Structure and function of the metal-binding protein S100B and its interaction with the receptor for advanced glycation end products

S TRUCTURE AND F UNCTION OF THE M ^ETAL - B ^INDING P ^ROTEIN S100B ^{AND ITS}

I NTERACTION WITH THE R ECEPTOR FOR

A ^DVANCED G ^LYCATION E ND PRODUCTS

DISSERTATION

Z

E

G

D

N

(Dr. rer. nat.)

IM

F

B

U

K

VON

Dipl.-Biol. T

D

O

KONSTANZ,NOVEMBER 2006

Konstanzer Online-Publikations-System (KOPS) URL: http://www.ub.uni-konstanz.de/kops/volltexte/2007/2375/

URN: http://nbn-resolving.de/urn:nbn:de:bsz:352-opus-23752

DISSERTATION DER UNIVERSITÄT KONSTANZ TAG DER MÜNDLICHEN PRÜFUNG:13.02.2007 REFERENT:PROF.DR.P.M.H.KRONECK

KOREFERENT:PROF.DR.C.W.HEIZMANN

„JEMAND HAT MIR MAL GESAGT, DIE ZEIT WÜRDE UNS WIE EIN RAUBTIER EIN LEBEN LANG VERFOLGEN.ICH MÖCHTE VIEL LIEBER GLAUBEN, DASS DIE ZEIT UNSER GEFÄHRTE IST, DER UNS AUF UNSERER REISE BEGLEITET UND UNS DARAN ERINNERT, JEDEN MOMENT ZU GENIEßEN,

DENN ER WIRD NICHT WIEDERKOMMEN.WAS WIR HINTERLASSEN IST NICHT SO WICHTIG WIE DIE

ART, WIE WIR GELEBT HABEN.DENN LETZTLICH, SIND WIR ALLE NUR STERBLICH.“

JEAN-LUC PICARD

DEDICATED TO PETRA,PETER, ANGÉLIQUE,CARSTEN,

AND MY BELOVED ARIANE

-TABLE OF CONTENTS-

T ABLE OF C ONTENTS

ZUSAMMENFASSUNG III

SUMMARY VI

1 INTRODUCTION 1

1.1 CALCIUM IN BIOLOGY 1

1.2 THE CA²⁺-BINDING PROTEINS 4

1.3 THE EF-HAND PROTEINS 6

1.4 THE S100 FAMILY 9

1.5 THE S100B PROTEIN 13

1.6 RAGE, THE RECEPTOR FOR ADVANCED GLYCATION END PRODUCTS 15

1.7 SCOPE OF THIS STUDY 18

1.8 ZIEL DER STUDIE 18

2 MATERIALS & METHODS 21

2.1 ANALYTICAL METHODS 21

2.2 CRYSTALLOGRAPHY 22

2.3 CRYO-CRYSTALLOGRAPHY 29

2.4 DATA COLLECTION AND PROCESSING 30

2.5 STRUCTURE SOLUTION, MODEL BUILDING AND REFINEMENT 31

3 RESULTS 33

3.1 PURIFICATION, CRYSTALLIZATION AND PRELIMINARY X-RAY DIFFRACTION STUDIES ON HUMAN CA²⁺-BINDING PROTEIN S100B

3.1.1 ABSTRACT 33

3.1.2 INTRODUCTION 34

3.1.3 MATERIAL AND METHODS 35

3.1.4 RESULTS AND DISCUSSION 36

3.1.5 FIGURES 38

3.1.6 SUPPLEMENTARY INFORMATION 41

I

3.2 EXPRESSION AND PURIFICATION OF THE SOLUBLE ISOFORM OF HUMAN

RECEPTOR FOR ADVANCED GLYCATION END PRODUCTS FROM PICHIA PASTORIS

3.2.1 ABSTRACT 45

3.2.2 INTRODUCTION 46

3.2.3 MATERIAL AND METHODS 47

3.2.4 RESULTS AND DISCUSSION 53

3.2.5 CONCLUSION AND OUTLOOK 58

3.2.6 FIGURES 60

3.3 THE CRYSTAL STRUCTURE OF HUMAN EF-HAND PROTEIN S100B REVEALS A MULTIMERIC ORGANIZATION: IMPLICATIONS ON RAGE SIGNALING

3.3.1 ABSTRACT 67

3.3.2 INTRODUCTION 68

3.3.3 MATERIAL AND METHODS 70

3.3.4 RESULTS 76

3.3.5 DISCUSSION 82

3.3.6 FIGURES 85

3.3.7 SUPPLEMENTARY INFORMATION 94

3.4 CRYSTAL STRUCTURES OF THE ZINC- AND CALCIUM-LOADED HUMAN S100B

3.4.1 ABSTRACT 96

3.4.2 INTRODUCTION 97

3.4.3 MATERIALS AND METHODS 99

3.4.4 RESULTS AND DISCUSSION 102

3.4.5 FIGURES 107

4 CONCLUSION 118

5 REFERENCES 127

6 APPENDIX 139

6.1 ABBREVIATIONS 139

6.2 AMINO ACIDS 141

6.3 INTERNATIONAL SYSTEM OF UNITS (SI) 142

7 ACKNOWLEDGMENTS 143

-ZUSAMMENFASSUNG-

Z USAMMENFASSUNG

1. S100B ist ein dimeres Ca²⁺ und Zn²⁺-bindendes EF-Hand Protein. Es ist auf dem Chromosom 21 codiert und wird überwiegend in Gliazellen exprimiert. S100B gehört zur Familie der S100 Proteine, welche 21 Mitglieder umfasst. Dieses Protein bindet neben Kalzium auch Zink und Kupfer mit hoher Affinität. Es ist bekannt, dass eine erhöhte Expression von S100B mit Krankheiten wie Alzheimer oder Krebs assoziiert ist. S100B kann sekretiert werden und hat sowohl intra- als auch extrazellulär die Funktion eines Signalproteins.

Rekombinantes humanes S100B wurde in Escherichia coli exprimiert und bis zur Homogenität in dieser Studie gereinigt. Die einzelnen Polypeptidketten haben eine molekulare Masse von 10,7 kDa. Erstmals wurden verschiedene S100B Spezies gereinigt, die ihrer molekularen Masse nach tetrameren, hexameren und oktameren S100B entsprachen. Untersuchungen mittels CD-Spektroskopie und Gelelektrophorese zeigten, dass alle drei Multimere korrekt gefaltet und nicht über Disulfidbrücken verbunden waren. Es wurden erstmals Kristalle von humanem S100B in Anwesenheit von Kalzium (Ca²⁺-S100B, pH 7,2) sowie in Anwesenheit von Zink und Kalzium (Zn²⁺- Ca²⁺-S100B) bei verschieden pH Werten (pH 6,5, 9,0 und 10,0) erzeugt.

Die Kristalle von Ca²⁺-S100B streuten bis zu einer Auflösung von 1,80Å. Die Analyse der Kristallstruktur ergab, dass a) sich je zwei Dimere zu einem Tetramer arrangiert hatten, und b) sich aus diesen zwei Tetrameren ein Oktamer bildete. Diese Architektur wird in dieser Arbeit erstmalig beschrieben. Die Bildung dieser tetrameren bzw.

oktameren Spezies wird über spezifische ionische, hydrophobe als auch durch zwei zusätzliche Kalzium Ionen gewährleistet. Diese zusätzlich vorhandenen Kalzium Ionen scheinen besonders für die Ausbildung des Oktamers nötig.

Erstmals wurden multimere Spezies von Ca²⁺-S100B im menschlichen Gehirn nachgewiesen. Ca²⁺-S100B existiert somit offensichtlich nicht nur in homodimerer Form beim Menschen, sondern auch organisiert in größeren, multimeren Komplexen. Diese III

Multimere scheinen vergleichbar mit der tetrameren bzw. oktameren Struktur, welche in den Ca²⁺-S100B Kristallen entdeckt wurden.

Die Zn²⁺-Ca²⁺-S100B Kristalle, wurden am SLS (Swiss Light Source) in Villigen (CH) vermessen. Die Kristalle streuten bis 1,60Å (pH 6,5), 1,55Å (pH 9,0) und 1,66Å (pH 10,0). Von allen drei Kristall-Formen wurde jeweils ein kompletter Datensatz aufgenommen. In allen drei pH Bedingungen kam humanes Zn²⁺-Ca²⁺-S100B ausschließlich als Dimer im Kristall vor. Dabei wurde das Zink Ion jeweils auf nahezu die gleiche Weise koordiniert (pH 6,5 und pH 10,0: Zn(NHis)3(OGlu) bzw. bei pH 9,0:

Zn(NHis)4. Aus der Struktur ist ersichtlich, dass von einem S100B Dimer vier Ca²⁺- und zwei Zn²⁺-Ionen gebunden werden. Diese Strukturen erklären auch, warum eine Multimerisation von Zn²⁺-Ca²⁺-S100B nicht möglich war. Da Zink an dem Imidazolring von dem für eine multimerisation essentiellen Histidin 15 koordiniert ist, ist die Bildung höherer Oligomere blockiert.

2. Der ´Receptor for Advanced Glycation End products´ (RAGE), ist ein Mitglied der Immunglobulin-Superfamilie von Zelloberflächenrezeptoren. Aufgebaut ist er aus einer kurzen zytosolischen und einer Transmembran-Domäne sowie aus drei extrazellulären Domänen (V-CI-CII). RAGE kommt besonderst häufig auf Endotheliengewebe vor, wird aber auch von anderen Geweben exprimiert. Eine gesteigerte Expression von RAGE wird mit Krankheiten wie Diabetes und Alzheimer in Verbindung gebracht. RAGE ist ein Multiligandenrezeptor und bindet neben den

´Advanced Glycation End products´ (AGEs) das Aβ Protein sowie verschiedene S100 Proteine. Neben der membranassoziierten Form existiert auch eine lösliche Form von RAGE, die durch alternatives `Splicing` entsteht. Diese Form von RAGE wird sRAGE (soluble RAGE) genannt und umfasst nur die drei extrazellulären Domänen.

Für den Nachweis von sRAGE wurden vorab drei verschiedene RAGE-spezifische Antikörper hergestellt, die jeweils gegen eine der drei extrazellulären Domänen (V-C1- C2) gerichtet waren.

Erstmals wurde humanes sRAGE in Pichia pastoris erfolgreich in ausreichenden Mengen exprimiert und aufgereinigt. Dieses so gewonnene sRAGE konnte für

-ZUSAMMENFASSUNG-

Interaktionsstudien in vitro als auch für Untersuchungen in vivo genutzt werden. Das gereinigte sRAGE zeigte posttranslationale Glycosylierungen.

In vitro Studien mit sRAGE und den im Zuge dieser Arbeit gereinigten S100B Spezies (Dimer, Tetramer und Multimer) zeigten erstmals in Immunoassays, dass alle gereinigten S100B Spezies mit hoher Affinität an sRAGE binden. Des weiteren wurde in ELISA- Studien gezeigt, dass die gereinigten multimeren S100B Spezies eine höhere Affinität zu sRAGE haben als die dimere S100B Form.

Die Ergebnisse dieser Arbeit dokumentieren, dass die Bindung von Ca²⁺-S100B an RAGE hoch spezifisch ist und dass multimere Ca²⁺-S100B Komplexe eine deutlich bessere Bindung aufweisen als das Ca²⁺-S100B Dimer allein. In „Surface Plasmon Resonance“ Studien wurde gezeigt das diese verbesserte Bindung damit zu erklären ist, dass die multimeren Ca²⁺-S100B Komplexe im Stande sind, mehr als nur einen RAGE- Rezeptor binden. Diese Möglichkeit für eine verbesserte Bindung an RAGE besteht somit für Zn²⁺-Ca²⁺-S100B nicht, da diese nur als Dimere vorkommen. Wie aus den Kristallstrukturen von Zn²⁺-Ca²⁺-S100B hervorgeht, ist die Bildung von Multimeren blockiert solange Zink gebunden ist.

Man kann also annehmen dass neben der spezifischen Aktivierung von RAGE durch extrazelluläre Ca²⁺-S100B Dimere, multimere Ca²⁺-S100B Komplexe auch eine wichtige Rolle bei der Modulation der Aktivierung von RAGE im Bezug auf Stärke und/oder Zeit spielen. Funktionen wie die Modulation von RAGE durch Rezeptor Multimerisation, scheinen für das nur als Dimer vorkommende Zn²⁺-Ca²⁺-S100B wohl nicht von Relevanz zu sein.

V

S UMMARY

1. S100B is a dimeric Ca²⁺ and Zn²⁺-binding EF-hand protein. It is encoded on the chromosome 21 and highly expressed in glial cells. S100B belongs to the family of the S100 of proteins, which covers 21 members. This protein binds zinc and copper with high affinity beside calcium. It is known, that an increased expression of S100B is correlated with diseases such as Alzheimer or cancer. S100B can be secreted and functions both intra- and extracellularly as a signal protein.

Recombinant human S100B was expressed in Escherichia coli and purified to homogeneity. The individual polypeptides have a molecular mass of about 10.7 kDa. For the first time, different species of S100B were purified, which according to their molecular mass corresponded to tetrameric, hexameric and octameric S100B.

Investigations by means of CD-spectroscopy and gel electrophoresis showed that the three multimeric species were correctly folded, and not connected via disulfide bridges.

For the first time crystals of human S100B in the presence of calcium (Ca²⁺-S100B, pH 7.2) as well as in the presence of zinc and calcium (Zn²⁺-Ca²⁺-S100B) have been obtained at differently pH values (pH 6.5, 9.0 and 10.0).

Crystals of Ca²⁺-S100B diffracted to a resolution of 1.80Å, and a complete dataset has been recorded. The analysis of the crystal structures revealed a) that two dimers were arranged to one tetramer, and b) two tetramers were arranged to one octamer. This architecture is described for the first time. The formation of these tetrameric and/or octameric species is stabilized by specific ionic and hydrophobic interactions and two additional non EF-hand calcium ions. These additional calcium ions seem to be necessary for the final formation of an octameric S100B species.

For the first time, multimeric species of Ca²⁺-S100B were found in human brain extracts.

Thus, Ca²⁺-S100B does not only exist as homodimer within humans, it is also organized in larger, multimeric complexes. These multimeric species in humans might be comparable with the tetrameric and/or octameric structure, which were discovered in the Ca²⁺-S100B crystal structure.

-SUMMARY-

The Zn²⁺-Ca²⁺-S100B crystals were measured at the SLS (Swiss Light SOURCE) in Villigen (CH). The crystals diffracted up to 1.60Å (pH 6.5), 1.55Å (pH 9.0) and 1.66Å (pH 10.0). Crystals of each condition were measured and complete datasets were recorded.

In the crystals of all three crystallization condition exclusively dimeric human Zn²⁺-Ca²⁺- S100B were found and the zinc ions are almost coordinated in the same way. These structures also explain why higher multimers of Zn²⁺-Ca²⁺-S100B were not observed.

Since zinc coordination at the imidazole of the histidine 15 is essential for multimerization, the formation of higher multimers is blocked. Independent of the pH at which they had been grown, all crystals contained exclusively dimeric Zn²⁺-Ca²⁺-S100B.

The zinc ions were coordinated nearly identical for pH 6.5 and pH 10.0: Zn(NHis)3(OGlu) respectively for pH 9.0: Zn(NHis)4. The solved structures reveled that four Ca²⁺ and two Zn²⁺ were bound per dimer.

2. The ' Receptor for to Advanced Glycation end products ' (RAGE), is a member of the immunoglobulin superfamily of cell surface receptors. It is composed of a small cytosolic and a short transmembrane domain, as well as of three extracellular domains (V-CI-CII). In addition, RAGE is highly expressed in endothelial cells but also in other tissues. An increased expression of sRAGE is associated with diseases such as diabetes and Alzheimer. RAGE is a multi-ligand receptor and binds besides the 'advanced glycation end products' (AGEs) also the Aβ protein and different members of the S100 proteins. In addition to the membrane associated form of RAGE a soluble form of RAGE was also found, an outcome of alternative `splicing`. This form of RAGE is called sRAGE (soluble RAGE) and consists only of the three extracellular domains. For detection of RAGE in our studies, three different specific antibodies were constructed which were directed against one of the three extracellular domains (V-C1-C2).

For the first time, human sRAGE was expressed and purified successfully in sufficient quantities, in Pichia pastoris. The obtained sRAGE could be used for interaction studies in vitro and for investigations in vivo. The purified sRAGE was posttranslationally glycosylated.

VII

In vitro studies with sRAGE with the various purified S100B species of this work (dimers, tetramers and multimers) revealed for the first time in immunoassays that all obtained S100B species did bind to sRAGE with high affinity. Moreover, in Elisa studies it was shown that the multimeric S100B species have a higher affinity towards sRAGE compared to the dimeric form of S100B.

The results described in this work show that the interaction of Ca²⁺-S100B with RAGE is highly specific and that multimeric Ca²⁺-S100B complexes exhibit a higher affinity to RAGE than the S100B dimers. Surface plasmon resonance studies point out that the improved binding of multimeric Ca²⁺-S100B complexes is favored due to the ability of multimeric S100B to bind more than one receptor molecule. This is not possible for zinc loaded S100B, since Zn²⁺-Ca²⁺-S100B can only form dimers. In this case the formation of higher multimers is blocked by Zn²⁺ which coordinates to a histidine which is essential for dimerization.

One can assume that apart from the specific activation of RAGE by extracellular Ca²⁺- S100B dimers, multimeric Ca²⁺-S100B complexes also play a key role in modulation of RAGE activity by receptor multimerization, with regard to strength and/or duration of activation. This function seems to be not important in the case of dimeric Zn²⁺-Ca²⁺- S100B.

1 I NTRODUCTION

1.1. Calcium in biology

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 calcium (Ca²⁺) in the blood and in the extracellular space or is stored intracellularly in distinct compartments (Carafoli, 2003). The significance of calcium in cell biology was first described by Ringer 1883 where he demonstrated that small amounts of the Ca²⁺ were necessary to maintain heart muscle contractility (Ringer, 1883).

Calcium is described as a second messenger liberated from intracellular stores (Carafoli, 2002). Almost 80 years after Ringer, physiologists assign calcium as a second messenger in analogy to cyclic adenosine 3', 5’-monophosphate (cAMP) which had been shown during the 1960's to be 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 stimuli leads to a change of the intracellular Ca²⁺ concentration, caused upon relocation of Ca²⁺

ions by an influx from the extracellular space and by the simultaneous release from the endoplasmatic reticulum. The cytoplasmatic Ca²⁺ concentration can rise from 100 nM in a rested cell to roughly 1 mM in an activated cell (Berridge et al., 2000). If Ca²⁺ acts as a signal, its intracellular concentration must be precisely regulated. This is granted by two major systems; the plasma membrane Ca²⁺ ATPases (PMCAs) and the sarco/endoplasmatic Ca²⁺ ATPases, which are capable of pumping Ca²⁺ against a large concentration gradient out of the cell or into the sarco/endoplasmatic reticulum. Those pumps maintain the Ca²⁺ concentration in resting cells at a level of 100-200 nM (Carafoli, 2003; Strehler and Treiman, 2004).

-INTRODUCTION-

Once Ca²⁺ is released intracellularly as a response to the external stimuli, it exerts its action by binding to specific Ca²⁺-modulated proteins. The first identified Ca²⁺-binding protein was troponin-C (TnC) which confers Ca²⁺ sensitivity to the adenosine triphosphatase (ATPase) driving the contractile system in muscle cells (Ebashi et al., 1967).

Today it is clear that Ca²⁺ ions are involved in the control of a vast range of cellular functions such as neurotransmitter release, muscle contraction, blood clotting, cell growth and cell cycle regulation as depicted in Figure 1.1. This high versatility is achieved by varying the spatial and temporal aspects of Ca²⁺-signaling (Berridge, 1997;

Lipp and Niggli, 1996). 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).

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

Why is Ca²⁺ so effective as a second messenger, and why was this particular ion selected through evolution for the important messenger function? Ca²⁺ is one of only four important alkali and alkaline earth metals in living systems, along with magnesium, sodium, and potassium, which might qualify for the same job. The evolutionary choice 2

for Ca²⁺ as an intracellular messenger was probably made when the switch to multicellular life forced the specialization of cells. Once cells have specialized, they have to communicate by exchanging molecular signals. The choice for Ca²⁺ might have been dictated by its binding flexibility regarding to its possible coordination numbers. This property enables reversible binding to complex molecules (i.e. proteins) (Carafoli, 2005).

Ca²⁺-modulated proteins must not only distinguish Ca²⁺ from other ions, but also be able to bind Ca²⁺ in presence of much higher concentrations of e.g. Mg²⁺. The particular combination of the ionic radius and charge distribution allows Ca²⁺ a high degree of selectivity, although it has the same charge as Mg²⁺ and almost the same radius as Na⁺ (see Table 1.1.).

Properties: Metals: Ca²⁺ Mg²⁺ K⁺ Na⁺

Ionic radius (in pm): 100 72 138 102

Preferred ligand number: 6-8 6 6-8 6

Preferred coordination atoms: O N,O O O

Ion to oxygen distance*: 2.2-2.4 Å 2.0-2.1 Å 2.46 Å 2.25 Å

Relative ratio of intracellular concentrations: with Ca²⁺ = 1

1 25 920 111

Table 1.1: Selected alkali and alkaline earth metals in human erythrocytes. Adapted from *Glusker, 1991; Kaim and Schwederski, 2004.

How can Ca²⁺ binding proteins distinguish between Ca²⁺ and Mg²⁺, which have the same overall charge? The EF-hand binding sites are able to discriminate between the two small, divalent cations, Mg²⁺ and Ca²⁺. Both Mg²⁺ and Ca²⁺ are closed-shell, spherical metal ions, making them relatively “hard”. However, Ca²⁺ has a substantially larger radius than Mg²⁺ and can achieve a higher coordination number. Mg²⁺, prefers a octahedral arrangement of its ligands with distances between ion and ligands in the 2.0-

-INTRODUCTION-

4

2.1 Å range vs. a distance of 2.2-2.4 Å for Ca²⁺-O. The coordination number in spherical metal ions is primarily a function of the size of the ion, because optimal coordination is achieved through close packing of many ligands as possible around the ions (Falke et al., 1994; Martin, 1990b). Additionally, preference for Ca²⁺ over Mg²⁺ is achieved by exploiting that calcium binds stronger to oxygen ligands whereas Mg²⁺ binds stronger to nitrogen ligands (Martin, 1990a).

Nevertheless, the most important aspect is the ligand exchange rate of Ca²⁺. In comparison to Ca²⁺ Mg²⁺ forms much tighter and more stable complexes. This means that exchange of Mg²⁺ from a protein cannot be achieved at the same velocity as with Ca²⁺; in other words Mg²⁺ modulated proteins would rather be slower and therefore Mg²⁺

as a second messenger would be less effective.

1.2. The Ca

binding proteins

The Ca²⁺ binding proteins (CaBPs)can be divided according to their function in two major subgroups. Those that bind Ca²⁺ in order to regulate the intracellular Ca²⁺

concentration (Ca²⁺ buffering and Ca²⁺ transporting proteins) and those that bind Ca²⁺to decode its signal (Ca²⁺ sensors).

Ca²⁺ buffering and Ca²⁺ transporting proteins: free Ca²⁺ buffering proteins in the cytosol and organelles are acidic proteins. They have in general a low Ca²⁺-binding affinity but can store a large amount 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 Ca²⁺ binding 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).

Most of the Ca²⁺ sensor proteins such as calmodulin and troponin C are members of the EF-hand protein family. They contain the high-affinity Ca²⁺ binding EF-hand motif (see next chapter). These Ca²⁺ sensor proteins have different functions but they are

structurally related. The binding affinities for Ca²⁺ are so low that the proteins are kept in their apo form (Ca²⁺ free state) in the resting cell but Ca²⁺ binding occurs when the Ca²⁺

concentration rises as a response to external stimuli (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 in Ca²⁺- binding proteins is the so-called C2-domain. This domain was described for the first time in protein kinase C (PKC). 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 binding of the Ca²⁺ ions leads to a change in the electrostatic surface charge of the C2-domain upon progressive binding of Ca²⁺ to the sites Ca1, Ca2 and Ca3 (Figure 1.2. B) (Ubach et al., 1998). This change in the electrostatic surface potential of the C2-domain in synaptotagmin I enables binding to the phospholipid membranes (Fernández Chacón et al., 2002).

Figure 1.2: A. Structure of the Ca²⁺ binding C2-typ domain of synaptotagmin I (Adapted from Rizo and Südhof, 1998) B. Change in the electrostatic surface potential of the C2-domain of synaptogamine 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.

-INTRODUCTION-

6

1.3. The EF-hand proteins

The EF-hand motif is the most common Ca²⁺ binding motif. Over 600 proteins of this family are known by now. They function both as Ca²⁺ sensor or Ca²⁺ buffer in eukaryotes, and contain one or more EF-hand motifs (Carafoli, 2002; Lewit-Bentley and Réty, 2000; Yap et al., 1999).

The name EF-hand is deduced from the structure of parvalbumin, whereby E and F matches up to the fifth and the sixth helix in this structure. Parvalbumin that is present in high amounts in fast twitching muscles was the first EF-hand protein which was discovered by Kretsinger in 1972. One year later in 1973, Kretsinger and Nockolds determined the three-dimensional structure of carp parvalbumin. It became clear that Ca²⁺ was bound by a particular helix-loop-helix structure in the molecule (Kretsinger and Nockolds, 1973). The term EF-hand was introduced in 1975 by Tufty and Kretsinger, the configuration of the helix-loop-helix motive resembles a right hand with stretched thumb and an index-finger with the loop represented by the middle finger bent around the Ca²⁺

ion at the centre (Tufty and Kretsinger, 1975) (Figure 1.3. A). This classical EF-hand motif is characterized by a sequence of usually 12 amino residues with the pattern X*Y*Z*–Y*–X**–Z. The positions X, Y, Z, –X, –Y and –Z represent the ligands, which participate in metal coordination, and the stars represent the intervening residues (Figure 1.3.B.). In most cases, the Ca²⁺ ion is coordinated in a pentagonal bipyramidally fashion by oxygen atoms of five residues and one water molecule. Usually a glutamic acid at the –Z position coordinates Ca²⁺ with both side chain carboxylate oxygen atoms.

Characteristic features are the –Y position that is normally occupied by main chain carbonyl oxygen, and the –X position, where a water molecule coordinates the Ca²⁺. The side chains of aspartate or asparagine form the position X and Y. This mode of Ca²⁺

binding was named the canonical EF-hand (Figure 1.3. B) (Lewit-Bentley and Réty, 2000).

Figure 1.3: Schematic diagrams 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 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 a water molecule (W) Adapted & modified from Branden and Tooze, 1991.

In most cases the EF-hand motifs occur in adjacent pairs like in calmodulin with four EF-hands or in case of parvalbumin, troponin C and the S100 proteins which represent the minimal motif with two EF-hands (Lewit-Bentley and Réty, 2000). An exception is calpain, a thiol protease regulated by Ca²⁺ which contains five EF-hand motifs (Blanchard et al., 1997; Lin et al., 1997; Strobl et al., 2000). As already pointed Ca²⁺

sensor proteins undergo a conformational change induced by Ca²⁺ binding. The binding of Ca²⁺ leads to an exposure of a hydrophobic surface which is buried in the Ca²⁺ free state. This hydrophobic surface serves as a target-binding and -recognition site. One of the best investigated EF-hand proteins is calmodulin (Carafoli, 2004a). Calmodulin is present in all cell types and is highly conserved as illustrated by 90% sequence identity in all multicellular eukaryotes (Moncrief et al., 1990). Studies on calmodulin-binding to target proteins revealed for the first time that hydrophobic residues are involved in target

-INTRODUCTION-

protein interactions (Zimmer et al., 2003). Calmodulin activates a large number of different target proteins. Several structures of the calmodulin-target complexes are known and a summary is shown in Figure 1.4. These structures give an idea about the diversity of possible conformations and how each conformation can be unique to the particular activation mechanism, which is characteristic for the corresponding target protein. 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). A quite different overall structure and mode of interaction of an EF-hand protein has been shown for the S100 proteins.

Figure 1.4: Comparison of different calmodulin target structures. Centre: calmodulin (CaM) is shown in yellow with its four Ca²⁺ sites occupied (Ca²⁺ shown as blue spheres); the arrow indicates the flexibility of the central helix linking the N- and C-terminal halves of CaM. Left: different modes of interaction of Ca²⁺ loaded CaM with CaM-binding peptides (shown in green) of CaM kinase II (CaMKII), CaM kinase kinase (CaMKK) and myosin light chain kinase (MLCK). Right: the dimeric structure of the CaM-binding domain of the K⁺ channels (shown in green and aquamarine) each bound to a molecule of CaM, with the two N-terminal sites occupied (Schumacher et al., 2001); and the structure of Bacillus anthracis edema factor (EF) shows how CaM, with Ca²⁺ bound to its two C-terminal sites, can activate the enzyme by remodeling the catalytic site (Drum et al., 2002). Adapted from Hoeflich and Ikura, 2002; Klee and Means, 2002.

8

1.4. The S100 family

The S100 proteins represent the largest subgroup within the EF-hand Ca²⁺ protein family.

Whereas calmodulin exists as a monomer with four very similar EF-hands the S100 proteins contains two different types of EF-hands. S100 proteins form dimers and bind besides Ca²⁺ (Kd = 20-500 µM), also Zn²⁺ (Kd = 0.1-2000 µM) and Cu²⁺ (Kd = 0.4-5 µM) with high affinities. A total of 21 members in human have currently been assigned to the S100 family with sequence identities ranging from 22% to 57% (Figure 1.5.) (Fritz and Heizmann, 2004; Heizmann et al., 2003; Marenholz et al., 2006).

Figure 1.5: Figure legend see next page.

-INTRODUCTION-

Figure 1.5: Multiple sequence alignment of human S100 proteins. The Ca²⁺-coordinating residues are highlighted in dark red (side chain coordination), and in light red (backbone oxygen coordination). The Zn²⁺-coordinating residues in S100A7 and the conserved binding site in S100A12 are highlighted in magenta. Hydrophobic residues that are essential for dimerization are highlighted in green. Residues that are putative Zn²⁺ ligands in other S100 proteins are highlighted in yellow (cysteine) and blue (histidine), respectively. Adapted & modified from Fritz and Heizmann, 2004; Marenholz et al., 2004.

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 100% ammonium sulfate (Moore, 1965). Therefore, he called the isolated molecule S100 protein. Later research has shown that Moore's original preparation primarily contained two polypeptides, which are now called S100A1 and S100B (Hilt and Kligman, 1991). S100 proteins are small acidic proteins with a size of 10-12 kDa and except calbindin D9k (S100G, only monomeric) they form non-covalent homo- and heterodimers. An example for a typical structure of a Ca²⁺-S100 protein is shown in Figure 1.6. (Otterbein et al., 2002).

Figure 1.6: Schematic representation of human Ca -S100A6 (PDB code 1K96) showing the homodimer and bound Ca ions (yellow). Adapted from Fritz and Heizmann, 2004.

2+

2+

10

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 an intermediate region, referred as the hinge region. 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 individual proteins (Donato, 2003). The plane for dimerization (~2500 Ų) of S100 proteins is composed by hydrophobic residues which are absent in the case for calbindin. 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 classical EF-hand motif with 12 residues at the C-terminus (Figure 1.5.) (Fritz and Heizmann, 2004). Interestingly, the N- terminal EF-hand of S100 proteins exhibits only small conformational changes upon Ca²⁺ binding. Helix HII undergoes a small rearrangement in direction of the Ca²⁺ binding site with virtually no change in the interhelical angle of helix HI and HII (Figure 1.7. A).

Whereas the C-terminal canonical EF-hand that represents the target interaction site of S100 proteins undergoes a dramatic change in its conformation. In the Ca²⁺ free state, the helices HIII and HIV flanking the EF-hand loop adopt an antiparallel conformation similar to the EF-hands in Ca²⁺ free calmodulin (Ishida et al., 2002). Upon Ca²⁺ binding to S100 proteins, there is a large change in the orientation of helix HIII (Figure 1.7. B) whereas helix HIV that is engaged in the dimer interface does not move (Fritz and Heizmann, 2004). One consequence of the different length in the different EF-hands is that Ca²⁺

binding to the individual EF hands occur with different affinities. With 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.

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Figure 1.7: Conformational change in 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 (Otterbein et al., 2002). Adapted from Fritz and Heizmann, 2004.

2+ 2+

2+

So far S100 proteins have been isolated only from vertebrates were 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 imply 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 the human chromosome region 1q21, which points towards multiple gene duplication (Marenholz et al., 2006; Fritz and Heizmann, 2004;

Ridinger et al., 1998).

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 contraction, cell cycle regulation, cell growth, cell differentiation, mobility, transcription, differentiation and secretion. Some S100 proteins are known to be associated with human diseases like cardiomyopathy and cancer. S100A4 has been implicated in cancer and metastasis, where it shows an increased expression. S100A8/A9 which forms a heterodimer is associated with rheumatoid arthritis. Moreover, certain diseases are associated with altered expression levels of S100 proteins and can be largely classified 12

into four categories: diseases of the heart, diseases of the central nervous system, inflammatory disorders, and cancer (Marenholz et al., 2004). The variation of these physiological functions of the S100 proteins is at least in part controlled by their cell- type specific expression. 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; Sturcheler 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 (Davey et al., 2001). A further diversity within the S100 proteins results from the ability to bind other metal ions in addition to Ca²⁺. Many S100 proteins can bind Zn²⁺ and Cu²⁺ at sites different from the EF-hand site (Ambartsumian et al., 2001).

1.5. The S100B protein

S100B is one of the most investigated members of the S100 protein family. It exists in solution as a homodimer and is known to bind besides Ca²⁺ also Zn²⁺ and Cu²⁺. In the brain, S100B is mainly expressed in glia cells like astrocytes and oligodentrocytes (Donato, 2001; Huttunen et al., 2000). It is proposed that this protein plays a crucial role in glia proliferation, neuronal differentiation and in the maturation of a variety of neurons (Park et al., 2004). Comparisons of available structures of S100B with some of its targets show different modes of binding for each ligand (Figure 1.8.). Besides hydrophobic interactions, there are also charge-charge interactions contributing to the stability of S100B/target complexes. Many of these interactions originate from residues in the linker region between the two EF-hands (Bhattacharya et al., 2003).

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Figure 1.8: Comparison of S100B/target peptide binding modes: S100B is shown in the open conformation. A: Ca -S100B/NDR62-87 peptide complex, 1PSB. B: Ca -S100B/p53367-388 peptide, 1DT7. C: Ca -S100B (human)/TRTK-12, D: Ca -S100B (rat)/TRTK-12, 1MWN. The different colors display the properties of the binding surface (yellow for hydrophobic, red for acidic, blue for basic).

Adapted from Bhattacharya et al., 2003.

2+ 2+

2+ 2+

S100B is unusual in the sense that it shows extracellular activities and exerts cytokine like functions, in addition to its intracellular function. S100B is highly abundant in the brain where it constitutes about 0.5 % of all soluble proteins (Fritz and Heizmann, 2004).

Intracellularly, S100B inhibits the phosphorylation of several target proteins such as annexin II (Garbuglia et al., 2000; Hagiwara et al., 1988), neurogranin (Sheu et al., 1995), p53 (Baudier et al., 1992) and the cytoplasmic domain of the myelin associated glycoprotein in a Ca²⁺ dependent manner (Kursula et al., 2000). Moreover, S100B is also involved in the organization of the cytoskeleton; it influences the integrity of the cytoskeleton by sequestrations of tubulin filaments or binding to the fibrillary protein CapZ (Inman et al., 2002; Kilby et al., 1997; Sorci et al., 2000).

Secretion of S100B to the extracellular space has been shown for astrocytes (Zimmer and Van Eldik, 1987). S100B is secreted from human glioblastoma cells upon an increase of 14

the intracellular concentrations of calcium or a decrease of zinc (Davey et al., 2001).

Once secreted, extracellular S100B exerts dose dependent neurotrophic or neurotoxic effects. 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 (AD) (Davey et al., 2001). On the other hand, it has been shown that toxic levels of glutamate in neurons reduce also the S100B secretion, induced by serum deprivation in hippocampal astrocytes. A glutamate concentration at 2 mM as well inhibits S100B release in brain slices (Tramontina. et al., 2006 ). Interestingly S100B is encoded on chromosome 21 unlike most other S100 proteins, which leads to life-long overexpression of S100B in Down’s syndrome (Griffin et al., 1989).

1.6. RAGE, the receptor for advanced glycation end products

By now only one multiligand cell surface receptor has been identified to mediate the extracellular signals of secreted S100 proteins, activating a signal cascade including the MAP-kinase or NF-κB pathway (Hofmann et al., 1999). This receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily. It is a multi-ligand, cell surface receptor expressed by neurons, microglia, astrocytes, cerebral endothelial cells, pericytes, and smooth muscle cells. At least three major types of the RAGE isoforms (full length, C-truncated, and N-truncated) are present in human brains 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. Differential expression of each isoform may play a regulatory role in the physiological and pathophysiological functions of RAGE (Lue et al., 2005). The inhibition of RAGE signaling by the secreted sRAGE might occur by sequestering circulating RAGE ligands or compete with full-length RAGE for binding to adhesion molecules such as β2-integrin Mac-1 (Chavakis et al., 2003). Several studies using animal models have shown that sRAGE is a

-INTRODUCTION-

pharmaceutically relevant therapeutic molecule with 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). Moreover, RAGE activation has also been implicated as a progressive factor in several disorders including chronic inflammation, cancer and Alzheimer’s disease (Schmidt et al., 2001). Interestingly it has been shown that both S100B and RAGE are expressed in elevated levels in Alzheimer’s disease (Arancio et al., 2004; Liu et al., 2005). It is proposed that larger S100 assemblies in the extracellular space may trigger an aggregation of RAGE, which then in turn activates the intracellular signaling cascade (Figure 1.9.). Multimeric forms of S100 proteins have been reported for S100A12, (Moroz et al., 2002), for S100A4 (Novitskaya et al., 2000) and S100A8/A9 (Brückner et. al., 2005). It has been shown that even in the presence of endogenous full-length RAGE, solely the expression of truncated DN- RAGE, acts in a dominant negative manner and abolishes RAGE-mediated cellular activation (Schmidt and Stern, 2001). This effect indicates that larger assemblies, like receptor dimers, of RAGE are necessary for signaling and that multimeric forms of S100 proteins can trigger the formation of these assemblies.

Figure 1.9: Schematic depiction of RAGE-S100B interaction, showing the three extracellular domains of the receptor: two C-type domains (conserved) 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 + transmembrane-spanning domain). Adapted & modified from Schmidt and Stern, 2001.

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1.7. Scope of this study

S100B is one of the most abundant proteins in the brain and exerts intra- as well as extracellular functions. It has been implicated to play a key role in many neurodegenerative diseases. Earlier studies showed that these functions might be controlled in a Ca²⁺ and / or Zn²⁺ dependent manner. A crucial step in the pathogenesis of neurodegenerative diseases is the activation of the cell surface receptor RAGE by extracellular S100B. Currently, only two NMR structures of human Ca²⁺-S100B has been described: dimeric S100B (1UWO) and a complex with the peptide TRTK12 (1MQ1). Furthermore only one crystal structure of bovine S100B (1MHO) has been solved so far, but there is no crystal structure of human S100B.

The focus and scope of this doctoral thesis was to collect more structural information on the human S100B protein and to investigate the interaction of S100B with its receptor RAGE.

1.7. Ziel der Studie

S100B ist eins der am häufigsten exprimierten Proteine im Gehirn wo es sowohl intra- als auch extrazellulare Funktion übernimmt. Es wird angenommen dass es eine Schlüsselrolle in der Entstehung verschiedener neurodegenerativer Erkrankungen einnimmt. Frühere Studien zeigten bereits, dass diese Funktionen Ca²⁺ und / oder Zn²⁺

abhängig gesteuert werden. Ein entscheidender Schritt in der Pathogenese bei neurodegenerativen Erkrankungen ist die Aktivierung des Zelloberflächenrezeptors RAGE durch extrazelluläres S100B. Bislang sind nur zwei NMR Strukturen des humanen Ca²⁺-S100B Protein bekannt. Eine Struktur von S100B allein (1UWO) und eine im Komplex mit dem Peptid TRTK12 (1MQ1). Die einzige Kristallstruktur zu S100B ist die des homologen S100B aus Rind (1MHO). Derzeit ist noch keine Kristallstruktur zum humanen S100B verfügbar.

Schwerpunkt und Ziel der hier vorgelegten Doktorarbeit war es detaillierte Strukturinformationen über das menschliche S100B Protein zu bekommen und die Interaktion von S100B mit seinem Rezeptor RAGE zu untersuchen.

2 M ^ATERIALS & M ^ETHODS

Those materials and methods, which have not been described in this section, will be presented separately in the publications in detail (chapter 3).

2.1. Analytical methods

Protein

S100B was determined spectrophotometrically using the extinction coefficient ε 278 nm = 1520 M^-1 cm^-1, calculated from the amino acid sequence (http://www.expasy.org/tools/#

proteom).

sRAGE was determined by the bicinchoninic acid method (Smith et al. 1985); 100 µl sample (5-20 µg protein) and 1 ml 50:1 (v/v) BCA / 4 % (w/v) CuSO4⋅5H2O were mixed and incubated for 15 min at 60 °C. After incubation, the samples were cooled on ice and centrifuged for 5 min at 12,000 g. The absorbance at 562 nm was measured on a HP 8452 A diode array spectrophotometer (Hewlett Packard) and the concentration was calculated using BSA (5-21 µg) for calibration.

Molecular mass

The native relative molecular mass of the S100B species was determined on a Superdex^TM 75 HiLoad^TM 26/60 gelfiltration column (2.6 × 60 cm; GE Healthcare), equilibrated in 50 mM Tris-HCl, pH 7.6, 150 mM NaCl. The column was calibrated with cytochrome c (12.4 kDa), carbonic anhydrase (29 kDa), conalbumin (76 kDa), and alcohol dehydrogenase (150 kDa) (Sigma).

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Mass spectrometry

Mass spectra of undigested purified recombinant human S100B were obtained on a PE SCIEX API 365 LC/MS electrospray ionizationmass spectrometer (Perkin-Elmer). The protein samples were dialyzedagainst water and diluted to a final concentration of 50 µg/mlin running medium containing 50 % acetonitrile and 0.1 % aceticacid. 20 µl of the protein solution wereinjected into the electrospray ionization mass spectrometer (ESI- MS). (The measurments were kindly conducted by Heinz Troxler; Department of Pediatrics, Division of Clinical Chemistry and Biochemistry, University of Zürich Switzerland)

UV/Vis spectroscopy

UV/Vis absorption spectra were obtained at room temperature with a Cary 50 spectrophotometer (Varian, Darmstadt), or with a HP 8452 A Diode Array spectrophotometer (Hewlett Packard).

2.2. Crystallography

The notations of the method in this subsection (2.2.) are taken in parts from the textbook X-Ray Crystallography of Biomacromolecules (Messerschmidt, A. 2006).

Theoretical background

Protein crystallography is used to produce magnified images of protein molecules.

Thereby, the maximum attainable resolution of any microscopic technique is limited by the applied wavelength. Consequently, the wavelength defines the fineness of detail that can be observed. In order to image the atomic structure of a molecule, for example the atomic distance of a carbon-carbon σ-bond is 1.54 Å, it is necessary to use wavelengths no larger than a couple of Ångström units. Electromagnetic waves of this wavelength are known as X-rays.

However, while light or electron microscopy uses lenses to merge the waves diffracted by an object into an enlarged image, there are no such lenses available for X-rays. Max von Laue realized in 1912, that the three-dimensionally ordered lattice arrangement of a crystal would cause interference of the diffracted photons resulting in discrete maxima whose intensity can be measured in an appropriate experimental set-up.

Crystal growth

The process of crystal formation is in principle thermodynamically favored, driven by the gain of entropy through the loss of the proteins ordered hydratation shell. Crystals grow if the solution that surrounds them is oversaturated. Then, the molecules of the protein crystal are deposited on the surface of the growing crystal, and come into exactly the correct orientation to continue the pattern of crystal lattice. Mechanistically, the crystallization process can be divided into two stages, seed formation and crystal growth.

Seed formation occurs in an equilibrium of formation and dissolvation of small aggregates, determined by the free energy ΔG. The critical cluster size for protein crystals is between 10 and 200 molecules.

Crystals

A crystal can be regarded as a three-dimensional repetition of a single building block, the unit cell. Within the unit cell, a crystal can contain further symmetry elements – rotation, reflection, and/or inversion – dividing it into several asymmetric units, which form the most basic structural element within the crystal. The geometry of the unit cell together with the possible symmetry operations defines the space group of the crystal. Although there are 230 space groups in seven crystal systems (Table 2.1.), only 65 are enantiomorphic and are thus feasible for chiral molecules such as proteins. Identification of the correct space group is essential for correct indexing of diffraction patterns and therefore the first step of understanding a crystal structure. A typical axial length of the unit cell in protein crystals can range from 30 Å to 200 Å.

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Crystal system

Minimum symmetry requirement

Conventional choice of axes

Constraints on interaxial angles and axial lengths

Triclinic None No constraints None

Monoclinic One 2-fold axis b parallel to 2-fold

a and c perpendicular to 2-fold

α and γ = 90°

Orthorhombic Three perpendicular 2-fold axes

a, b, and c parallel to 2-fold axes

α, β, and γ all 90°

Trigonal One 3-fold axis c parallel to 3-fold

a and b perpendicular to 3-fold

β = 120°, α and γ = 90°

a and b equal length Tetragonal One 4-fold axis c parallel to 4-fold

a and b perpendicular to 4-fold

α, β, and γ all 90°

a and b equal length Hexagonal One 6-fold axis c parallel to 6-fold

a and b perpendicular to 6-fold

β = 120°, α and γ = 90°

a and b equal length Cubic Four 3-fold axes a, b, and c related by 3-fold axis α, β, and γ all 90°

a, b and c equal length

Table 2.1: Protein crystal systems.

X-ray diffraction by crystals

If an X-ray photon encounters an electron, it may be absorbed and sets the electron into vibration at the X-ray frequency. This vibrating electron then emits an X-ray photon in a random direction, of the original energy and wavelength but with a phase difference of 180° (coherent scattering). Since the atoms are localized within the crystal lattice in a regular, periodically repeated spatial arrangement, the single waves originating from any point of finite electron density sum up to a total intensity of the secondary radiation of zero (destructive interference), except if the path difference between the waves is an integer multiple of their wavelength (constructive interference). Given the correct orientation of the crystal, this condition is fulfilled for corresponding positions in all unit cells. Diffraction of X-rays on the real lattice of a crystal thus creates another three- dimensional lattice of diffraction maxima. As the geometric properties of this lattice are inverse to those of the real crystal, it is referred to as the reciprocal lattice.

A convenient way to describe diffraction by a crystal lattice is to imagine every single diffraction spot to be a reflection of the incident beam on an imaginary lattice plane. The

position of the lattice planes and their corresponding reflections are identified by the Miller indices (h,k,l) (see Figure 2.1. A) which result from the points of intersection between the lattice plane and the unit cell edges. The values of (h,k,l) correlate to the reciprocal coordinates of the points of intersection whereas the lengths of the unit cell edges are standardized to 1.

Regarding coherent diffraction on a set of lattice planes with distance d, constructive interference will occur at an angle θ, if the path difference between the diffracted waves is an integer multiple of the wavelength λ. This relation between reflection angle and lattice plane distance is known as Bragg’s law (see Figure 2.1. B):

λ θ n d_hklsin = 2

The Ewald sphere (see Figure 2.2.) is a tool for constructing reciprocal lattice points based on Bragg’s law. It is a sphere of radius 1/λ with the crystal in its centre.

A B

Figure 2.1: Reciprocal lattice planes and Bragg’s law. A) Lattice planes that allow for constructive interference of diffracted waves are those that divide the unit cell edges into integer fractions. The number of those fractions is used to index the plane. The group of lattice planes shown would have the Miller indices (2,3,3). B) Two waves that are reflected by two adjacent lattice planes with distance d have a difference in path length that is equal to 2d sin θ, as it can be derived from the scheme. A prerequisite for constructive interference is, that this difference in path is an integer multiple of the wavelength used:

2d sin θ = n λ (Bragg’s law). Both schemes are taken from Blow, 2002.

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The point where the incident beam enters the sphere and the origin O of the reciprocal lattice are on opposite sides of the centre. The normal vectors

sro

Sr

of the imaginary lattice planes build up the reciprocal lattice, their length reflecting the reciprocal distance of the planes. Bragg’s law is fulfilled for every reciprocal lattice point that lies on the Ewald sphere. A rotation of the crystal rotates the reciprocal lattice in the same way, allowing different reciprocal lattice points to intersect with the sphere. For the given orientation of the crystal, those points are the ones that can be recorded on an X-ray detector.

As every recorded diffraction spot represents one lattice plane (h,k,l), the measurement of the positions of the spots is sufficient to deduce the geometry of the crystal and in most cases also the space group, as additional symmetry elements can manifest in the form of systematic extinctions of reflections. On the other hand, the content of the unit cell influences the intensity of the diffraction spots. A crystal is described as the convolution of the unit cell content with the three-dimensional lattice. This means that the diffraction pattern of this crystal will be the product of the diffraction pattern of the lattice with that of the unit cell content. Since the lattice diffraction pattern is zero except at the reciprocal lattice of points, the diffraction pattern of the crystal formed by convolution with this lattice is also zero except at the reciprocal lattice points.

C O

1 / λ Sr

d θ

Figure 2.2: The Ewald construction. In reciprocal space, the crystal (C) is placed in the centre of a sphere (here, in two dimensions, a circle) with radius 1/λ, called the Ewald sphere. The origin of the reciprocal lattice, i.e. reflection (0,0,0), is placed in (O). The reciprocal lattice (grey dots) will rotate as the crystal does and only those reciprocal lattice points that intersect with the Ewald sphere will be in diffraction condition (red dots) and will be recorded on an image plate detector in real space.

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