carbonic anhydrase, 29 kDa; cytochrome c, 12.3 kDa). For a large-scale purification, the supernatant from a 400 mL-culture was obtained as described above. After concentration to 30 mL by ultrafiltration using a 30,000 cut-off membrane and dialysis against 10 mM Na- acetate, pH 5.0, for 16 h at 4°C, the solution was applied to a cation-exchange Mono S HR5/5 column (GE Healthcare) equilibrated with 10 mM Na-acetate, pH 5.0 and connected to an ÄKTA-FPLC system (GE Healthcare). After column washing with the same buffer, elution was performed with a 0– 1 M NaCl gradient in the same buffer. Fractions were analyzed by SDS–PAGE.

2.2.2.4 Isothermal titration calorimetry (ITC)

Isothermal titration calorimetry (ITC) is a widely used technique to study biological processes at the molecular level, especially to characterize binding affinities of ligands to a protein. Thereby, ITC measures small changes of temperature during the reaction and allows the simultaneous determination of thermodynamic parameters such as heat of binding (enthalpy; ∆H), number of binding sites (n) or the calculation of the binding constant K (Santos et al. 2007). ITC experiments and the instrumental set-up (Figure 25) are described below. The solution in the syringe containing the test compound was titrated into the sample cell (25 °C) with the protein solution. Every injection causes a change in heat, resulting in a change in temperature in the sample cell in comparison to the reference cell. The change in power, which was required to return the sample cell to 25 °C, was recorded with every injection until all binding sites of the protein in the sample cell were saturated (heat signal diminishes).

Figure 25 : Representative diagram of typical ITC experiment.

An ITC instrument consists of two identical cells surrounded by an adiabatic jacket. Reference heater (green), cell feedback heater (blue) detect temperature differences between the reference and the sample cell upon ligand (Q-compound) titration into the sample cell containing the protein solution (S100A9).

2.2.2.4.1 ITC titrations of S100A9 with Q-compounds

ITC experiments were carried out at the institute of Biochemistry (Laboratory of Prof. Dr. Oliver Einsle) using a MicroCal VP-ITC calorimeter. All samples were centrifuged and degassed for 15 min to remove air bubbles prior to the titration experiment. The titration curves were corrected using buffer controls and analyzed using Origin software (MicroCal), NITPIC and SEDPHAT.

For the preparation of protein samples with a concentration of 20µ, S100A9 was diluted in 20 mM Tris- HCl (pH 7.6), 100 mM NaCl, 2 mM CaCl2, 4 mM MgCl2 and 20

µM ZnCl2.S100A9 was injected with a glass syringe into the cell just before the run. The samples containing 300 µM Q-compounds were diluted in the same buffer but without ZnCl2 and transformed into the MS syringe. All titrations were performed at 25°C, and an injection volume of 6-13 µL was used for all subsequent titrations, with a 120 s initial equilibration delay and 120-270 s delay in the intervals between the injections.

2.2.2.5 Micro scale thermophoresis (MST)

In this method, the physical effect referred to as thermophoresis, which describes the directed movement of molecules through temperature gradients, is exploited to analyse molecular binding reactions. The thermophoretic properties of a molecule depend on its size, charge, and hydration shell.

Microscale thermophoresis analyses were carried out with a Monolith NT.115 instrument (NanoTemper, Munich, Germany). Monolith NT.115 hydrophilic capillaries (Monolith NT.115) were loaded with the samples. The surface of the capillaries is covalently coated with a dense brush of a hydrophobic polymer and all measurements were performed in duplicate. Data evaluation was performed with the Monolith software (Nano Temper, Munich, Germany). MST experiments were carried out at The Max Planck Institute of Immunobiology and Epigenetics (MPI-IE) in 53

Freiburg (Laboratory of Dr. Andrea Pichler) (Figure 26). In MST, the binding of the tested molecule to an interacting partner influences the thermal migration behavior of the fluorescently labeled molecule. The subsequent fluorescence depletion in a heated spot of the protein solution is measured as a function of increasing interacting partner concentration, with Kd values being derived from the depletion curves

Figure 26: Overview of MicroScale Thermophoresis (MST).

A: Schematic overview how to prepare the MST-Experiment. B: schematic overview how to load samples into vials and in the slots. (The pictures were adapted from User Manual for the

Monolith NT.115). C: MST of VC1243 and S100A9. Fluorescently labeled VC1 are initially distributed evenly and diffuse freely in solution. By switching on the heating with a focused IR-Laser, the molecules experience the thermophoretic force in the temperature gradient and move out of the heated spot. In the steady state, this movement is counterbalanced by ordinary mass diffusion. After turning off the laser, the particles diffuse back towards a homogeneous distribution.

2.2.2.5.1 MST of VC1243 and S100A9

Protein interaction studies using microscale thermophoresis were performed according to Duhr and Braun(Duhr & Braun 2006). VC1243 was labeled according to the protocol by NanoTemper using the Monolith™ NT.115 Protein Labeling Kit Blue- NHS (Amine Reactive). Experiments were performed using Hydrophilic capillaries in the NanoTemper Monolith™ NT instrument for blue dye fluorescence in buffer [50 mM HEPES pH 7.5, 100 mM NaCl 2 mM CaCl2, 5 mM MgCl2]. The samples of 20 µM

VC1 were diluted in the same buffer but without ZnCl2. The buffer was exchanged to the labeling buffer which was provided by NanoTemper Company. Running buffer of these experiments was also the same but without ZnCl2 and with 1 mg/ml BSA and 0.005% Tween. VC1 was mixed in a 1:1 ratio with Blue-NHS Hydrophilic dye (Monolith NT™ Protein Labeling Kit BLUE-NHS (Amine Reactive)) which was purchased from the NanoTemper Company. After 30 minutes incubation time, for optimal results unreacted free dye was eliminated by NAPs column, then serial dilution of labeled VC1 was prepared as shown in the Figure 26 A. For sample loading the hydrophobic glass capillaries were used, and capillaries were put in the slots on the sample tray. The highest concentration of S100 A9 (first vial) is placed in the front of the tray (Figure 26 B). The titration curves were analyzed using NT Control Software.

2.2.2.6 Surface plasmon resonance (SPR)

SPR is a label-free technology which utilizes the properties of certain metals (e.g., gold) that can generate plasmons. In defined conditions, the photons of an incident light will generate a resonance phenomenon in the plasmons of the metal, resulting in surface plasmon resonance and the generation of evanescent waves by the metal. The sensor chips are constituted by a piece of glass covered by a thin layer of gold which is modified with diverse chemical groups. The matrix of NTA chip is carboxymethylated dextran pre-immobilized with nitrilotriacetic acid (NTA). Histidine-

tagged molecules are immobilized via Ni2+/NTA chelation. SPR experiments were carried out at the institute of Pharmacology and Toxicology (Laboratory of Prof. Dr. Klaus Aktories).

2.2.2.6.1 SPR for VC1243 and S100 proteins

All buffer solutions were prepared with Milli-Q water. After preparation, the solutions were filtered and degassed prior use. The following figures (Figure 27 and Figure 28) represent the mechanism of SPR in the presence of immobilized ligand His6-VC1243 and its binding partners, which are usually called analytes. Glycosylated His-VC1243 was also immobilized on SPR chip to examine effect of glycosylation on VC1 ligand interactions. Glycosylated His-VC1243 was expressed in eukaryote Pichia pastoris and then purified by our collaboration partner Dr. Genny Degani at the University of Milan (Chapter 2.2.2.3).

Running buffer was 20 mM Tris- HCl (pH 7.6), 300 mM NaCl, 2 mM CaCl2, and 0.01% Tween. 1 mg/ml BSA was added to the running buffer for the experiments with S100A8/A9 or S100A9 to avoid unspecific binding of analytes to the reference part of the chip. Regeneration of chip was done with 350 mM EDTA in water, pH8.3 and the 2+ chip was charged with Ni using a 0.5 mM NiCl2 solution. After docking the chip in the instrument and priming the machine, both parts of the chip were regenerated for 60 seconds with 350 mM EDTA and then nickel solution was injected for 60 seconds on the active part of the chip. Regeneration and loading with Ni2+ was performed at flow rates of 10 µl/min. The blank surface was used as a reference, and nickel solution was not injected over the reference surface. Ligand (His6-VC1243) was injected at a concentration of 50µg/ml during 1 to 3 min at a flow rate of 10 µl/min until certain value of loading was reached.

Figure 27: Overview of SPR experiments using NTA chips.

A: Chemical structure of Nitrilotriacetic acid (NTA) B: Scheme SPR biosensor. This NTA surface is used to capture histidine-tagged molecules His6-VC1243. Light reflected from a glass induces an evanescent field in both the gold and glass, with the field being greater in the latter. Light is then reflected out of the glass and a detector records the angle at which resonance is satisfied. C: Sensor Chip NTA

Figure 28: Schematic of a SPR measurement for VC1243 and S100 proteins

VC1 immobilization via a His tag to the sensor NTA chip. As analyte (S100 protein) flow over the sensing layer and binds to the immobilized ligand molecules, the angle of reflectivity that satisfies the resonance condition will change accordingly, until it reaches saturation and all binding sites have been occupied.

2.2.2.7 Protein X-ray crystallography

Protein X-ray crystallography is a biophysical method to obtain a three-dimensional detailed model of proteins structures with a resolution of individual atoms. Approximately 90% of all protein structures in the protein data bank (PDB) are resolved by X-ray crystallography. Moreover, X-ray crystallography is an important tool in drug discovery. It should be noted the PDB was started to set up in 1970’s and according to its statistical reports in up to date more than 90000 protein structures have been determined by X–ray crystallography while around 11000 proteins structures were determined by other experimental methods (Berman et al. 2000). The main steps in X-ray crystallography can be summarized as crystal growth, harvesting, soaking, freezing the crystals, diffraction data collection, phase determination and

model building. Each step requires special efforts and is composed of several sub- steps. In this part of my thesis, I will shortly describe each step.

2.2.2.7.1 Concept of protein X-ray crystallography

In principle, in X-ray crystallography a model is built based on diffraction images of a single protein crystal by Fourier methods. Note again that crystallography does not image atoms or molecules.

The X-ray beam with intense photons of an energy around 5-20 KeV hits the mounted crystal on a rotatable goniometer. Diffraction images are recorded on an area detector and are combined into a diffraction data set. Using Fourier Transform (FT) the reciprocal diffraction spaces are transformed into molecular spaces which reconstruct the electron density of all the molecules that form the protein crystal. Growing protein crystals are the first challenge to obtaining the protein structure which can take days to years. Before growing protein crystal, one needs good amounts of homogenous end and pure protein.

2.2.2.7.2 Crystallization

The crystallization process can be demonstrated by a phase diagram (Figure 29). Certain parameters such as the concentration of protein, precipitant(s) and additive(s) effect on crystallization process which consists of four zones or phases. Physically, crystallization is the separation of fluid phases that contain different concentrations of common components (phase separation) in a thermodynamically metastable supersaturated system under the control of kinetic parameter. The thermodynamic parameters can be divided into wide-ranging and intensive Factors. The extensive parameters are the protein and reagent concentration. For intensive factors temperature and pH value have to be mentioned.

The first phase after precipitation is spontaneous nucleation which is followed by the metastable zone where crystals are stable and may grow but no further nucleation will take place. However, these processes do not necessarily always lead to protein crystals that are suitable for diffraction. This is why optimization of crystal growth conditions is particularly important to obtain better protein crystals.

Figure 29 : Protein crystallization phase diagram.

Protein crystallization diagram dependent on protein and the precipitant concentration. In the clear phase the protein is complete soluble. With increasing concentration of protein or/and precipitant the metastable phase is reached. In this phase the protein solution becomes saturated and crystal can grow. Further increasing of concentrations cause precipitation of the protein and unstable decomposition can occur. (Chayen 2004)

2.2.2.7.3 The vapor diffusion technique

The vapor diffusion technique was used in all performed crystallization screenings. The working process of this method is illustrated with the hanging drop model in Figure 30. The protein solution was mixed with the mother liquor (ML) in different ratios. The protein-ML mixture exhibits a lower vapor diffusion pressure than the reservoir solution and therefore water can evaporate from the protein-ML drop and is 60

taken up by the reservoir solution. Consequently the concentration of protein and precipitant in the drop slowly increases until the solubility limit of protein is exceeded. The solution becomes supersaturated and enables nucleation, phase separation and crystal growth (Krauss et al. 2013)(Figure 30).

Figure 30: Vapor diffusion method in a hanging drop system.

A drop containing unsaturated precipitant and protein solution is placed in a well containing a reservoir with precipitant in higher concentrations. The well is hermetically sealed to prevent droplet evaporation and to allow vapor equilibration of the droplet and the reservoir. Equilibration of water vapor from the protein-containing droplet to the reservoir solution causes the protein solution to reach a supersaturation level, where nucleation and initial growth occur. A: Hanging drop technique. The droplet is placed on a siliconized glass that is used to close the well; B: Sitting drop technique. The droplet is placed on a small bridge inside the well (Krauss et al. 2013).

2.2.2.7.4 Crystallization of S100A9-Ca2+-Zn2+

The crystallization of S100A9-Ca2+-Zn2+ was performed by with 5 mg/ml S100A9, 10 mM CaCl2, and 0.375 mM ZnCl2. The excessive addition of zinc chloride led to immediate precipitation of the protein. Therefore the addition of exactly two zinc ions per S100A9 molecule had to be performed for each crystallization setups. The first

S100A9-Ca2+-Zn2+ crystals were obtained by Dr. Stephan Giesler (working group Fritz) at the following condition: 0.1 M Mes/NaOH pH 6.5, 35% PEG 400. Reproducing the S100A9 crystals was performed in several 15 wells hanging drop plates with the conditions that mentioned and with 10 mM CaCl2 and 0.375 mM

ZnCl2. Drop ratio was 1 µl protein into 1, 5 µl reservoir. Crystals of S100A9 grew within 5 to 7 with size of size of 0.3 × 0.2 × 0.3 mm.

2.2.2.7.5 Soaking of compounds into S100A9

One method to prepare protein-ligand complexes in presence of small molecules is to soak a crystal in mother liquor containing an excess of ligand. It can be harvested after a short time or incubate the crystal for a longer time. Moreover, a diluted ligand in mother liquor can play a role as cryoprotectant if desirable and practical. The concentration of ligand is depending on the dissociation constant (Kd). If protein molecules pack in the crystal in such a way that they obscure the ligand-binding site, or if crystals do not tolerate extended soaking without cracking or dissolving, then co- crystallization with ligand is attempted.

2.2.2.7.6 Crystallization of S100A9 and of a VC1243-S100A9 complex

The crystallization of S100A9 homodimer and complex of VC1243 with S100A9 homodimer were started with initial screens that were kindly performed by Anita Arnold and Dr. Spephan Giesler using a Phoenix Liquid Handling System (Art Robbined Instruments) with 96well INTELLI-PLATES®. Sitting drop vapor diffusion method was used. All protein samples were prepared freshly just before pipetting. The plates were sealed with films and stored at 20°C. Drop ratios were 0.3:0.3 µL, 0.15:0.3 µL or 0.3:0.15 µl. After identification of initial hits, the crystallization conditions were refined changing the conditions with regard to buffer composition, pH, additives and protein concentrations. 62

For fine and seed screens EasyXtal 15 well DG Tool plates (Qiagen) were used with a reservoir volume of 1ml and drop capacity of 6 µl. Crystal plates were stored in a temperature controlled cabinet. For harvesting, of crystals CryoLoopsTM (Hampton research) with different sizes were used.

PEG 400, PEG 4000, 20% Glycerol were used as cryoprotectants for harvested crystals of VC1243 with S100A9. All cryoprotectants were diluted in the mother liquor. In the case of S100A9 crystals, the crystallization condition with 0.05 M MES pH 6.3 and 37% PEG 400 turned out as the best cryoprotectant.

To soak the Q-compounds into S100A9 crystals, Q-compounds were diluted in mother liquor to concentrations ranging from 1mM to 10mM. Then S100A9 crystals grown in 0.05 M MES pH 6.3 and 37% PEG 400 were transferred to soaking buffers and incubated for different times ranging from 1 hour to 3 weeks and harvested afterwards. The harvested crystals were immediately flash frozen in liquid nitrogen and stored in it until measured at the synchrotron.

2.2.2.7.7 Data collection, and X-ray diffraction data analysis

Data collection is the last physical experiment that deserves much attention to obtain the high-quality final structure.Proteins themselves have a regular structure but in the crystal lattice, they are very irregularly arranged. Proteins do not tessellate and therefore it has huge gaps within the structure. The structure of a correctly grown protein crystal can be complicated but is always made up of identical repeating 3D motifs (or unit cells). In a perfect crystal, there are no gaps between unit cells. Protein molecules in a lattice have rotational symmetry in a different rotational axis.

Unit cell in the crystal is the smallest bit which contains one or more repeating motives. Bravais lattices have higher symmetry with more than one motive per unit cell. asymmetric units of chiral molecules within a crystal motive involve only simple rotations and rotations around a screw. Indexed spots name with integer numbers called Miller indices (h, k, l).

The electrons in the crystal (real space) diffract X-rays to form a diffraction pattern (reciprocal space). Both are related to each other through a Fourier Transform resulting from a diffraction described by Bragg's Law.

The scattered photons can either interact with each other in constructive interference, when they are in phase and amplify each other, or in destructive interference, when they have different phases, which will lead to extinction of the beam. Only the constructive interference of the scattered waves can be detected as reflection on the X-ray detector. The interaction of scattered X-rays is described by Bragg´s law (1), which was formulated by William Lawrence and William Henry Bragg in the year 1912. According to Bragg’s law (Equation 1) where λ is the wavelength of the X-ray wave, the inter-planar distances between two lattice planes are called d with h, k, l as the Miller Indices of the lattice planes, θ as the X-ray scattering angle and n as a positive integer (Fultz & Howe 2007) (Figure 31).

Equation 1 ,, = ʎ

Figure 31: Schematic of a wave scattered at crystal lattice planes.

Two planes separated by spacing, d. Two X-ray beams with identical phase and wavelength hit crystal lattice planes and the waves are scattered off two different atoms within it. The lower beam must travel the extra distance (Fultz & Howe 2007).

Oscillation method is the most common data collection method in macromolecular crystallography that involves small slices of rotation of the crystal around a single axis around 0.1° to 1° while the crystal is exposed to X-rays and a detector records reflections as diffraction images which are often called frames (Figure 32). To analyze and compute the direction of the diffracted X-rays from all sets of parallel planes of the lattice, a lattice in the reciprocal space is used. As the crystal, and thus its reciprocal lattice is a rotated, most reciprocal lattice points will pass through the Ewald sphere. The scattering is no longer featureless but forms sharp spots on a photographic film or area detector. This collection of spots is called a diffraction pattern. In an oscillation method (Figure 32) some reciprocal lattice points are fully and some are only partly recorded on one image and continue on the next one.

Figure 32: Schematic depiction of an oscillation method diffraction pattern.

When the reciprocal lattice oscillates in a small angle around the rotation axis, small areas of different reciprocal planes will cross the surface of Ewald’s sphere, reaching diffraction condition. Thus, the detector screen will show diffraction spots from the different reciprocal planes forming small "lunes" on the diagram (figure on the right). A "lune" is a plane figure bounded by two circular arcs of unequal radii, i.e., a crescent.

After integration, the integrated values of the different images are combined into one set of structure factors and normalized, this process is called scaling. The result of the correct assignment of the structure factors is an electron density map. An atomic model of the structure should be built into the electron density map. The correct building of the structure is used during refinement to improve the (approximate) phases obtained earlier. The aim of the experiment is to obtain the electron density map, which is the Fourier transformation of the diffraction data.

One-dimensional Fourier transform (Equation 2) equation related real and reciprocal space. This equation describes that for any function () there is another function called (ℎ) that is the Fourier transform (FT) for ().

Equation 2

(ℎ) = () 66

Several available softwares can turn structure factors into electron density maps by using general three-dimensional Fourier sum equation (Equation 3), where is the volume of the unit cell and (, , ) is a structure factor representing a single X-ray reflection. Each structure factor is a complete description of a diffracted ray recorded as reflection ℎ .

Equation 3

1 (, , ) = ()

The amplitude can be calculated from the intensity. The square root of the measured 1 intensity of reflection ℎ and frequency is equal to . To obtain complete structure factors, the phase of each diffracted ray must be obtained. Clearly, each reflection has its own phase.

The diffraction pattern is the measured intensities of the structure factors. As the phase information of the structure factors cannot be measured, the electron density is not obtained by Fourier Transform, this fact is known as the phase problem.

There are several methods to obtain initial phases including direct method for small molecules, molecular replacement, isomorphous replacement, anomalous dispersion and combination of all methods.

As a general rule, low-resolution data >4.0 Å can still be useful for phasing experiments, however, there are insufficient to refine a quality model and a resolution about 2.0-2.5 Å is suitable for model building.

After obtaining and improving estimated phases, the electron density map becomes clearer and it is easier to construct a molecular model. Refinement is the last step to obtain a structure of proteins that is in good agreement with the original data.

There are many software suites that can be used to analyze protein X-ray diffraction data. Described here is data analysis procedures based on the programs XDS, Refmac implemented in CCP4, PyMol, and Coot.

3 RESULTS

3.1 Expression and purification of His6-VC1243

3.1.1 Expression of His6-VC1243 out of Origami B (DE3)

His6-VC1243 was expressed in E.coli Origami B (DE3) with a yield of 45 g wet weight out of 8 L medium. The expression was carried out for 16 hours at 25°C (Figure 33). The SDS-PAGE analysis revealed a clear overexpression band at 26 kDa.

Figure 33: Recombinant expression of His6-VC1243.

The expression of His6-VC1243 was analyzed with Coomassie-stained SDS-PAGE.

3.1.2 Purification of His6-VC1 243

After recombinant protein expression, the bacterial cells were harvested and lysed with a Microfluidizer. Cell breakage was monitored by SDS-PAGE (Figure 34). After

Microfluidizer treatment and ultracentrifugation the His6-VC1243 was detected in the supernatant. About 50% of His6-VC1243 expressed protein was soluble whereas the other half was insoluble most likely due to aggregation.

Figure 34: SDS-PAGE of cell extracts of His6-VC1243.

SDS-PAGE of cell extracts of His6-VC1243 after Microfluidizer treatment and ultracentrifugation. Around 10 μl of each sample was loaded onto the gel. After Microfluidizer treatment His6-VC1243 was detected in the supernatant.

2+ The His6-VC1243 was purified by affinity chromatography on a Ni -Sepharose column. The chromatogram is shown in Figure 35.The elution fractions containing

His6-tagged proteins were collected and analyzed by SDS-PAGE as shown in Figure 36. The flow through showed only minor amounts of protein at the expected size of

26 kDa for His6-VC1243, whereas the elution fraction 2 (E2) shows a major band at the expected size, but it also contained a further band around 55 kDa. Also, another elution fraction (E1) revealed a band at 26 kDa for His6-VC1243. The wash fractions displayed only a low amount of a 26 kDa band.

2+ Figure 35: Purification of His6-VC1243 by Ni -Sepharose (IMAC)

The y-axis shows the absorption units (AU) at 280 nm and the x-axis display the elution volume in ml. The lines indicate the points of collection from different washing and elution steps. FT= flow through, W2= washing fraction, E1= elution fraction 1, E2= elution fraction 2.Elution peak has one shoulder (Fraction E2) which is load on the SDS-PAGE separately.

Figure 36: SDS-PAGE of the fractions from Ni2+-Sepharose run.

Coomassie-stained SDS-PAGE of the washing and elution fractions. 5 µl of flow through (FT), 10 µl of washing fraction (W2) and 8 µl of the elution fractions (E1 and E2) were loaded onto the SDS-PAGE. E1 shows clear band around 26 KDa, however E2 has one additional band around 55 KDa which correspond to dimer state of VC1.

For SPR experiments the flexible His6-tag was not removed by thrombin cleavage.

His6-VC1243 was concentrated to a volume of 10 ml with a final concentration of 7 mg/ml. As a second purification step, size exclusion chromatography (SEC) was applied the chromatogram is shown in Figure 37.

Figure 37: Size exclusion chromatography of His6-VC1243.

The protein was detected at 280 nm (solid line) and putative DNA contamination at 260 nm (dash line). The y-axis shows the absorption units (AU) and the x-axis displays the elution volume in ml. Two main peaks eluted from the size exclusion chromatography.

Two main peaks eluted from the size exclusion chromatography at volumes of 152 ml and 166 ml. The two peaks correspond to molecular mass of about 58±10 and 30±10 kDa which might represent a homodimer and monomer of VC1243.

The eluted fractions from the two peaks were analyzed on SDS-PAGE that is shown in Figure 38. The His6-VC1243 protein is identified as a band with a size of 26 kDa. Fractions 54 to 62 contained pure protein. The eluted fractions 54 to 62 were also combined and concentrated to a final concentration of 13 mg/ml.

Figure 38: SDS-PAGE from the SEC purification step of His6-VC1243.

5 µl of each fraction were loaded onto the gel. The second elution peak containing fraction 54-62 were combined for concentration.

3.2 Expression and purification of sRAGE

3.2.1 Expression of sRAGE in E.coli Rosettagami B (DE3)

His-sRAGE expressed in E.coli Rosettagami B (DE3). The harvested cells wet weight around 27 g out of 8 L medium. The expression procedure was the same like His6-

VC1243 as described in chapter 2.2.1.5 Samples from before induction and after induction were analyzed on SDS-PAGE and showed bands around 36 kDa.

3.2.2 Purification of sRAGE

Cell were resuspend and lysed, with Microfluidizer as described in chapter 2.2.2.1.Crude extract was ultracentrifuged at 100.000 g for 1 hour at 4 °C. The analysis of the isolation was done on SDS-PAGE and is shown in Figure 39.

Figure 39: SDS-PAGE of cell extracts of sRAGE.

SDS-PAGE of cell extracts of sRAGE after 4 times Microfluidizer treatment and ultracentrifugation. Around 10 μl of each sample was loaded onto the gel.

Supernatant was diluted in. 20 mM HEPES,300 mM NaCl and 30 mM Imidazole and was loaded on Ni2+-Sepharose column.Then column was washed with same buffer until OD280 reached approximately to the baseline. Afterwards protein was eluted with 20 mM HEPES,300 mM NaCl and 500 mM Imidazole (Figure 40). Protein was

collected in fractions and protein purity of fractions was checked by SDS-PAGE. Fractions containing almost pure proteins were combined (Figure 41).

Figure 40: Ni2+-Sepharose (IMAC) chromatography result of sRAGE.

The y-axis shows the absorption units (AU) at 280 nm and the x-axis display the elution volume in ml. The lines indicate the points of collection from different washing and elution steps. FT= flow through, W1= washing fraction 1, W2= washing fraction 2, E1= elution fraction 1, E2= elution fraction 2.

Figure 41: SDS-PAGE of the fractions from Ni2+-Sepharose run.

Coomassie-stained SDS-PAGE of the washing and elution fractions. 5 µl of flow through (FT), 10 µl of washing fractions (W1 and W2) and 8 µl of the elution fractions (Elute1 and Elute2) were loaded onto the SDS-PAGE.

For crystallization trials the flexible His-tag was removed by thrombin cleavage (Figure 42) After 3 h over 90% of the His-tag was removed from sRAGE and the pH was adjusted to 5.2 to increase the solubility of sRAGE without the His-tag. SDS- PAGE showed some bands in a range from 70 and 130 kDa which belonged to oligomerization states of sRAGE.

Figure 42: Thrombin cleavage of the His-sRAGE for removal of the His6-tag.

The analysis was done SDS-PAGE of thrombin cleavage at different time points until 60 min. 5 µl for each time point were loaded onto the gels. The digestion was performed until over

90% of the N-terminal His6-tag was removed.

As a second purification step size exclusion chromatography (SEC) was applied and shown in Figure 43. One main peak eluted from the size exclusion chromatography at a volumes of 144 ml as well as a small peak at 110 ml. The eluted protein from the first peak was analyzed by SDS-PAGE is shown in Figure 44. The sRAGE protein is depicted by the band size of 35 kDa and pure in fractions 50 to 54. The fractions 50 to 54 were combined and concentrated to a final concentration of 19 mg/ml with a total volume of 500 µl.

Figure 43: Purification of sRAGE by size exclusion chromatography.

The protein was detected at 280 nm (solid line) and putative DNA contaminations at 260 nm (dashed line). They axis shows the absorption units (AU) and the x-axis displays the elution volume in ml.

Figure 44: SDS-PAGE from the SEC purification step of sRAGE.

5 µl of each fraction were loaded onto the gel. The main elution peak containing out of fractions 50-54 were combined for concentration.

3.3 Expression and purification of S100A9

3.3.1 Expression of S100A9-C3S out of E.coli BL21 (DE3)

Cell lysates of E.coli with recombinant S100A9-C3S was kindly provided by Christine Betz (working group Fritz 2010). The supernatant of cells lysates was dialysed overnight at 4°C using cut-off of 6000-8000Da in lysis buffer. All buffers are described in 2.1.3.4.

3.3.2 Purification of S100A9-C3S

The first step of purification of S100A9-C3S was ion exchange chromatography (IEX) using a 15 mL column Source Q15 resin. The Chromatogram showed one major peak at elution volumes 150 ml to 225 ml. Further protein eluted between 250 ml to 750 ml (Figure 45).

Figure 45: Purification of S100A9 by ion exchange chromatography.

Proteins were detected at 280 nm (black). The left y-axis shows the absorption units (AU) at 280 nm, and the right y-axis shows NaCl concentration. The x-axis displays the elution volume in ml, and the concentration of high salt buffer is green colored.

The five pooled fractions were analyzed by SDS-PAGE. Only pool 1 shows a clear band at a molecular mass of 13 kDa and second pooled showed bands in higher molecular mass and in the third and fourth peak very low amounts of S100A9 were 81

detected (Figure 46). After SDS-PAGE the corresponding fractions were combined as indicated.

Figure 46: SDS-PAGE of S100A9 corresponding fractions from IEX run.

A 10 µl sample of each fraction was loaded on 10% SDS-PAGE. Pool 1 showed a high concentration of S100A9-C3S.

The pool one of five IEX runs were combined and had a concentration of 6 mg/ml. As a final purification step size exclusion chromatography was applied. The chromatogram showed an elution peak at an elution volume from 100-120 mL, which corresponds to 28 kDa and represents a homodimer of S100A9-C3S. (Figure 48)

Figure 47: Purification of S100A9-C3S by size exclusion chromatography.

The protein was detected at 280 nm (solid line) and putative DNA at 260 nm (dashed line). The y-axis shows the absorption units (AU) at 280 nm

The fractions 100-120 mL were analyzed on a SDS-PAGE gel. On SDS-PAGE the fractions of 105-115 mL showed distinct bands at a molecular mass of 13 kDa (Figure 48). The elution peak showed no shoulders or additional peaks, which lead to the conclusion that multimeric states were not present in the injected sample. The S100A9 protein was concentrated and the final concentration was adjusted to 20 mg/ml.

Figure 48: Coomassie-stained SDS-PAGE of the second SEC run.

5 µl of 1:10 diluted samples were loaded onto the gel. In comparison with sample before SEC, all additional bands were absent after size exclusion chromatography.

3.4 Co-crystallization of S100A9 with VC1243

The aim of this part was to characterize the structure a complex between S100A9 and VC1243. There was no record about structure a complex of these two proteins recently (Chang et al. 2016). Initial screening was performed with commercial and non-commercial initial screens which were described in chapter 2.1.9. Co- crystallization was performed with 2.7 mg/ml VC1243 and 2.9 mg/ml S100A9. Two equivalents of ZnCl2 per S100A9 protein dimer and 10 mM CaCl2 were added to the buffer to ensure high-affinity complex formation. Crystals of 15 different conditions were analyzed at the synchrotron. Crystals out of 5 conditions diffracted to a resolution of 8-12 Å. These 5 crystallization conditions were refined. The X-ray

analysis of the putative VC1-S100A9 complex, crystallization conditions and further details are summarized in Table 3.

Table 3: Crystallization conditions and diffraction data of a putative VC1243-S100A9 complex.

Screen name-Drop Crystallization Diffraction Shape-Evaluation Number condition 0.1 M Bis-Tris pH5.5 >8 Å Angular crystals

25%PEG 3350 Index –G10.I 0.2 M Magnesium chloride 0.1 M Tris/HCl pH8.5 >8-9 Å Angular crystals

Index –G5.I 25%PEG 3350 6-7 Å Needle crystals

0.2M Lithium sulfate 11 Å 0.015 M Tiricine 8-10 Å Needle crystals pH8.5 MemGold-A3.I

24% PEG4000 0.1 M Bis-Tris 8.5 Å Needle crystals propane pH7 MemGold-H12.I

3 M sodium chloride 0.1 M HEPES pH7.5 8-10 Å Angular crystals PEG I-F4.I 25% PEG 3350

The proteins were diluted in 30 mM Tris/HCl pH 7.5 supplied with 10 mM CaCl2,

0.218 mM ZnCl2 and a final NaCl concentration of 200 mM. The pictures of crystallization drops were taken at day 29 from initial screens (Figure 49).

Figure 49: Crystallization of a putative S100A9-VC1 complex

Five crystallization conditions yield crystals (A-E) which are tested at the synchrotron. A: 0.1 M Bis-Tris pH5.5+ 25% PEG 3350+ 0.2 M Magnesium chloride. B: 0.1 M Bis-Tris propane pH 7+ 3 M Sodium chloride. C: 0.01 M Tricine pH 8.5 + 24% PEG4000. D: 0.1 M HEPES pH 7.5+ 25% PEG 3350. E: 0.1 M Tris/HCl pH 8.5+25% PEG 3350+0.2 M Lithium sulfate.

3.4.1 Optimizations of crystallization of VC1-S100A9

Five conditions of initial screen yielded crystals. Based on original conditions one fine screen with varying PEG concentrations and varying pH values were prepared. Moreover, seed stocks for further crystallization experiments were prepared. Seeding improves nucleation during crystallization.

The micro-seeding was used to reproduce the crystals of VC1-S100A9 which diffract to a resolution of 8-24 Å or with too thin and too fragile crystals not measurable with X-rays. The seed screenings were performed in 15well plates (EasyXtal Tool, QIAGEN) and the conditions with protein concentrations are mentioned in chapter 2.1.9. For the reproduction of already analyzed crystals, these were transferred out of the nylon loop into a new tube containing 20 µl of the original reservoir solution and

strongly mixed 3 times for 30 sec. From this stock solution different dilutions of seeds were prepared and added in a 1:10 to final drop size. The drop size was the original drop ratio between protein and mother liquor. The seed stock and the dilutions were stored at -20°C. For seeding of not measurable crystals the whole drop volume was transferred into a new tube containing the original reservoir solution and vortexed 3 times for 30 sec. Again different dilutions were prepared and the procedure was the same as mentioned above.

Crystals of VC1-S100A9 grew within 6 to 12 days and had different shapes (Figure 50). Crystals were harvested, cryo-cooled in different cryoprotectant solutions and analyzed at the synchrotron. The crystals found in these conditions diffracted to 2.3 to 10 Å resolution. Analysis of the data by molecular replacement revealed that all crystals contained only S100A9 proteins.

Figure 50: Crystals obtained after fine seed screening of VC1-S100A9.

Seed screenings were developed based on the conditions from the original crystallization solutions. A: 0.1 M Tricine pH 8.7+ 20% PEG 4000 (50%) B: 0.2 M malate-imidazole pH

5.3+11% PEG 3350 (50%). C: 0.1 M bis-tris pH 5.3+ 0.2 M MgCl2+ PEG 3350 [50%] + Azide

0.02%. D: 0.1M Bis-Tris pH 5.7+ 0.2 M MgCl2+ PEG 3350 [50%]. . E: 0.05 M Sodium Citrate Citric Acid pH 4.7+ 0.07 M NaCl + 18% PEG 400 (100%). F: 0.1 M MES pH 6.5+ 15% PEG MME 5000.

3.5 Monitoring interactions of S100A9 with VC1 domain using MST

MST study on S100A9 interaction with VC1 domain performed as described in chapter 2.2.2.5.1. A constant concentration of the S100A9 was incubated with different concentrations of the labeled VC1 in presence and absence of Zn2+ ions in buffer. Afterward, samples were loaded into a glass capillary and thermophoresis analysis was performed. Fluorescently labeled VC1 was incubated with increasing concentrations of S100A9 and MST analysis was performed using the NanoTemper Monolith apparatus. Quantitative analysis of the interaction of VC1 and S100A9-Zn2+ (0-10 µM) yields a binding affinity coefficient (Kd) of around 4 µM (Figure 51). In contrast, MST results for interaction of S100A9 with VC1 without Zn2+ showed that S100A9 does not interact with VC1 in absence of Zn2+.

S100A9 binding to VC1 domain by MST 960

950

940

Fnorm[1/1000] 930

920 1 100 10000 Concentration[nM]

Figure 51: Analysis of S100A9 binding to RAGE-VC1 by MST.

Unlabeled S100A9 homodimer was titrated into a fixed concentration of labeled VC1 The binding curve revealed a Kd of 4 µM.

3.6 Monitoring binding of inhibitors and Zn2+ to S100A9 using ITC

3.6.1 Calorimetric titrations of Zn2+ ions into S100A9 homodimer

SPR and MST results showed that S100A9 binds to VC1 only in presence of Zn2+ ions. Therefore the affinity of S100A9 towards Zn2+ was explored by ITC. Moreover, It was analyzed whether Ca2+ or Mg2+ influence Zn2+ binding.

The experimental design, consisted of three different injections of the Zn2+ into S100A9 placed in the ITC cell, in 3 different conditions based on presence of Ca2+ or Mg2+ in the buffer (Figure 52).

Figure 52: Schematic illustration of calorimetric titrations of Zn2+ into S100A9.

A: Injecting Zn2+ into S100A9 when Ca2+ ions are already bound to S100A9. B: Titration of Zn2+ ions into S100A9 homodimer while negative charged surface of proteins was covered by Magnesium ions. C: Adding Zn2+ into S100A9 homodimer in absence of Ca2+.

Titrations were started by injecting 100 µM Zn2+ (21 times, 8 µl aliquots) into 10 µM S100A9 homodimer in Tris buffer that contains Ca2+ (20 mM Tris, 100 mM NaCl and

2 mM CaCl2). This experiment was repeated with 30 µM S100A9 and 300 µM ZnCl2. Results of the titration curves were analyzed using Origin software, NITPIC, and SEDPHAT. Figure 53 shows the global evaluation of these two titrations with a Kd around 4 µM.

2+ Figure 53: Isotherm corresponding to the binding of Zn to S100A9/A9 at 25 °C.

2+ The raw data of the titration of Zn to S100A9 are shown in the upper panel. The lower panel shows the integrated data obtained by subtracting the heat of dilution. The concentrations of Zn2+ was 100 µM and S100A9 10 µM. (Red line), or S100A9 30 µM and Zn2+ 300 µM (Blue line) respectively. The changes in the heat during each injection of Zn2+ into a solution of the S100A9 domain fit a single-site binding model with a KD of approximately 4 µM and ΔG = - 7.373 kcal/Mol. The titrations were performed in 20 mM Tris-HCl (pH 7.6) containing 2 mM Ca2+.

It was assumed that some Zn2+ ions might bind to some other parts of surface and not only to the specific binding sites in S100A9 (2 binding sites per homodimer)

Therefore ITC measurement for Zn2+ binding to S100A9 were repeated in presence of Mg2+. Moreover, the influence of Ca2+ on the Zn2+ affinity to S100A9 was investigated (Figure 54).

2+ Figure 54: ITC titrations of the ZnCl2 domain with S100A9 in presence of Mg .

The concentrations of the Zn2+ and S100A9 used in the ITC experiments were 20 µM and 200 µM, respectively. The changes in the heat during each injection of Zn2+ into a solution of the S100A9 fit a single-site binding model with a Kd of approx. 3 µM. The titrations were performed in 20 mM Tris-HCl pH 7.6 containing 2 mM CaCl2 and 5 mM MgCl2.

Next ITC measurement was performed in absence of calcium ions in the buffer to know more details about the effect of calcium on S100A9 zing binding (Figure 55). This ITC experiment was repeated more than 5 times with different concentration of

ZnCl2 and at the end with titration 600 µM of ZnCl2 into 30 µM S100A9 (41 injections, 6 µl aliquots) the following binding curve was obtained (Figure 55) with 3 inflection points around 1.5 molar ratio, 2.5 molar ratio and 4 molar ratio which corresponds to saturation of each binding sites or maybe aggregation/ precipitation of S100A9 with high concentration of titrated Zn2+.

Figure 55: Binding of zinc by titrating S100A9 with Zinc without calcium.

The changes in the heat during each injection of Zn2+ into a solution of the S100A9 fit a single-site binding model with a Kd of approx. 3.6 µM. The titrations were performed in 20 mM Tris-HCl pH 7.6 containing 5 mM MgCl2 without CaCl2.

3.6.2 Calorimetric titrations of Q-compounds into S100A9 homodimer

As a next step a wide set of inhibitors of S100A9 were characterized by ITC. Since binding events in the ITC result in a change of released or absorbed heat, 21 numbers of injections is sufficient to make a binary decision regarding a binding event. Figure 56 shows binding curves resulting from ITC titration experiments of different Q-compounds into S100A9. ITC data demonstrate specific and high-affinity binding with Kds in nanomolar to micromolar range for all compounds tested. However, it was not possible to obtain reasonable affinity binding curves for all Q- compounds tested. A reason for that might be the low solubility of some Q- compounds in ITC buffer.

In all ITC experiments, a 300 µM solution of Q-compound was placed in a syringe as the protein ligand and titrated into a sample cell containing a 20 µM solution of the S100A9 homodimer. All titrations were performed at 25°C.

A quick look at the datasets reveals that each Q-compound exhibits a different behavior. Indeed, whereas most of them show a low endothermic reaction with approximately the same magnitude of energy change and the amount of heat released after each injection decreases (Figure 56). Some of Q-compounds like ABR-240010 (Figure 56 D) show a relatively strong exothermic reaction accompanied by a decrease in the heat change magnitude. This behavior is correlated with a decrease in the number of molecules capable of binding to the newly injected protein. However, the same effect is not observed in the Isomer of this compound (ABR-240011 Figure 56 A). The summary is given in Table 4 listing the thermodynamic parameters of each ITC experiments.

Table 4: Thermodynamic parameters of each ITC experiments for Q-compounds against the binding for S100A9

Q- ΔG Kd[µM] Ka [M-1] dH[kcal/Mol] dS[cal/Mol*K] compounds [kcal/Mol]

ABR-240011 4.566 -5.922 2.190e+04 -2870.923 -9609.263

ABR-240007 11.76 -6.724 8.482e+04 -14.080 -24.674

ABR-239709 593 -4.402 1.687e+03 -19.744 -51.457

ABR-240010 4.70 -7.268 2.124e+05 -5.388 6.305

ABR-239979 11.76 -6.392 4.842e+04 -64.098 -193.54

ABR-249974 7.62 -6.982 1.311e+05 -8.000 -3.415

ABR-215050 14.25 -6.611 7.013e+04 -84.073 -259.809

ABR-239071 36.31 -6.057 2.754e+04 -13841.758 -46405.168

ABR-239981 28.33 -6.204 3.529e+04 -3996.242 -13382.654

A B C

D E F

G H I

Figure 56: ITC titrations of the Q-compounds into S100A9.

The upper panels represent the raw data, the bottom panels are the integrated plot of the amount of heat liberated per injection as a function of the molar ratio of the Q-compound to S100A9. The concentrations of Q-compounds and S100A9 used in the ITC experiments were 300 µM and 20 µM, respectively. The changes in the heat during each injection of inhibitors into a solution of S100A9 fit a single-site binding model in 20 mM Tris-HCl pH 7.0 2+ containing 4 mM CaCl2. For S100A9 samples 20 µM Zn was added to the buffer. A: The raw thermogram and binding isotherm of ABR-240011 binding to the S100A9 homodimer. B: The raw thermogram and binding isotherm of ABR-240007 binding to the S100A9 homodimer. C: The raw thermogram and binding isotherm of ABR-239709 binding to the S100A9 homodimer. D: The raw thermogram and binding isotherm of ABR-240010 binding to the S100A9 homodimer E: The raw thermogram and binding isotherm of ABR-239979 binding to the S100A9 homodimer. F: The raw thermogram and binding isotherm of ABR- 249974 binding to the S100A9 homodimer G: The raw thermogram and binding isotherm of ABR-215050 binding to the S100A9 homodimer H: The raw thermogram and binding isotherm of ABR-239071 binding to the S100A9 homodimer. I: The raw thermogram and binding isotherm of ABR-239981 binding to the S100A9 homodimer

3.7 SPR measurements for S100 proteins to immobilized VC1

In order to understand how S100 proteins interact with RAGE and to understand what effect glycosylation might have the interactions, I studied the binding of S100A9, S100A12 and S100A8/A9 to VC1 prepared from E.coli or from Pichia Pastoris by SPR. SPR results show micromolar affinity between S100 proteins and the VC1-domain and strict calcium and zinc dependency for the binding.

3.7.1 Binding of S100A9, S100A8/A9, S100A12 to immobilized VC1

A detailed analysis of SPR measurements for S100 proteins binding to immobilized VC1 revealed affinity constants in the nM to the µM range. I have exploited SPR technique to analyze the binding of S100 proteins to immobilized VC1243 in presence of Ca2+ and Zn2+ in greater detail. The results show that the interaction of these molecules depends both on Ca2+ and Zn2+.

Figure 57 shows the sensogram after injection of five concentrations of S100A9- Zn2+ which was analyzed and fitted by BIAevaluation Biacore software. The Kd obtained by SPR in this measurement was the same as by MST measurement around 4 µM. The interaction between S100A9 and RAGE was also strictly dependent on the presence of physiological concentrations of both Ca2+ and Zn2+.

RU 350 300 250 200 150 100 Response 50 0 -50 -200 0 200 400 600 800 1000 1200 1400 1600 Tim e s

Figure 57: SPR binding curves and analysis for S100A9-VC1.

Sensorgram shows association and dissociation with varying concentration of Zn-S100A9 (31.3 nM, 62.5 nM, 125 nM and 250 nM from left to right) which was perfectly fit by a 1:1 binding model (black line; BIAevaluation software; Biacore). The Kd, the value was calculated to 4.19 µM.

The second study performed had the aim to examine the effect of glycosylation on the VC1-S100A9 binding (Figure 58). The Kd value was calculated to 20 nM which was much less than the Kd of non-glycosylated VC1 and S100A9. In comparison dissociation phase (koff) for glycosylated VC1 and S100A9 is much slower than for non-glycosylated VC1 (Table 5) which shows long-lasting target binding for VC1 in the glycosylated state. Furthermore, glycosylation of VC1 increases speed of association phase (higher kon) as shown in Table 5.

RU 2500

2000

1500

1000

500 Response

-500 -200 0 200 400 600 800 1000 1200 1400 1600 Tim e s

Figure 58: SPR binding curves and analysis for Zn-S100A9 and glycosylated VC1.

Sensorgram shows association and dissociation with varying concentration of Zn- S100A9 (31.3 nM, 62.5 nM, 125 nM and 250 nM from left to right) which was perfectly fit to a 1:1 binding model (black line; BIAevaluation software; Biacore). The Kd, was calculated to 20 nM.

SPR experiments of S100A8/A9 and His-VC1243 showed that S100A8/A9 binds too to the His-VC1243 in a concentration-dependent way (Figure 59). His-VC1243 was immobilized at a density of 1565 RU. Binding of S100A8/A9 to His-VC1243 appeared to be strictly Ca2+ and Zn2+ dependent like S100A9 binding, as determined by SPR. More detailed studies on affinity and on/off rates could be calculated from these SPR curve as they fit into any reliable 1:1 binding model. The affinity of the S100A8/A9- -6 VC1 interaction, which is in the µM range [4.82×10 M], and calculated kon [1247 -3 1/(M×s)] and koff [6.007×10 1/s] were obtained after fitting the response curve to a 1:1 Langmuir model.

RU 2500

2000

1500

1000

500 Response

-500 -200 0 200 400 600 800 1000 1200 1400 1600 Tim e s

Figure 59: SPR binding curves and analysis for Zn-S100A8/A9 and VC1.

The sensorgram shows association and dissociation with varying concentration of Zn- S100A8/A9 (0.25 µM, 0.5 µM, 1 µM, 2 µM and 4 µM from left to right) which was perfectly fit to a 1:1 binding model (black line; BIAevaluation software; Biacore). The Kd, the value was calculated to 4.82 µM.

In the case of interaction of S100A8/A9 with glycosylated VC1 the affinity constant -6 was decreased to [1.16×10 ] (Figure 60). His-VC1243 was immobilized to around 1500 RU. Analytes concentration, association and dissociation time, flow rate and other parameters were the same like in SPR experiments of S100A8/A9 and non- -3 glycosylated His-VC1243. kon [8026 1/(M×s)] and koff [9.284×10 1/s] and were calculated values after fitting the response curve to a 1:1 Langmuir model.

S100A8/A9 binds to glycosylated VC1 with higher kon (faster), however there is no large difference in the dissociation kinetics (koff) of glycosylated VC1-S100A8/A9 and non-glycosylated VC1-S100A8/A9. S100A8/A9 dissociates slightly faster from glycosylated VC1 than from non-glycosylated VC1, which means that the lifetime of the complexes is very similar. However, the response units of each binding reaction were higher for glycosylated versus non-glycosylated VC1 including better stoichiometric binding (Table 5).

RU 4000 3500 3000 2500 2000 1500 1000 Response 500 0 -500 -200 0 200 400 600 800 1000 1200 1400 1600 Tim e s

Figure 60: SPR binding curves and analysis for Zn-S100A8/A9 and glycosylated VC1.

Sensorgrams generated after injection of Zn-S100A8/A9 (0.25 µM, 0.5 µM, 1 µM, 2 µM and 4 µM from left to right) (120 s at 30 µl/min) over glycosylated His-VC1 coupled to the sensor chip (black line; BIAevaluation software; Biacore). The Kd value was calculated to 1.16 µM.

SPR studies of binding affinities between glycosylated-VC1 (Figure 62) and non- glycosylated VC1 (Figure 61) with S100A12 revealed, although there is no significant difference in Kd of these two experiments. However, they have the same amount of immobilized ligands (around 1300RU), the response units for injection of S100A12 into glycosylated VC1 shows earlier saturation and higher responses.

RU 800 700 600 500 400 300 200 Response 100 0 -100 -200 0 200 400 600 800 1000 1200 1400 1600 Tim e s

Figure 61: SPR binding curves and analysis for S100A12 and VC1.

Sensorgrams generated after injection of S100A12 (0.0625 µM, 0.125 µM, 0.25 µM, 0.5 µM, 1 µM, and 4 µM from left to right) (120 s at 30 µl/min) over the indicated ligand His-VC1 coupled to the sensor chip (black line; BIAevaluation software; Biacore). The Kd the value was calculated to 0.349 µM.

RU 1200

1000

800

600

400

Response 200

-200 -200 0 200 400 600 800 1000 1200 1400 1600 Tim e s

Figure 62: SPR binding curves for interaction between S100A12 and glycosylated VC1.

Sensorgrams generated after injection of S100A12 (0.0625 µM, 0.125 µM, 0.25 µM, 0.5 µM, 1 µM, and 4 µM from left to right) (120 s at 30 µl/min) over glycosylated VC1 coupled to the sensor chip (black line; BIAevaluation software; Biacore). The Kd, the value was calculated to 0.320 µM.

Following Table 5 summarizes affinity and on/off values, that could be calculated from the SPR curve as they fit into any reliable 1:1 binding model. Noteworthy, all SPR measurements revealed a 1:1 interaction between S100 proteins and VC1.

Table 5: More details on kinetic and binding parameters from SPR measurements

SPR experiments kon (1/Ms) koff (1/s) KD (M) Chi² (RU²)

S100A9-His6-VC1243 7341 0,002206 4,19E-06 15

S100A9-Pichia His-VC1243 6,74E+04 0,001346 2,00E-08 398

S100A8/A9-His6-VC1243 1247 0,00601 4,82E-06 2970

S100A8/A9-Pichia His-VC1243 8026 0,009284 1,16E-06 3920

S100A12-His6-VC1243 8,36E+08 260,7 3,12E-07 691

S100A12-Pichia His-VC1243 2,44E+05 0,07799 3,20E-07 442

3.8 Crystallization and structure of S100A9-Ca2+-Zn2+

The crystallization of S100A9-Ca2+-Zn2+ was performed by Anita Arnold and Dr. Stephan Giesler (working group Fritz). The best diffracting crystals were obtaining from the following condition: 0.1 M Mes/NaOH pH 6.5, 35% PEG 400. The obtained crystals showed a bow-tie like shape (Figure 63). The crystals diffracted to a maximum resolution of 1.7Å. These crystals belong to space group P212121 with unit cell parameters a=63.2 Å, b=90, c=105.7 Å, and α=β=γ=90°. Table 6 summarizes the data collection statistics.

Figure 63: S100A9 Crystals in 0.1 M Mes/NaOH pH 6.5, 35% PEG 400.

The scale bars represent 100 µm.

100

There are three homodimers of S100A9 in the asymmetric unit. After refinement, the structure obtained from crystals of space group P212121 showed a Rwork of 18.5 % and a Rfree of 21.6 % (Table 7).

Table 6: Data collection statistics of S100A9-Ca2+-Zn2+ crystals.

Space group P212121

Unit cell lengths a=63.2; b=89.66; c=104.79

Unit cell angles 90°

resolution 50-1.67 (1.85-1.67)

Wavelength (Å) 0.9763

Completeness 95.8 (84.3)

Redundancy 12.2 (9.8)

R sym (%) 14.8 (238)

R meas (%) 15.5 (249)

CC* 99.9 (39.0)

I/(i) 11.4 (1.1)

No. of molecules per ASU 3 homodimers

101

Table 7: Refinement statistics of S100A9-Ca2+-Zn2+ crystals.

Rwork (%) 18.48 (35.9)

Rfree (%) 21.55 (37.4)

RMS bonds (Å) 0.006

RMS angles (°) 0.95

Ramachandran favored (%) 99

Ramachandran allowed (%) 1

Ramachandran outliers (%) 0

Clashscore 3.16

Molecular replacement, refinement, and model building were performed by me and Günter Fritz. First molecular replacement trials were performed using the known structure of S100A9-Ca2+ as a search model (PDB ID: 1IRJ) (Itou et al. 2002). However, no clear solution was found. Further molecular replacement was performed with a model of a homodimer S100A9 derived from the S100A8/A9 structure (PDB ID: 4GGF) (Damo et al. 2013). The homodimer S100A9-Ca2+-Zn2+ is stabilized by the hydrophobic interactions between both subunits. Each subunit contains two helix- loop-helix EF-hand motifs, which are connected by a further loop. The structure revealed two zinc ions and four calcium ions bounds per homodimer (Figure 64A)

The structural alignment of human S100A9-Ca2+-Zn2+ and S100A9-Ca2+ illustrated that zinc binding induced large conformational changes. A significant change was observed for the C-terminal classical specific EF-hand motifs. Here, the interhelical angles were changed which lead to the suggestion that the zinc binding at S100A9- Ca2+ induces a more open conformation of the molecule. The crystal structure of S100A9-Ca2+-Zn2+ revealed an elongation in helix IV compared to the S100A9-Ca2+ crystal structure. This elongation resulted from the movement of amino acids which are involved in the coordination of zinc ions at the dimer interface (Figure 65).

102

A B

Figure 64: Cartoon representation of S100A9 homodimer.

A: Homodimer S100A9 loaded with Ca2+- (orange spheres) and Zn2+ (green spheres). The S100A9 subunits are in yellow and blue colors. B: The zinc ions bound at the interface of both subunits. Zn2+ is bound by three His and one Asp residues originating from both monomers.

The amino acid side chains which are responsible for the binding of zinc ions are three histidine residues (His95, His91, and His20) and one aspartate residue (Asp30). The zinc binding was mediated by two amino acids of both S100A9 subunits and the binding site is close to a bound calcium ion of the S100 specific EF-hand. The binding site is illustrated in Figure 64B.

103

A B

Figure 65: Comparison of the surfaces of S100A9-Ca2+and S100A9-Ca2+-Zn2+.

A: Overlay Surface representation of S100A9-Ca2+ (colored in blue and yellow), S100A9- Ca2+-Zn2+ (colored in forest green) B: cartoon diagrams of comparison between S100A9-Ca2+ and S100A9-Ca2+-Zn2+ structures.

3.8.1 Structure of S100A9 in complex Q-compounds

For the discovery and characterization of the compound binding sites, the crystals depicted in Figure 63 were used for soaking experiments. The soaking was performed using different compound concentrations from 1 mM to 5 mM and soaking time was varied from 1 hour to 3 weeks. The crystals containing S100A9-Ca-Zn with soaked compounds were measured at a wavelength of 0.91 Å to confirm the position of Br in some compounds. The binding sites of 8 different Q-compounds were characterized.

Figure 66 to Figure 73 show Individual compounds binding to the hydrophobic pocket. The binding of the most of the Q-compounds led to some structural rearrangements in S100A9-Ca2+-Zn2+. In particular, the hinge region of the EF-hand shifts, to accommodate the compounds. Dependent on the type of Q-compounds

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from three residues or up to nine residues are participating in the stabilization of the compounds mostly with hydrophobic interactions. Overall, the compound is stabilized mainly by hydrophobic interactions.

A B

Figure 66: Hydrophobic binding pocket for ABR 239979 in S100A9-Ca2+-Zn2+.

A: The hydrophobic pocket for ABR 239979. ABR 239979 blocks a hydrophobic pocket between helix III and helix IV. The subunit is shown in magenta B: The tight closure of the hydrophobic protein interaction site of S100A9-Ca2+-Zn2+. The fo-fc electron density map is shown in grey at 2-3 σ.

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A B

Figure 67: Hydrophobic binding pocket for ABR 239338 in S100A9-Ca2+-Zn2+.

A: Hydrophobic surface and interactions of S100A9-Ca2+-Zn2+coordinating ABR 239338 binding. B: The surface representation of S100A9-Ca2+-Zn2+ is colored magenta and the compound ABR 239338 is showed in balls and stick to indicate their position in the cavity.

A B

Figure 68: Binding cavity of (Tasquinimod) ABR 215050 in S100A9-Ca2+-Zn2+.

A: Hydrophobic surface and deep binding cavity of tasquinimod on S100A9-Ca2+- Zn2+surface. B: The surface representation of chain A of S100A9 homodimer loaded with Ca2+-Zn2+ is colored in magenta and the compound Tasquinimod is showed in balls and stick to indicate their position of three fluorine atoms which nicely fit into the cavity.

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A B

Figure 69: Binding cavity of ABR 239508 in S100A9-Ca2+-Zn2+.

A: Hydrophobic surface and interactions of S100A9-Ca2+-Zn2+coordinating ABR 239508 binding. B: The surface representation of S100A9-Ca2+-Zn2+ is colored magenta and the compound ABR 239508 is showed in balls and stick to indicate their position of three fluorine atoms in the cavity. Bromine atom faces to His 61 (distance is around 2.7 Å) and Asp 65. Phenyl ring points to outside of structure.

A B

Figure 70: surface representation of binding pocket for ABR 239397 in S100A9-Ca2+- Zn2+.

A: ABR 239397 binding pocket on S100A9-Ca2+-Zn2+ chain A. B: the electron density map of ABR 239397 inside of the hydrophobic binding pocket. One of the Cl atoms faces to His 61 with distance around 3.0Å.

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A B

Figure 71: surface representation of binding pocket for ABR 240007 in S100A9-Ca2+- Zn2+.

A: ABR 240007 binding pocket on S100A9-Ca2+-Zn2+ chain A. B: the electron density map of ABR 240007 inside of the hydrophobic binding pocket.

A B

Figure 72: Binding cavity of ABR 240010 in S100A9-Ca2+-Zn2+.

A: ABR 240010 binding pocket on S100A9-Ca2+-Zn2+ chain A. B: the electron density map of ABR 240010 inside of the hydrophobic binding pocket. Side chain attached to phenyl ring points to outside of structure and three fluorine atoms fit perfectly in hydrophobic binding pocket.

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A B

Figure 73: Hydrophobic binding pocket for ABR 239105 in S100A9-Ca2+-Zn2+.

A: surface representation of binding cavity for ABR 239105 compounds on S100A9-Ca2+- Zn2+. B: the electron density map of ABR 239105 on S100A9-Ca2+-Zn2+.

helix III 90°

helix IV

Figure 74: All compounds bind to the same site in S100A9.

Overlay of different compounds complexed with S100A9 homodimer. The subunits are shown in magenta and blue, compounds are shown as ball-and-stick model. For clarity only binding to one subunit is shown. The compounds block a hydrophobic pocket between helix III and helix IV of S100A9 and thereby block binding of S100A9 to its receptors.

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4 DISCUSSION

4.1 Conformational changes upon Zn2+ binding

Zn2+ binding S100 proteins can be classified into two categories: His-rich and Cys- rich. Sequence alignments and high-resolution structures revealed a conserved binding motif for S100A9 with His-rich sites each consisting of 3 His and 1 Asp residues, at the dimer interface. Since the S100A9 proteins exist as a dimer, each protein binds two Zn2+ ions at the two symmetrically disposed sites (Figure 64B). Zn2+ concentrations inside cells are very low (e.g. 2–10 nmol L-1 in the cytoplasm). However, outside of the cell the Zn2+ concentration can reach 2-20 µM. S100A9 is capable of binding Zn2+ ions have affinity around 4 µM. This means that S100A9 is able to bind Zn2+ in the extracellular space but not inside the cell. ITC data for binding of Zn2+ to S100A9 in the absence and presence of Ca2+ and other metal ions like Mg2+ showed an interplay between Zn2+ and Ca2+ binding to S100A9 as. However, this interplay does not have a huge effect on the affinity of S100A9 towards Zn2+. In fact, the mechanism of Zn2+ binding in absence of calcium ions is changed and binding is less driven by entropic changes.

The amino acid side chains which are responsible for the binding of Zn2+ are three histidine residues (His95, His91, and His20) and one aspartate residue (Asp30). The binding site is illustrated in Figure 64B. The zinc binding was mediated by two residues of each S100A9 subunit, i.e. a monomer alone is not able to bind Zn2+. The binding site is close to a bound Ca2+ in the S100 specific EF-hand. Upon Zn2+ binding helix IV is elongated by two helical turns, moreover, the interhelical angle between helix III and helix IV is slightly increased by 5 ° opening the hydrophobic cleft (Figure 65)

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4.2 Analysis of RAGE–S100 interactions

The receptor for advanced glycation end products (RAGE) plays a central role in the innate immune system and serves as a pro-inflammatory ligand receptor, which induces important inflammatory signaling cascades leading to NFκB activation. The detailed binding mechanism between RAGE and its different ligands remains still unclear. The structure determination of a formed complex would be a groundbreaking result for basic research and also for the development of therapeutic approaches in the field of autoinflammatory diseases. To bring the topic one step forward co- crystallization trials between the ligand binding domain VC1 and the S100A9 were performed.

S100A9 is specifically involved in the phagocyte-derived immune response and is a high-affinity binding ligand for RAGE (Heizmann et al. 2002; Kierdorf & Fritz 2013; B. Chen et al. 2015). For the investigation of co-crystallization between RAGE and S100A9, the VC1 tandem domain of RAGE was used. The co-crystallization approaches were performed with approx. 5 mg/ml of each protein. For high affinity and stable complex formation 10 mM CaCl2 and 200 mM NaCl was added to the proteins. The protein solution of S100A9 was supplied with two equivalents of ZnCl2 per S100A9 molecule. However, until now, no S100A9-VC1 complex could be resolved at high resolution. Well diffracting crystals obtained from protein mixtures contained only S100A9 but not VC1.

Moreover, I aimed to understand how S100A9 and other proteins interact with RAGE, I have applied different techniques and analyzed the interaction of S100A9, S100A8A9 and S100A12 with VC1 domain of RAGE by SPR and MST techniques.

According to previous studies (Xie et al. 2013; Riva et al. 2012; Yatime & Andersen 2013; Rauvala & Rouhiainen 2010), the strength of the signaling cascade inside the cell is dependent on the affinity between the interaction partners and the duration of the active signaling complex. Therefore, it is necessary to resolve the association and dissociation phase of the binding process. In particular, the dissociation phase is of

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significant interest because it directly reflects the lifetime of a signaling-competent complex. The common factor linking these S100 proteins is their tendency to oligomerize. Preassembly has substantial implications for the mechanism of RAGE activation by its diverse ligands (Donato 2007; Koch et al. 2010). Indeed, earlier studies have demonstrated the formation of dimers among receptors. For instance, S100A12 dissociates from VC1 much faster than other tested S100 proteins and probably induces less pronounced receptor activation than others. Moreover, SPR results also revealed faster association for S100A12 than for S100A9, S100A8A9 which is in good agreement with the hexameric state of S100A12. However, these results are also contradicting the proposed prolonged binding of multimeric ligands to the receptor.

4.3 Effect of N-glycosylation on ligand binding affinity of RAGE

Native RAGE is N-glycosylated at two potential sites in the V domain, but the bacterially expressed proteins are not glycosylated. RAGE glycosylation has a minimal effect on the affinity of binding of some S100 proteins but a large effect on some others. In summary there is evidence from the SPR results (Figure 57 to Figure 62 and Table 5) that glycosylated VC1 binds S100 proteins with a different mechanism than non-glycosylated VC1. Incomplete dissociation of glycosylated VC1 and S100 proteins indicate stronger binding. Likewise, higher responses ofA100s towards glycosylated VC1 than to non-glycosylated VC1 support the hypothesis that glycosylation of RAGE affects ligand binding and downstream signaling (Osawa et al. 2007; Dattilo et al. 2007).

SPR allowed me to follow the interaction between same S100 proteins (analyte) with glycosylated and non-glycosylated VC1 in real time. That way I could investigate in great detail the effect of glycosylation on the interaction of several S100 proteins with VC1. The initial study was performed with S100A9, S100A8/A9 and S100A12 and showed nanomolar to micromolar affinity to non-glycosylated VC1. Using the same approach, I showed that S100 proteins bind with the same affinity but not same association and dissociation behaviors to glycosylated VC1 suggesting that

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glycosylation might not affect the affinity to its ligands but for sure the mechanism of binding that is clearly not the same. In addition, SPR results revealed that the presence of calcium and zinc ions is essential for the binding.

4.4 Analysis of S100A9-inhibitors interactions

I have analyzed and characterized a large series of different compounds that inhibit S100A9-receptor interaction. These compounds were provided by our collaboration partner (Active Biotech Company, Lund, Sweden) and have been explored as treatments for autoimmune/inflammatory diseases in humans by block the interaction of RAGE with S100A9. Q-compounds were have tested in many clinical trials and S100SA9 is a novel target for these compounds (Björk et al. 2009). So it was one major goal of my Ph.D. thesis to learn more on the mechanism of inhibition of these compounds.

Investigation of inhibitors was started with 40 different Q-compounds. The first step was measuring the affinity binding between them and S100A9 (2.2.2.4.1). ITC measurements were not successful since some cases Q-compounds were so weaker binder to S100A9 or were not soluble in ITC buffer. As shown in Figure 56, a monotonic decrease in the heat evolved when increasing amounts of Q- compounds were added. That suggests that the small molecules bound only to one type of binding sites. However, a different trend in the isotherms for Q-compounds can be observed when changing the concentrations. The best experimental conditions in this study were keeping the concentration of Q-compounds was around 300 µM. Increasing the compounds concentration higher than 300 µM caused early saturation of S100A9.

For Q-compounds in the first few injections, S100A9 is much in excess of the added compounds, and an almost constant heat release is observed. The plateau region reveals that the added Q-compounds are bound completely to S100A9, however some of them are a weak binder and some of them are a strong binder. As the titration continues (the concentration of Q-compounds increases), the free

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compounds concentration in the reaction cell decreases and the heat flow also decreases. Clearly each compound displays individual heat decrease curves. Even highly similar Isomer compounds, show a different course of titration.

Due to an increase in the endothermic contribution, the peaks suddenly become smaller and this point of change is called reflection point. Obviously, reflection points from one compound to another one were different.Accordingly to theory, at higher Q- compounds concentrations, no heat of interaction should be observed (i.e. the heat value is constant and around zero). However, for several compounds no saturation of S100A9 was observed. So it was not possible to determine exactly at what molar the compound bound to S100A9.

Although it was expected that Q-compounds of similar chemical structure might be led to similar binding isotherms or similar thermodynamic parameters, marked differences are observed in the shape of the curves and reflection and saturation points which show different mechanism of binding.

4.5 Characterization of Q-compounds binding sites on S100A9.

Q-compounds represent an important class of molecules for the treatment of autoinflammatory and autoimmune diseases, which are tested in many clinical trials from phase I-III (Björk et al. 2009; Isaacs 2010). The structural characterization of the binding sites of Q-compounds in S100A9-Ca2+-Zn2+ provides first insights into the binding mode and the effects on S100A9.

Co-crystallization and soaking experiments of one of the Q-compounds (ABR- 215757) were started by Dr. Christine Betz (Betz 2013) (working group Fritz) and no electron density for this compound was observed at that time. The project to characterize compound binding sites was continued by Dr. Stephan Giesler. In his Ph.D. thesis, he characterized the binding sites for compound ABR-239652 in S100A9 proteins by co-crystallization (Giesler 2015).

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The best results were obtained from crystals grown in 0.05 M MES/NaOH pH 6.3 with 37% PEG400 and soaked with 1 mM to 5 mM different Q-compounds from 24 hours to 48 hours after adding up to 10% of DMSO to the soaking buffer. However, some Q-compounds were not well soluble in the soaking buffer as indicated by crystals of compounds formed during incubation. In total, more than 30 different compounds were analyzed in this work, resulting in 8 new structures of S100A9-compound complexes. It turned out that addition of DMSO was crucial for successful soaking of several compounds. Addition of up to 10 %of DMSO to soaking buffer was strictly required to obtain a structure.

Strikingly, compounds with different core structure bind to the same site in S100A9. Common to all compounds is the tight closure of the hydrophobic pocket of S100A9 that serves as interaction site with RAGE. Interestingly, preferentially only one binding pocket was occupied by the compounds which might be due to crystal packing

The crystal packing is important for compound binding during soaking experiments. Diffusion of the compounds occurs through the solvent channels in the crystals. ITC data showed that the compound is able to bind both monomers of S100A9 dimer, but in the crystal only one monomer is accessible. The quinolone heterocycle in all bound Q-compounds remains on the top of the pocket and the side chains point into the cavity. This binding mode is associated with a few structural changes that were observed after binding of compounds. However, these conformational changes were not similar for all compounds binding to S100A9. For example, the binding cavity was much deeper upon ABR 215050 binding than for the other compounds and more residues were involved in binding (Figure 74). In summary all compounds block a hydrophobic pocket between helix III and helix IV of S100A9 and thereby block binding of S100A9 to its receptors.

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6 Supplements

6.1 List of figures

Figure 1: Primary structure of RAGE and domain organization...... 8

Figure 2: Structure of RAGE and ligand-binding sites...... 10

Figure 3: Structure of the Tandem VC1 Domains of RAGE ...... 11

Figure 4 : Activation of RAGE and signaling pathway...... 13

Figure 5: Structural organization of EF-Hands...... 16

Figure 6: Conformational changes of S100 protein upon ca2+ binding and target binding. ....16

Figure 7: The cartoon representation of S100A9 dimer...... 17

Figure 8: SPR sensograms of S100A9-RAGE and S100A8/A9-RAGE interaction...... 19

Figure 9: Chemical structure of Q-compound ABR 215050...... 20

Figure 10: Chemical structure of Quinoline-3-carboxamide ABR 21215062...... 20

Figure 11: Scheme of blockage of RAGE-S100A9 interaction by inhibitors...... 21

Figure 12: Chemical structure of Quinoline-3-carboxamide ABR 215050...... 26

Figure 13: Chemical structure of Quinoline-3-carboxamide ABR 239071...... 27

Figure 14: Chemical structure of Quinoline-3-carboxamide ABR 239981...... 27

Figure 15: Chemical structure of Quinoline-3-carboxamide ABR 239105...... 28

Figure 16: Chemical structure of Quinoline-3-carboxamide ABR 239709...... 28

Figure 17: Chemical structure of Quinoline-3-carboxamide ABR 240007...... 29

Figure 18: Chemical structure of Quinoline-3-carboxamide ABR 249974...... 29

Figure 19: Chemical structure of Quinoline-3-carboxamide ABR 240011...... 30

Figure 20: Chemical structure of Quinoline-3-carboxamide ABR 239979...... 31

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Figure 21: Chemical structure of Quinoline-3-carboxamide ABR240010 ...... 31

Figure 22: Chemical structure of Quinoline-3-carboxamide ABR239397 ...... 32

Figure 23 : Chemical structure of Quinoline-3-carboxamide ABR239338 ...... 32

Figure 24: Chemical structure of Quinoline-3-carboxamide ABR239508 ...... 33

Figure 25 : Representative diagram of typical ITC experiment...... 52

Figure 26 : Overview of MicroScale Thermophoresis (MST)...... 54

Figure 27 : Overview of SPR experiments using NTA chips...... 57

Figure 28: Schematic of a SPR measurement for VC1243 and S100 proteins ...... 58

Figure 29 : Protein crystallization phase diagram...... 60

Figure 30: Vapor diffusion method in a hanging drop system...... 61

Figure 31: Schematic of a wave scattered at crystal lattice planes...... 65

Figure 32: Schematic depiction of an oscillation method diffraction pattern...... 66

Figure 33: Recombinant expression of His6-VC1243...... 69

Figure 34: SDS-PAGE of cell extracts of His6-VC1243...... 70

2+ Figure 35: Purification of His6-VC1243 by Ni -Sepharose (IMAC) ...... 71

Figure 36: SDS-PAGE of the fractions from Ni2+-Sepharose run...... 72

Figure 37: Size exclusion chromatography of His6-VC1243...... 73

Figure 38: SDS-PAGE from the SEC purification step of His6-VC1243...... 74

Figure 39: SDS-PAGE of cell extracts of sRAGE...... 75

Figure 40: Ni2+-Sepharose (IMAC) chromatography result of sRAGE...... 76

Figure 41: SDS-PAGE of the fractions from Ni2+-Sepharose run...... 77

Figure 42: Thrombin cleavage of the His-sRAGE for removal of the His6-tag...... 78

Figure 43: Purification of sRAGE by size exclusion chromatography...... 79

Figure 44: SDS-PAGE from the SEC purification step of sRAGE...... 80

Figure 45: Purification of S100A9 by ion exchange chromatography...... 81

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Figure 46: SDS-PAGE of S100A9 corresponding fractions from IEX run...... 82

Figure 47: Purification of S100A9-C3S by size exclusion chromatography...... 83

Figure 48: Coomassie-stained SDS PAGE of the second SEC run...... 84

Figure 49: Crystallization of a putative S100A9-VC1 complex ...... 86

Figure 50 : Crystals obtained after fine seed screening of VC1-S100A9...... 87

Figure 51: Analysis of S100A9 binding to RAGE-VC1 by MST...... 88

Figure 52: Schematic illustration of calorimetric titrations of Zn2+ into S100A9...... 89

2+ Figure 53: Isotherm corresponding to the binding of Zn to S100A9/A9 at 25 °C...... 90

2+ Figure 54: ITC titrations of the ZnCl2 domain with S100A9 in presence of Mg ...... 91

Figure 55: Binding of zinc by titrating S100A9 with Zinc without calcium...... 92

Figure 56: ITC titrations of the Q-compounds into S100A9...... 95

Figure 57: SPR binding curves and analysis for S100A9-VC1...... 96

Figure 58: SPR binding curves and analysis for Zn-S100A9 and glycosylated VC1...... 97

Figure 59: SPR binding curves and analysis for Zn-S100A8/A9 and VC1...... 97

Figure 60: SPR binding curves and analysis for Zn-S100A8/A9 and glycosylated VC1...... 98

Figure 61: SPR binding curves and analysis for S100A12 and VC1...... 99

Figure 62: SPR binding curves for interaction between S100A12 and glycosylated VC1...... 99

Figure 63: S100A9 Crystals in 0.1 M Mes/NaOH pH 6.5, 35% PEG 400...... 100

Figure 64: Cartoon representation of S100A9 homodimer...... 103

Figure 65: Comparison of the surfaces of S100A9-Ca2+and S100A9-Ca2+-Zn2+...... 104

Figure 66: Hydrophobic binding pocket for ABR 239979 in S100A9-Ca2+-Zn2+...... 105

Figure 67: Hydrophobic binding pocket for ABR 239338 in S100A9-Ca2+-Zn2+...... 106

Figure 68: Binding cavity of (Tasquinimod) ABR 215050 in S100A9-Ca2+-Zn2+...... 106

Figure 69: Binding cavity of ABR 239508 in S100A9-Ca2+-Zn2+...... 107

Figure 70: surface representation of binding pocket for ABR 239397 in S100A9-Ca2+-Zn2+...... 107 126

Figure 71: surface representation of binding pocket for ABR 240007 in S100A9-Ca2+-Zn2+...... 108

Figure 72: Binding cavity of ABR 240010 in S100A9-Ca2+-Zn2+...... 108

Figure 73: Hydrophobic binding pocket for ABR 239105 in S100A9-Ca2+-Zn2+...... 109

Figure 74: All compounds bind to the same site in S100A9...... 109

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6.2 List of tables

Table 1: Name of chemicals and companies...... 23

Table 2: Name and source of Initial screens...... 43

Table 3: Crystallization conditions and diffraction data of a putative VC1243-S100A9 complex...... 85

Table 4: Thermodynamic parameters of each ITC experiments for Q-compounds against the binding for S100A9 ...... 93

Table 5: More details on kinetic and binding parameters from SPR measurements ...... 100

Table 6: Data collection statistics of S100A9-Ca2+-Zn2+ crystals...... 101

Table 7: Refinement statistics of S100A9-Ca2+-Zn2+ crystals...... 102

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6.3 Abbreviations

A8 S100A8 A9 S100A9 AGE Advanced glycation endproducts Asp Aspartate Aβ Amyloid β BSA Bovine serum albumin Ca Calcium cryoEM Cryo-electron microscopy CV Column volume DAMPs damage-associated molecular pattern proteins DESY Deutsches Elektronen Synchrotron DMSO Dimethyl sulfoxide DYT Double yeast tryptone EDTA Ethylenediaminetetraacetic acid GdnHCl Guanidinium hydrochloride HEPES 2-(4-(2-Hydroxyethyl)-1-piperazinyl)- Ethan sulfoacid His Histidine HMGB1 High-mobility group protein B1 IEX Ion exchange chromatography Ig Immunoglobulin IL-1 β Interleukin-1 β IMAC immobilized metal ion affinity chromatography IPTG Isopropyl β-D-1 -thiogalactopyranoside ITC Isothermal titration calorimetry Jak/STAT Janus kinase/signal transducers and activators of transcription. kDa kilo Dalton M Molar MAP mitogen-activated protein MS Multiple sclerosis MW molecular weight NF-κB Nuclear factor 'kappa-light-chain-enhancer' of activated B-cells NMR Nuclear magnetic resonance spectroscopy PAMPs pathogen-associated molecular patterns PEG Polyethyleneglycol PRR pattern recognition receptor PSI Paul Scherrer Institute Q-compounds Quinoline-3-carboxamide compounds RAGE receptor for advanced glycation end products S100A9-Ca S100A9 with supplemented Ca2+ S100A9-Ca-Zn S100A9 with supplemented Ca2+ and Zn2+ SDS sodium dodecyl sulfate SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis SEC size exclusion chromatography SPR Surface plasmon resonance SN supernatant TMM Transition metal mix TNFα tumor necrosis factor α TLR toll-like receptor Tris Tris(hydroxymethyl)-aminomethane Zn Zinc

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6.4 Acknowledgments

Firstly, I would like to express my sincere gratitude to my advisor Dr. Günter Fritz for the continuous support of my Ph.D. study and related research, for his patience, motivation, and immense knowledge. His guidance helped me in all the time of research and writing of this thesis. I could not have imagined having a better advisor and mentor for my Ph.D. study. Besides my advisor, I would like to thank Prof. Dr. Oliver Einsle, for his insightful comments and encouragement, but also for the hard question which incented me to widen my research from various perspectives.

My sincere thanks also goes to Prof. Susana Andrade, who provided me an opportunity to join her team’s office, and who gave access to the laboratory and research facilities. Without her precious support it would not be possible to conduct this research. I would also like to thank my thesis committee Prof. Dr. Andreas Bechthold for serving as my committee member.

I gratefully acknowledge the funding received towards my Ph.D. from International Graduation academy (IGA), University of Freiburg (The State low on Graduate Funding –Landesgraduiertenförderung-(LGFG)).

I greatly appreciate the support received through the collaborative work undertaken with Thorsten Brink at the institute of Pharmacology and Toxicology university of Freiburg (laboratory Prof. Dr. Dr. Klaus Aktories), Dr. Genny Degani at University of Milan, Dr. Nathalie Eisenhardt at Max Plank Institute of Immunobiology and Epigenetics, Freiburg.

This Ph.D. study would not have been possible without the corporation and continuous support by Active Biotech Company.

I would especially like to thank my colleagues and lab mates Dr. Stephan Giesler, Anita Arnold, Prachi Dabhade, Maria Agostina di Renzo, Philipp Lewe, Pauline Bentz, and Fabian Frank at the institute of biochemistry, the University of Freiburg for all the fun we have had in the last three years. All of you have been there to support me during my Ph.D. thesis.

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I would also like to thank my family. Words cannot express how grateful I am. To my beloved mother (Farideh), your prayer for me was what sustained me thus far. To my beloved sisters (Dr. med. Reyhaneh Michael, Rana Tadayon, and Rayeheh Deutsch), my supportive brother in laws (Dr. rer. nat. Joachim Michael and Matthias Deutsch) and, my beautiful nieces (Melodie and Emily), Thank you for supporting me for everything, and especially I can’t thank you enough for encouraging me throughout this experience. You are the most important people in my world.

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