Structural insights into signal-transducing proteins : Regulation of the EF-hand protein S100A2 and activation of the Receptor for Advanced Glycation Endproducts

S TRUCTURAL I NSIGHTS INTO S IGNAL - TRANSDUCING

P ROTEINS : R EGULATION OF THE EF- HAND P ROTEIN

S100A2 AND A CTIVATION OF THE R ECEPTOR FOR

A DVANCED G LYCATION E NDPRODUCTS

D ISSERTATION

Z

E

G

D

N

(Dr. rer. nat.)

I

F

B

U

K

VORGELEGT VON

Dipl. Biol. M

K

DISSERTATION DER UNIVERSITÄT K^ONSTANZ TAG DER MÜNDLICHEN PRÜFUNG:06.11.2007 REFERENT:PROF.DR.P.M.H.KRONECK

REFERENT:PROF.DR.C.W.HEIZMANN

REFERENT:PROF.DR.W.WELTE

„I am among those who think that Science has great Beauty. A Scientist in his Laboratory is not only a Technician: he is also a Child placed before natural Phenomena which impress him like a fairy Tale.”

Marie Curie (1867-1934)

DEDICATED TO LORENZ,MELANIE, MIRIAM AND FRIEDERIKE

-TABLE OF CONTENTS-

T ABLE OF C ONTENTS

ZUSAMMENFASSUNG III

SUMMARY VII

INTRODUCTION 1

1.1 CA²⁺ IN BIOLOGICAL SYSTEMS 1

1.2 CA²⁺-BINDING PROTEINS 3

1.3 EF-HAND PROTEINS 4

1.4 ZN²⁺ IN BIOLOGICAL SYSTEMS 8

1.5 THE S100 PROTEIN FAMILY 10

1.6 THE S100A2 PROTEIN 15 1.7 RAGE, THE RECEPTOR FOR ADVANCED GLYCATION ENDPRODUCTS 16

1.8 SCOPE OF THIS STUDY 20

MATERIALS & METHODS 21

2.1 CRYSTALLOGRAPHY 21

2.2 SURFACE PLASMON RESONANCE 30

RESULTS 33

3.1 IMPLICATIONS ON ZINC BINDING TO S100A2 33

3.1.1 ABSTRACT 33

3.1.2 INTRODUCTION 34

3.1.3 MATERIALS AND METHODS 36

3.1.4 RESULTS 40

3.1.5 DISCUSSION 49

3.1.6 FIGURES AND TABLES 52

3.1.7 SUPPLEMENTARY DATA 61

3.2 PURIFICATION AND CRYSTALLIZATION OF THE HUMAN EF-HAND TUMOR SUPPRESSOR PROTEIN

S100A2 66

3.2.1 ABSTRACT 66

3.2.2 INTRODUCTION 66

3.2.3 MATERIALS AND METHODS 67

3.2.4 RESULTS AND DISCUSSION 70

-TABLE OF CONTENTS-

3.3.1 A^BSTRACT 74

3.3.2 INTRODUCTION 75

3.3.3 MATERIALS AND M^ETHODS 77

3.3.4 RESULTS AND DISCUSSION 78

3.3.5 FIGURES AND TABLES 83

3.4 CRYSTAL STRUCTURE OF CA²⁺-LOADED S100A2 AT 1.3 Å RESOLUTION 87

3.4.1 ABSTRACT 87

3.4.2 INTRODUCTION 88

3.4.3 MATERIALS AND M^ETHODS 89

3.4.4 RESULTS AND DISCUSSION 90

3.4.5 FIGURES AND TABLES 97

3.5 THE CRYSTAL STRUCTURE OF HUMAN RAGELIGAND-BINDING DOMAIN 106

3.5.1 ABSTRACT 106

3.5.2 INTRODUCTION 107

3.5.3 MATERIALS AND METHODS 108

3.5.4 RESULTS 109

3.5.5 FIGURES AND T^ABLES 114

CONCLUSIONS 119

4.1 STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF S100A2 119

4.1.1 METAL BINDING TO S100A2 119

4.1.2 IMPLICATIONS OF METAL BINDING ON THE CELLULAR FUNCTION OF S100A2 122 4.2 STRUCTURE AND LIGAND-BINDING PROPERTIES OF RAGE 124

4.2.1 LIGAND BINDING TO RAGE 124

4.2.2 MECHANISMS FOR ACTIVATION AND BLOCKING OF RAGE 126

REFERENCES 129

APPENDIX 146

6.1 ABBREVIATIONS 146

6.2 AMINO ACIDS 148

6.3 INTERNATIONAL SYSTEM OF UNITS (SI) 148

6.4 CURRICULUM VITAE 149

6.5 CONFERENCE POSTERS AND TALKS 151

6.6 PUBLICATIONS 152

ACKNOWLEDGEMENTS 153

-ZUSAMMENFASSUNG-

Z USAMMENFASSUNG

1. S100A2 gehört zur Familie der S100 Proteine, welche die größte Untergruppe der Ca²⁺-bindenden EF-Hand Proteine darstellt. S100 Proteine bestehen aus einer modifizierten S100-spezifischen EF-Hand am N-Terminus und einer C-terminalen klassischen EF-Hand.

Unter physiologischen Bedingungen liegen sie als Homo- oder Heterodimere vor. S100A2 wurde als Tumorsuppressor identifiziert, und kürzlich wurde gezeigt, dass S100A2 Ca²⁺

abhängig p53 bindet und aktiviert. In mehreren Tumorarten wurden jedoch erhöhte S100A2 Konzentrationen nachgewiesen, was auf eine komplexere Funktion des Proteins während der Tumorgenese hindeutet. Diese Beobachtungen zeigen die zentrale Bedeutung von S100A2 bei der Regulierung des Zellzyklus.

Neben Ca²⁺ bindet S100A2 auch Zn²⁺ mit hoher Affinität. Bisher gab es keine übereinstimmenden Studien bezüglich Stöchiometrie, Affinität, Geometrie und Lage der Zn²⁺

Bindestellen.

In der vorliegenden Arbeit wurden rekombinantes S100A2 und verschiedene S100A2 Cystein Æ Serin Varianten in Escherichia coli exprimiert und zur Homogenität aufgereinigt. Die korrekte Faltung der S100A2 Varianten wurde durch Circular Dichroismus Spektroskopie nachgewiesen.

Die hochaffine Bindung von Zn²⁺ an S100A2 wurde anhand verschiedener spektroskopischer Methoden mit Cd²⁺ und Co²⁺ als intrinsische Sonden analysiert. Die Untersuchungen zeigten zwei verschiedene Zn²⁺ Bindestellen mit jeweils tetraedrischer Koordination. Die erste Bindestelle liegt an der Kontaktfläche der beiden Untereinheiten, wohingegen die zweite Bindestelle durch die Zusammenlagerung zweier S100A2 Dimere gebildet wird. Dies führt zur Bildung eines Tetramers, wobei Zn²⁺ die zwei Dimere verbrückt.

In dieser Studie wird erstmals eine derartige Zn²⁺-abhängige Oligomerisierung für S100 Proteine vorgestellt. Die apparente Konstante Kd für Zn²⁺ wurde mit Kompetitionsexperimenten mit dem Zn²⁺ Chelator 4-(2-Pyridylazo)-Resorcinol bestimmt und beträgt 25 nM.

Die Ca²⁺ Affinitäten wurden durch intrinsische Tyrosinfluoreszenz und durch Titrationsexperimente mit dem kompetitiven Fluorophor BAPTA-5N bestimmt. Mit Kd

Werten von ~245 µM für die erste und ~60 µM für die zweite EF-Hand bewegen sich die

-ZUSAMMENFASSUNG- wurde. Zn²⁺ Bindung in der zweiten Bindestelle führt jedoch zu einer deutlichen Abnahme

(300-fach) der Ca²⁺ Affinität der einen EF-Hand und einer geringeren Abnahme (3-fach) der anderen EF-hand. Daher wird die Aktivität von S100A2 durch Ca²⁺ und Zn²⁺ kontrolliert, wobei Zn²⁺ Bindung die Ca²⁺ Affinität negativ reguliert.

S100A2 wurde in seiner inaktiven Ca²⁺-freien Form und in seiner aktiven Ca²⁺- gebundenen Form kristallisiert, um Erkenntnisse über die strukturelle Reorganisation während der Aktivierung des Proteins zu erhalten. Die Kristalle beugten bis zu einer Auflösung von 1.6 Å bzw. 1.3 Å. Beide Strukturen zeigen eine für S100 Proteine typische dimere Organisation. Eine Untereinheit besteht aus einer N-terminalen EF-hand, die von Helix I und II flankiert wird, sowie eine C-terminale EF-hand, die von Helix III und IV flankiert wird. Die zwei EF-Hände werden durch die sog. ‘Hinge’ Region verbunden. In den Elektronendichten ließ sich interessanterweise ein Na⁺ in der N-terminalen EF-Hand mit oktaedrischer Koordination nachweisen. Dies zeigt, dass in der Abwesenheit von Ca²⁺ bereits eine funktionelle Ca²⁺ Bindestelle vorgebildet ist, wohingegen die C-terminale EF-Hand eine offene Konformation ohne eine derartige Vorbildung aufweist. In der Ca²⁺-gebundenen Struktur wird das Ca²⁺ pentagonal bipyramidal koordiniert, wobei jeweils ein Wassermolekül die Koordination vervollständigt. Durch eine Änderung der Position von Helix III nehmen Helix III und Helix IV eine erheblich unterschiedliche Orientierung (Δ92°) im Vergleich zur Ca²⁺-freien Struktur ein. Diese ausgeprägte Umorganisierung der molekularen Architektur wird von der Aufhebung und Neubildung hydrophober Wechselwirkungen begleitet. Durch die Bewegung von Helix III öffnet sich eine hydrophobe Einbuchtung zwischen Helix III und Helix IV, die die Bindestelle für Zielproteine wie z. B. p53 darstellt. Diese Bindestelle zeichnet sich im Vergleich zu anderen Ca²⁺-gebundenen S100 Proteinen durch einzigartige Eigenschaften aus: sie weist eine signifikant grössere und tiefere hydrophobe Einbuchtung auf.

2. Der ‘Receptor for Advanced Glycation Endproducts’ (RAGE) ist ein Zelloberflächenrezeptor, welcher zur Familie der Immunoglobuline gehört. Der extrazelluläre Teil des Rezeptors besteht aus drei immunoglobulin-ähnlichen Domänen: einer V-Typ Domäne und zwei C-Typ Domänen. Eine einzelne transmembranäre Helix verbindet den extrazellulären Teil mit der kurzen zytosolischen Domäne. RAGE kommt häufig in Endothelgewebe vor, wird aber auch in anderen Geweben exprimiert. Zwei weitere Isoformen von RAGE entstehen durch ‘alternative Spleissvorgänge’: DN-RAGE (dominant negatives

-ZUSAMMENFASSUNG- repräsentiert die lösliche Form des Rezeptors und enthält nur die extrazelluläre Domäne (V-

C1-C2). sRAGE ist die weitaus häufigere Isoform von RAGE. Der Rezeptor bindet eine große Anzahl verschiedener Moleküle, wie ‘advanced glycation endproducts’ (AGEs), β- Faltblatt Fibrillen und mehrere S100 Proteine. Aufgrund des breiten Spektrums an Liganden wird RAGE auch als ‘pattern recognition receptor’ bezeichnet, der eher Strukturmotive als spezifische Liganden erkennt. Die Aktivierung von RAGE spielt eine zentrale Rolle in zahlreichen Krankheiten wie Diabetes, chronischen Entzündungen, Krebs und in der Alzheimer’schen Krankheit. Daher wird RAGE als ein potentielles therapeutisches Zielmolekül in einer Vielzahl von Krankheiten angesehen.

Drei verschiedene Domänen von RAGE (V-Domäne, V-C1-Domänen und C2- Domäne) wurden exprimiert und zur Homogenität aufgereinigt, um die Ligandenbindedomäne zu identifizieren. Fluoreszenztitrationen mit N^ε-Carboxymethyllysin (CML) zeigten, dass die V-C1-Domänen notwendig und ausreichend für Ligandenbindung an RAGE sind. Die kinetischen Analysen der Bindung von S100A12 und von ageBSA an V-C1- Domänen weisen eine hochaffine Bindung mit schnellen Assoziationsraten auf, wohingegen die Bindung von Amyloid-β durch eine langsame Assoziation gekennzeichnet ist. Amyloid-β dissoziiert allerdings nicht mehr von RAGE, was zu einer irreversiblen Bindung führt.

Darüber hinaus akkumuliert Amyloid-β am Rezeptor.

Kristalle der Ligandenbindedomäne von RAGE (V-C1-Domänen) beugten bis zu einer Auflösung von 1.85 Å. Die Struktur wurde mit der ‘multiple anomalous dispersion’ (MAD) Methode bestimmt, wobei Zn²⁺ als anomaler Streuer diente. Die elektrostatischen Oberflächenpotentiale der V-C1-Domänen zeigten, dass große positiv geladene Bereiche die Oberfläche dominieren. Daher zieht das Oberflächenpotential von RAGE negativ geladene Moleküle wie AGEs, S100 Proteine oder Amyloid-β an. Die Vielfalt zellulärer Reaktionen könnte durch verschiedene Bindestellen für die verschiedenen Liganden erklärt werden.

Bisher ist nicht vollständig verstanden wie RAGE aktiviert wird, und wie Signale ins Zellinnere übertragen werden. Eine Signalübertragung über die C2-Domäne und die transmembranäre Helix zur intrazellulären Domäne erscheint unwahrscheinlich. Daher wird vermutet, dass die Signalübertragung durch Rezeptoroligomerisierung vermittelt wird. Ein Aktivierungsmechanismus durch Dimerisierung von RAGE stimmt mit der Anordnung der V- C1-Moleküle im Kristall überein. Zwei Moleküle sind über die C1-Domäne seitlich aneinander angeordnet, wobei ein verbrückendes Zn²⁺ an der Kontaktstelle beteiligt ist.

Aufgrund dieser Wechselwirkung von zwei V-C1-Molekülen im Kristall wurden ‘surface

-ZUSAMMENFASSUNG- Wechselwirkung zwischen zwei V-C1-Molekülen. Auf der Grundlage dieser Ergebnisse wird

folgendes Modell für die Aktivierung von RAGE vorgeschlagen: Ligandenbindung an RAGE stabilisiert einen dimeren oder höher multimeren Zustand des Rezeptors. Dadurch nähern sich die zytosolischen Domänen einander an, und induzieren die Bildung einer Signalplattform, die die intrazellulären Signalkaskaden aktivieren kann.

-SUMMARY-

S UMMARY

1. S100A2 belongs to the family of S100 proteins which constitutes the largest subgroup of Ca²⁺-binding EF-hand proteins. They contain a modified S100-specific EF-hand at the N- terminus and a classical C-terminal EF-hand. Under physiological conditions they occur as homo- or heterodimers. S100A2 was identified as a tumor suppressor and was recently shown to bind and activate p53 in a Ca²⁺-dependent manner. However, in several tumors also elevated levels of S100A2 are reported, which point to a more complex role in tumorigenesis.

Altogether, the data show that S100A2 plays a central role in cell cycle regulation.

Besides Ca²⁺ S100A2 binds Zn²⁺ with high affinity. Up to now there was no consensus concerning stoichiometry, affinity, geometry, and location of the Zn²⁺-binding sites.

Recombinant human S100A2 and variants of S100A2 with cysteine to serine exchanges were expressed in Escherichia coli and purified to homogeneity. The proper fold of the S100A2 variants was proven by circular dichroism spectroscopy.

High affinity Zn²⁺ binding to S100A2 was investigated by different spectroscopic methods with Cd²⁺ and Co²⁺ as intrinsic probes. The investigations revealed two different Zn²⁺-binding sites with a tetrahedral coordination. Site 1 is located at the dimeric interface at the surface of S100A2, whereas site 2 is formed by the association of two S100A2 dimers resulting in a Zn²⁺-bridged tetrameric species. This is the first time that such a Zn²⁺-dependent oligomerization could be shown in S100 proteins. The apparent Kd for Zn²⁺ constitutes 25 nM as determined by competition with the Zn²⁺-chelator 4-(2-pyridylazo)-resorcinol.

By intrinsinc tyrosine fluorescence and titrations with the competitive fluorophore chelator BAPTA-5N the Ca²⁺ affinities of S100A2 were determined. With Kd values of

~245 µM for the first EF-hand and ~60 µM for the second EF-hand the Ca²⁺ affinities reside in a similar range as observed in other Ca²⁺-sensor S100 proteins. Zn²⁺ binding to site 2 resulted in a dramatic drop (~300-fold) in Ca²⁺ affinity of one EF-hand and a smaller drop (~3-fold) of the other EF-hand. Thus, the activity of S100A2 is controlled by Ca²⁺and Zn²⁺, with Zn²⁺ negatively modulating the Ca²⁺ affinity of S100A2.

In order to get insights into the structural rearrangement during activation, S100A2 was crystallized in its inactive Ca²⁺-free and in its active Ca²⁺-loaded form. Crystals diffracted to 1.6 Å and to 1.3 Å, respectively. Both structures showed a dimeric organization typical for

-SUMMARY- C-terminal EF-hand flanked by helix III and IV. The two EF-hands are connected by the so-

called hinge region. Interestingly, the electron density maps revealed a Na⁺ bound to the N- terminal EF-hand with an octahedral coordination. This demonstrates the pre-formation of an intact Ca²⁺-binding sphere, whereas the C-terminal EF-hand adopts an open non pre-formed conformation in the absence of Ca²⁺. In the Ca²⁺-loaded structure the Ca²⁺ ions are coordinated in a pentagonal bipyramidal manner, whereby an additional water molecule completes the coordination. Compared to the Ca²⁺-free form, helix III and helix IV show a significantly different orientation (Δ92°) as a result of a repositioning of helix III. This drastic molecular reorganization is associated with the breaking and formation of new intramolecular hydrophobic interactions. The movement of helix III opens a hydrophobic cavity between helices III and IV which constitutes the binding site for targets like p53. This binding site exhibits unique features compared to other Ca²⁺-bound S100 proteins: it is characterized by a significantly larger and deeper hydrophobic cavity.

2. The ‘Receptor for Advanced Glycation Endproducts’ (RAGE) is a member of the superfamily of immunoglobulin type cell surface receptors. The extracellular moiety of the receptor is composed of three immunoglobulin-like domains: one V-type domain and two C- type domains. A single transmembrane helix connects the extracellular part with the short cytosolic domain. RAGE is highly expressed by endothelial cells but also by other tissues. As a result of alternative splicing two further isoforms of RAGE exist: (i) DN-RAGE (dominant negative RAGE) is devoid of the intracellular signaling domain and the major isoform, (ii) sRAGE (soluble RAGE) is a soluble form of the receptor containing only the extracellular region (V-C1-C2). RAGE has the unusual property to bind a large range of different molecules such as advanced glycation end products (AGEs), ß-sheet fibrils , and several S100 proteins. Due to its broad repertoire of ligands RAGE is considered as a pattern recognition receptor which recognizes rather a structural motif than a specific ligand. RAGE activation has been implicated as a progressive factor in several disorders including diabetes, chronic inflammation, cancer and Alzheimer’s disease. Therefore RAGE is regarded as a potential therapeutic target in a various number of diseases.

In order to map the ligand-binding domain of RAGE, three different domains (V- domain, V-C1 domains and C2 domain) were expressed and purified to homogeneity. By fluorescence titrations with S100A12 and N^ε-carboxymethyllysine (CML) it was demonstrated that the V-C1 domains are necessary and sufficient for ligand binding to RAGE.

-SUMMARY- fast association rates for S100A12 and glycated BSA, whereas binding of amyloid-β is

characterized by slow association. However, amyloid-β did not dissociate from RAGE resulting in an irreversible binding. Moreover, amyloid-β accumulates on the receptor.

Crystals of the ligand binding domain of RAGE (V-C1 domains) diffracted to 1.85 Å and the structure was determined by multiple anomalous dispersion (MAD) using Zn²⁺ as anomalous scatterer. The electrostatic surface potential of V-C1 domains revealed that the surface is dominated by large positively charged patches. Thus, the surface potential of RAGE can act like a trap in attracting negatively charged molecules, such as AGEs, S100 proteins or amyloid-β. The large variety of cellular responses might be explained by different binding sites for the different ligands.

Up to now it is rarely understood how RAGE is activated and transfers signals into the cell. It is unlikely that the signal is transduced via the C2 domain and the transmembrane helix to the intracellular domain. Therefore it is proposed that RAGE signaling is mediated by ligand-induced receptor oligomerization. An activation mechanism via RAGE dimerization is consistent with the arrangement of V-C1 molecules in the crystal. Two molecules arrange side-by-side via C1 domain which involves a bridging Zn²⁺ ion at the interface. Based on this interaction of two V-C1 proteins in the crystal, ‘surface plasmon resonance’ experiments were performed which revealed a tight interaction between two V-C1 molecules. Based on these findings the following model for RAGE activation is proposed: ligand binding to RAGE stabilizes a dimeric or even higher multimeric state of RAGE. Thereby, the cytosolic domains of RAGE approximate which induces the formation of a signaling platform which can activate the intracellular signal-transducing cascade.

-INTRODUCTION-

1. I NTRODUCTION

1.1 Ca

in biological systems

In the late 19^th century Sidney Ringer already recognized the significance of calcium ions (Ca²⁺), when he demonstrated that small amounts of Ca²⁺ were necessary to maintain heart muscle contractility (Ringer, 1883). Today, it is well established that calcium is one of the most important metals in life. Of the approximately 1400 g of calcium in the human body most of it is immobilized in bones and teeth as hydroxyapatite. Only a minor part circulates as free Ca²⁺ in the blood and in the extracellular space or is stored intracellularly in distinct compartments (Carafoli, 2003).

Almost 80 years after the discovery by Ringer, physiologists assigned calcium as a second messenger in analogy to cyclic adenosine 3', 5’-monophosphate (cAMP) which had been shown to act as an important mediator of hormonal responses (Rasmussen, 1970; Scott, 1993; Sutherland et al., 1965). The term second messenger implies that Ca²⁺ is released intracellularly as an intermediary and transient signal produced upon excitation of the cell by external stimuli. The first messenger is the extracellular signal that may be a hormone, cytokine or a neurotransmitter originating from an endocrine gland or a neighboring nerve cell (Alberts et al., 2002; Carafoli, 2002). Thus, an external stimulus must modulate the intracellular Ca²⁺ concentration whereby the Ca²⁺ signal is generated. The Ca²⁺ signal itself is uniform and simple: in resting cells the Ca²⁺concentration is 100 nM but increases to

≥ 1000 nM upon activation (Berridge et al., 2000). The machinery which provides the Ca²⁺, senses the ion and removes it afterwards, represents a wide network of different components.

This interplay of components, the so-called Ca²⁺ toolkit, makes the Ca²⁺ signal enormously versatile. Variation of the toolkit can result in individual modulation and adaptation in response to a Ca²⁺ signal in a given cell.

The low Ca²⁺ concentration in the cytoplasm is strictly controlled and maintained by Ca²⁺ transporters which pump Ca²⁺ against a large electrochemical gradient into the extracellular space or into the endoplasmatic reticulum (ER). The Ca²⁺ concentration in the cytoplasm in the resting cell is maintained at approximately 100 nM whereas the extracellular

-INTRODUCTION- respectively. In the plasma membrane the Ca²⁺ ATPases and Na⁺/Ca²⁺ exchangers extrude the

Ca²⁺ to the outside whereas the sarco-endoplasmatic reticulum ATPases pump Ca²⁺ into the internal stores. However, more components are required to maintain homeostasis, if the Ca²⁺

concentration has largely increased during signaling. In this case the transport capacity of the pumps is too low to return fast enough to the basal resting level of Ca²⁺. For this purpose the Ca²⁺ is rapidly bound by buffer proteins which slowly release the Ca²⁺ to the cytoplasm after the signal (Carafoli, 2003; Strehler and Treiman, 2004).

The global Ca²⁺ signal is initiated by an amplification of a series of small subcellular Ca²⁺ signals which are generated by the opening of single or clustered Ca²⁺ channels.

Dependent on channel and cell type the signals last between a few milliseconds and seconds and have dimensions of a few hundred nanometers to several micrometers. At a certain level of Ca²⁺ the signals are largely amplified and a Ca²⁺ wave sweeps through the cell. When gap junctions connect cells, these intracellular waves can jump to the neighboring cells producing intercellular Ca²⁺ waves, which activate simultaneously a large number of cells (Berridge et al., 2003). Further variation is achieved by different sets of specific Ca²⁺-binding proteins (CaBPs), which change their conformation upon Ca²⁺-binding, and thus enables them to bind to specific effector proteins (Heizmann et al., 2002).

Today it is clear that Ca²⁺ ions represent an ubiquitous cellular signal regulating a plethora of cellular processes as depicted in Figure 1.1 (Berridge, 2005).

Figure 1.1: Calcium-modulated cellular functions (Carafoli, 2004).

-INTRODUCTION-

1.2 Ca

-binding proteins

Generally, Ca²⁺-binding proteins can be divided into two major subgroups according to their function. The first group comprises proteins which regulate and modulate the intracellular Ca²⁺ concentration (Ca²⁺-buffering and Ca²⁺-transporting proteins). The second group includes the Ca²⁺-sensor proteins which decode the Ca²⁺ signal into a molecular response that triggers the different molecular events leading to the specific cellular answer.

Soluble Ca²⁺-buffering and Ca²⁺-transporting proteins are localized in the cytosol and distinct organelles. In general, they are acidic proteins and exhibit low Ca²⁺-binding affinities.

Nevertheless, they are able to store large amounts of Ca²⁺. Two important Ca²⁺-buffering proteins are calsequestrin in the sarcoplasmatic reticulum and calreticulin in the endoplasmatic reticulum. The predicted numbers of Ca²⁺-binding sites for a calsequestrin monomer vary between 18 and 50 (Ikemoto et al., 1974; MacLennan and Wong, 1971;

Slupsky et al., 1987). These binding sites are thought to require only a pair of acidic residues, in contrast to the more complex structures found in the Ca²⁺-sensor proteins (Beard et al., 2004). Exceptions are parvalbumin and calbindin D28K, which both show typical features for sensor proteins but they serve as Ca²⁺-buffer /-shuttle proteins in the cytosol (Carafoli, 2005).

The most common calcium-binding motif found in Ca²⁺-sensor proteins, like troponin C, calmodulin or the S100 proteins, is the EF-hand motif. Despite a large range of functions, these Ca²⁺-sensor proteins are structurally closely related. The binding affinities for Ca²⁺ are adjusted in such a sophisticated manner, that the proteins are kept in their apo form (Ca²⁺-free state) in the resting cell. However, when the Ca²⁺ concentration rises as a response to external stimuli, Ca²⁺ binding occurs (Cormier, 1983). The Ca²⁺ binding is driven by a gain in entropy when water molecules surrounding the Ca²⁺ are liberated as Ca²⁺ binds to its target (Krause et al., 1991). A second type of Ca²⁺-binding site is the C2-domain. This domain was described in protein kinase C for the first time. It is formed by a stack of two four-stranded β- sheets, whereby the Ca²⁺ ions are coordinated by the residues of two or three loops (Figure 1.2 A) (Rizo and Südhof, 1998). The progressive binding of Ca²⁺ leads to a change in the electrostatic surface charge of the C2-domain (Figure 1.2 B) (Ubach et al., 1998). This change in the electrostatic surface potential of the C2-domain of synaptotagmin I enables binding to the phospholipid membrane (Férnandez-Chacón et al., 2002).

-INTRODUCTION-

Figure 1.2: A: Structure of the Ca²⁺-binding C2-type domain of synaptotagmin I (adapted from (Rizo and Südhof, 1998). B: Change in the electrostatic surface potential of the C2-domain of synaptogamin I upon progressive binding of Ca²⁺ to three Ca²⁺ sites. The electrostatic surface potential of the C2-domain in the Ca²⁺-binding region is shown before Ca²⁺ binding and after Ca²⁺

binding to the 1^st , 2^nd and 3^rd site (adapted from Ubach et al., 1998).

1.3 EF-hand proteins

To date 1162 different proteins carrying the EF-hand motif are known which are divided into 45 subfamilies (Haiech et al., 2004). In the human genome alone, 230 proteins containing one or more EF-hands have been identified. They function both, as Ca²⁺ sensor or Ca²⁺ buffer in eucaryotes and contain one or more EF-hand motifs (Carafoli, 2002; Lewit-Bentley and Réty, 2000; Yap et al., 1999).

The term EF-hand was introduced by Kretsinger based on the first three-dimensional structure of a protein from this family, that of parvalbumin (Kretsinger and Nockolds, 1973).

They found that Ca²⁺ is bound by a helix-loop-helix structure, whereby the helices involved in parvalbumin were helices E and F. The structure resembles a hand with stretched thumb and forefinger representing the helices and the middle finger representing the connecting loop (Tufty and Kretsinger, 1975) (Figure 1.3 A). This classical EF-hand motif is characterized by a sequence of 12 amino acid residues with the pattern X*Y*Z*–Y*–X**–Z. The positions X,

-INTRODUCTION- stars represent the intervening residues. This mode of Ca²⁺ binding was named the canonical

EF-hand (Lewit-Bentley and Réty, 2000) (Figure 1.3 B and Figure 1.6).

Figure 1.3: Schematic illustrations of the EF-hand. A: The EF-hand motif is symbolized by a right hand. Helix E (red) runs from the tip to the forefinger. The flexed middle finger corresponds to the loop region (green) of 12 residues that binds calcium (pink). Helix F (blue) runs to the end of the thumb. B:

The structure of the Ca²⁺-loaded EF-hand of troponin C. Ca²⁺ is coordinated by seven oxygen atoms, three from the side chains of Asp (D)9, Asn (N)11 and Asp (D)13, two from the side chain of Glu (E)20, one from the main chain (-y) and one from an additional water molecule (W) (adapted &

modified from Branden and Tooze, 1991).

The arrangement of the coordinating ligands allows binding of Mg²⁺ as well as of Ca²⁺. However, the coordination for both metal ions is different: Mg²⁺ is bound by six ligands in an octahedron whereas Ca²⁺ is bound by 7 ligands in a pentagonal bipyramid. This results in an high selectivity of the EF-hand for Ca²⁺, despite a large background of Mg²⁺ ions in the cell (0.5–1.0 mM).

EF-hands almost always occur in pairs in a face to face manner like in calmodulin or troponin C with four EF-hands arranged in two pairs. Parvalbumin and the S100 proteins represent the minimal motif with two EF-hands per functional unit (Lewit-Bentley and Réty, 2000). The pairing of the sites presumably stabilizes the protein fold and increases the Ca²⁺

affinity of the sites by different protein-protein interactions. An exception is calpain, a thiol protease regulated by Ca²⁺ which contains five EF-hand motifs (Blanchard et al., 1997; Lin et

-INTRODUCTION- The EF-hand containing Ca²⁺-sensor proteins undergo a large conformational change

upon Ca²⁺ binding. Coordination of the Ca²⁺ triggers a change in shape and surface charge of the protein which leads to binding and regulation of target proteins like e.g. protein kinases, phosphatases, adenylyl cyclases, metabolic enzymes, cytoskeleton proteins, and transcription factors. The binding and regulation of target proteins is illustrated by the action of calmodulin which is one of the best characterized Ca²⁺-sensor EF-hand proteins. Calmodulin is present in all cell types and is highly conserved as illustrated by 90% sequence identity in all multicellular eucaryotes (Moncrief et al., 1990). Calmodulin regulates a large number of different target proteins. The protein consists of two globular domains which are connected by a flexible helix. Both globular domains are composed of two adjacent EF-hands which bind Ca²⁺ with different affinities. In the Ca²⁺-free state the helices of the EF-hands are located in an antiparallel manner and the domain remains in a so-called closed conformation. Ca²⁺

binding induces a movement of the helices outwards, now enclosing an angle of about 90°.

The whole domain opens and exposes a hydrophobic surface patch for target-protein interaction (Bhattacharya et al., 2004). These structural changes are well documented in the crystal structure of Ca²⁺-loaded calmodulin as well as in several structures of calmodulin- target complexes. Figure 1.4 shows the structures of Ca²⁺-loaded calmodulin and two calmodulin-target complexes. These structures illustrate the diversity of possible conformations and how each conformation is unique to the particular, target-dependent activation mechanism. Consideration of these available structures suggests that the calmodulin specificity is inherent in the calmodulin-binding motif of the target proteins rather than in calmodulin itself (Klee and Means, 2002).

-INTRODUCTION-

Figure 1.4: Different modes of interaction of calmodulin with target proteins. Calmodulin is shown in blue and cyan; the bound Ca²⁺ ions are shown as yellow spheres. A: The structure of Ca²⁺-loaded calmodulin resembles a dumbbell whereby the central helix between the N- and C-terminal lobes is stretched (Chattopadhyaya et al., 1992). B: Complex of calmodulin with a fragment of the myosin light chain kinase (MLCK) shown in magenta. The two lobes wrap around the helical peptide forming a molecular envelope whereby several contacts are made through the hydrophobic surface patch (Meador et al., 1992). C: Complex of calmodulin with the regulatory domain of the Ca²⁺-dependent K⁺ channel. The structure exhibits two calmodulin molecules in an extended conformation which engage two regulatory domains of the K⁺ channel leading to dimerization and opening of the channel (Schumacher et al., 2001).

-INTRODUCTION-

1.4 Zn

in biological systems

Zinc represents an essential trace element for eucayrotic as well as for procaryotic organisms.

In the human body the total zinc amounts 2-4 g. Thereby, it is the most abundant transition metal occurring in humans after iron (Folkers et al., 2001). Despite Zn²⁺ is highly abundant in the cell (1-10 µM), it is tightly bound to different molecules and only a minor fraction is present as free Zn²⁺. The free Zn²⁺ concentration of eucaryotic cells in healthy tissue is assumed to range between 10⁻¹³ to 10⁻⁹ M (Choi and Koh, 1998; Outten and O'Halloran, 2001). The majority of the Zn²⁺ is bound to zinc finger proteins (Laity et al., 2001), metallothioneins (Vasak and Hasler, 2000), and reduced glutathione (Krezel et al., 2003;

Perrin and Watt, 1971). Compared to iron or copper Zn²⁺ is redox inactive because of its full 3d¹⁰ shell (Vallee and Auld, 1992). Therefore, Zn²⁺ is a stable ion in biological systems, where redox potentials frequently change. Protein-bound Zn²⁺ exhibits catalytic, structural as well as regulatory functions.

Catalytic Zn²⁺: The coordination number in catalytic Zn²⁺-binding sites is four or five.

This leads to a distorted-tetrahedral or trigonal-bipyramidal coordination sphere, whereby a water molecule is involved throughout. The water molecule is ionized or polarized by basic amino acid residues depending on the arrangement of the remaining ligands. This provides hydroxide ions for a nucleophilic attack on the substrate at neutral pH. Due to its properties as lewis acid Zn²⁺ participates in acidic catalysis, whereby the water ion is replaced by the substrate (Bertini et al., 1985). Zn²⁺-containing enzymes are for example the well established alcohol dehydrogenase, carboxypeptidase or carboanhydrase (overview in (Auld, 2001).

Structural Zn²⁺: A major task of Zn²⁺ is the induction of a particular conformation or the stabilization of a quaternary structure. This includes the assistance in protein folding by reducing the enthalpy (Blasie and Berg, 2002). The first characterization of a structural Zn²⁺

was in the alcohol dehydrogenase more than 30 years ago (Eklund et al., 1974). However, the most prominent structure with a structural Zn²⁺ is the zinc finger domain. It was first described in 1985 in the transcription factor TFIIIA from Xenopus laevis as a repetitive motif (Miller et al., 1985). Proteins containing a zinc finger motif are involved in protein folding, recognition of DNA-target sequences as well as in lipid binding. The Cys2His2 zinc finger is the most common DNA-binding motif encoded in the human genome and in the genome of other multi-cellular eucaryotes (Figure 1.5). The human genome is estimated to contain approx. 700 Cys2His2 zinc finger coding genes (Papworth et al., 2006). Today, the main focus is the engineering of customized zinc fingers capable of binding specific DNA sequences, and

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Figure 1.5: Schematic representation of the zinc finger of the embryonic protein Xfin from Xenopus laevis. The domain is composed of an antiparallel β-hairpin (red) connected by a loop (blue) and a following α-helix. The Zn²⁺ is coordinated in a tetrahedral fashion by a Cys₂His₂ motif (Branden and Tooze, 1991).

Regulatory Zn²⁺: There is emerging evidence for the role of Zn²⁺ as a key regulator in cellular processes (Frederickson, 2003; Maret, 2002). Intracellularinformation transferred by Zn²⁺ binding to proteins, in a manner analogous to Ca²⁺, was already discussed more than a decade ago (O'Halloran, 1993). The large number of Zn²⁺-dependent DNA-binding and regulatory proteins implies that changes in free Zn²⁺ are directly translated into altered gene expression (Berg and Shi, 1996). Actually, rapid changes in free Zn²⁺ are observed upon opening of Ca²⁺ channels during signaling events (Frederickson, 2003; Li et al., 2001a; Li et al., 2001b) or as a result of oxidative stress (Maret, 2004; Tatsumi and Fliss, 1994; Turan et al., 1997). Within signaling microdomains the transient Zn²⁺ concentrations have been reported to rise very fast up to 0.1-10 µM (Devinney et al., 2005; Frederickson, 2003). This elevated Zn²⁺ concentration in the cytoplasm is rapidly decreased by Zn²⁺-specific transporters which pump the Zn²⁺ into vesicles (McMahon and Cousins, 1998). Moreover, a recent study shows that proteins with lower affinity could bind Zn²⁺ by the formation of a ternary complex with another Zn²⁺-binding protein. This finding further documents that Zn²⁺

transfer can occur rapidly in the virtual absence of free Zn²⁺ (Heinz et al., 2005). Thus, the changes in free Zn²⁺ have many parallels to the classical Ca²⁺ signaling: an initial signal leads

-INTRODUCTION- regulating their biological activity. The signal is terminated by removal of the metal ion into

cellular compartments.

1.5 The S100 protein family

S100 proteins form the largest subgroup within the superfamily of EF-hand Ca²⁺-binding proteins. The first S100 protein was discovered in 1965 by Moore. He investigated proteins specific for the nervous system in higher animals and isolated a protein from bovine brain that turned out to be completely soluble in a 100% saturated ammonium sulfate solution (S100) (Moore, 1965). Meanwhile, in human 21 different S100 proteins have been identified. Their sequence identities range from 22% to 57% showing that the diversity within the family is quite large (Figure 1.6). The length of the proteins varies between 78 residues (S100G) and 114 residues (S100A9). They are small acidic proteins with molecular masses between 10- 12 kDa. Except S100G (calbindin D9k) all S100 proteins form non-covalent homo- and heterodimers (Fritz and Heizmann, 2004; Heizmann et al., 2003; Marenholz et al., 2006). So far, S100 proteins have been isolated only from vertebrates where they serve intracellularly as Ca²⁺ sensors, except S100G, which serves as a Ca²⁺ buffer. Other EF-hand proteins like troponin C and calmodulin are also found in invertebrates, which implies that the S100 family is an evolutionary rather young group within the EF-hand superfamily. Interestingly, most of the S100 proteins (16 out of 21) are found on one gene cluster that is highly conserved in mammalia, which points towards multiple gene duplication. In human the gene cluster is located on chromosome 1, region q21 (Marenholz et al., 2004; Ridinger et al., 1998). Besides Ca²⁺ (Kd = 20-500 µM) many S100 proteins bind Zn²⁺ (Kd = 0.1-2000 µM) and Cu²⁺ (Kd = 0.4-5 µM) with high affinities which regulate their signaling activity.

Each S100 subunit consists of two EF-hands, an N-terminal EF-hand and a C-terminal EF-hand, which is followed by a C-terminal extension. The two EF-hands are connected by a short region, referred to as the hinge region (Figure 1.7). The S100 members differ from one another mostly in the length and sequence of the hinge region and the C-terminal extension, which are thus suggested to determine the biological activity of the individual proteins (Donato, 2003). The plane for dimerization (~2500 Ų) of S100 proteins is composed of hydrophobic residues which are absent in S100G. Generally, S100 proteins are characterized by the presence of two Ca²⁺-binding sites of the EF-hand type (i.e., helix-loop-helix), whereby the N-terminal EF-motif is composed of 14 residues which is specific for S100 proteins and a

-INTRODUCTION- 2004). One consequence of the different lengths of the different EF-hands is that Ca²⁺ binding

to the individual EF-hands occurs with different affinities: a lower affinity in the case of the N-terminal site and a ~100-times higher affinity in the case of the C-terminal site.

Figure 1.6: Multiple sequence alignment of human S100 proteins. The Ca²⁺-coordinating residues are highlighted in dark-red (side chain coordination), and light-red (backbone oxygen coordination). The Zn²⁺-coordinating residues in S100A7 and the Cu²⁺-binding site in S100A12 are highlighted in magenta. Hydrophobic residues that are essential for dimerization are highlighted in green. Residues which are putative Zn²⁺ ligands in other S100 proteins are highlighted in yellow (cysteine) and blue (histidine), respectively.

-INTRODUCTION- The N-terminal EF-hand flanked by helices I and II exhibits only small conformational

changes upon Ca²⁺ binding. Helix II undergoes a small rearrangement in direction of the Ca²⁺- binding site with practically no change in the interhelical angle of helix I and II (Figure 1.8 A). The C-terminal canonical EF-hand represents the target interaction site of the Ca²⁺- sensor S100 proteins. The EF-hand is characterized by the typical pattern X*Y*Z*–Y*–X**–

Z and classified as a classical or canonical EF-hand (Figures 1.6 and 1.8 B). In the Ca²⁺-free state the helices III and IV flanking the EF-hand loop adopt an antiparallel conformation (Figure 1.8 B) similar to the EF-hands in Ca²⁺-free calmodulin (Ishida et al., 2002).

Figure 1.7: Schematic representation of human Ca²⁺-loaded S100A6 (pdb code 1K96) (Otterbein et al., 2002) showing the homodimeric organization of S100 proteins. The subunits are coloured in red/orange and blue/cyan; bound Ca²⁺ ions are shown as yellow spheres. The two EF-hands of each subunit are connected by the hinge region (adapted from Fritz and Heizmann, 2004).

In the Ca²⁺-loaded state of S100 proteins there is a large change in the orientation of helix III (Figure 1.8 B), whereas helix IV that is engaged in the dimer interface does not move. This movement of helix III results in a perpendicular orientation of helices III and IV, changing the interhelical angle by approximately 90°.

-INTRODUCTION-

Figure 1.8: Conformational changes in the EF-hands of S100 proteins. The S100-specific EF-hand is depicted in A, the canonical EF-hand in B. The Ca -free (apo) protein is shown in blue, the Ca - loaded form in red. Ca ions are shown as yellow spheres. The coordinates were taken from the crystal structures of human S100A6

2+ 2+

2+

(pdb codes 1K8U, 1K96) (Otterbein et al., 2002) (adapted from Fritz and Heizmann, 2004).

The S100 family has received increasing interest in recent years due to their cell- and tissue-specific expression and their involvement in widely different processes such as cell cycle regulation, cell growth, cell differentiation, mobility, transcription, differentiation and secretion. Several S100 proteins are known to be associated with human diseases. Certain diseases are associated with altered expression levels of S100 proteins and can be largely classified into four categories: diseases of the heart, diseases of the central nervous system, inflammatory disorders, and cancer (Marenholz et al., 2004). S100A4 has been implicated in cancer and metastasis, where it shows an increased expression. At nanomolar concentrations S100B stimulates neurite outgrowth (Huttunen et al., 2000), whereas micromolar concentrations of S100B exerts deleterious effects (Rothermundt et al., 2003), e.g. high levels of extracellular S100B have been implicated in glia activation, which is a prominent feature in Down syndrome and Alzheimer’s disease (Davey et al., 2001). S100A8/A9 which forms a heterodimer is associated with rheumatoid arthritis.

Besides their versatile intracellular functions, several S100 proteins such as S100A4, S100A8/A9, S100A12, S100A16 and S100B can be secreted and act extracellularly in a cytokine-like manner (Heizmann et al., 2003; Marenholz et al., 2004; Sturchler et al., 2006).

For example, the S100A8/A9 heterodimer acts as a chemotactic molecule in inflammation (Newton and Hogg, 1998), whereas S100B exhibits neurotrophic activity as mentioned above.

-INTRODUCTION- Within the last years it was recognized that some S100 proteins undergo

oligomerization and form multimeric species. Such S100 multimers have been shown to play a major role in extracellular and intracellular signaling. Very recently it has been shown that only a multimeric form of S100A4 has neurite sprouting activity (Kiryushko et al., 2006). The crystal structure of human S100B revealed an octameric organization whereby four dimers form a tight assembly (Figure 1.9 A) (Ostendorp et al., 2007). For S100A12 it was shown that additional bound Ca²⁺ ions trigger the formation of a hexameric species (Figure 1.9 B) which was proposed to be the active species in RAGE receptor signalling (Moroz et al., 2002). A common feature of these two multimeric structures of S100 proteins is the occurrence of Ca²⁺

ions in addition to those bound in the EF-hands. These additional Ca²⁺ ions are bound at the interface of the subunits, presumably stabilizing the larger structures (Figure 1.9). This finding implies that high Ca²⁺ concentrations might be required for the formation of the S100 protein multimers. In particular high Ca²⁺ concentrations are present in the extracellular space where S100A8/A9, S100A12, and S100B act as pro-inflammatory signaling molecules via the receptor RAGE (Donato, 2003).

Figure 1.9: Multimeric structures of S100 proteins. A: Human S100B forms a tight octameric assembly which is partially stabilized by Ca²⁺ ions bound at the interface of S100B homodimers (pdb code 2H61) (Ostendorp et al., 2007). B: Three S100A12 dimers (shown in green, blue and magenta) form a hexamer. The Ca²⁺ ions bound to the EF-hands are shown as bright yellow spheres. Ca²⁺ ions at the interface stabilizing the multimers are shown as orange spheres (pdb code 1GQM) (Moroz et al., 2002).

-INTRODUCTION-

1.6 The S100A2 protein

S100A2 is expressed in lung and kidney in high amounts, whereas it is also found in liver, heart, and in skeletal muscle (Glenney et al., 1989). Like other S100 proteins S100A2 occurs as homodimer and binds four Ca²⁺ per dimer. Besides Ca²⁺ S100A2 also binds Zn²⁺ with high affinity (Franz et al., 1998). Each S100A2 subunit harbors four cysteine residues (Cys2, Cys21, Cys86, Cys93) that are presumably involved in the coordination of Zn²⁺. Interestingly, the S100A2 ortholog in chimpanzee is identical to human S100A2 and contains four Cys residues, whereas the orthologs from dog and cow contain only one Cys residue. The Zn²⁺- binding properties of S100A2 have been the subject of three investigations, with virtually no consensus as to stoichiometry, affinity, location and structure (Franz et al., 1998; Randazzo et al., 2001; Stradal et al., 2000).

S100A2 is unique among the S100 proteins because it is predominantly localized in the nucleus (Mandinova et al., 1998). Besides the interaction with tropomyosin which might modulate the organization of the actin cytoskeleton (Gimona et al., 1997), S100A2 is involved in cell cycle regulation. S100A2 was identified as a novel tumor suppressor in human mammary epithelial cells (Lee et al., 1992). Drastic downregulation of S100A2 is also observed in several tumor tissues like prostate adenocarcinoma (Gupta et al., 2003), lung cancer (Feng et al., 2001) and breast carcinoma (Wicki et al., 1997). Recently, it was shown that S100A2 binds and activates p53 Ca²⁺-dependently (Mueller et al., 2005). This finding directly links the tumor suppressing activities of S100A2 and p53 and suggests a positive regulation of p53 through S100A2. Intriguingly, other studies report an oncogenic involvement of S100A2 in the tumorigenesis of different squamous cell carcinoma (Villaret et al., 2000; Xia et al., 1997). This might be due to interactions of S100A2 with ΔNp63 which has oncogenic and growth-stimulating activities in the development of tumors (Hibi et al., 2003). These findings point towards a complex picture of S100A2 concerning cell cycle regulation.

-INTRODUCTION-

1.7 RAGE, the receptor for advanced glycation endproducts

RAGE is a member of the superfamily of immunoglobulin type cell surface receptors (Neeper et al., 1992; Schmidt et al., 1992). It is expressed by neurons, microglia, astrocytes, cerebral endothelial cells, pericytes, and smooth muscle cells. The extracellular moiety of the receptor is composed of three immunoglobulin-like domains: one V-type domain and two C-type domains followed by a single transmembrane helix and a 43 amino acid long cytosolic domain (Schmidt et al., 1994). At least three major types of RAGE isoforms (full length and two C-terminally truncated) are present in human brain as a result of alternative splicing. One isoform of RAGE is DN-RAGE (dominant negative RAGE). It is devoid of the intracellular signaling domain and prevents activation of full length RAGE by its ligands. The second major isoform sRAGE is a soluble form of the receptor containing the extracellular region only (Figure 1.10) (Schlueter et al., 2003; Yonekura et al., 2003). Differential expression of each isoform may play a regulatory role in the physiological and pathophysiological functions of RAGE (Lue et al., 2005). RAGE has the unusual property to bind a large range of different molecules such as advanced glycation end products (AGEs) (Basta et al., 2002), ß-sheet fibrils (Du Yan et al., 1997; Yan et al., 1996), amphoterin (HMGB1) (Hori et al., 1995) and several S100 proteins (Hofmann et al., 1999; Marenholz et al., 2004). RAGE was identified first for its AGE binding activity and termed according to this property. AGEs are formed by the so-called Maillard reaction: protein amine-containing residues are unspecifically modified by sugars in a Schiff base reaction. This reaction is usually followed by an Amadori rearrangement and further oxidation and fragmentation reactions, which yield cross-linked proteins and lower molecular weight fragments (Bierhaus et al., 1998). The receptor is strongly activated by cross-linked AGE-modified proteins. Due to its broad repertoire of ligands RAGE is considered as a pattern recognition receptor (PRR) (Schmidt et al., 2001).

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Figure 1.10: Schematic depiction of RAGE and its activation, showing the three extracellular domains of the receptor: two C-type domains (constant) and the V-type (variable) domain, which might be critical for ligand binding. sRAGE: soluble RAGE (extracellular domain of RAGE); DN-RAGE:

dominant negative RAGE (extracellular domain + membrane-spanning domain) (adapted & modified from Schmidt and Stern, 2001).

Binding of ligands to RAGE activates various signaling pathways, whereby most of them lead to the activation of the proinflammatory transcription factor NF-κB (Bierhaus et al., 2005). NF-κB regulates the expression of proteins, such as cytokines, adhesion molecules and RAGE itself. Additionally, a number of anti-apoptotic genes are also under the control of NF- κB (Barnes and Karin, 1997; Li and Schmidt, 1997). One unique feature of RAGE-mediated NF-κB activation is the prolonged time course of the signal. Since RAGE expression is induced by NF-κB, sustained activation of NF-κB results in upregulation of the receptor and further maintains and amplifies the signal (Bierhaus et al., 2001). Actually, RAGE expression is induced in situations where either ligands accumulate and/or transcription factors regulating RAGE are activated. Under physiological conditions cells do not express significant amounts of RAGE (Bierhaus et al., 2005). Therefore, RAGE has the potential to convert a transient proinflammatory response, evoked by an inflammatory stimulus, into sustained cellular dysfunction (Schmidt et al., 2001).

Hence, RAGE attracted most interest by its involvement in a number of pathophysiologic situations. AGEs have been shown to form in disorders like in diabetes or in vascular diseases (Schleicher et al., 1997). The AGE adduct N^ε-carboxymethyllysine (CML) leads to the upregulation of a range of proinflammatory molecules via RAGE. Thus, AGEs may mediate the proinflammatory phenotype of diabetic vessels (Bierhaus et al., 2001).

-INTRODUCTION- stimulating tumor cell migration, invasion, proliferation, and the generation of matrix

metalloproteinases (Taguchi et al., 2000). Recently, it could be shown that RAGE mediates amyloid-β transport across the blood-brain barrier and leads to accumulation in the brain (Deane et al., 2003). Extracellular deposits of amyloid-β, which accumulate in the brain in Alzheimer’s disease as a component of neuritic plaques, bring about neuronal damage and dysfunction (Selkoe, 2001). Moreover, amyloid-β binding to RAGE generates oxidative stress and activates NF-κB which results in a proinflammatory pathway in Alzheimer’s disease (Du Yan et al., 1997). Extracellular S100 proteins like S100A12 and S100B were found especially at sites of chronic immune/inflammatory responses, as in cystic fibrosis and rheumatoid arthritis. They have been shown to activate endothelial cells, vascular smooth muscle cells, monocytes and T-cells via RAGE, resulting in the generation of cytokines and proinflammatory adhesion molecules (Hofmann et al., 1999; Yan et al., 2003).

Due to the involvement in this plethora of different pathophysiologic situations, RAGE is a designated pharmaceutically relevant therapeutic molecule. Several studies using animal models exhibit possible applications in cancer (Taguchi et al., 2000), chronic autoimmune inflammatory diseases (Yan et al., 2003) or diabetic disorders (Wendt et al., 2006; Wendt et al., 2003).

The impact of RAGE on multiple chronic disease states has largely focussed attention away from physiologic roles of the receptor. HMGB1-RAGE interaction may contribute to axonal sprouting which accompanies neuronal development (Fages et al., 2000). This corresponds with the high expression of RAGE during embryonic development, whereas it is downregulated in most tissues during adult life (Kokkola et al., 2005). Furthermore, activation of RAGE by HMGB1 and S100B can promote cell survival through increased expression of anti-apoptotic proteins (Huttunen et al., 2000). Nowadays, RAGE is recognized as a central receptor in the innate immune system. Engagement of RAGE induces NF-κB-dependent upregulation of the endothelial adhesion molecules VCAM-1 and ICAM-1 (Schmidt et al., 1993; Schmidt et al., 2001). In addition, RAGE directly acts as a counter receptor for leukocytes by binding β2-integrins (Chavakis et al., 2003; Chavakis et al., 2004), and thus promotes and perpetuates inflammatory cell recruitment.

Up to now it is rarely understood how RAGE is activated by its ligands and how it functions in cellular signaling. In fact, binding of ligands to RAGE has been shown to activate multiple cellular signaling cascades including ERK1/2, SAPK/JNK, MAP kinases as well as rho-GTPases and phosphoinositol-3-kinase. As mentioned above, many of these signaling

-INTRODUCTION- (Bierhaus et al., 2005). Nevertheless, it remains unclear how the signal is transferred from the

extracellular ligand-binding site to the cytosolic domain and how the different cellular signaling cascades are initiated upon ligand binding.

The current hypothesis for RAGE activation is ligand induced receptor dimerization.

For S100 proteins it is proposed that larger assemblies in the extracellular space may trigger an aggregation of RAGE, which then in turn activates the intracellular signaling cascade.

Multimeric forms of S100 proteins have been reported for S100A12 (Moroz et al., 2002), for S100A4 (Novitskaya et al., 2000), S100A8/A9 (Korndoerfer et al., 2005), and for S100B (Ostendorp et al., 2007). Recently, it was shown that multimeric species of S100B bind with high affinty to RAGE. A putative RAGE complex induced by binding of a Ca²⁺-loaded tetrameric form of S100B is depicted in (Figure 1.11) (Ostendorp et al., 2007).

Figure 1.11: RAGE activation by dimerization through S100B binding. S100B is shown in red, the two RAGE receptors are shown in blue and green, respectively. S100B binds to both V-type domains of RAGE, thereby inducing receptor dimerization and activation (Ostendorp et al., 2007).

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1.8 Scope of this study

1. S100A2 is predominantly localized in the nucleus. In the beginning, it was described as tumor suppressor, but now there is increasing evidence that it exerts oncogenic functions as well. The protein attracted most attention by its interaction with the tumor suppressor p53 in a Ca²⁺-dependent manner (Mueller et al., 2005). Besides Ca²⁺ S100A2 binds Zn²⁺ with high affinity. Up to now there is no consensus concerning stoichiometry, affinity, geometry, and location of Zn²⁺-binding sites. Additionally, there are no structural data of S100A2 available.

In this study Zn²⁺-binding to S100A2 is examined by spectroscopic methods. Furthermore, the influence of Zn²⁺-S100A2 on Ca²⁺ affinity will be addressed. In order to gain structural insights into molecular rearrangements upon Ca²⁺ binding, crystallization and X-ray diffraction experiments with Ca²⁺-bound and Ca²⁺-free S100A2 will be performed.

Subsequently, high resolution X-ray structures of both states of S100A2 will provide the structural basis for S100A2 activation upon Ca²⁺ binding. This study is designed to elucidate the cellular regulation of S100A2 by metal binding and to characterize structural consequences upon Ca²⁺ binding concerning activation and target recognition.

2. The receptor for advanced glycation endproducts (RAGE) is a multiligand receptor which recognizes tertiary structures rather than amino acid sequences. Therefore it is considered as pattern recognition receptor (PRR). RAGE attracted large interests because it is involved in a variety of diseases such as cancer, chronic autoimmune inflammatory diseases, diabetic disorders, and Alzheimer’s disease. Up to now there is only rare knowledge about target recognition, ligand-binding sites, and mechanisms of receptor activation. In this study the ligand-binding domain will be mapped by surface plasmon resonance (SPR) experiments with different ligands. With the identified domain crystallization experiments and X-ray diffraction experiments will be performed. The focus of this study is to explain the multiple ligand binding on a structural basis. Additionally, a three dimensional structure of the ligand- binding domain will provide further insights into possible mechanisms of activation.