Structural characterization of the novel 34 in complex with its cognate receptor CSF-1R

Erwin PANNECOUCKE

Master’s dissertation submitted to obtain the degree of Master of Biochemistry and Biotechnology Major Biochemistry and Structural Biology Academic year 2011-2012

Promoter: Prof. Dr. Savvas Savvides Scientific supervisor: Drs. Jan Felix Department of Biochemistry and Microbiology Laboratory for Biochemistry & Biomolecular Engineering Unit for Structural Biology and Biophysics

Structural characterization of the novel cytokine in complex with its cognate receptor CSF-1R

Erwin PANNECOUCKE

Master’s dissertation submitted to obtain the degree of Master of Biochemistry and Biotechnology Major Biochemistry and Structural Biology Academic year 2011-2012

Promoter: Prof. Dr. Savvas Savvides Scientific supervisor: Drs. Jan Felix Department of Biochemistry and Microbiology Laboratory for Protein Biochemistry & Biomolecular Engineering Unit for Structural Biology and Biophysics

Acknowledgements At the start of this academic year, I had already spend several afternoons and even several days in a laboratory, experiencing how the sometimes abstract theory from the lessons could be translated to useful applications in the real scientific world. There is however one practical session that I will probably remember for the rest of my life, and that is our excursion to the synchrotron facility in Hamburg. Not because of the excellent steak house, the overwhelming Chinese buffet nor because of the, euhm, let’s say intriguing Reperbahn. I will remember it because of that old, industrial-looking hutch were we were locked into and, away from all daylight, were most of the time staring at three monitors with black dots on it. I can safely say that that specific day, in which concepts like ‘Bravais lattice’, ‘Bragg-spacing’, ‘diffraction symmetry’ and ‘phasing’ were almost literally thrown at us, was one of the most intellectual challenging days of my life. Never before I had to process so much mental material during such a long time, but I enjoyed literally every minute of it. At that very moment I realized not only my head was going to blow, but also that I wanted to explore this mystical world of X-ray crystallography. At the end of this Master’s Dissertation, that same world is now a bit less mystical, although that I have the feeling that I’ve only seen a tip of the ice berg. I’ve had the opportunity to participate in this interesting project and despite the long days at the lab (I thought that the university was closed after 18h and on Sunday, but boy was I proven wrong) and the both intellectually and physically exhausting synchrotron night shifts, I enjoyed it so much that I’m hoping to be able to continue in it.

I would like to express my gratitude to several people, of which in the first place professor Savvides. Not only has he given me this opportunity to participate in this interesting project, but the way he inspires and challenges both students and lab members, is truly enriching. Next, I would like to thank my supervisor Jan Felix for allowing me to join in his project. Despite the professional setbacks, he succeeded every time to see the bright side and never hesitated a single moment in patiently helping me around. Even now, 600 km away and occupied a whole day, he still wants to correct my thesis! Furthermore, I would like to thank the reading commission for reading this thesis. I also would like to thank Kenneth an Bjorn for the little chat sessions at the Senseo machine, and all other members of L-ProBE for their pleasant intercourse and open-minded mentality. Also my collegues a.k.a. fellow students earn my gratitude for their pleasant talks, their sharing of experiences and for helping me with stupid questions I didn’t dare to ask anyone else. A big thank you for my parents and my two sisters as well. Even after five years they have still no clue about what I’m studying, but nevertheless keep supporting me. I still don’t understand how they succeeded in bearing my horrible mood twice a year, during the exams. Finally, I want to thank Lien for her support and her willingness to listen, and the surprising hearty way she kept welcoming me when I was for the umpteenth time too late on our appointment because I miscalculated my lab work.

i Table of Contents

Table of Contents Acknowledgements ...... p i Table of Contents ...... p ii List of Figures ...... p vi List of Tables ...... p viii List of Abbreviations ...... p ix Samenvatting ...... p xii Summary ...... p xiii

Part1: Introduction p 1 I. Hematopoiesis ...... p 2 1.1 Blood – a complex tissue in a liquid form ...... p 2 1.1.1 Hematopoiesis follows a strictly regulated hierarchy ...... p 2

II. Receptor tyrosine kinases ...... p 5 2.1 Phosphorylation is the key to almost every cellular signal transduction ...... p 5 2.1.1 Receptor tyrosine kinases are an important class of receptors ...... p 5 2.2 Receptor Tyrosine Kinases – a large and various family ...... p 5 2.1.2 The RTK ligand elicits receptor dimerization ...... p 6 2.1.3 Receptor:receptor interactions trigger the post-oligomerization events ..... p 7

III. Class III receptor tyrosine kinases ...... p 9 3.1 RTK-III are structurally homologues ...... p 9 3.1.1 RTK-IIIs are characterized by a modular structure ...... p 9 3.1.2 All RTK-III have extracellular Ig-like domains ...... p 10 3.1.3 Most RTK-III have a conserved motif for homotypic receptor interactions ...... p 11 3.1.4 KIT, Flt3 and CSF-1R bind a ligand with a short-chain four-helical bundle cytokine fold ...... p 12 3.2 The Platelet-derived receptor PDGFR and its ligands ...... p 12 3.2.1 The Platelet-derived growth factor family involves five isomers, binding two receptors ...... p 12 3.2.2 PDGFs show a cysteine knot fold ...... p 13 3.2.3 The PDGF:PDGFR interactions obeys the classical 2:2 stoichiometry ...... p 13

ii Table of Contents

3.3 The receptor tyrosine kinase KIT and its ligand, SCF ...... p 15 3.3.1 – key player in a wide range of (patho)physiological mechanistics ...... p 15 3.3.2 SCF:KIT – the structural paradigm for ligand-induced dimerization of RTK-III ...... p 15 3.4 Fms-like tyrosine kinase-3 Flt3 and its ligand, Flt3 Ligand ...... p 17 3.4.1 Flt3L and signaling through the Flt3:Flt3L complex is indispensable for hematopoietic homeostasis ...... p 17 3.4.2 The Flt3L:Flt3 interaction – an ensemble of surprising features ...... p 18 3.5 Colony-stimulating factor-1 and its ligand, CSF-1 ...... p 19 3.5.1 Colony-stimulating factor-1 is essential for the immune system ...... p 19 3.5.2 An unexpected twist in CSF-1R signaling: interleukin 34 ...... p 20 3.6 Interleukin 34 and CSF-1 – binding partners of the promiscuous CSF-1R ...... p 20 3.6.1 IL-34 and CSF-1 functionally overlap but show a differential expression pattern ...... p 20 3.6.2 both CSF-1 and IL-34 bind CSF-1R ...... p 21 3.6.3 The complex of mIL-34 and mCSF-1R is similar to mCSF-1 bound to mCSF-1R, but nevertheless does have his own features ...... p 22

Part 2: Goal p 25 IV. Aim of the Research Project ...... p 26

Part 3: Results p 28 V. Time course limited proteolysis of full-length hIL-34 in complex with hCSF-1RD1-D5 ...... p 29

VI. Small-scale co-expression of different hIL-34:hCSF-1R complexes ...... p 31 6.1 Co-expression of IL-34 and its receptor, CSF-1R ...... p 31

VII. Large scale expression and crystallization of hIL-34p180 in complex with hCSF-1RD1-D3 ...... p 33 7.1 Cobalt Immobilized Metal Affinity Chromatography of hIL-34p180:hCSF-1RD1-D3 ...... p 33

7.2 Gel filtration of hIL-34p180:hCSF-1RD1-D3 ...... p 33

7.3 Crystallization screening of hIL-34p180:hCSF-1RD1-D3 ...... p 35

7.4 Deglycosylation and purification of the hIL-34p180:hCSF-1RD1-D3 complex ...... p 35

7.5 Crystallization trials of deglycosylated hIL-34p180:hCSF-1RD1-D3 ...... p 37

iii Table of Contents

VIII. Large scale expression and crystallization of hIL-34p180 in complex with hCSF-1RD2-D4 ...... p 41 8.1 Cobalt Immobilized Metal Affinity Chromatography of hIL-34p180:hCSF-1RD2-D4 ...... p 41

8.2 Gel-filtration of hIL-34p180:hCSF-1RD2-D4 subsequent to Co IMAC ...... p 43

8.3 Crystallization trials of hIL-34p180:hCSF-1RD2-D4 ...... p 45

Part 4: Discussion and Conclusions p 48 IX. Discussion...... p 49 9.1 Time course limited proteolysis experiment of full-length hIL-34 in complex with hCSF-2RD1-D5 ...... p 49 9.2 Small-scale co-expression of different hIL-34:hCSF-1R complexes ...... p 49 9.3 Large-scale expression and crystallization of hIL-34p180 in complex with hCSF- 1RD1-D3 ...... p 50 9.4 Large-scale expression and crystallization of hIL-34p180 in complex with hCSF- 1RD2-D4 ...... p 52

X. Conclusions ...... p 54 10.1 Large-scale expression and crystallization of deglycosylated hIL-34p180 in complex with hCSF-1RD1-D3 ...... p 54 10.2 Large-scale expression and crystallization of hIL-34p180 in complex with hCSF- 1RD2-D4 ...... p 54

XI. Future perspectives ...... p 55

Part 5: Material and Methods p 56 XI. Material and methods ...... p 57 12.1 Preparation of hCSF-1R plasmid DNA ...... p 57 12.1.1 Transformation to electrocompetent cells ...... p 57 12.1.2 Transformation of electrocompetent E. coli MC 1061 cells and preparation of expression constructs ...... p 57 12.2 Expression and purification of complexes between recombinant hIL-34 and hIL-34p180 and recombinant hCSF-1R in HEK293S GnTI-/- cell lines ...... p 57 12.2.1 Tetracycline-induced expression of recombinant hIL-34 or hIL-34p180, simultaneously to transfection with hCSF-1R constructs in HEK293S GnTI-/- cell lines ...... p 57

iv Table of Contents

12.2.2 Cobalt immobilized metal affinity chromatography (Co IMAC) of hIL- 34:receptor complexes ...... p 58 12.2.3 Purification of ligand:receptor complexes by size-exclusion chromatography ...... p 58 12.2.4 Analysis of the protein contents in Co IMAC and gel-filtration fractions . p 59 12.3 Time-course limited proteolysis of full-length hIL-34 in complex with hCSF-1RD1-D5 ...... p 59 12.4 Small-scale co-expression of different hIL-34:hCSF-1R complexes...... p 59

12.5 Deglycosylation and purification of hIL-34p180:hCSF-1RD1-D3 complex ...... p 60

12.5.1 Deglycosylation of hIL-34p180:hCSF-1RD1-D3 ...... p 60

12.5.2 Purification of deglycosylated hIL-34p180:hCSF-1RD1-D3 ...... p 61 12.6 Crystallization trials ...... p 61

12.6.1 Crystallization of hIL-34p180:hCSF-1RD1-D3 ...... p 61

12.6.2 Crystallization of deglycosylated hIL-34p180:hCSF-1RD1-D3 ...... p 61

12.6.3 Crystallization of hIL-34p180:hCSF-1RD2-D4 ...... p 63 1.7 Crystallographic experiments ...... p 63 12.7.1 Data collection from crystals containing deglycosylated hIL-34p180:hCSF-1RD1-D3 ...... p 63

12.7.2 Data collection from hIL-34p180:hCSF-1RD2-D4 crystals ...... p 63 12.7.3 X-ray data analysis ...... p 63

Part 6: References p 64 XII. Reference list ...... p 65

Part 7: Appendices p 70 Appendix 1. Preparing electrocompetent E. coli MC 1061 cells ...... p 71

Appendix 2. Transformation of electrocompetent E. coli MC 1061 cells and preparation of plasmid DNA ...... p 72

Appendix 3. Time course limited proteolysis of full-length hIL-34 in complex with hCSF-1RD1-D5 ...... p 73

Appendix 4. Calculated molecular masses and extinction coefficients of hIL-34 and hIL-34p180 alone and in complex with diverse hCSF-1R constructs ...... p 75

v List of figures

List of Figures

Figure 1.1. Stem cells give rise to the cellular repertoire of the hematopoietic and immune system...... p 3 Figure 2.2: Ligand-induced receptor dimerization induces transphosphorylation and subsequent receptor activation...... p 7 Figure 3.1: Architecture of the RTK-III receptors...... p 9 Figure 3.2: Classification of the Immunoglobulin-like fold ...... p 10 Figure 3.3: Most RTK-III engage in homotypical interactions upon ligand binding...... p 11 Figure 3.4: The four-helical bundle cytokine fold...... p 12 Figure 3.5: PDGFs fold into a cysteine knot structure...... p 13 Figure 3.6: PDGF-BB dimerizes PDGFR-β by an RTK-III characteristic 2:2 stoichiometry ... p 14 Figure 3.7: SCF interacts with three sites on KIT and subsequently undergoes conformational changes...... p 16 Figure 3.8: The structure of Flt3L in complex with Flt3 shows some unanticipated features...... p 18 Figure 3.9: Despite the difference in sequence, mCSF-1 and mIL-34 adopt a similar fold ...... p 21 Figure 3.10: CSF-1R interacts in a very similar way with CSF-1 and IL-34 ...... p 23 Figure 3.11: IL-34 and CSF-1 have overlapping binding sites on CSF-1R ...... p 24

Figure 5.1: Limited proteolysis of full-length hIL-34 in complex with hCSF-1RD1-D5 ...... p 30 Figure 6.1: (co)expression of hIL-34p180 and different hCSF-1R constructs ...... p 31 Figure 6.2: Western blot analysis of the (co)expressed IL-34p180 and divers hCSF-1R constructs ...... p 32

Figure 7.1: Cobalt IMAC elution profile of hIL-34p180 in complex with hCSF-1RD1-D3, derived from a large-scale expression in the HEK293S GnTI-/-cell line ...... p 33

Figure 7.3: Amylose affinity chromatography of the hIL-34p180:hCSF-1RD1-D3 after being incubated overnight with EndoHf...... p 36 Figure 7.4: Optimization screen of the initial crystal condition gave few hits ...... p 37 Figure 7.5: Silver Bullets™ screen with the best scoring crystal condition form the grid screen as basis, gave multiple hits ...... p 38 Figure 7.6: Silver stained gel of a crystal from the Silver Bullets™ screen, analyzed by SDS-PAGE ...... p 39

Figure 7.7: Representative diffraction pattern from crystals of hIL-34p180:hCSF-1RD1-D3 . p 40

vi List of figures

Figure 8.1: Cobalt IMAC purification of medium containing hIL-34p180:hCSF-1RD2-D4 ...... p 42

Figure 8.2: Superdex™ 200 gel-filtration of hIL-34p180:hCSF-1RD2-D4 and subsequent analysis of key fractions on a SDS-PAGE gel ...... p 44 Figure 8.3: Condition C4 of the ProPlex™ screen containing spherulite crystals ...... p 45

Figure 8.4: hIL-34p180:hCSF-1RD2-D4 containing crystal drop with C10 of the ProPlex™ screen ...... p 46

Figure 8.5: Representative diffraction pattern of hIL-34p180:hCSF-1RD2-D4 ...... p 47

vii List of Tables

List of Tables Table 1.1: Overview of the best characterized hematopoietic cytokine functions...... p 4

Table 7.1: Key experimental details of data collection on crystals of deglycosylated hIL-34p180:hCSF-1RD1-D3 ...... p 40

Table 8.1: Key experimental details of data collection on crystals of hIL-34p180:hCSF-1RD2-D4 ...... p 46

Table A.1: Calculated molecular masses and extinction coefficients of hIL-34 and hIL-34p180 alone and in complex with diverse hCSF-1R constructs...... p 75

viii Abbreviations

List of Abbreviations ALL Acute lymphoblastic leukemia AML Acute myeloid leukemia Arg Arginine Asn Asparagine AU Absorbance units BME Beta-mercaptoethanol CLP Common lymphoid progenitor CMP Common myeloid progenitor Co IMAC Cobalt immobilized metal affinity chromatography

CO2 Carbon dioxide CSF-1 Colony-stimulating factor-1 CSF-1R Receptor for Colony-Stimulating Factor-1 CSF1-RDx Extracellular domain x of CSF-1R CSF-1RDx-DX Extracellular domains x to y of CSF-1R Cys Cysteine D Aspartic acid DC Dendritic cell DMEM Dulbecco's Modified Eagle Medium DNA Deoxyribonucleic acid E Glutamic acid EGF epidermal growth factor EPO ERK Extracellular signal-regulated kinase Flt3 Fms-like tyrosine kinase receptor-3

Flt3Dx Extracellular domain x of Flt3 Flt3Dx-Dx Extracellular domains x to y of Flt3 Flt3L Ligand for Flt3 G Glycine G-CSF Granulocyte colony-stimulating factor GlucNAc N-acetylglucosamine Gly Glycine GM-CSF Granulocyte/macrophage colony-stimulating factor GMP Granulocyte- progenitor GnTI N-acetylglucosaminyltransferase-I hCSF-1 human Colony-stimulating factor-1 hCSF-1R human Colony-stimulating factor-1 receptor HEK Human embryonic HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid hIL-34 human Interleukin 34 HSC Hematopoietic stem cells I Isoleucine Ig Immunoglobulin

ix Abbreviations

IL-11 IL-3 IL-34 Interleukin 34 IL-5 Interleukin 5 ITC Isothermal titration calorimetry IUP Inherently unstructured protein(region) Kd Dissociation constant KIT Stem cell factor receptor KITDx Extracellular domain x of KIT KITDx-Dy Extracellular domains x to y of KIT L leucine LIF Leukemia inhibitory factor LT-HSC long-term hematopoietic stem cells Man Mannose mAU Mille-absorbance units MBP Maltose-binding protein M-CSF Macrophage colony-stimulating factor CSF-1 mCSF-1 mouse Colony-stimulating factor-1 mCSF-1R mouse Colony-stimulating factor-1 receptor MEP Megakaryocyte-erythroid progenitor MES 2-(N-morpholino)ethanesulfonic acid mIL-34 mouse Interleukin 34 MPP Multipotente progenitor mRNA Messenger ribonucleic acid N-glycosylation Asparagine-linked carbohydrate structure NK cells Natural killer cells OD600 Scattering intensity for with a wavelength of 600 nm O-glycosylation Hydroxyl-linked carbohydrate structure PDB RCSF Protein Data Bank PDGF Platelet-derived growth factors PDGFR Receptor for platelet-derived growht factors PEG Polyethylene glycol ph Photons PK Protein kinase PTB Phosphotyrosine-binding R Arginine RONN Regional Order Neural Network RTK Tyrosine Kinase Receptor RTK-III Receptor Tyrosine Kinase Class III SCF Stem cell factor SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis SH2 Src homology-2 ST-HSC Short-term hematopoietic stem cells Tyr Tyrosine

x Abbreviations

UV280 Ultraviolet light with a wavelength of 280 nm VEGFR Vascular endothelical growth factor receptors v- Feline sarcoma viral oncogene x In the contexts of an sequence: 'every possible amino acid' φ Hydrophobic amino acid

xi Samenvatting

Samenvatting Hematopoëse, de ontwikkeling van mature bloedcellen, is een essentieel fysiologisch proces in het behoud van de homeostase. Het proces wordt zeer strikt gereguleerd door een complexe samenwerking van hematopoëtische die hun effect uitoefenen door te binden aan specifieke receptoren aan het celoppervlak. Een subgroep van dergelijke receptoren is klasse III van de Receptor Tyrosine Kinases (RTK-III) receptorfamilie. Men heeft reeds kunnen aantonen dat signalisatie doorheen deze receptoren essentieel is voor zowel de ontwikkeling als het behoud van het cellulaire repertoire binnen het immuun- en hematopoëtische systeem. Deze receptoren zijn in de afgelopen 40 jaar dan ook al het onderwerp geweest van talloze onderzoeken. Dat er echter nog veel werk is binnen dit domein, wordt weerspiegeld door de recente ontdekking van interleukine 34 (IL-34) als tweede, natuurlijk voorkomende ligand voor de Colony-Stimulating factor-1 receptor (CSF-1R). Terwijl celbiologische studies proberen te achterhalen wat het nut is van twee functioneel redundante cytokines, zijn er recent twee structurele studies verschenen die de kristallografische structuur tonen van zowel humaan als muis IL-34 in complex met hun receptor. Maar ondanks de waardevolle inzichten die deze studies opleveren, is geen van beiden in staat om de globale architectuur van het IL-34:CSF-1R complex te belichten, noch kunnen zij duidelijkheid scheppen omtrent de voorspelde homotypische receptor- interacties. Nochtans zou deze kennis niet enkel bijdragen tot de fundamentele kennis over CSF-1R signalisatie, maar ook onontbeerlijk zijn voor het rationeel ontwerpen van geneesmiddelen gericht tegen de extracellulaire domeinen.

In deze Masterproef worden de eerste stappen gezet om met behulp van X-stralen kristallografie de ternaire structuur van hL-34 in complex met hCSF-1R te bepalen. Ook de betrokkenheid van de receptor in homotypsiche interacties behoort tot de doelstellingen. HEK293S GnTI-/- cellijnen die hetzij recombinant hIL-34, hetzij recombinant een deel van hIL-34 stabiel expresseren, zijn reeds voor deze doeleinden ontwikkeld. Deze cellijnen zullen nu gebruikt worden om hCSF-1R constructen te coëxpresseren met hIL-34. Het hIL-34:hCSF-1R complex dat hierdoor gesecreteerd wordt, zal met behulp van kobalt- affiniteitschromatografie en gelfiltratie uit het medium gezuiverd worden en worden onderworpen aan kristallisatietesten. Om de kans op succesvol kristallisatie-experiment te verhogen, zullen verschillende constructen van de extracellulaire receptordomeinen op deze manier getest worden. Ook deglycosylatie van proteïnecomplexen als een mogelijke piste voor het verkrijgen van kristallen, zal in deze Masterproef onderzocht worden.

Deze studie toont aan dat deglycosylatie van proteïnecomplexen inderdaad kan aangewend worden als kristallisatiestrategie. Daarnaast werden ook kristallen verkregen van het complex met een domain 4 bevattend hCSF-1R construct. Ondanks geen van de kristallen geleid heeft tot een structuurbepaling, kan gesteld worden dat deze scriptie nieuwe pistes blootgelegd heeft die mogelijks tot inzichten kunnen leiden in de structurele mechanismen van de hIL-34:hCSF-1R interactie. Hoewel deze studie zelf niet beantwoordt aan de vooropgestelde doelstellingen, leiden de getrokken conclusies naar nieuwe strategieën om een hoge-resolutiemodel te verkrijgen van het ternaire complex, en duidelijkheid te scheppen over de mogelijkse homotypische receptor-interacties.

xii Summary

Summary The development of mature blood cells, termed hematopoiesis, is a process that is essential for the maintenance of the homeostasis. This process is very strictly regulated by a complex pattern of hematopoietic cytokines that bind and signal trough their cognate cell-surface expressed receptors. One such class of receptors is the Receptor Tyrosine Kinase class III (RTK-III) receptor family. Signalization trough these receptors is proven to be indispensable for both development and maintenance of the hematopoietic and the immune cellular repertoire, and has already been the subject of numerous studies over the past 40 years. However, indicative for the work that has yet to be done, is the recent discovery of interleukine 34 (IL-34) as a second, naturally occurring ligand of Colony-Stimulating Factor-1 Receptor (CSF-1R). Whilst cell biological studies are trying to understand the relevance of two CSF-1R-binding cytokines, two recent publications were able to show the crystallographic structure of both human and mouse IL-34 in complex with their receptor. Despite the valuable insights that they deliver, both structures are still failing to show the global architecture of the IL-34:CSF-1R complex and lack structural insights in the predicted homotypical receptor interactions. These insights would not only contribute significantly to our knowledge about CSF-1R signaling, but are also indispensable in rational design of drugs targeting the extracellular domains.

During this Master’s Dissertation, we set out towards the ternary structure determination of human IL-34 in complex with human CSF-1R by X-ray crystallography. Also the determination of possible CSF-1R homotypical interactions is part of the objectives. Stable HEK293S GnTI-/- cell lines that secrete recombinant hIL-34 or truncated hIL-34 in milligram amounts, are already developed. These cell lines are now used to co-express human CSF-1R with hIL-34. Subsequent to expression, the resulting hIL-34:hCSF-1R complex is purified by a combination of cobalt affinity and size exclusion chromatography, and subjected to diverse crystallization trials. To enlarge the likelihood of success, different constructs of the extracellular domains of hCSF-1R are assayed. Moverover, deglycosylation of the complex is tested as a possible crystallization strategy.

At the end of this study, deglycosylation of the protein complex appeared to be a successful crystallization approach, and also crystals from complexes with hCSF-1R, including domain 4, were obtained. However, none of the diffracting crystals could lead to a structure. Nevertheless one can conclude that this Master’s Dissertation has revealed novel possible approaches to gain insights in the structural mechanisms underlying hIL-34 recognition by hCSF-1R. Although no direct results are derived from this study, its conclusions lead to new strategies for achieving a high-resolution model of the human ternary complex, providing clarity about the homotypical receptor interactions.

xiii

Part 1 Introduction

Part 1: Introduction

I. Hematopoiesis 1.1 Blood – a complex tissue in a liquid form Blood is a special form of binding tissue (S. Silbernagl & Rothenburger, 2008) that unifies an enormous diversity of compounds. The five liters of blood of an average individual consists roughly of 55 % fluid plasma - being water, , metabolites and ions - and 45 % cells. The latter, known as the cellular compartment, is formed by three groups of cells: the erythrocytes, the leukocytes and the platelets. The most abundant cells in the cellular blood compartment are the erythrocytes. They have a biconcave disk-shaped morphology and contain hemoglobin molecules that coordinatively bind O2 and CO2, transporting it from the longs to tissues and from the tissues to the longs, respectively. Also the binding of immunocomplexes and their transportation to the and the for their removal is part of their task description (Murphey et al, 2008). Leukocytes are a collection of diverse cell types that crucially contribute to humoral and peripheral immunity. The macrophages and neutrophils are cells with phagocytic activity that engulf and destroy pathogens. Also dendritic cells can perform phagocytosis, but only in their immature state; after maturation, these cells migrate to the nearest lymph nodes and present pathogen antigens to the T-lymphocytes. The T- and B-lymphocytes then act together to establish a humeral resistance to that specific pathogen. Eosinophils, basophils, neutrophils and mast cells are the so called granulocytes, cells that release pre-formed content upon activation. The natural killer cells, finally, are able to recognize and kill virally infected or even transformed cells. As both innate and adaptive immune responses depend upon the activities of the lymphocytes, these cell types are indispensable for keeping an organism in a healthy state. Platelets finally, the third group of cells in the cellular blood compartment, are irregularly shaped fragments of megakaryocytes that by clotting hinder blood loss in case of injuries. Platelets are also known to be the major source of growth factors. Blood cells are continually produced and renewed during an individual’s lifetime, each cell type with its own typical half-life value. For example, 2.4 million new erythrocytes are being produced per second and released in the blood. Leukocytes, on the other hand, can exist for several years, although their number can increase a hundredfold during an infection. Keeping in mind the great diversity in blood cell morphology and functioning, it is an astonishing fact that all these cells are derived from common hematopoietic stem cells and progenitor cells that reside in the bone marrow. This process, in which hematopoietic progenitor cells are directed to differentiate and mature out of a self-renewal pool of multipotent hematopoietic stem cells (HSCs), is termed hematopoiesis. It is a complex and strictly regulated process that is essentially in the steady-state homeostasis and covers important processes, such as the removal and renewal of old and damaged blood cells, and the modulation of blood cells to their need.

1.1.1 Hematopoiesis follows a strictly regulated hierarchy The formation of all blood cells starts with the long-term hematopoietic stem cells (LT-HSC), which reside in the bone marrow (Figure 1.1) (Passegue et al, 2003). These cells have a life- time expanding self-renewal capacity, in which each cell division gives rise to two daughter

2 Part 1: Introduction cells. One of them will stay in an undifferentialized state – being a LT-HSC – whilst the other can differentiate into a short-term HSC (ST-HSC). In contrast with the LT-HSC, these ST-HSC retain their self-renewal capacity only for about 8 weeks, in which they produce a cellular subset of multipotente progenitor (MPP) cells.

Figure 1.1: Stem cells give rise to the cellular repertoire of the hematopoietic and immune system. LT-HSCs have a life-time self-renewal capacity, and give rise to the ST-HSCs, that retain their self-renewal capacity for only 8 weeks. During that period, they produce the MPPs that in their turn give rise to the CLP cells and the CMP cells. The CMPs develop further to MEP and GMPs, which eventually give rise to the erythrocytes, the platelets, the granulocytes and the macrophages. CLP on the other hand, will lead to the development of the T-, B- and NK cells. Dendritic cells can develop from both CMP and CLP. For the explanation of the abbreviations, the reader is referred to the text. Figure adapted from Bryder, Rossi, & Weissman, 2006.

The MPPs have only a brief self-renewal capacity, and they are the last subpopulation of hematopoietic cells from which descendants are still capable of differentiating in both myeloid and lymphoid cells. The MPPs differentiate into two oligolineage progenitors: common lymphoid progenitor (CLP) cells and common myeloid progenitor (CMP) cells. CLPs give rise to the cells that mediate the adaptive immunity – that is pro-T-cells, pro-NK-cells and pro-B-cells. CMPs on the other hand differentiate into megakaryocyte-erythroid progenitor (MEP) cells and granulocyte-monocyte progenitor (GMP) cells, which in their turn mature into erythrocytes, platelets, and the cells responsible for the innate immunity, being granulocytes and macrophages. Interestingly, dendritic cells can mature from both CLP and CMP lineages, suggesting that there is at least one alternative commitment pathway to the mutually exclusive developmental pathways as explained above.

3 Part 1: Introduction

The continual production of hematopoietic cells and their complex maturation patterns are controlled by a number of glycoproteins that act on every cell population in hematopoiesis. These proteins, generally called hematopoietic cytokines, are a diverse group of glycoproteins that are released in the bloodstream by a variety of cells, and act on their targets by binding on cell-surface exposed receptors (Metcalf 2008). In general, binding of the cytokine to its receptor triggers intracellular signal cascades and evokes a very specific outcome, going from cell growth to inflammation. An overview of the physiological functions of the best characterized hematopoietic cytokines can be found in Table 1.1.

Hematopoietic cytokine Abbreviation Major hematopoietic function Humoral regulator of erythrocyte Erythropoietin EPO development Macrophage colony- Development of , CSF-1 stimulating factor macrophages and osteoclasts Granulocyte colony- Development of granulocyte G-CSF stimulating factor colonies Granulocyte/macrophage Development of a divers set of GM-CSF colony-stimulating factor granulocyte & macrophage colonies Development of macrophages, Interleukin 3 IL-3 granulocytes, megakaryocytes and erythroid colonies Major regulator of eosinophil Interleukin 5 IL-5 production

Interleukin 11 IL-11 Stimulator of megakaryocytes

Cofactor in stimulating the Leukemia inhibitory factor LIF megakaryocyte colony formation Induces proliferation of Stem cell factor SCF hematopoietic stem cells and progenitor cells. Gives an active proliferation Fms-like tyrosine kinase 3 Flt3 stimulus for stem cells and developing dendritic cells Functionally redundant to CSF-1, Interleukin 34 IL-34 but ought to play a complementary role.

Table 1.1: Overview of the best characterized hematopoietic cytokine functions. Hematopoietic cytokines are glycoproteins that regulate the hematopoietic pathway by activating their cognate cell surface receptors.

4 Part 1: Introduction

II. Receptor tyrosine kinases 2.1 Phosphorylation is the key to almost every cellular signal transduction One of the best-understood post-translational modifications of proteins is phosphorylation, in which a phosphate group is reversibly transferred from an activated nucleotide to the hydroxyl group of tyrosine, serine and/or threonine in a reaction catalyzed by a diverse group of enzymes, the protein kinases (PKs). The addition (and removal, in the case of dephosphorylation) of this negatively charged group causes a significant change of the proteins ternary structure due to Coulomb repulsion and/or attraction between the phosphate group and atoms of the nearby amino acids. This initial local change in conformation is translated throughout the whole protein chain, affecting the total protein structure and leading to an alternation of protein activity and/or substrate specificity. The resulting change in three-dimensional structure can be very dramatic, and is exploited by every kingdom of life to control the function of proteins in a wide variety of processes (Manning et al, 2002).

2.1.1 Receptor tyrosine kinases are an important class of receptors A total of 518 protein kinases can be found in the , consisting of serine, threonine and tyrosine kinases. The latter is the smallest subgroup, counting only 90 tyrosine kinases, of which 58 are of the receptor type (Manning et al, 2002; Robinson et al, 2000). Those receptors, known as the Tyrosine Kinase Receptors (RTKs), represent a significant 0.19% of all human genes and exert functions as diverse as the constituents of the RTK family itself, playing key roles in regulation of essential cell-to- processes, including signals leading to proliferation, commitment, differentiation, motility, adhesion, cell survival, metabolism, cell cycle control and death of cells (Manning et al, 2002; Lemmon & Schlessinger, 2010). Inevitably, controlling such a multitude of fundamental cellular processes comes with a price of unambiguously being linked to a variety of clinical disease states, developmental abnormalities and cancer. Needless to say that this family of receptors has been the scope of countless studies, which have provided valuable insights in their structure, regulation and function over the last 20 years.

2.2 Receptor Tyrosine Kinases – a large and various family The human RTKs form a large group of 58 membrane-spanning proteins that fall under 20 subfamilies (Figure 2.1). All RTKs are endowed with an intrinsic protein kinase activity. Upon activation, they are capable of transferring the γ-phosphate group of ATP to the hydroxyl group of tyrosines on target proteins, initiating specific signal transduction cascades. In order to go from an inactive protein in the membrane to an active receptor, that is competent to transduce molecular signals, at least two events are necessary. First, the ligand must be bound by its receptor. Second, this binding event must be followed by a transition of receptor conformation, going from an inactive to an activated state.

5 Part 1: Introduction

Figure 2.1: The receptor tyrosine kinases are a diverse group of twenty subfamilies. The 20 families with all their known members are schematically shown. All RTKs are Type I trans- membrane proteins and show a modular build, here presented with accordance to the key. Figure from Lemmon and Schlessinger 2010.

2.2.1 The RTK ligand elicits receptor dimerization In general, the cellular outcome of RTK signaling is mediated by binding of a diverse family of polypeptide ligands, mostly growth factors, to their cognate RTK receptors. This binding mostly induces dimerization of the receptor, which is the trigger for specific intracellular signaling cascades. Early structural studies of RTKs have provided us with the insight that the ligand-induced receptor dimerization typically goes through the conceptually most straight- forward mechanism: in most cases, two monomers of a dimeric ligand interact simultaneously but independent of each other with the ectodomain of a receptor monomer, in which each ligand:receptor interaction is identical and involves the same amino acids. Evidence for this ‘ligand-mediated’ receptor dimerization can be deduced from the crystal structure of diverse RTKs in complex with their ligands, for example in the case of the stem cell factor receptor (KIT), the nerve growth factor TrkA, the structure of Ax1, Tie2 and Eph receptors and the Flt1 vascular endothelial growth factor receptor. It must however be emphasized that although this activation mechanism is shared among most of the RTKs, there are important exceptions on this concept. It is, for example, long known that the insulin receptor is present in the membrane as a heterotetramer (Van Obberghen, 1994), but in which two cross-linked heterodimer receptor units can be recognized. Binding of insulin or the insulin-like growth factor-1 causes conformational changes in both cross-linked receptors, triggering its tyrosine kinase (Ward et al, 2007). Next

6 Part 1: Introduction to this obvious case of a pre-dimerized receptor, there is also evidence that that the epidermal growth factor (EGF) receptor is present in the membrane as a preformed dimer. Moreover, it has been suggested that activation of the Tie2 RTK by its angiopoietin ligands requires tetramerization of the receptor, whilst the ephrin receptor in complex with its ligand can even form high-order oligomers (Barton et al, 2006; Himanen & Nikolov, 2003; Moriki et al, 2001).

2.2.2 Receptor:receptor interactions trigger the post-oligomerization events Regardless the mono- or oligomeric state of the inactive receptor in the membrane, the general principles of the post-ligand binding receptor activation are more or less conserved among members of the RTK family. Bridged by their ligand, the receptor oligomerization event brings both the receptors extracellular and intracellular domains in close proximity to each other, mostly triggering the receptors to engage in homotypical interactions. This will finally lead to the formation of a stable, physiological active receptor complex that is held together by both non-covalent ligand:receptor and receptor:receptor interactions.

Figure 2.2: Ligand-induced receptor dimerization induces transphosphorylation and subsequent receptor activation. (A) In absence of the ligand, the kinase domain is inactive (red) through the inhibitory interactions of the juxtamembrane domain (orange) and the C-terminal tail (blue) with the kinase activation loop (yellow). (B) Bridged by their ligand (fuchsia), the cytoplasmatic domains are juxtaposed in such a manner that transphosphorylation of the tyrosine residues (shown as circles) is stimulated. (C) Transphosphorylation of the Tyr residues (black dots) causes the juxtamembrane domain and the C-terminal tail to swing away from the kinase complex, and stabilizes the activation segment in a catalytically active conformation. This results in a kinase domain (green) that is capable of phosphorylating recruited downstream signaling proteins. Figure adapted from Hubbard 2004.

It is thought that homotypical interactions, conformational changes and/or a reduced distance of the receptors cytoplasmatic domains, trigger transphosphorylation of the Tyr residues in the cytoplasmatic domains of the dimer (Figure 2.2). In all RTKs, three of these segments are targeted by this initial phosphorylation event: the activation loop, which resides in the catalytic center of the kinase domain, the juxtamembrane region and the

7 Part 1: Introduction

C-terminal tail of the kinase domain (Hubbard, 2004). The juxtamembrane domain and the C-terminal kinase tail adopt in all studied RTKs an autoinhibitory conformation, preventing a correct positioning of the activation loop or of some key residues in the substrate binding sites. Transphosphorylation of these two segments induces a conformational change that makes them both swing away from the kinase domain. Together with the transphosphorylation of the activation domain, this event is the key stimulus for the enzymatic tyrosine kinase activity. The transphosphorylation of the receptor does not only stimulate its enzymatic activity, but also generates recruitment sites for proteins with a src homology-2 (SH2) or a phosphotyrosine-binding (PTB) domain. These domains enable the protein to recognize and dock at the phosphotyrosine domains that the activated receptor provides. These so-called adaptor proteins will on their turn get phosphorylated and activated by the receptor, and will pass on the activation signal by a transduction chain, mostly leading to the activation and/or inhibition of transcription.

However, a recent survey of structural, biochemical and biophysical studies on RTK has pointed out that the general principles underlying RTK activation are more diverse than previously thought (Lemmon & Schlessinger, 2010; Hubbard, 2004). As more structural data on receptor activation came available, it appeared that the events down-stream of receptor oligomerization - being the activation of the kinase domains, the link between receptor phosphorylation and the cellular signaling and the positive and negative feedback loops – are as diverse as the receptor family itself. Despite the interesting challenge in understanding the different mechanisms of how the presence of an extracellular ligand can induce such structural changes in a receptor so that it can be seduced to trigger its signalization cascade, such a comprehensive study falls way beyond the scope of this project. Instead, this introduction will focus on the ligand binding events of a specific subfamily of the RTK receptors that play key roles in the hematopoiesis, the Class III RTKs (RTK-III).

8 Part 1: Introduction

III. Class III receptor tyrosine kinases 3.1 RTK-III are structurally homologues The Class III of the RTK family (RTK-III) is a subclass of the RTK receptors and harbors four receptors: - KIT, receptor for stem cell factor (SCF), - The fms-like tyrosine kinase receptor 3 (Flt3), receptor for Flt3L, - the colony-stimulating factor-1 receptor (CSF-1R), receptor for CSF-1 and interleukin 34 (IL-34), - the PDGFR-α and PDGFR-β, receptors for PDGF. The only two cytokines receptors that are known to act directly on hematopoietic stem cells, being KIT and Flt3, are categorized in this receptor family. This observation is indicative for the key role that RTK-III members play in hematopoiesis. Signalization trough KIT, Flt3 and CSF-1R is essential for stimulating the proliferation of hematopoietic stem cells and their early progenitor cells, and for the generation of macrophages, monocytes, natural killer cells, dendritic cells, melanocytes and mast cells (Rettenmier & Roussel, 1988; Fröhling et al, 2007; Suzukia et al, 1995). In other words, the cellular repertoire of the hematopoietic and the immune system depends upon signalization trough these receptors. PDGFRs are known for their indispensable role in angiogenesis, both in new blood vessel formation and in the growth of already-existing blood vessel tissue. However, by the discovery that tumor derived PDGFR-β signalization can induce expression of erythropoietin (Xue et al, 2011), this receptor subfamily can be linked to hematopoietic activities as well.

3.1.1 RTK-IIIs are characterized by a modular structure The RTK-III are type I membrane receptors and are characterized by their multi-domain structure, featuring 5 extracellular immunoglobulin (Ig) –like domains, followed by a single transmembrane helix, a juxtamembrane domain and an intracellular split kinase domain (Figure 3.1). Molecular phylogeny and chromosomal synteny clearly shows that the RTK-IIIs share a common ancestor with the RTK-V family, which consist of the vascular endothelial growth factor receptors VEGF-R1, VEGF-R2 and VEGF-R3. All RTK-V members have the same membrane-spanning multi-domain structure, but possess seven instead of five Ig-like domains (Grassot et al, 2003).

Figure 3.1: Architecture of the RTK-III receptors. RTK-III are type I membrane proteins featuring 5 Ig-like domains (blue), a transmembrane domain (cyan), a juxtamembrane domain (orange) and a kinase domain (red and purple) that is split by the kinase insert (yellow). Figure adapted from Hamilton, 2008.

9 Part 1: Introduction

3.1.2 All RTK-III have extracellular Ig-like domains The immunoglobulin-like fold is a structural protein fold that can be found in a very heterologous group of proteins, exerting a wide range of biological roles going from epitope recognition in antibodies to ligand binding in surface receptors (Halaby & Mornon, 1998; Halaby et al, 1999; Harpaz & Chothia, 1994; Bork et al, 1994). Membership to the Ig-like fold is based on the conserved three-dimensional structure of the domain, rather than at the sequence level (Bork et al, 1994). Typically, an Ig-like domain is about 100 amino acids long and contains seven to nine β-strands (denominated A to G) that are distributed between two face to face packed β-sheets, forming a very characteristic β-sandwich. All Ig-like modules have a common structural core of four β-strands (B, C, E and F), embedded in the β-sandwich with a total of three to five additional strands (A, A’, C’, C”, D and G). The specific strand topology and the strand number forms the basis of the Ig-like domain classification (Figure 3.2).

Figure 3.2: Classification of the Immunoglobulin-like fold. Classification is based on the strand number and strand topology and results in 5 classes: V, C1, C2, I1 an I2 (Yuzawa et al. 2007 and Asasnovas et al. 1998). Typically, an Ig-like protein fold contains 7-9 strands (A-G) embedded in a beta-sandwich. Schemes (dark green) for the V-type and C2 type Ig-domains are taken from Sun et al, 2004. Structures (light green,

generated with Pymol), for C1, I1 and I2 types are CSF-1RD1 , KITD3 and Flt3D1 respectively (PDB entries 3EJJ, 2E9W and 3QS9).

The β-sandwich scaffold can be further decorated by additional strands and helices. It shows a wide variation in the length and structure of the loop regions, needed to foresee each domain of its distinct biological activity. A hallmark of the Ig-like domain is the intersheet disulfide bridge in the domain core, but its presence is not strictly needed, as illustrated by domains 1 and 4 of Flt3.

10 Part 1: Introduction

3.1.3 Most RTK-III have a conserved motif for homotypic receptor interactions That the Ig-like domains not only play crucial roles in ligand binding, but also in the subsequent transition of the receptor to an activated receptor complex, can be deduced from the highly conserved D4-D4 interaction that takes place after receptor dimerization. As the structure of the KIT ectodomain in complex with SCF became available (Yuzawa et al, 2007), it was noticed that binding of SCF to two receptor monomers invoked a large conformational change in the membrane-proximal regions of both receptors, providing the ideal goniometry for an interreceptor D4:D4 interface (Figure 3.3B and 3.3C). Key residues for this interaction are provided by the βE-βF loop, in which the hydrogen bounds between a conserved Arg of the one receptor and Gly of the second receptor are forming the core of the interaction, zipping up the D4:D4 interface.

A

PDGF-Alpha QEIRYRSKLKLIRAKEEDSGHYTIVAQNEDAVKSYTFELL--- PDGF-Beta SETRYVSELTLVRVKVAEAGHYTMRAFHEDA------KIT SNIRYVSELHLTRLKGTEGGTYTFLVSNSDVNAAIAFNVYVN- CSF-1R DTYRHTFTLSLPRLKPSEAGRYSFLARNPGGWRALTFELTLR- Flt3 -GLDNGYSISKFCNHKHQPGEYIFHAENDDAQFTKMFTLN--- B C

Figure 3.3: Most RTK-III engage in homotypical interactions upon ligand binding. All RTKIII except from Flt3 carry a well conserved sequence motif in domain 4 which plays a key role in receptor dimerisation and the subsequent transition to an activated complex. (A) Sequence alignment of the human RTK-III, showing the conserved motif. (B) Overall and (C) close-up view of the interactions at dimerized KITD4. Arg381 and Gly386 are shown as sticks, domain 4 as a ribbon and H-bonds as dots. Figures are generated with Pymol (PDB entry 2E9W).

Structure based analysis of the relevant sequences revealed that this sequence motif, being L/IxRφxxxD/ExG (Figure 3.3A), is not only conserved in the KIT receptor of all known participants of the animal kingdom, but can be found in all RTK-III family members but Flt3, and even in the seventh Ig-like domain of all type V RTK, where it probably fulfills a similar role. Even more interesting, is the finding that in PDGFR-β and in the VEGF receptor, these interactions appear to be unneeded for ligand binding, but are however indispensable for receptor activation and tyrosine phosphorylation of the kinase domains (Yang et al, 2010; Yuzawa et al, 2007).

11 Part 1: Introduction

It is thought that receptor dimerization with the use of D4:D4 mediated homotypical interactions is a general concept in RTK-III/RTK-V receptor activation. However, as suggested above, there is already a significant exception on this concept. Structure analysis of the full length ectodomain of Flt3 in complex with Flt3L points out that no extracellular homotypical receptor interactions are present or even needed for the receptor activation, nor by the D4 domains, nor by any other ectodomains (Verstraete et al, 2011). But as for the first, this finding only strengthens the hypothesis of a general concept, as Flt3 lacks the conserved interaction motif.

3.1.4 KIT, Flt3 and CSF-1R bind a ligand with a short-chain four-helical bundle cytokine fold.

With the exception of PDGFR (see below), all RTK-III bind a ligand that is categorized under the so-called four-helical bundle cytokine superfamily of the short-chain type (Figure 3.4). However, despite this classification into one family, its members show low sequence similarity and their classification is rather based on their overall characteristic fold (Hill et al, 2002). As the name suggests, the core of this fold consist of four α-helices, all about 15 amino acids long, that are arranged in a up-up-down-down topology with overhand connected Figure 3.4: The four-helical bundle loops. The AB loop is positioned underneath the CD-loop, cytokine fold. The core of this fold and both loops can contain short α- or β-structures and consists of four helices, arranged in disulfide bridges. SCF, Flt3L, CSF-1 and IL-34 are all an up-up-down-down topology. biologically active as dimers. Figure adapted from Hill et al., 2002

3.2 The Platelet-derived growth factor receptor PDGFR and its ligands 3.2.1 The Platelet-derived growth factor family involves five isomers, binding two receptors The platelet-derived growth factor (PDGF) is a family of homo- and heterodimers that are linked by a disulfide bound (Reigstad et al, 2005; Heldin & Westermark, 1999). Currently, there are five different known isoforms of PDGF: PDGF-A, PDGF-B, PDGF-C, PDGF-D and PDGF-AB, with only the latter being a heterodimer. Despite their name, the PDGFs are not only being expressed by platelets, but their mRNA can be found in a wide range of cell types, including fibroblasts, myoblast, smooth muscle cells, retinal pigment epithelial cells, and this throughout the whole body. They all bind on the same set of receptors, PDGFR-α and PDGFR-β. Binding of PDGF causes the receptor to homo- or heterodimerize, forming a PDGFR-αα, PDGFR-ββ or PDGFR-αβ dimeric receptor. All three receptor complexes have different affinities towards the 5 PDGFs, and their expression pattern is of great significance in the physiological actions of the PDGFs. Taken together, all 5 PDGF isoforms exert important functions in both the embryonic and the adult body. During embryogenesis, they play key roles in de development of the kidneys, the blood vessels, the alveoli, the central nervous system, the eyes and the cochlear hair

12 Part 1: Introduction cells of the inner ear. In adults, their main role is tissue remodeling and wound healing by inducing the formation of connective tissue, stimulating angiogenesis and the disposition of extracellular matrix, and acting as a mitogen for smooth muscle cells and fibroblasts. Playing such important roles in tissue development and angiogenesis, it is no surprise that PDGFs are associated with a diversity of pathologies of which cancer is the most important one. For all PDGF, it is shown that they play part in the autocrine transformation of various cells types, thus contributing to oncogenesis. Elevated levels of PDGFs are found in a divers set of malignancies, sarcomas and fibrosis.

3.2.2 PDGFs show a cysteine knot fold All PDGFs are cataloged under the superfamily of the growth factors due do their typical cysteine knot fold (Figure 3.5). This type of protein fold is however not shared with the other ligands that bind on RTK-III receptors, being CSF, Flt3L, CSF-1 and IL-34. More-over, all four PDGFs show a high sequence similarity with the vascular endothelial growth factors (VEGF), which bind on the RTK-V family of receptors (Reigstad et al, 2005). The PDGF cysteine knot structure contains 4 tightly twisted antiparallel paired β-strands. One of each β-strand pair is linked to the other by two disulfide bridges, thereby forming a knot-like structure with an opening in the middle (Reigstad Figure 3.5: PDGFs fold into a cysteine knot et al, 2005; Vitt et al, 2001). A third disulfide structure. Shown is the homodimeric PDGF-B, bridge penetrates the created ring, forcing the with both protomers in a different color, and protein structure to partially expose some the disulfide bounds presented as sticks. Each hydrophobic residues to the surrounding. Those protomer consists of 4 beta-strands, connected hydrophobic residues than are the driving force with intrachain disulfide bounds and adopting for dimerization of two monomers, in which a knot-like structure. Two interchain disulfide those residues are successfully buried in the bridges are covalently connecting both homo- or heteromere structure. In these homo- protomers. Figure generated with Pymol (PDB or heteromers, two monomers are connected by entry 1PDG). interchain disulfide bounds.

3.2.3 The PDGF:PDGFR interactions obeys the classical 2:2 stoichiometry Following the general concept of the RTK-III family receptor activation, binding of each PDGF induces dimerization of the receptor, triggering the transition to an activated state. Whereas PDGFR-α can be activated by PDGF-AA, PDGF-BB, PDGF-CC and PDGF-AB, PDGFR-β can only be activated by PDGF-BB and PDGF-DD. Of all possible PDGF:PDGFR complexes, currently only the structure of PDGFR-BB in complex with the first three ectodomains of PDGF-ββ is solved (Figure 3.6A) (Shim et al, 2010). As expected, these domains all exhibit the Ig-like fold, of which two domains of each receptor are involved in ligand binding. The structure reveals that the ligand:receptor interaction is mainly of hydrophobic nature, in which a total of 2870 Ų of solvent-accessible surface is

13 Part 1: Introduction buried (Figure 3.6B). This relatively large binding interface is a continuous site, in which PDGFR-βD2 and PDGFR-βD3 equally contribute to the ligand binding interface. Although it has been predicted that subsequent to ligand binding, the PDGFR-βD4 domains would engage into homotypical interactions, there is to date still no structural evidence for this to happen. On the contrary, binding experiments involving PDGF-C and the ectodomain of PDGFR-α, revealed that binding of PDGF-C on the full length ectodomain is slightly less favorable compared to the binding PDGF-C on the first three domains (a Kd of 1.04 µM and 0.47 µM, respectively). This counter-intuitive finding clearly indicates the work that has yet to be done on this topic.

A B

Figure 3.6: PDGF-BB dimerizes PDGFR-β by an RTK-III characteristic 2:2 stoichiometry. (A) The overall structure of the PDGF-BB:PDGFR-βD1-D3 complex. (B) The large ligand:receptor interaction site. PDGFR-βD2 and

PDGFR-βD3 participate equally to this continuous interaction site, which involves both PDGF-BB protomers. Both figures generated with Pymol (PDB entry 3MJG).

Despite the fact that the two PDGF receptors only share about 30% sequence identity, most of the residues involved in ligand binding are conservative substitutes. Analysis of those substitutes however shows that 6 out of the 7 aromatic residues present in the PDGFR-β ligand-binding site are substituted by smaller ones. As smaller residues provide a larger conformational plasticity due the lower likelihood of sterical clashes, these substitutions can possibly explain why PDGFR-β is more selective than PDGFR-α when it comes to ligand binding. One the ligand site, one can see an interesting similar trend in amino acid substitution: the residues at the edge of the receptor-binding interface are long-chain hydrophilic in the case of PDGF-B, but small-chain hydrophilic in PDGF-A. Here however, a smaller side chain length increases the selectivity of the ligand for one receptor type: as a smaller chain is less suited to make hydrophilic and salt interactions, PDGF-A will be more dependent on complementary interactions of the receptors side, making PDGF-A more selective for the PDGFR-α. This pious behavior of PDGF-A is indeed experimentally observed (Hart et al, 1988; Gilbertson et al, 2001).

14 Part 1: Introduction

3.3 The receptor tyrosine kinase KIT and its ligand, SCF 3.3.1 Stem cell factor – key player in a wide range of (patho)physiological mechanistics Stem cell factor (SCF), also known as steel factor or mast cell growth factor, is a hematopoietic cytokine that binds on hematopoietic stem cells and the early erythroid progenitors (Ashman, 1999). As in the case for PDGF, SCF also exists in more than one isoform, though constituting a less extensive family with only two members, a soluble one and a membrane-bound one (Broudy, 1997). Both isoforms are expressed from a single gene of which the pre-mRNA is differentially spliced. Subsequent to translation, both proteins are then proteolytically cleaved, although the exact site and kinetics of cleaving depends on the specific isoform. Both isoforms are differentially expressed and have a dissimilar effect on the survival and proliferation of the hematopoietic stem cells.

SCF was first discovered and purified out of rat liver-conditioned medium, where it was shown to have a prominent action on mast cell proliferation (Zsebo et al, 1990). However, subsequent studies could show that when SCF was used in combination with other hematopoietic growth factors, a powerful stimulus was provided for the proliferation of hematopoietic stem cells and the early hematopoietic progenitor cells (Mcniece & Briddell, 1995). Acting in synergy with other cytokines, SCF is able to induce erythroid colony formation and plays an important role in mast cell development and maturation (Ashman, 1999). SCF/KIT signaling is present in several clinically important pathologies. At the protein level, KIT was first identified as a cell-surface marker of human acute myeloid leukemia (AML). Also, KIT was identified as a viral oncogen (v-kit) responsible for the transforming activity of the Hardy-Zuckermann IV feline sarcoma virus (Besmer et al, 1986; Gadd & Ashman, 1985). Several studies show that gain-of-function mutations in KIT can be the underlying cause of both acute and chronic myeloid leukemia and of dysplasia and leukemia of the mast cell lineage (Lerner et al, 1986; Bühring et al, 1991; Goselink et al, 1994; Muroi et al, 1995). Furthermore, it has been shown that deregulated KIT activation is involved in various other cancers, e.g. in gastrointestinal stromal tumors and small cell cancer (Lux et al, 2000; Hirota et al, 1998; Krystal et al, 1996). On the other hand, loss-of-function mutations of SCF or KIT has been shown to be causative for piebald’s disease, which is characterized by a white forelock, heterochromiairidis and pigmentary changes (white nose bridge, white eyelashes and a depigmentation of the ventral chest and abdomen).

3.3.2 SCF:KIT – the structural paradigm for ligand-induced dimerization of RTK-III SCF is biologically active as a homodimer, formed by two head-to-head packed protomers connected by interchain disulfide bonds. A SCF monomer adopts the typical short-chain four- helical bundle cytokine fold and contains two antiparallel interacting β-strands, formed by the αAαB and αCαD loop region (Zhang et al, 2000). Of all RTK-III ligand:receptor complexes, the structure of SCF bound to the full-length ectodomain of KIT (Figure 3.7A) is the first one that has been solved (Yuzawa et al, 2007). Since then, this structure has been used as some kind of a template to which all further structural studies on the RTK-III family have been compared to. The KIT ectodomain has a

15 Part 1: Introduction serpentine-like shape, of which all domains have the typical Ig-like fold. All domains interact tighy which each other (mean surface buried area of about 827 Ų), thus ensuring the upward directed topology of the receptor. All domains but KITD5 are classified under the I- subset of the Ig-like fold, KITD5 itself belongs to the C2-subset. Following the general concept of the RTK-III ligand:receptor interaction, the structure of CSF:KIT shows a 2:2 stoichiometry, in which the ligand is scissored between the two receptor ectodomains. Each protomer interacts directly with KITD1, KITD2 and KITD3, thus providing three interaction sites with a surface buried area of 4120 Ų for the entire ligand:receptor complex (Figure 3.7B). An interesting fact is that when the structure of unbound KIT is compared with the structure of KIT bound to SCF, almost no notable conformational changes can be seen. SCF on the other hand undergoes important structural changes when bound to KIT (Figure 3.7C). These structural changes can be found in the connecting loops, mainly the αC-β2 loop locking in place for establishing interactions at site I, and the flexible N-terminus, going from a random coil to a helical element. Finally, also a change in the angle between the two SCF protomers can be detected, which is about 5° increased when SCF is bound to KIT.

A B

C

Figure 3.7: SCF interacts with three sites on KIT and subsequently undergoes conformational changes. (A) Overal view of the SCF:KIT interaction. SCF (dark blue) shows a typical short-chain four-helical bundle fold. KIT (both protomers shown in green) is build up out of 5 Ig-like domains. The interaction site is located on KITD1-D3. Figure generated with Pymol (PDB entry 2E9W). (B) View on the SCF:KIT interface, showing the interaction regions of both molecules. Acidic amino acids are shown in red, basic amino acids in blue, polar amino acids in orange and hydrophobic amino acids in yellow. Figure adopted from Yuzawa et al. 2007. (C) The structure of free SCF (green) compared to KIT-bound SCF (magenta). Upon receptor binding, SCF undergoes conformational changes at the αC-β2 loop and at its N-terminus. Also the angle between the two protomers increases about 5°. Figure adopted from Yuzawa et al. 2007.

16 Part 1: Introduction

As was predicted from the conserved L/IxRφxxxD/ExG motif that can be found in KITD4, homotypical interactions take place between opposite D4 domains. Upon ligand binding, a reorientation of KITD4 and KITD5 relatively to the ligand binding-region takes place. This reorientation aligns KITD4 and KITD5 on neighboring KIT receptors, thereby making homotypical receptor-receptor contacts possible to occur. Both types of interactions are of electrostatic nature, and it is proposed that the homotypical interactions at KITD4 are aid by the once at KITD5 to convert the relatively flexible SCF bound KIT-monomer to a rigid dimeric structure in which the transmembrane and intracellular parts of the receptor are brought in close proximity to each other. These domains can then interact and start the structural procedure that gives rise to the downward signaling cascade.

3.4 Fms-like tyrosine kinase-3 Flt3 and its ligand, Flt3 Ligand 3.4.1 Flt3L and signaling through the Flt3:Flt3L complex is indispensable for hematopoietic homeostasis. As already stated at the start of this chapter, Flt3L is a cytokine that is known to act directly on stem cells (Fichelson, 1998). Resembling SCF, Flt3L is expressed both as a type I transmembrane protein and as a soluble protein generated by proteolytic activities, and in which both the free and membrane bound form are both biological active. Surprisingly, the mRNA of Flt3L is ubiquitously expressed in both hematopoietic and non-hematopoietic tissues, but only in the stromal fibroblasts of the bone marrow Flt3 can be found (Wodnar- Filipowicz, 2003). In contrast to the ligand, the expression of the receptor is restricted to ST-HSCs and LT-HSCs, and it is believed that not the ligand but the receptor is the limiting factor in the Flt3:Flt3L signaling system. Flt3L is the cognate ligand of Flt3, and a series of in vivo studies in animals and in humans have supplied a body of evidence that Flt3L:Flt3 signaling plays an important role in maintaining the steady-state condition of hematopoiesis (Wodnar-Filipowicz, 2003; Drexler & Quentmeier, 2004). But perhaps the most interest in Flt3 signalization comes from its effect on dendritic cells: Flt3 plays a key role in the development of DCs, both from the lymphoid and from the myeloid branch of the hematopoiesis. The most illustrative example in this view is the notice that a remarkable expansion of the DCs has been achieved in healthy human volunteers that received Flt3L for 10 or 14 days (Maraskovsky et al, 2000). Given the recent developments in dendritic cell therapy as a method to treat cancer, this latter observation has already resulted in clinical trials (Stanford medicin cancer Institute). Unfortunately, constitutive Flt3 signaling caused by oncogenic forms of Flt3 or by an overexpression of the wild-type receptor, is clearly linked to a number of hematopoietic malignancies, of which acute myeloid and lymphoblastic leukemia (AML and ALL, respectively) are the most established ones. It has been shown that in 70% to 100% of AML cases, Flt3 is expressed at high levels, and this also applies to a high percentage of ALL patients. In AML patients, somatic mutations leading to a constitutively active Flt3 have been identified in the juxtamembrane domain and in the kinase domain. Also, point mutations and driver mutations in the juxtamembrane domain and in the extracellular domain have been identified in clinical cases (Fröhling et al, 2007; Stirewalt & Radich, 2003).

17 Part 1: Introduction

3.4.2 The Flt3L:Flt3 interaction – an ensemble of surprising features Flt3L is a non-covalently linked homodimer that adopts the typical short-chain helical-bundle fold (Savvides et al, 2000). Similar to SCF, the two protomers dimerize head-to-head, and two β-strands present in the αAαB-loop and αCαD-loops form an antiparallel interacting β- sheet. An interesting feature is the presence of a 310-helix which is continuous with αC. The 310-helix is linked with the N-terminal unstructured segment trough a biological indispensable disulfide bridge.

Recently, the structure of Flt3L in complex with the ectodomain of Flt3 (Flt3D1-D5) has been determined (Figure 3.8A) (Verstraete et al, 2011). This structure exposed some very unanticipated assembly features, making this a unique ligand:receptor interaction among the RTK-III receptor family. Looking at the crystal structure, the first feature that catches the eye is the overall topology of the ligand:receptor complex. Instead of having two upright oriented receptor monomers bridged by their ligand, one can now see a horseshoe-like ring structure in which Flt3D1 is perpendicularly oriented away from the plane of the ring assembly, whilst Flt3D2 is stowed underneath Flt3L in the ring opening (Figure 3.8A and 3.8B). A second striking feature, which is in fact very unique for the Flt3L:Flt3 complex, is that the ligand binding epitope involves only Flt3D3. This interaction site comprises a total surface buried aria of not even half the SCF:KIT interaction site (~900 Ų versus ~2060 Ų, respectively), but is yet able to establish a binding interaction with a Kd of a remarkable 8.7 nM for the full length ectodomain. In contrast to SCF bound to KIT, comparison of free and Flt3-bound Flt3L does not reveal any changes in conformation. However, just as in the case with KIT, one can again clearly see that the angle between the two Flt3L protomers increases with 5°-6°. Unfortunately, a structure of free Flt3 is not available at this moment to investigate the receptors plasticity upon ligand binding.

A B Figure 3.8: The structure of Flt3L in complex with Flt3 shows some unanticipated features. (A) Ribbon representation of Flt3L in complex with Flt3D1-D5. The epitope binding site of Flt3 is compact, and only

involves amino acids from Flt3D3. (B) Top view of the structure, showing that Flt3D1 and Flt3D2 protrude perpendicularly from the plane of the C interaction, which is unique in the RTK-III family, as clarified by (C) the same top view of the SCF:KIT complex. Figures generated with Pymol (PDB entries 3QS9 and 2E9W).

18 Part 1: Introduction

Despite the conserved residues in the Flt3D3-D4 linker region, the binding of Flt3L apparently does not induce the same alignment of the two neighboring Flt3D4 domains as seen in the SCF:KIT structure. In contrast, the two domains are clearly oriented away from each other, thus making homotypical interactions impossible. Although this was more or less expected as Flt3D4 lacks the conserved L/IxRφxxxD/ExG motif, the absence of homotypical interactions is unique amongst the RTK-III and even the RTK-V receptor family.

3.5 Colony-stimulating factor-1 and its ligand, CSF-1 3.5.1 Colony-stimulating factor-1 is essential for the immune system The colony-stimulating factors (CSFs) are a family of four cytokines, all active on granulocyte- macrophage populations: macrophage-CSF (M-CSF or CSF-1), granulocyte-CSF (G-CSF), granulocyte/macrophage-CSF (GM-CSF) and interleukin 3 (IL-3) (Barreda et al, 2004). Taken together, all four cytokines are essential for survival, proliferation, differentiation and functional modulation of a wide range of mature blood cells and their precursors (Metcalf, 2008). Of all CSFs, CSF-1 was the first to be discovered and purified on the basis of its capacity to induce in vitro development and proliferation of macrophage colonies from myeloid precursors (Bradley & Metcalf, 1966; Pluznik & Sachs, 1966). As is the case for all previously discussed cytokines, more than one isoform of CSF-1 exists: although CSF-1 is expressed from a single gene, at least 5 different splice variants of the mRNA can be found, ranging in size from 1.6 kb to 4 kb (Douglass et al, 2008). All different transcripts can give rise to a soluble isoform of CSF-1 but are dependent on the activity of α or β convertase for their maturation. However, two of the five mRNA’s decode for a CSF-1 isoform with a transmembrane domain that is able to exert its biological functions without being cleaved. This formation of structurally different CSF-1 variants can regulate cell functioning thus by autocrine, juxtacrine, paracrine or endocrine mechanisms as well as by direct cell-cell contact.

By binding its receptor, which is only expressed by the mononuclear phagocytic cell lineage, CSF-1 stimulates survival, proliferation and differentiation of mononuclear phagocytes, consisting of monocytes and macrophages (Pixley & Stanley, 2004; Chitu & Stanley, 2006). CSF-1 signaling starts during the early myeloid differentiation in the late hematopoiesis, where it obligatorily synergizes with SCF and IL-3 to produce the so called mononuclear phagocyte progenitor cells. From that moment on, CSF-1:CSF-1R signaling acts independently to regulate the proliferation and differentiation of progenitor cells to fully maturated monocytes and macrophages. Furthermore, CSF-1:CSF-1R signaling seems to be involved in the clearance of lipoproteins, fertility, pregnancy osteoclasts formation and bone morphology (Pixley & Stanley, 2004; Chitu & Stanley, 2006; Shimano et al, 1990; Naz & Stanley; Pollard et al, 1987). However, CSF-1 is also implicated in a wide range of (auto)inflammatory diseases such as Crohn’s disease, ulcerative colitis, osteopetrosis, rheumatoid arthritis, atherosclerosis and asthma. Furthermore, exaggerated CSF-1R signaling is associated with the rejection of skin xenografts and renal and bone marrow allografts, and increased levels of circulating CSF-1 has been found in a relative wide range of malignancies, including ovarian cancer, breast cancer, endometrial cancer and giant tumor cells of the bone.

19 Part 1: Introduction

3.5.2 An unexpected twist in CSF-1R signaling: interleukin 34 The specific role of CSF-1:CSF-1R signaling in the mononuclear phagocyte lineage is mostly characterized by studying CSF-1 deficient op/op mice and CSF-1R knock-out mice. Both mice have a low body weight, a reduced growth rate and skeletal abnormalities. They are toothless and show a severe reduction in most tissue macrophage populations (Chihara et al, 2010). However, when op/op mice are directly compared to SCF-1R-/- mice, more severe phenotypes are being noticed in the case of the latter, thus pointing to one or more alternative factors that signal trough CSF-1R (Dai, 2002). It is only recently that this hypothesis was confirmed by the discovery of a new cytokine, interleukin 34 (IL-34) (Lin et al, 2008). This novel cytokine binds CSF-1R in a very specific and CSF-1 independent manner, but shows surprisingly no sequence similarity with CSF-1 or any other known protein. The presence of a second, naturally occurring ligand with absolutely no sequence similarity is a unique feature within the RTK-III receptor family. This example clearly shows that even in long-studied systems, our knowledge regarding fundamental (patho)physiological processes might only be the tip of the iceberg.

3.6 Interleukin 34 and CSF-1 – binding partners of the promiscuous CSF-1R 3.6.1 IL-34 and CSF-1 functionally overlap but show a differential expression pattern. Human IL-34 is expressed in two isoforms, that only differs at the presence or absence of Gln81, resulting a dimeric glycoprotein of respectively 241 or 242 amino acids. Both the mouse IL-34 (mIL-34) and human hIL-34 (hIL-34) sequences start with a 20 amino acid long signal, and end with a 50 amino acid long C-terminal part which is predicted to be an inherently unstructured region (IUP). Also, hIL-34 is predicted to have one N-glycosylation site on Asn76, and eight O-glycosylation sites clustered in the C-terminal IUP. IL-34 functionally overlaps with CSF-1 by promoting the survival and proliferation of peripheral blood monocytes and stimulating macrophage colony formation out of precursors in the bone marrow. Similar to CSF-1 signaling, IL-34 stimulates the phosphorylation of extracellular signal-regulated kinase-1 and -2 (ERK1/2) in human monocytes. Despite these functional similarities, some striking differences between IL-34 and CSF-1 can be observed as well: the binding affinity of hIL-34 towards hCSF-1R is higher than hCSF-1, with a Kd of 1 pm versus 34 pM respectively (Lin et al, 2008). Moreover, mutation studies indicated that hIL-34 and hCSF-1 have overlapping but yet distinct binding sites on CSF-1R. Noteworthy, tyrosine phosphorylation of hCSF-1R and downstream molecules induced by hIL-34 binding, declines faster than hCSF-1 induced receptor phosphorylation. This indicates that despite the stronger binding affinity, hIL-34 activates its receptor more transiently (Chihara et al, 2010).

CSF-1 is expressed by many different cell types (Ryan et al, 2001), and numerous stimuli increases its synthesis to effectively regulate immune responses and pregnancy. Dramatic increases in the level of CSF-1 have been reported in the uterus during pregnancy, an also during embryonic development CSF-1 expression is scaled up (Ryan et al, 2001; Wei et al, 2010). IL-34 mRNA can be found in a diverse range of tissues and IL-34 follows a more or less similar expression pattern as CSF-1, although the levels of expression mostly differ between the two cytokines. Furthermore, it has been shown that when IL-34 is expressed under CSF-1

20 Part 1: Introduction specific spatiotemporal conditions, it is capable of rescuing the phenotype of op/op mice. In contrast to this seemingly similar expression pattern in adult tissues, clear differences exist during embryonic development (Wei et al, 2010). For example, a recent study could now show that both CSF-1 and IL-34 are indispensable for the development of the brain, but are however expressed at other regions (Nandi et al, 2012). Taken all together, IL-34 is functional redundant with CSF-1, although their differential spatiotemporal expression points towards a complementary functioning in both adult and embryonic organisms.

CSF-1R activation demonstrates an interesting restriction in species specificity. Thus is shown that mCSF-1R can be well activated by both mCSF-1 and hCSF-1 (Elegheert et al, 2011; Wei et al, 2010), but only very inefficiently by hIL-34 (Chihara et al, 2010; Wei et al, 2010). hCSF-1R however shows a contrary species specificity, in which mCSF-1 binds the human receptor with only a low affinity (Elegheert et al, 2011).

3.6.2 both CSF-1 and IL-34 bind CSF-1R. The mCSF-1 structure (Figure 3.9A), already solved in 1992 (Pandit et al, 1992), greatly resembles the topology of SCF and Flt3L, although the three structures differ in the number of disulfide bridges: a SCF dimer has only two of them, Flt3L has three, while CSF-1 has seven, of which one (Cys31 – Cys31) is used to covalently link the two monomers at their interaction site.

A B mCSF-1 mIL-34

Figure 3.9: Despite the difference in sequence, mCSF-1 and mIL-34 adopt a similar fold. Both (A) mCSF-1 and (B) mIL-34 show the typical short-chain four-helical bundle fold. Compared to mCSF-1 or other cytokines, mIL-34 shows a unique feature, namely two alpha helices at the top of the structure, which are partially substituting for the two beta-sheets found in mCSF-1. Topologically, the structure of hIL-34 and mIL-34 are very much alike, exhibiting the same features. The disulfides are presented as yellow sticks. Both structures are generated with Pymol (PDB entries 3EJJ and 4EXN for (A) and (B) respectively)

Although IL-34 has no sequence similarity with any other protein, it was expected to adopt a four-helical bundle fold based on structure prediction algorithms (Jan Felix, unpublished data). Recently, the crystallographic structure of a truncated version of hIL-34 and mIL-34 were solved (Ma et al, 2012; Liu et al, 2012). Both hIL-34 and mIL-34 indeed appear to fold into a four-helical bundle, but their structures exhibit some unique features compared to other four-helical bundle cytokines (Figure 3.9). One can already see at first sight that mIL-34 shows a short-chain four-helical bundle cytokine core of four helices (αA, αB, αC and

21 Part 1: Introduction

αD) in an up-up-down-down topology. Next to the four-helical bundle, one can observe two antiparallel interacting β-strands, characteristic for all RTK-III binding cytokines. However, in comparison to mCSF-1, these β-strands in the IL-34 structures are much shorter and are partially substituted by 2 additional α-helices. Furthermore, IL-34 contains two intrachain disulfide pairs, both located at the pole of each protomer, which do not show any structural resemblance to other short-chain helical bundle cytokines. Interestingly, the Cys177-Cys191 disulfide bound seems to lock the position of the C-terminus on αA and αD, whilst the C-terminus of mCSF-1 is at the other side of the protomer. Unlike CSF-1, the head-to-head dimerization of IL-34 does not involve a covalent bound. Each hIL-34 protomer buries 656 Ų of solvent accessible area, which is relatively small when compared to CSF-1 (1711 Ų), SCF (1690 Ų), Flt3L (1640 Ų) and even its rodent counterpart mIL-34 (1300 Ų). Despite this smaller binding area, a strong hydrophobic patch in the dimeric interaction platform may obligate IL-34 to dimer formation.

3.6.3 The complex of mIL-34 and mCSF-1R is similar to mCSF-1 bound to mCSF-1R, but nevertheless does have his own features The crystal structure of mCSF-1 in complex with the first three extracellular domains of mCSF-1R (Figure 3.10A and 3.10B) shows that mCSF-1 interacted with its receptor in a 2:1 molar ratio, forming a binary complex (Chen et al, 2008). Presented ITC data suggested that the presence of domains 4 and 5 of the CSF-1 receptor where necessary for ternary complex formation. The authors concluded that the crystal structure showed an intermediary complex, and that receptor activation occurs in a 2-step mechanism. Furthermore, they proposed that binding of one mCSF-1 dimer forms a binary complex with a lowered affinity for a second mCSF-1 receptor ‘leg’ (negative cooperativity). However, a recent study including Small-Angle X-Ray scattering and ITC data proved unambiguously that both hCSF-1:hCSF-1RD1-D3 and mCSF-1:mCSF-1RD1-D3 can form stable ternary complexes in solution (Elegheert et al, 2011). Hence, a mechanism was proposed where both mCSF-1 and hCSF-1 can offer two binding sites leading to complex formation, and that the assembly of a ternary complex is enhanced by homotypical receptor-receptor interactions mediated by domain 4 of CSF-1R (positive cooperativity).

Surprisingly, a binary complex was also observed for the hIL-34:hCSF-1RD1-D3 complex. The authors could however show that this appearance of a binary hIL-34:hCSF-1RD1-D3 complex in the crystal structure is probably a crystallographic artifact. This was proven by performing a silver stain of the solution in the crystal drops, revealing the presence of excess receptor. Also, ITC data presented in this paper shows the formation of ternary complexes for both hIL-34:hCSF-1RD1-D3 and hIL-34:hCSF-1RD1-D5. These studies strengthen the hypothesis that despite the binary crystal structures of the mCSF-1 and hIL-34 complexes, ternary complex formation does not occur through the two-step mechanism proposed by Chen et al., 2008. This hypothesis is finally confirmed by the recent publication of a crystallographic complex featuring mIL-34 forming a ternary complex with the first three extracellular domains of mCSF-1R (Liu et al, 2012). This structure thus provides some valuable insights, but as it is shown that hCSF-1R binds mCSF-1 only with low affinity, it has yet to be elucidated what the relevance of this complex structure is towards its human counterpart. Also the finding that

22 Part 1: Introduction the surface buried area at the two interaction sites (see further) differs between the mIL- 34:mCSF-1R and hIL-34:hCSF-1R, points to possible differences in structural features.

A B

C D

Figure 3.10: CSF-1R interacts in a very similar way with CSF-1 and IL-34. Although that the crystal structure suggest a 2:1 binary stoichiometry, the biological active complex is probably a ternary 2:2 complex. The ribbon presentation of mCSF-1RD1-D3 in complex with (A) mCSF-1 and (C) mIL-34 is shown. (B) Lateral view of the mCSF-1:mCSF-1R complex, showing that similar to the SCF:KIT complex, mCSF-1RD1-D3 is bend between mCSF-1RD1 and mCSF-1RD2. A similar bend in the structure can be seen in (D) the lateral view of mIL-34 in complex with mCSF-1R. All images are generated with Pymol (PDB entries 3EJJ and 4EXP). For the ease of comparison, the second mCSF-1R molecule has been left out of the mIL-34:mCSF-1R complex.

Looking at the crystal structure of mCSF-1 in complex with its receptor (Figure 3.10A and 3.10B), one can immediately see the resemblance with the SCF:KIT complex regarding the topology of mCSF-1RD1: the structure of mCSF-1RD1-D3 is bend ~100° between mCSF-1RD1 and mCSF-1RD2. This bending of CSF-1RD1-2 can also be observed for mIL-34 and hIL-34 in complex with their receptor (Figure 3.10C and D), and is more pronounced then SCF:KIT. But while the KIT receptor is wrapped around its ligand and uses domains 1, 2 and 3 for ligand binding, CSF-1R binds both CSF-1 and IL-34 with the use of only two domains, namely D2 and D3.

Interestingly, hCSF-1RD2 can be structurally superimposed to KITD2, and one can notice that both ligands bind on opposite sides of their receptor. Even more interesting is the observation that CSF-1RD1 is oriented away from the ligand and adopts an opposite orientation compared to KITD1. This can however be partially explained by the reorientation of CSF-1RD2 to provide its binding site: as CSF-1RD1 is not involved in ligand binding, the

23 Part 1: Introduction

reorientation of CSF-1RD1 is probably translated to the orientation of CSF-1RD1 due to the tight CSF-1RD1-D2 interface (1240 Ų buried surface area).

It is quite intriguing how two different ligands can bind on more or less the same docking sites of a receptor (Figure 3.10 & Figure 3.11A). For both cytokines, the interaction site can be described as a two-site binding mode, in which the surfaces contributed by CSF-1RD2 and CSF-1RD3 are discontinuous. But where the mCSF-1:mCSF-1R buries 1740 Ų of solvent accessible area (900 Ų and 840 Ų for site I and site II respectively), the interaction sites for mIL-34 provide 900 Ų for site I and 1300 Ų for site II. The hIL-34 buries even more surface area, as the sites provide 1280 Ų and 1160 Ų respectively. Thus the IL-34:CSF-1R interaction site is larger than the CSF-1:CSF-1R interaction, delivering a possible reason for its higher Kd compared to the Kd of CSF-1:CSF-1R interaction. A B

Figure 3.11: IL-34 and CSF-1 have overlapping binding sites on CSF-1R. (A) Surface representation of mCSF-1R showing the binding interfaces of mCSF-1 and mIL-34. Shared (light pink), IL-34 (dark blue) and CSF-1 (green) specific contacts are indicated. Figure generated with Pymol (PDB entry 4EXT) and data from Liu et al, 2012. (B) Three charged Arg residues (labeled and colored red) with a high intrinsic flexibility are located on a protrusion of CSF-1RD2. This conformational freedom allows them to accept different patterns of negative charges, presented by IL-34 and CSF-1. Figure generated with Pymol (PDB entry 3EJJ).

Taken all together, two synergistic mechanisms are proposed to explain how CSF-1R can recognize and bind two completely different surfaces. The first strategy is to utilize a combination of both overlapping and cytokine-specific receptor interfaces to recognize the two ligands. It is ought that the flexible CSF-1RD2-D3 linker region greatly contributes to this effect by allowing both domains to rotate, thus complementing the specific needs of its ligand. It is indeed seen in the mIL-34:mCSF-1R interaction that mCSF-1RD3 is 20° rotated as compared to the mCSF-1:mCSF-1R complex. Furthermore, as IL-34 has slightly larger dimensions then CSF-1, it is able to interact with residues that are unattainable for CSF-1. For the second strategy, it is postulated that CSF-1R makes use of the intrinsic flexibility of positively charged long-chain residues of which the side chain rotamer conformations allows to form salt bridges with different patterns of negative charges. This is especially the case for Site I, where Arg142, Arg146 and Arg150 are positioned on a protrusion of the CD-loop and thus are able to accept electrons from a wide range of directions (Figure 3.11B).

24

Part 2 Goal

Part 2: Goal

IV. Aim of the Research Project The Receptor Tyrosine Kinase class III receptor family plays key roles in mammalian organisms by being responsible for the cellular repertoire and maintenance of hematopoietic and immune system. Improper signaling of these receptors can cause severe clinically important pathologies, of which some are affecting literally thousands of patients all across the world. Despite the fact that a great number of clinically important mutations is already known, the development of effective drugs is a long and challenging task. In recent years, there has been a growing interest in inhibiting protein-protein interactions with the use of small molecules, but the development in this field is hindered by the extent of the average protein-protein interaction epitope on the one hand, and the lack of knowledge of functional hotspots at the other hand (Wells & McClendon, 2007). From the perspective of RTK-III pathophysiologies, knowledge about ligand binding and subsequent receptor activation could contribute significantly to the rational development of highly specific agonists and antagonists.

This master thesis started with the intention to contribute towards the structure determination of IL-34 in complex with its receptor, CSF-1R, and to understand how CSF-1R is able to bind different ligands with no sequence similarity. During this thesis, the structure of hIL-34 with the first three ectodomains of one hCSF-1R molecule was published. Also the structure of a ternary mIL-34 in complex with the three ectodomains of mCSF-1R has become available now. However, high resolution information about the global architecture of hIL-34:hCSF-1R complex is still missing, and none of the latest publications could address the issue of possible homotypical receptor interactions.

The main goal of this master thesis is to set first steps in determining the ternary structure of human CSF-1R in complex with human IL-34 by X-ray crystallography. Secondly, we want to define the role of CSF-1RD4 in possible homotypical interactions, as not only the ligand:receptor but also the receptor:receptor interface could be a possible drug target. To provide answers to these structurally oriented questions, a combination of X-ray crystallography and mammalian recombinant protein production will be used: - Recombinant protein production will be performed in HEK293S GnTI-/- cell lines. These cells secrete glycoproteins with a homogenous glycosylation pattern and thus a possible increased likelihood of crystallization (Reeves et al, 2002; Chang et al, 2007).

- Two stable HEK293S GnTI-/- cell lines for the tetracycline inducible production of either full-length hIL-34 or truncated hIL-34 are already developed. Both cell lines express and secrete hIL-34 in milligram amounts, suitable for X-ray crystallographic studies.

- Multiple receptor ectodomain constructs will be co-expressed in stable HEK293S GnTI-/- cell lines. Co-expression increases the yield of protein production by efficient sequestering of hIL-34 and preventing its aggregation.

26 Part 2: Goal

- Different receptor constructs will be evaluated for their complex crystallizability and multiple hIL-34:hCSF-1R crystals will be tested at synchrotron radiation facilities. Finally, protein deglycosylation as a crystallization strategy will be performed as well.

This newly acquired information would contribute significantly to fundamental knowledge about hCSF-R signalization. Moreover, such new insights would allow precise intervention when CSF-1R signaling is associated with pathophysiological processes. Characterizing the binding of hIL-34 to hCSF-1R and the subsequent hCSF-1R receptor activation thus could lead to new insights in pathologies and hopefully aid the development of new and specific drugs.

27

Part 3 Results

Part 3: Results

V. Time course limited proteolysis of full-length hIL-34 in complex with hCSF-1RD1-D5 A limited proteolysis experiment of full-length hIL-34 was designed to investigate the predicted unstructured character of the C-terminus. If this region is indeed unstructured, it should be more susceptible to a low amount of proteases than the folded core of the protein. For this experiment, two different proteases, elastase and chymotrypsin, were chosen. Full-length hIL-34 in complex with the full-length ectodomain of hCSF-1R (hCSF-1RD1-D5) was co-expressed and purified with Cobalt Immobilized Metal Affinity Chromatography (Co IMAC) followed by gel-filtration (Methods 13.2). For each protease, two different concentrations were used (1 µg and 0,1 µg in 50 µl) and added to the protein complex in solution (Material & Methods, 13.3). For each sample, a negative control was provided in which no protein complex was present. Fractions were taken after 1 min, 10 min, 30 min, 1 hour and 2,5 hours and incubated with Laemmli buffer and beta-mercaptoethanol (BME) at 95° C. The samples were stored in -20 °C and loaded on an SDS-PAGE gel the next day (Figure 5.1).

Starting from the first time point (Figure 5.1A), one can clearly see the receptor in lanes two, four, seven and nine (upper band, apparent molecular weight of 75 kDa). Also hIL-34 (apparent molecular weight of 37 kDa) is present in those lanes, although visibly at a lower concentration. Incubation with 1 µg elastase already affects hIL-34, as can be seen that the band at 37 kDa is already less intense compared to the other lanes, and some new bands are already appearing at lower molecular weight. Looking at lane seven, it appears that 1 µg of chymotrypsin is already capable of processing both the receptor and the ligand after one minute, as both the band at 75 kDa and at 32 kDa are starting to disappear, and a ladder in the gel is becoming visible. Throughout the experiment (Figure 5.1), the band at 37 kDa, accounting for hIL-34, is cut by the proteases and shifts to a band around 22 kDa. The band of the receptor is however only fading away in the presence of 1 µg chymotrypsin, and is almost unaffected in the three other samples. These results indicate that IL-34 is being processed to a product with an apparent weight of 22 kDa. In this view, it is interesting to note that the apparent molecular weight of hIL-34p180 is also approximately 22 kDa.

29 Part 3: Results

Figure 5.1: Limited proteolysis of full-length hIL-34 in complex with hCSF-1RD1-D5. Two different proteases, elastase (E) and chymotrypsin (C), were added in two concentrations (1 µg and 0.1 µg per 50 µl) to the full- length hIL-34 in complex with the full ectodomain of its receptor (+P). For each sample a negative control was prepared in which no protein complex was present (-P). Samples were taken at (A) 1 min, (B) 10 min, (C) 30 min, (D) 1 hour and (E) 2,5 hours and loaded on a SDS-PAGE gel. The ectodomain of the receptor has an apparent weight of 75 kDa, whilst the apparent weight of full-length hIL-34 is about 37 kDa. The molecular weight of the marker (M) proteins is indicated and expressed in kDa.

30 Part 3: Results

VI. Small-scale co-expression of different hIL-34:hCSF-1R complexes 6.1 Co-expression of IL-34 and its receptor, CSF-1R Plasmid DNA of four different hCSF-1R constructs (Elegheert et al, 2011) were prepared from transformed electrocompetente MC 1061 E. coli and purified by a QIAGEN Plasmid Giga Kit (Material & Methods, 13.1). All four plasmids contained two or more extracellular domains of hCSF-1R, cloned in the pHL-sec vector: - pHL_hCSF-1R_D1-D5 contains the sequence for the full ectodomain of hCSF-1R, - pHL_hCSF-1R_D1-D3 expresses for the first three ectodomains of hCSF-1R, - pHL_hCSF-1R_D2-D3 involves sequences of only domain two and three of hCSF- 1R, - pHL_hCSF-1R_D2-D4 codes for ectodomains two to four of hCSF-1R. Two six-well plates were seeded with hIL-34p180 HEK293S GnTI-/- cells and grown to 90% confluence (Material & Methods, 13.4). In duplicate, hIL-34p180 expression was induced and/or the cells were transfected with a specific pHL_hCSF-R plasmid (Figure 6.1). After five days of expression, the medium in each well was harvested and subjected to a Western blot analysis (Figure 6.2).

Figure 6.1: (co)expression of hIL-34p180 and different hCSF-1R constructs. All wells were seeded with IL-34p180 HEK293S GnTI-/- cells. At 90% confluence, expression of hIL-34p180 was induced in well 1 to 5. In wells 2 to 5, this induction was performed simultaneously with transfection of the indicated hCSF-1R sequence containing plasmids. In well six, transfection with pHL_hCSF-1R_D2-D3 DNA was not accompanied with induction of hIL-34p180 expression.

As can be seen in Figure 6.2, expression medium of wells one to five indeed show the presence of hIL-34p180, which has an apparent weight of 22 kDa. Furthermore, in the expression medium of well two, three, five and six, the receptor ectodomains are visible at 45 kDa (hCSF-1RD1-D3), 75 kDa (hCSF-1RD1-D5), 45 kDa (hCSF-1RD2-D4) and again 45 kDa (hCSF-1RD1-D3) respectively. Transfection with pHL_hCSF-1R_D2-D3 (lane four) does not lead to detectable mounts of CSF-1RD2-D3 in the expression medium. In lanes three to five, a signal can be seen at a molecular weight of 20 kDa. The nature of this band is unknown, but can be caused by non-glycosylated hIL-34p180, as this results in a protein product with an apparent weight of 20 kDa (see further).

31 Part 3: Results

Figure 6.2: Western blot analysis of the (co)expressed IL-34p180 and divers hCSF-1R constructs. Media from a six-well plate, transfected and/or induced as indicated in Figure 6.1, was harvested and loaded on a SDS-PAGE gel. After completion of the run, the proteins were blotted overnight on a nitrocellulose membrane. Visualization of bands was carried out using a C-terminal Anti-His HRP antibody. Lane 1: induced medium containing hIL-34p180 (22 kDa). Lanes 2-5: induction of hIL-34p180 with simultaneous transient expression of the first three ectodomains (lane 2, 45 kDa), the full ectodomain (lane 3, 75 kDa), ectodomains two and three (lane 4, band not visible) or ectodomains two to four (lane 5, 45 kDa) of hCSF-1R. Lane 6: non-induced cells transfected with ectodomains one to three of hCSF-1R (band only visible in the duplicate).

32 Part 3: Results

VII. Large scale expression and crystallization of hIL-34p180 in complex with hCSF-1RD1-D3

7.1 Cobalt Immobilized Metal Affinity Chromatography of hIL-34p180:hCSF-1RD1-D3

In order to obtain high amounts of hIL-34p180:hCSF-1RD1-D3 and allow screening for crystallization conditions, pHL_hCSF-1R_D1-D3 DNA was transfected in stable hIL-34p180 HEK293S GnTI-/-cells for the co-expression of both proteins (Material & Methods, 13.2.1). The expression medium was overnight loaded on a 25 ml TALON column packed with cobalt to establish a first purification (Material & Methods, 13.2.2). The resulting chromatogram can be seen in Figure 7.1.

2000,00

1500,00

1000,00

500,00 Absorbance at 280 nm (mAU) nm 280 at Absorbance

0,00 0,00 20,00 40,00 60,00 80,00 100,00 120,00 140,00 160,00 Elution volume (ml)

Figure 7.1: Cobalt IMAC elution profile of hIL-34p180 in complex with hCSF-1RD1-D3, derived from a large- -/- scale expression in the HEK293S GnTI cell line. Expression medium containing hIL-34p180:hCSF-1RD1-D3 was loaded on cobalt packed TALON column according to the Methods section (Material & Methods, 13.2.2). After washing with equilibration buffer (not shown), a second wash step at 10.43 ml was performed with 15 mM imidazole to elute tightly but non-specific binding contaminants. Their elution is detected by the first

increase in absorbance at 280 nm. When the UV280 absorbance was returned to baseline (at 74.93 ml), the

complex was eluted, resulting in a peak of UV280 absorbance between 85 and 110 ml.

7.2 Gel filtration of hIL-34p180:hCSF-1RD1-D3 To obtain a higher monodispersity of the sample, the Co IMAC fractions between 85 ml and 110 ml were pooled, concentrated to 2 ml, centrifuged and injected on a Superdex™ 200 gel-filtration column, equilibrated with HEPES buffer (Material & Methods, 13.2.3). An elution profile with a total of three peaks was observed (Figure 7.2A), of which the first corresponds to the void volume. Analysis of the peakfractions are shown in Figure 7.2B. The “shoulder” at the left of the second peak indicates the presence of two protein populations eluting between 72 ml and 86 ml. The first population, causing the

33 Part 3: Results shoulder, appears to be a protein of 22 kDa, identified as hIL-34p180 on the SDS-PAGE. This protein elutes before the protein complex, containing truncated hIL-34 at 25 kDa and the receptor at a molecular weight of 45 kDa. The earlier elution volume of hIL-34p180 in the shoulder peak points to aggregation of uncomplexed hIL-34.

Finally, SDS-PAGE analysis reveals that the last peak in UV280 absorbance is caused by the elution of the excess of hCSF-1RD1-D3 (45 kDa).

Figure 7.2: Gelfitration of the pooled IMAC elutionpeak fractions, containing hIL-34p180 in complex with hCSF-1RD1-D3. Pooled peak fractions of cobalt IMAC purification were concentrated to 2 ml and injected on the Superdex™ 200 gel-filtration column. (A) Gel-filtration profile when eluting with HEPES buffer. (B) Fractions of the eluens, indicated by the gray overlay boxes on the chromatogram, were analyzed on a SDS-PAGE gel. The shoulder of the first peak contains (aggregated) uncomplexed hIL-34p180 (22 kDa band), whereas the top of the first peak clearly contains the hIL-34p180:hCSF-1RD1-D3 complex (22 and 45 kDa band). In the second gel, only receptor bands (45 kDa band) are visible, indicating that this last peak only contains hCSF-1RD1-D3. The first lane of each gel contains the molecular weight marker (M), of which the molecular weights are indicated in kDa.

34 Part 3: Results

7.3 Crystallization screening of hIL-34p180:hCSF-1RD1-D3 To ensure that the protein content in the final sample was as homogeneous as possible, only the top peak fractions (62 ml to 66 ml) of the second elutionpeak of the gel-filtration were pooled. The sample was concentrated to 5,9 mg/ml, and the Mosquito Crystal® (TTP Labtech) robot was used to set up crystal screens with 0.2 µl drops using a 1:1 protein- reservoir solution ratio (Material & Methods 13.6.1). Different commercially available crystallization screens were tested, including the Index, Crystal Screen Lite, PEG ION-1, PEG ION-2 (Hampton Research, Aliso Viejo, CA, USA) and the Proplex (Molecular Dimensions, Suffolk, UK) screen. After one day, 1/3th of the drops showed precipitation, which points to a protein concentration that is desirable for crystallization. Follow-up of all cyrstalization conditions indicated that in most conditions in which the precipitant or the salt contained a 0.1 M – 0.2M sodium acetate or sodium citrate based component, either phase separation or severe precipitation could be seen. Conditions of the Index screen in which polyethylene glycol (PEG) 3500 was present, gave on first sight some interesting “spots” in the drop, but in time revealed only very fine precipitate. Interestingly, a same development could be seen in several drops of the PEG ION-1 and, in lesser extent, the PEG ION-2 screen in conditions with a sodium based salt and a pH between 6.0 and pH 7. In both screens, PEG 3350 is the precipitant for all conditions. However, no further correlation between all these conditions could be found. After several weeks, most drops contained amorphous precipitate. Only very few conditions remained clear or showed larger (typically 52 µm x 10 µm) and more structured precipitate with a very irregular surface. However, no clear correlation than an intermediate pH (pH 6 – pH 8) could be found between those conditions.

7.4 Deglycosylation and purification of the hIL-34p180:hCSF-1RD1-D3 complex

Despite the homogeneous glycosylation of the hIL-34p180:hCSF-1RD1-D3 complex, no positive crystallization ‘hits’ were observed. To increase the chance of yielding protein crystals, deglycosylation of the protein complex was tried as a possible crystallization method. Deglycosylation was performed following the protocol of Chang et al. (Chang et al, 2007) as described in the Methods section (Material & Methods, 13.5.1). Endoglycosidase EndoHf was added to purified hIL-34p180 in complex with hCSF-1RD1-D3 and incubated overnight. A fraction was taken and stored to load on a SDS-PAGE gel later (Figure 7.3B). Next, the sample was injected on a column, packed with amylose/agarose beads (Material & Methods 13.5.2). EndoHf, which is covalently tagged with a C-terminal maltose-binding protein (MBP), should be withheld on the column due to interaction with the amylase resin matrix, whereas the hIL-34p180:hCSF-1RD1-D3 complex is not retained by the matrix and can be captured in the flow-through. Next, EndoHf can be eluted from the column using a buffer containing 10 mM of maltose. The elution profile after amylose affinity chromatography and the corresponding analysis of the fractions on a SDS-PAGE, are presented in Figure 7.3.

35 Part 3: Results

As can be seen, the complex is indeed not withheld by the column, and both the truncated hIL-34 (apparent molecular weight of 20 kDa) and the receptor construct (apparent molecular weight of 32 kDa) are present in the flow trough. If one compares the SDS-PAGE analysis of the peak fractions after gel-filtration (Figure 7.2B) to the apparent molecular weight of the complex constituents after incubation with EndoHf (Figure 7.3B, second and third lane), a clear shift in bands can be seen. The receptor construct is shifted from a molecular weight of 45 kDa to 32 kDa after gel-filtration and deglycosylation respectively. The same is true for hIL-34p180, as a shift from 22 kDa to 20 kDa can be seen.

A

Figure 7.3: Amylose affinity chromatography of the hIL-34p180:hCSF-1RD1-D3 after being incubated overnight with EndoHf. Truncated hIL-34 in complex with the first three extracellular domains of hCSF-1R was overnight incubated with the EndoHf. Next, the sample was injected on a 2.5 ml amylose resin column. (A) Elution profile of the amylose affinity chromatography. The first peak corresponds to hIL-34p180:hCSF-1RD1-D3 , which is unretained by the amylose matrix. At 10 ml, elution with HEPES buffer containing 10 mM maltose was started, resulting a peak at 12 ml. (B) SDS-PAGE analysis of the different elution fractions. Lane 2 (‘Before’) contains sample before injection. Three protein populations are present in the sample: EndoHf (band at 75 kDa), hCSF-1RD1-D3 (band at 32 kDa) and hIL-34p180 (band at 20 kDa).

Lane 2 (‘FT’) corresponds to the top peak fraction of the flow-through and contains hCSF-1RD1-D3 (upper band) and hIL-34p180 (lower band). As expected, the top peak fraction after elution (Lane 3, ‘Elution’) only contains EndoHf. The lanes with the molecular marker are indicated with M, the molecular weights are expressed in kDa.

36 Part 3: Results

7.5 Crystallization trials of deglycosylated hIL-34p180:hCSF-1RD1-D3 The flow-through fractions of the amylose affinity chromatography were pooled and concentrated to 4.5 mg/ml. After centrifugation, a pellet was visible and the protein concentration was reduced to 1.68 mg/ml, indicating that the maximum solubility was reached. Several crystallization trials were performed using the Mosquito® Crystal (TTP Labtech) robot in 0.2 µl (1:1 protein-reservoir ratio) drop setups (Material & Methods 13.6.2). In one of the crystallization drops (0.66 M ammonium sulfate, 0.066 M HEPES pH 7 and 0.33% PEG 8000), needle-like crystals could be discovered that were however, based on morphology, of pour quality: all were small (50 µm x 10 µm), and most had very pour defined edges and a rough-appearing surface. Based on this initial crystallization condition, an optimization grid screen was designed in which both the pH and the salt concentration were step-wise varied. This optimization screen resulted in multiple hits, all containing slightly bigger crystals (about 100 µm x 20 µm). One condition however, with 0.7 M ammonium sulfate and HEPES buffer pH 6, yielded bigger crystals (200 µm x 50 µm) which showed clear birefringence under polarized light (Figure 7.4).

A B

Figure 7.4: Optimization screen of the initial crystal condition gave few hits. Based on the initial crystallization hit, a grid screen was designed in which the pH and the salt concentration were stepwise varied. The screen was set up automatically with a reservoir of 75 µl and drops of 0.2 µl (1:1). Several crystallization conditions with birefringent crystals were identified. (A) and (B) both show drops containing 0.7 M ammonium sulfate, 0,066 HEPES buffer pH 7 and 0,33% PEG 8000. The crystal in condition A was, based on the crystal morphology, chosen as the best one.

In order to further optimize the hits from the grid screen, the condition that gave the morphologically best scoring crystals was used as a basis for a Silver Bullets™ screen (Hampton Research). The conditions of this screen are composed of a wider variety of small compounds, which may enhance crystal packing contacts and crystallization in conditions initially yielding small crystals. This screen, finally, gave several bigger and hopefully good diffracting crystals, of which the two best are shown in Figure 7.5.

37 Part 3: Results

Crystallization condition B3 (Figure 7.5A) (0.25% w/v 5-Sulfoisophthalic acid monosodium salt, 0.25% w/v Cystathionine, 0.25% w/v Dithioerythritol, 0.25% w/v L-Citrullineand 0.02 M HEPES sodium pH 6.8) showed baulk-like crystals of 50 µm x 50 µm x 80 µm. The crystals at condition B7 (Figure 7.5B) (0.33% w/v 1,4-Cyclohexanedicarboxylic acid, 0.33% w/v 2,2'-Thio-diglycolic acid, 0.33% w/v 5-Sulfoisophthalic acid monosodium salt and 0.33% w/v HEPES sodium pH 6.8) were more elongated and thinner, but were about four times longer (30 µm x 30 µm x 200 µm).

A B

Figure 7.5: Silver Bullets™ screen with the best crystallization condition form the grid screen as basis, gave multiple hits. All conditions of the grid screen were scored on basis of the crystal morphology. The condition in which to the best crystals was found, is used as basis for a Silver Bullets™ screen (Hampton Research). The screen was set up automatically with a reservoir of 75 µl and drops of 0.2 µl (1:1). Several hits were found, of which the two best are shown. (A) Condition B3, showing orthorhombic shaped crystals of a favorable thickness (about 30 µm -50 µm). Condition: 0,25% w/v 5-Sulfoisophthalic acid monosodium salt, 0,25% w/v Cystathionine, 0,25% w/v Dithioerythritol, 0,25% w/v L-Citrullineand 0,02 M HEPES sodium pH 6,8. (B) Condition B7 with more elongated rod-shaped crystals. Condition: 0.33% w/v 1,4-Cyclohexanedicarboxylic acid, 0.33% w/v 2,2'-Thio-diglycolic acid, 0.33% w/v 5-Sulfoisophthalic acid monosodium salt and 0.33% w/v HEPES sodium pH 6.8.

Condition B7 was reproduced and crystals from three drops were harvested and analyzed by SDS-PAGE (Material & Methods, 13.6.2). After the run, the bands of the gel were visualized by silver staining (Figure 7.6). Two crystals from two different drops (Figure 7.6, ‘D1’ and ‘D2’) were washed two times (Figure 7.6, ‘W1’ and ‘W2’), and the supernatant of both wash steps clearly indicate the presence of both hIL-34p180 (20 kDa) and hCSF-1RD1-D3 (32 kDa). Both bands are also visible when the remaining crystals are resuspended and loaded on the gel (Figure 7.6, lane 7 and 8). Furthermore, when a crystal from a third drop is resuspended without being washed (Figure 7.6, lane 9), also here the bands for both deglycosylated proteins are visible. For all samples, including the positive control, also a band at 75 kDa is visible, probably indicating the presence of EndoHf.

38 Part 3: Results

Figure 7.6: Silver stained gel of crystals from the Silver Bullets™ screen, analyzed by SDS-PAGE. Condition B7 of the Silver Bullets™ screen was reproduced. Crystals from three drops (D1-D3) were analyzed according to the Methods section (Material & Methods 13.6.2). Supernatant of the first wash (W1) and second wash (W2) were analyzed. The remaining crystals were resuspended and loaded (lane 7 and lane 8). Crystals from the third drop (D3) were loaded without being washed (lane 9). All crystals contain deglycosylated hCSF-1RD1-D3 (upper band; 32 kDa) and deglycosylated hIL-34p180 (lower band; 20 kDa). Also

EndoHf is visible (band at 75 kDa). The sample from the deglycosylated complex before the start of the crystallization screening is used as a positive control (+). Bands of the molecular weight marker (M) are indicated in kDa.

Several crystals from multiple Silver Bullets™ B3 and B7 conditions were harvested from the drop, cryoprotected with several cryoprotectant solutions and flash-frozen as described in the Methods section (Material & Methods, 1.6.2). The crystals were tested at beamline IO2 at the Diamond Light Source (Harwell Science and Innovation Campus, Oxfordshire, UK). A dataset of 180 ° was recorded using 100% beam intensity and 2 seconds exposure time. A representative diffraction image is shown in Figure 7.7. More experimental details about data collection can be found in Table 7.1 and in the Methods section (Material & Methods, 13.7.1).

39 Part 3: Results

Deglycosylated hIL-34p180:hCSF-1RD1-D3 Source Diamond Light Source, beamline I02 Wavelength 0.97949 Å Detector ADSC Q315r Number of recorded images 180 Oscillation degree 1 ° Resolution 40 Å – 8 Å

Table 7.1: Key experimental details of data collection on crystals of deglycosylated hIL-34p180:hCSF-1RD1-D3. Additional experimental parameters can be found in the Methods sections (Material & Methods, 13.7.1).

Figure 7.7: Representative diffraction pattern from crystals of hIL-34p180:hCSF-1RD1-D3. The datasets were recorded at the I02 beamline at DLS. Upper right corner: hIL-34p180:hCSF-1RD1-D3 crystal inside a cryoloop centered on the beam (red circle; 120 µm x 80 µm). Center: representative diffraction image of the recorded data set with resolution shells indicated. The beam center is indicated as a red cross, the beam stop by the white shadow. Single diffraction spots (black) are clearly visible, but only at low resolution.

40 Part 3: Results

VIII. Large scale expression and crystallization of hIL-34p180 in complex with hCSF-1RD2-D4 As previous attempts to gain good diffracting crystals of truncated hIL-34 in complex with the first three extracellular domains of its receptor remained unsuccessful, a different construct of the receptor ectodomain was to be tested as a crystallization target. Previous attempts to crystallize hIL-34 in complex with the full receptor ectodomain did not yield any crystals. Therefore, an attempt was made to co-express and crystallize truncated hIL-34 in complex with the extracellular domains two to four of hCSF-1R (hCSF-1RD2-D4).

To obtain high amounts of this protein complex, a similar approach was followed as for the expression and purification of hIL-34p180:hCSF-1RD1-D3 (Methods, 13.2). Based on the expression test (Figure 6.2), a low expression of hCSF-1RD2-D4 was expected. In order to optimize the expressed ligand to receptor ratio, 50 % more pHL_hCSF-1R_D2-D4 plasmid DNA was used for the transfection. After five full days of expression, the expression medium was harvested, centrifuged, filtered and stored at -20 °C.

8.1 Cobalt Immobilized Metal Affinity Chromatography of hIL-34p180:hCSF-1RD2-D4 The expression medium was thawed and loaded on a 25 ml TALON column, freshly packed with cobalt (Methods, 13.2). After extensively washing with equilibration buffer until the UV280 absorbance had returned to baseline, a second wash step was performed with 15 mM imidazole. When no contaminants eluted from the column and the absorbance at 280 nm was returned to baseline, elution was initiated. The resulting chromatogram is presented in Figure 8.1A. As can be seen, the resulting yield was significantly lower than observed for the purification of hIL-34p180:hCSF-1RD1-D3 (Figure 7.1). To check the constituents and purity of the elutionpeak, the fractions were analyzed on a SDS-PAGE. The results are presented in Figure 8.1B.

Both hIL-34 (22 kDa) and hCSF-1RD2-D4 (45 kDa) are present in the last elutionpeak, although hIL-34 is cleary overexpressed when compared to the receptor. Previous work has shown that uncomplexed IL-34p180 tends to form aggregates in solution, possibly due to the c-terminal his tag. The overabundance of hIL-34p180 causes formation of aggregates next to complexed hIL-34p180, and this can possibly explain the presence of a band at higher molecular weight (150 kDA). Both the hIL-34p180 and hCSF-1RD2-D4 bands are accompanied by bands of lower molecular weight, which are possibly glycosylation variants of hIL-34p180 (Figure 8.1, band at 20 kDa) and hCSF-1RD2-D4 (Figure 8.1, band at 40 kDa)

41 Part 3: Results

A

B

Figure 8.1: Cobalt IMAC purification of medium containing hIL-34p180:hCSF-1RD2-D4. In total, 2.5 l of expression medium was loaded on a TALON column packed with cobalt. (A) Cobalt IMAC elution profile of hIL-34p180:hCSF-1RD2-D4. After loading, the column was extensively washed with equilibration buffer (not shown). Next, tightly interacting contaminants were washed off with 15 mM imidazole, resulting in the first two peaks of the shown chromatogram. When the UV280 absorbance was returned to baseline (at 240 ml), the complex was eluted, resulting in the last peak. The third peak in the chromatogram is caused by pausing the elution several times due to mechanical problems. (B) Fractions of the expression medium (Pre), the flow-through while loading the expression medium (FT) and the first wash step with only equilibration buffer (0%B) were taken and stored at -20° C. When the Co IMAC was completed, several fractions and the previously stored samples were analyzed by SDS-PAGE. The elution fractions that were analyzed and their corresponding lanes in the SDS-PAGE gel are indicated by the gray boxes on the elution profile and the black lines guiding them to their corresponding place on the gel. Of the wash step with 15 mM imidazole (second peak on the elution profile), only the top peak fraction was analyzed. The molecular weights of the marker (M) bands are expressed in kDa.

42 Part 3: Results

8.2 Gel-filtration of hIL-34p180:hCSF-1RD2-D4 subsequent to Co IMAC Despite the presence of multiple unfavoured proteins species in the elutionpeak of the Co IMAC elution profile, all fractions between 265 ml and 280 ml were pooled conservatively due to the low protein yield. The fractions were concentrated to 2 ml, centrifuged and injected on a Superdex™ 200 column for gel-filtration (Material & Methods, 13.2.3). The resulting elution profile is presented in Figure 8.2A. Based on the small-scale expression test and the low UV280 absorbance of the Co IMAC elutionpeak, the yield was expected to be low. Nonetheless, the resulting peak of only 43 mAU was a quite disappointing result. SDS-PAGE analysis of the peak elution fractions (Figure 8.2B) revealed the presence of a higher molecular weight protein species in the first peak, which possibly corresponds to aggregated hIL-34p180, next to an unknown contaminant at 50 kDa. The third peak contained hIL-34p180 and hCSF-1RD2-D4, each showing multiple bands (22 and 25 kDa for hIL-34p180, 40 and 45 kDa for hCSF-1RD2-D4) possibly due to variations in glycosylation.

43 Part 3: Results

A

B

Figure 8.2: Superdex™ 200 gel-filtration of hIL-34p180:hCSF-1RD2-D4 and subsequent analysis of key fractions on a SDS-PAGE gel. The Co IMAC elutionpeak of truncated hIL-34 in complex with the ectodomain two to four of hCSF-1R was conservatively pooled, concentrated, centrifuged and injected on a Superdex™ 200 gel-filtration column. (A) The elution profile of the gel-filtration run, indicative for only a very low yield of the possible receptor complex (peak of 43 mAU). (B) Fractions, indicated by the gray box on the elution profile, were loaded on the corresponding lanes of a SDS-PAGE gel that was Coomassie stained after the run. Analysis points out that the high-moleculair weight species, seen in the peak fractions of the Co IMAC, elute between 57 ml and 61 ml (2). The fractions of 4, 5 and 6 show that in the peak of the elution profile, both constituents of the receptor are present in two populations, hIL-34p180 at 25 kDa and 22 kDa, hCSF-1RD2-D4 as 45 kDa and 40 kDa. The molecular weight of the molecular marker (M) bands are expressed in kDa.

44 Part 3: Results

8.3 Crystallization trials of hIL-34p180:hCSF-1RD2-D4 Prior to crystallization, a conservative pooling of the top elution peak was performed to increase the purity of the sample. The pooled fractions (75.5 ml to 81.52 ml) were concentrated to 4.5 mg/ml, resulting in only 15 µl of protein solution. First, the Mosquito® Crystal (TTP Labtech) robot was used to set up a Crystal Screen Lite (Hampton Research, Aliso Viejo, CA, USA) of only 48 conditions in a 0.2 µl drop size setup with a 1:1 ratio of protein-reservoir solution (Method, 13.6.3). Although this screen gave no crystal hits, it showed after one day precipitation in roughly 1/3th of the conditions indicating a usable concentration of the protein sample. Afterwards, the mosquito was used to set up a second screen (Proplex™; Molecular Dimensions, Suffolk, UK) (Material & Methods 13.6.3) with the remaining protein. After a few days, small spherulite crystals were discovered in condition C4 (0.2 M lithium sulfate; 0.1 M MES buffer pH 6; 20% PEG 4000). After 14 days, the crystals reached a size of 52 µm and 39 µm diameter (Figure 8.3, arrow 1 and 2 respectively).

Figure 8.3: Condition C4 of the ProPlex™ screen containing spherulite crystals. Truncated hIL-34 in complex with ectodomains 2 to 4 of hCSF-1R was purified by Co IMAC and a consecutive gel-filtration. The gel-filtration elutionpeak was pooled, concentrated to 4.5 mg/ml and used to setup a ProPlex™ crystal screen (75 µl reservoir and 0.1 µl drops with a 1:1 protein to reservoir solution ratio). After 14 days, condition C4 showed two spherulite crystals, with a diameter of 52 µm (red arrow 1), and 39 µm (red arrow 2) respectively. Condition: 0,2 M lithium sulfate; 0,1 M MES buffer pH 6; 20% PEG 4000.

After six days, a second condition (C10; 0.15 M ammonium sulfate, 0.1 M MES buffer pH 5.5 and 25 % PEG 4000) also showed similar but smaller (10 µm - 15 µm diameter) spherulite crystals of the same morphology as condition C4 (Figure 8.4). The composition of this condition shows some interesting similarities with condition C4.

45 Part 3: Results

Figure 8.4: hIL-34p180:hCSF-1RD2-D4 containing crystal drop with C10 of the ProPlex™ screen. Truncated hIL-34 in complex with ectodomains 2 to 4 of hCSF-1R was purified by Co IMAC and a consecutive gel-filtration. The gel-filtration elutionpeak was pooled, concentrated to 4.5 mg/ml and used for an automated ProPlex™ crystal screen (75 µl reservoir and 0,1 µl (1:1) drops), of which the drop with condition C10 after 14 days is shown. In the center, a brown-ish precipitate was already visible after one day. The spherulite crystals (red arrows) started to became visible after six days. Condition: 0.15 M ammonium sulfate; 0.1 M MES buffer pH 5.5; 25 % PEG 4000.

The two crystals of condition C4 were cryoprotected with 15% PEG 400 and subsequently flash-frozen as described in the Methods sections (Material & Methods, 13.6.3). The crystals were tested on beamline PXIII at the Swiss Light Source (Paul Scherrer Institute, Villigen). Two datasets of 360° and 180 ° were recorded with exposure times and oscillation ranges as indicated in Table 2 and in the Methods section (Material & Methods, 13.7.2). The resulting dataset clearly indicates a protein-like diffraction of the crystal (Figure 8.5), but shows only spots in the low-resolution range. Moreover, symmetry between the diffraction spots seems to be abscent, which is indicative for a crystal of bad quality.

hIL-34p180:hCSF-1RD2-D4 Source Swiss Light Source, beamline PXIII Wavelength 1.00001 Å Detector Pilatus 2M-F Detector First dataset Number of recorded images 360 images Oscillation degree 0.1 ° Exposure time per frame 0.1 seconds Second dataset Number of recorded images 180 images Oscillation degree 1° Exposure time per frame 2 seconds

Table 8.1: Key experimental details of data collection on crystals of hIL-34p180:hCSF-1RD2-D4. Additional experimental parameters can be found in the Methods sections (Material & Methods, 13.7.2).

46 Part 3: Results

Figure 8.5: Representative diffraction pattern of hIL-34p180:hCSF-1RD2-D4. The dataset of the two crystals of ProPlex™ condition C4 and one crystal of condition C10 were recorded at the PXIII beam at SLS. Upper right corner: the largest crystal from condition C4 inside a cryoloop and centered on the beam (red square, 80 µm x 45 µm). Center: representative diffraction image of the recorded data set with the resolution shells indicated as circles. The beam center is shown as a red cross, the beam stop as a white shadow. Diffraction spots (black spots) are visible, although no symmetry between the diffraction spots can be deduced

47

Part 4 Discussion and Conclusions

Part 4: Discussion and conclusions

IX. Discussion 9.1 Time course limited proteolysis experiment of full-length hIL-34 in complex with hCSF-2RD1-D5 Prior studies has shown that a segment of 60 amino acids at the C-terminus of human IL-34, including two cysteines, are dispensable for the biological activity of IL-34 (Chihara et al, 2010). In addition, analysis of the sequence with, for example, Regional Order Neural Network (RONN) (Yang et al, 2005) shows that the 55 C-terminal long region of hIL-34 has more than 50% probability to be disordered. Nevertheless, it could well be that this region adopts a secondary or tertiary structure when IL-34 is bound to its receptor. This information was the basis for a construct of IL-34 that lacked the predicted C-terminal IUP region, and possibly enlarges the chances of getting a well diffracting crystal of unbound hIL-34 or hIL-34 in complex with its receptor. However, if that region is involved in receptor binding, not only would the use of such a construct create a gap in knowledge about the ligand:receptor binding mechanistics, it would probably also decrease the stability of the hIL-34:CSF-1R complex. To determine if the C-terminus of hIL-34 remains unstructured when bound to the receptor ectodomain, a limited proteolysis experiment was designed. This experiment is based on the observation that a folded protein without any unstructured region is a sub-optimal substrate for proteases, as its peptide bonds are protected by the fold of the structure. However, when (parts of) the protein are unfolded, the proteases will be able to digest the peptide bonds more efficiently and thus these regions are more susceptible for a limited protease activity (Na & Park, 2009). hIL-34 has in total 49 predicted digestion sites for elastase and chymotrypsin, of which seven in the predicted IUP region.

Throughout the experiment, SDS-PAGE analysis (Figure 5.1) revealed that bands of hIL-34 shifted to lower molecular weights whilst bands of hCSF-1RD1-D5 stayed untouched when using 0.1 µg elastase or chymotrypsin. At 1 µg chymotrypsin however, both ligand and receptor are processed, indicating that this concentration of chymotrypsin is capable of cleaving them both. At the end of the experiment, for all four added protease concentrations, hIL-34 was processed to a protein with a molecular weight of 22 kDa. This indicates that only a part of hIL-34 is more susceptible towards proteolytic cleavage, as otherwise a ladder pattern would be visible. Based on the knowledge that our truncated hIL-34 construct that lacks the predicted IUP region (hIL-34p180), runs at exactly the same molecular weight as this proteolytically cleaved product, this experiment indicates that the C-terminal part of the protein possibly remains unstructured when hIL-34 is bound to the extracellular domains of its receptor and is more susceptible to proteolytic cleavage then the rest of the protein complex.

9.2 Small-scale co-expression of different hIL-34:hCSF-1R complexes Early studies by Jan Felix (unpublished data) have pointed out that hIL-34, being expressed as a full-length or as a truncated protein, was difficult to purify and aggregated at concentrations that are needed for structural studies (typically higher than 1 mg/ml). It was however shown that when hIL-34 is co-expressed with the extracellular domains of the

49 Part 4: Discussion and conclusions receptor, a stable ligand:receptor complex was formed that is physicochemically distinct from unbound hIL-34. This complex allows to concentrate hIL-34 to > 8 mg/ml, which is sufficiently high to allow crystallization trials. Co-expression of the complex is established by transient expression of hCSF-1R in stable hIL-34 or hIL-34p180 HEK293S GnTI-/- cell lines, simultaneously with induction of hIL-34 expression by adding tetracycline in the transfection medium. However, two choices had to be made: first, we had to decide which hIL-34 construct should be selected to work on. Secondly, a receptor construct had to be chosen to co-express hIL-34 with. Based on the limited proteolysis experiment (Results, 5), we decided that is was both biologically and structurally justified to focus on the truncated construct of hIL-34 (hIL-34p180), as the IUP region seems to remain unstructured in the ligand:receptor complex. In addition, absence of this unstructured region would possibly (but not surely) increase the likelihood of crystallization.

To address the second choice that had to be made, the small-scale co-expression experiment was performed in which four distinct receptor constructs were co-expressed with hIL-34p180. On all four tested ectodomain constructs, the IL-34:CSF-1R binding interface (domain 2 and domain 3) is present. The Western blot (Figure 6.2) shows that for all cells that were induced with tetracycline, a strong signal of hIL-34p180 was present. Also a lower band at about 20 kDa is detected, of which the nature is unknown. We however hypothesize that this band is could be caused by a non-glycosylated form of hIL-34p180. It is known that overexpression of proteins in mammalian cells can cause a saturation of the glycosylation machinery, thus leading to the secretion of underglycosylated or even non-glycosylated proteins. As can be deduced out of the Western blot (Figure 6.2), the full length ectodomain of hCSF-1R seemed to give the strongest signal, followed in intensity by CSF-1RD1-D3 and finally CSF-1RD2-D4. The plasmid coding for hCSF-1RD2-D3 did not lead to a detectable amount of this receptor construct in the expression medium, indicating that this plasmid is badly expressed or that this receptor construct is prematurely degraded. Although only one Western blot is shown, this experiment was performed in duplicate, confirming the results.

Based on this expression test, the full length ectodomain of hCSF-1R should be picked to co-express hIL-34p180 with. However, the approach in which hCSF-1RD1-D5 is co-expressed and purified in complex with hIL-34 is already extensively tested by Jan Felix, but ended in series of crystallization screens which remained unsuccessful. Co-expression of hIL-34p180 with the first three ectodomains of hCSF-1R (hCSF-1RD1-D3) was tried, as this was the second- best expressing receptor construct.

9.3 Large-scale expression and crystallization of hIL-34p180 in complex with hCSF-1RD1-D3

After five days expression of hIL-34p180 and hCSF-1RD1-D3, the cells were visibly shrunken and showed a star-like morphology, but most were still attached to the flask surface, which is indicative for their viability. While loading the expression medium overnight on a cobalt packed TALON column, some stripping of the cobalt was observed. This is possibly caused by the presence of sodium butyrate or other compounds in the expression medium. However,

50 Part 4: Discussion and conclusions at least 2/3th of the column remained unstripped, which is sufficiently high to withhold the total amount of protein complex. After the second wash had removed some tightly but non- specific interacting proteins, elution was started. This resulted in a sharp peak of UV280 absorbance, probably caused by elution of the complex. It is indeed seen that in de fractions of the subsequent gel-filtration (Figuur 7.2) both hIL-34p180 and hCSF-1RD1-D3 are present. Furthermore, analysis of fractions from that gel- filtration is a bit representative for the insoluble character of the hIL-34p180 construct: unbound hIL-34p180 seems to elute as a high molecular weight species, seen as a shoulder at the left of the complex-containing peak. So despite having the smallest molecular weight compared to the complex and the free receptor, hIL-34p180 elutes first. This is only possible when unbound hIL-34p180 is present as a high-order aggregate. At the end of the purification, about 1 mg hIL-34p180:hCSF-1RD1-D3 is yielded.

As the subsequent crystallization screens did not result in a protein crystal, it was decided to deglycosylate the protein complex. It is postulated that the chemical and conformation heterogeneousity of large carbohydrate structures can hinder protein crystallization. Despite -/- the expression of both hIL-34p180 and hCSF-1RD1-D3 in HEK293S GnTI cells, which produce glycoproteins with a homogeneous Man5GlcNAc2 carbohydrate structure, still some variance in occupancy and conformation can exist. The deglycosylation of the protein complex is performed at a concentration of 0.5 mg/ml, as losing the highly hydrophilic carbohydrate structures often leads to a diminished solubility of proteins. It is indeed seen that after deglycosylation and a well-succeeded purification of the complex by amylose affinity chromatography (Figure 7.3), the complex can be concentrated to only 1.68 mg/ml. EndoHf is an endoglycosydase that cleaves within the chitobiose core of high mannose oligosaccharides from N-linked glycoproteins, leaving only an N-acetylglucosamine covalently attached to Asn. hIL-34 has one predicted N-glycosylation site, and its apparent molecular weight on a SDS-PAGE gel is shifted from 22 kDa to 20 kDa after deglycosylation (Figure 7.2B and Figure 7.3B respectively). A shift of 1 kDa can be explained by the loss of the Man5GlnNAc structure. The remaining 1 kDa can be attributed to a reduction in apparent molecular weight, as large carbohydrate structures are known to retard migration of proteins during gel electrophoresis. The apparent molecular mass of CSF-1RD1-D3, which has four potential N-linked glycosylation sites, is reduced with 13 kDa after incubation with EndoHf (from 45 kDa after gel-filtration to 32 kDa after incubation with EndoHf; Figure 7.2B and Figure 7.3B respectively).

Of all the crystallization trials that were set up, crystallization condition C11 of the index screen (0.66 M ammonium sulfate, 0.066 M HEPES pH 7 and 0.33 % PEG 8000) showed crystals of about 50 µm x 10 µm. Although the crystals were, based on morphology, of bad quality (small, pour defined edges and a rough-appearing surface), they were tested by Jan Felix at the PXI beamline of the Swiss Light Source (Paul Scherrer Institute, Villigen) in combination with the Micro-Crystal-Diffractometer (focusing the beam size to 25 µm x 5 µm), resulting in a dataset of 180 images, recorded with a 1° oscillation range. However, the crystals only diffracted to about 8 Å, which was too low to be indexed.

51 Part 4: Discussion and conclusions

With this knowledge, an optimization grid screen was set up in which we applied minor changes to the pH and salt concentration. Such small changes could possibly have a great effect on the crystal packing and yield larger and/or better diffracting crystals. It was indeed seen that in conditions with a slightly higher concentration of ammonium sulfate (0.7 M versus 0.66 M in the initial screen) and a lower pH (HEPES buffer pH 6 versus HEPES buffer pH 7 in the initial screen), larger crystals developed with dimensions of about 200 µm x 50 µm (Figure 7.4). In an attempt to yield ever bigger crystals, this optimized condition was used as a basis for the additive Silver Bullets™ screen. This screen contains a large combinatorial library of small molecules with diverse characteristics that could possibly enhance crystal packing even more by providing additional stabilizing interactions to the protein complex in the crystal. Two conditions showed indeed larger crystals of about 30 µm x 30 µm x 200 µm and 50 µm x 50 µm x 80 µm (Figure 7.5). Subsequent analysis of crystals from reproduced additive screen conditions could show that they indeed contained hIL-34p180 and hCSF-1RD1-D3 (Figure 7.6). However, a band at the molecular weight of EndoHf was detected as well, although the signal was much weaker compared to the signal of the complex constituents. This band is probably the result of trace amounts of EndoHf that was not retained by the amylose affinity chromatography nor removed by subsequent washing steps of the crystal. Thus it was concluded that the new crystals indeed contained the hIL-34p180:hCSF-1R complex. The crystals from the additive screen were not only bigger, but also more easily manageable and less fragile than the crystals from the initial Index screen. In general, such features indicate a higher quality of the crystal packing. But despite this seemingly higher crystal quality, the crystals again only diffracted to 7.5 Å - 8 Å (Figure 7.7).

This observation led to the hypothesis that the low diffraction quality of the crystal was perhaps inherently to the constituting protein identities. Thus it could well be that bad crystal packing interactions, inherently to one or both proteins, would always lead to crystals that only diffract in the low-resolution range.

9.4 Large-scale expression and crystallization of hIL-34p180 in complex with hCSF-1RD2-D4 Based on previous the expression analysis (Results, 6.1), we decided to co-express and crystallize hIL-34p180 ectodomains two to four (CSF-1RD2-D4). Truncated hIL-34 in complex with these specific receptor ectodomains, could yield some interesting features:

- CSF-1RD1 is not involved in ligand binding and could be oriented away from the ligand:receptor interaction site as seen in the mCSF-1:mCSF-1R complex. If so, CSF-1RD1 could possibly exhibit higher flexibility than the rest of the complex and influence crystal packing in an unfavorable way. Co-expression of a complex in which CSF-1RD1 is not present, could thus possibly yield a more compact complex with more favorable characteristics towards crystallization.

52 Part 4: Discussion and conclusions

- Domain 4 of CSF-1R contains a conserved sequence motive for homotypical receptor:receptor interactions. However, the existence of such interaction between two CSF-1 receptors has not yet been shown by high-resolution structural studies. If domain 4 is present in the crystal structure, chances are that these homotypical interactions will be detected.

- Homotypical receptor:receptor interactions would possibly further lock the IL-34:CSF-1R complex in a specific topology, reducing the flexibility of the receptor ectodomain.

Because of the low detection of hCSF-1R2-4 in the Western blot, we decided to transfect the hIL-34p180 HEK293T GnTI-/- cells with 50% more pHL_hCSF-1R_D2-D4 plasmid DNA, but with retention of the DNA/PEI ratio. Despite this raised amount of DNA, the yield after Co IMAC was only about 20% of Co IMAC hIL-34p180:hCSF-1RD1-D3 yield (Figure 8.1A). Moreover, in contrast to previous purifications, one could see multiple bands accompanying the ones of hIL-34p180 and hCSF-1RD2-D4. Although it Is hypothesized that these bands are proteins with a different glycosylation pattern, their exact identity is unknown. Also high-order complexes in the elutionpeak could be detected (Figure 8.1B). The yield of the Co IMAC purification was that low that we decided to pool conservatively, despite the presence of multiple undesirable protein populations in the elutionpeak. This was rationalized by the knowing that we still had to perform a second chromatographic purification. The high-order complexes in the Co IMAC elutionpeak could be easily separated from the rest of the protein complex by the gel-filtration (Figure 8.2). However, bands below the apparent molecular weight of hIL-34p180 and hCSF-1RD2-D4 were still present in the elutionpeak (Figure 8.2B). We could not perform any other subsequent separation method as the yield of this complex was too low: another chromatographic experiment would inevitably lead to a loss of protein content. But luckily, after setting up only two crystallization screens, a condition was found that allowed crystallization of the hIL-34p180:hCSF-1RD2-D4 complex. The two resulting spherulite crystals (Figure 8.3) could be tested at beamline PXIII of the Swiss Light Source (Paul Scherrer Institute, Villigen) with a Pilatus 2M-F Detector, which has the advantage to work in a shutterless fashion and thus doesn’t show any read-out noise. The resulting dataset shows diffraction to about 7.5 Å (Figure 8.5). Moreover, the diffraction pattern indicates that the crystal content is indeed of protein nature, but a silver stain of one such crystal to confirm presence of the complex, has yet to be done. Despite the absence of a clear symmetry in the diffraction pattern, these results are encouraging, and further optimization of the crystallization condition could yield better results.

A crystallization optimization screen had to be set up, but despite our efforts, we did not succeed in purifying a second batch of hIL-34p180 in complex with hCSF-1RD2-D3. This is due to the low amounts of hCSF-1RD2-D4 that are secreted in the medium during transient expression. One possible reason is that expression plasmid contains some detrimental sequences due to cloning errors. However, recent experiences in the lab by Nico Callewaert have somewhat led to the notion that the N-terminal sequence of a protein can act as a fold initiator. When the protein is missing the N-terminal sequences (as in the case with

53 Part 4: Discussion and conclusions

hCSF-1RD2-D4), it would be biased towards protein degradation by the fold quality check of the endoplasmatic reticulum. However, this hypothesis is only very preliminary and has yet to be further investigated.

X. Conclusions 10.1 Large-scale expression and crystallization of deglycosylated hIL-34p180 in complex with hCSF-1RD1-D3 Despite the observation that the crystals of deglycosylated truncated hIL-34 in complex with the first three deglycosylated ectodomains of hCSF-1R were larger and less fragile than previously obtained crystals, their diffraction stayed in the low-resolution range. As all deglycosylated hIL-34p180:hCSF-1RD2-D4 crystals were derived from two subsequent optimization screens of crystallization conditions, it is unlikely that there is room for further optimization of those derived conditions. Moreover, with the publication of the crystallographic structure featuring a binary truncated hIL-34:hCSF-1RD1-D3 complex (Ma et al., 2012), one could wonder if another, slightly less truncated hIL-34p180:hCSF-1RD1-D3 structure would still contribute significantly to the knowledge in this field.

10.2 Large-scale expression and crystallization of hIL-34p180 in complex with hCSF-1RD2-D4

The crystal structure of hIL-34p180 in complex with hCSF-1RD2-D4 could lead to some significant insights in ligand:receptor binding and the subsequent receptor activation mechanics. Possibly, a ternary complex would be derived with a clear view on homotypical receptor interactions at domain 4. Although predicted by the conserved D4-D4 interaction motive, high-resolution data for such interaction has not yet been derived for CSF-1R. Despite the fact that expression and purification of hIL-34p180 in complex with ectodomains two to four of hCSF-1R is troublesome and far from efficient, protein-containing crystals could be derived. X-ray exposure of those crystals, of which the contents has yet to be confirmed, reveals a bad diffraction pattern that is however clearly of protein nature. Indexing of the spots gave a very untrustworthy solution. As these crystals are derived from conditions that haven’t had even the slightest optimization but readily diffract to about 7.5 Å, it shows that this crystallization approach is at least promising. Further progression on this complex is however prevented by the extreme low purification yields of hIL-34p180:hCSF-1RD2-D4, which is on his turn the result of a low hCSF-1RD2-D4 expression. This low expression could be due to the quality of the expression plasmid, of by the lack of its N-terminal domain which possibly acts as a fold initiator. However, the relevance of the latter to protein expression, has yet to be determined.

54 Part 4: Discussion and conclusions

XI. Future perspectives For the crystallization of truncated hIL-34 in complex with the first three ectodomains of hCSF-1R, a new initial crystallization screen can be initiated. Alternatively, crystals from the Silver Bullets™ screen can be optimized themselves, by for example dehydration of the crystal. Also a more extensive screen on the cryoprotectants can possibly lead to better diffraction of the crystals.

Co-expression and purification of hIL-34p180 in complex with hCSF-1RD2-D4 as described in this Master’s Dissertation is far from efficient. The low expression yield of hCSF-1RD2-D4 can be addressed by developing a new expression vector. Alternatively, a cell line that stable expresses hCSF-1RD2-D4 could be developed, as it is reported that a stable expression can lead to higher expression yields. Also other sequence tags could be tested, as these can exert an effect on the proteins stability. If however the low expression yield relies in the absence of the N-terminal domain, the above suggested solutions would only exert a minor effect on hCSF-1RD2-D4 expression. One possible way to overcome this problem, is to design a hCSF-1R ectodomain construct with a recognition site for a protease in the D1-D2 linker. In this way, domain 1 could be proteolytically cleaved off after the efficient expression of the ectodomain construct.

55

Part 5 Material and Methods

Part 5: Material & Methods

XII. Material and methods 12.1 Preparation of hCSF-1R plasmid DNA 12.1.1 Transformation to electrocompetent cells

Escherichia coli MC 1061 cells were grown at 37 °C until an OD600 of 0.6 was reached. Next, the medium was centrifuged at 4000 rpm for 5 minutes at 4° C. The supernatant was discarded, the pellet resuspended in 10% autoclaved glycerol and centrifuged at 4000 rpm for 5 minutes at 4° C. This was repeated three times, and after the third time only a part of the supernatant was discarded, leaving 1 mm solution left above the pellet. The electrocompetent cells were resuspended in the remaining solution, allocated in fractions of 60 µl and stored at -80° C. A detailed protocol can be found in Appendix 1.

12.1.2 Transformation of electrocompetent E. coli MC 1061 cells and preparation of expression constructs

1 µl pure DNA of the expression construct was diluted 1/100 in H2O, added to electrocompetent E. coli MC 1061 cells and both were subsequently transferred to an ice- cooled cuvette. The cells were pulsed using an E. coli Pulser™ (Bio-Rad, USA) at 2.5 kV. After electroporation, the cells were quickly resuspended by adding 1 ml SOC medium, and incubated at 37° C for 15 minutes. Next, this regeneration medium was plated out at different concentrations on 100 µg/ml carbenicillin containing LB-plates and incubated overnight at 37° C. A single colony was picked up and used to inoculate a 50 ml of fresh LB medium that was again grown overnight at 37 °C. The next day, this preculture was used to inoculate 10 l fresh LB medium containing 100 µg/µl carbenicillin. When an OD600 of about 2.5 was reached (typically by overnight growth), the plasmid DNA was purified using a QIAGEN Plasmid Giga Kit (Qiagen, Venlo, The Netherlands). The purified plasmid DNA was stored at -20° C. A detailed protocol can be found in Appendix 2.

12.2 Expression and purification of complexes between recombinant hIL-34 and hIL- 34p180 and recombinant hCSF-1R in HEK293S GnTI-/- cell lines 12.2.1 Tetracycline-induced expression of recombinant hIL-34 or hIL-34p180, simultaneously to transfection with hCSF-1R constructs in HEK293S GnTI-/- cell lines HEK293S GnTI-/- are suspension adapted HEK293 cells that are deficient in N-acetylglucosaminyltransferase-I. As this enzyme is essential for the modification of N-linked carbohydrate structures in the cis-Golgi stacks, glycoproteins are secreted with homogenous (Mannose)5-(N-acetylglucosamine)2 N-linked glycans (Reeves et al). Stable full-length hIL-34 expressing HEK293S GnTI-/- or stable truncated hIL-34p180 expressing HEK293S GnTI-/- cell lines (Jan Felix, unpublished data) were grown in growth medium (Dulbecco's Modified Eagle Medium DMEM F12 with 10% heat-inactivated fetal calf serum, 6 10 units/l penicillin and 1 g/l streptomycin) in incubators maintaining a 5% CO2 atmosphere at 37° C. In order to obtain milligram amounts of the receptor complexes, the cells were cultivated in 175 cm² large culture flasks with 50 ml growth medium until a confluency of 90% was reached. At that moment, the medium was removed and replaced with 42 ml induction medium, which is serum-free DMEM F12 medium, containing penicillin G and

57 Part 5: Material & Methods streptomycin. Also 2 µg/ml tetracycline and 5 mM sodium butyrate were added to induce expression of either hIL-34 or hIL-34p180 via the TET-ON dependent expression promotor. The actual transfection was performed according to the protocol by Aricescu et al. (Aricescu et al, 2006). 100 µg prepared plasmid DNA (Section 13.1) was added to 150 µg polyethylenimine (PEG). The solution was pipetted up and down and serum-free DMEM F12 medium with penicillin G, streptomycin, tetracycline and sodium butyrate was added until 8 ml. This transfection solution was vortexed for 10 seconds and added to the induced HEK293S GnTI-/- cells after 10 minutes incubation at room temperature. After five days of expression, the medium was harvested and clarified from cellular debris by centrifugation at 6000 x g and a subsequently filtered using a 0.22 µm cut-off bottle top filter (Cornoring B.V., Amsterdam, The Netherlands). Optionally, the medium could be stored at – 20° C before purification.

12.2.2 Cobalt immobilized metal affinity chromatography (Co IMAC) of hIL-34:receptor complexes All ligand:receptor complexes studied during this Master’s Dissertation were purified from the collected medium by cobalt immobilized metal affinity chromatography (Co IMAC). A 25 ml TALON column was used (TALON matrix: Clontech, USA; column: GE Healthcare, Diegem, Belgium), connected to a semi-automated liquid chromatography system (AKTA purifier 10 or AKTA purifier 100, GE Healthcare, Diegem, Belgium). First, the TALON column was regenerated by washing with 10 bed volumes of 0.2 ml EDTA pH 7.0. Ten bed volumes of distilled H2O (dH2O) were loaded on the column, after which the column was charged with 10 bed volumes of a 50 mM CoCl2 solution. Again the column was washed, this time with an excess of dH2O (15 bed volumes) to make sure that all non-bound cobalt was washed off. After the regeneration, the tubings of the chromatographic system were cleaned with equilibration buffer, containing 300 mM NaCl and 50 mM NaH2PO4 pH 7.0. The column was equilibrated with 15 bed volumes of equilibration buffer, after which the medium was loaded overnight. When all medium was loaded, the column was extensively washed with at least 20 bed volumes equilibration buffer until the UV280 absorbance was returned to baseline. To wash off tightly but non-specific interacting proteins, a wash step was performed by mixing 97% equilibration buffer with 3% wash buffer (300 mM NaCl, 50 mM NaH2PO4 pH 7.0 and 500 mM imidazole), resulting in equilibration buffer containing 15 mM imidazole. When the UV280 absorbance was again returned to baseline, the complex was finally eluted by washing the column with 100% wash buffer (500 mM imidazole). After the chromatography, captured protein fractions (1.25 ml) were stored at 4° C.

12.2.3 Purification of ligand:receptor complexes by size-exclusion chromatography To achieve a higher purity of the receptor complex, the fractions containing the elution peak of the Co IMAC were pooled and concentrated to 2 ml by the use of a Vivaspin 15R concentrator (Sartorius Stedim Biotech, Vilvoorde, Belgium). Meanwhile, the pumps, the tubings and the 2 ml injection loop of the semi-automated liquid chromatography system and the 2.5 ml syringe (Hamilton Company, Bonaduz, Switzerland) were washed with HEPES buffer (150 mM NaCl, 20 mM HEPES, pH 7.5). The Superdex™ 200 gelfilteration column (GE

58 Part 5: Material & Methods

Healthcare, Diegem, Belgium) was equilibrated with one bed volume HEPES buffer, followed by injection of the protein sample on the column. Fractionated capturing of the eluens was started at 40 ml and the fractions were stored at 4° C.

12.2.4 Analysis of the protein contents in Co IMAC and gel-filtration fractions. After Co IMAC and gel-filtration, the protein content and purity of the several fractions were investigated by SDS-PAGE. Based on the elution profile, interesting fractions were chosen of which 7 µl was incubated for 10 minutes at 95° C with 8 µl Laemmli buffer containing 5% beta-mercaptoethanol (BME). Next, the samples were loaded on a 15% acrylamide/ bisacrylamide containing gel with a 6% acrylamide/bisacrylamide stacking gel and subjected to SDS-PAGE at 160V. After the run was completed, the protein bands were stained with Coomassie Bright Brilliant Blue for at least 3 hours (dependant on the protein concentration), after which the non-bound Coomassie solution was washed of the gel with Coomassie destain solution. Alternatively, a silver stain was used to detect protein bands with a 50 times higher sensitivity than when Coomassie staining. To do so, the gel from the SDS-PAGE was stained using the PlusOne™ protein silver stain kit (GE Healthcare, Diechem, Belgium).

12.3 Time-course limited proteolysis of full-length hIL-34 in complex with hCSF-1RD1-D5 To purify the full-length hIL-34 in complex with the first three ectodomains of hCSF-1R (hCSF-1RD1-D5), the pHL_hCSF-1R_D1-D5 expression construct (Elegheert et al, 2011) was -/- prepared and co-expressed with hIL-34 in stable hIL-34 HEK293S GnTI cells (Section 13.1 and 13.2.1). The expressed construct was purified with Co IMAC and subsequent gel- filtration (Section 13.2.2 and 13.2.3). After purification, the protein was present in HEPES buffer (150 mM NaCl, 20 mM HEPES, pH 7.5). Two proteases were used, elastase and chymotrypsin (Sigma-Aldrich, Bornem, Belgium), and of each protease a 500 µg/ml and 50 µg/ml stock was made in 0.1 M TRIS-HCl pH 8. Two microliter of these stock solutions were used to incubate 42 µg hIL-34:hCSF-1RD1-D5. For each of the conditions, a control was prepared in which the protein solution was replaced with dH2O. Of every condition and control, 10 µl samples were taken after 1 min, 10 min, 30 min, 1 hour and 2.5 hours. The samples were directly incubated for 10 minutes at 95°C with 10 µl Laemmli buffer containing 5% beta-mercaptoethanol (BME), after which they were stored at -20 °C. The next day, all samples were thawed, subjected to SDS-PAGE and stained with Coomassie- blue as described in Section 13.2.4. The detailed protocol can be found in Appendix 3.

12.4 Small-scale co-expression of different hIL-34:hCSF-1R complexes. Four different expression constructs of hIL-34p180 (pHL-hCSF-1R_D1-D5, pHL-hCSF-1R_D1- D3, pHL-hCSF-1R_D2-D4 and pHL-hCSF-1R_D2-D3; Elegheert et al., 2011) were prepared (Section 13.1). Two six-well plates were seeded with stable hIL-34p180 HEK293S GnTI-/- cells and grown in two milliliter growth medium (Section 13.2.1) under 5% CO2 atmosphere at

59 Part 5: Material & Methods

37° C. When the cells were grown to 90 % confluency, the medium was removed and replaced with 1 ml induction medium (Section 13.2.1). Subsequently, 1 ml medium of the following prepared solutions was added to each corresponding well of the two plates: - Well 1: induction medium with no receptor DNA was added. - Well 2: 4 µg pHL-hCSF-1R_D1-D3 DNA with 6 µg PEI was added to 1 ml of induction medium - Well 3: 4 µg pHL-hCSF-1R_D1-D5 DNA with 6 µg PEI was added to 1 ml of induction medium - Well 4: 4 µg pHL-hCSF-1R_D2-D3 DNA with 6 µg PEI was added to 1 ml of induction medium - Well 5: 4 µg pHL-hCSF-1R_D2-D4 DNA with 6 µg PEI was added to 1 ml of induction medium - Well 6: 4 µg pHL-hCSF-1R_D1-D3 DNA with 6 µg PEI was added to 1 ml of serum- free medium, that only contains DMEM F12, 106 units/l penicillin G and 1 g/l streptomycin. All of the above medium preparations were prepared, well mixed and incubated for 10 minutes at room temperature before they were added to the cells. After five days of expression, the medium was harvested and analyzed by SDS-PAGE (Section 13.2.4). The proteins from the gel were transferred to a nitrocellulose membrane by overnight electroblotting at 30 V. Next, the blot was treated with the Western Blot Signal Enhancer kit (Thermo Scientific Pierce), after which the membrane was blocked. The membrane was washed and incubated for one hour with an anti-His (C-terminal) Horse Radish Peroxidase (HRP) antibody (Invitrogen, Carlsbad, CA, USA) at room temperature. Finally, the protein content was visualized by chemoluminescence using a Lumi-light Western Blotting Substrate kit (Roche, Vilvoorde, Belgium), containing a luminol solution and a peroxidase solution.

12.5 Deglycosylation and purification of hIL-34p180:hCSF-1RD1-D3 complex

12.5.1 Deglycosylation of hIL-34p180:hCSF-1RD1-D3 pHL-hCSF-1R_D1-D3 expression plasmids were prepared (Section 13.1) and co-expressed with hIL-34p180 in stable hIL-34p180 HEK293S GnTI-/- cell lines. After five days of expression, the medium was harvested and the complex was purified by Co IMAC and gel-filtration followed by an analysis of the elutionpeak fractions (Section 13.2). The concentration of the complex in the pooled fractions was determined by ultraviolet–visible spectrophotometry (Nanodrop 1000 Spectrophotometer; Thermo Scientific, Erembodegem-Aalst, Belgium) using calculated molecular weights and extinction coefficients (Appendix 4) of both proteins. The complex was concentrated to 0.5 mg/ml using a Vivaspin 4R concentrator (Sartorius Stedim Biotech, Vilvoorde, Belgium) and 20 kU EndoHf (New England BioLabs, Hitchin, UK) was added. The sample was incubated overnight in temperature controlled conditions at 25° C using a thermoblock (Thermomixer comfort; Eppendorf, Rotselaar, Belgium). The next day, the sample was centrifuged for 30 minutes at 13200 rmp to remove aggregated and/or degraded protein fragments.

60 Part 5: Material & Methods

12.5.2 Purification of deglycosylated hIL-34p180:hCSF-1RD1-D3 Amylose affinity chromatography was performed by using a 2.5 ml Amylose-Resin column (Amylose Resin: New England BioLabs, Hitchin, UK; Column: GE Healthcare, Diegem, Belgium) connected to a semi-automated liquid chromatography system (AKTA purifier 10 or AKTA purifier 100, GE Healthcare, Diegem, Belgium). After the pumps and tubings of the chromatography system were washed with dH2O, the amylose-resin column was connected and washed with 3 bed volumes equilibration buffer (20 mM HEPES pH 7.4 and 150 mM NaCl). Meanwhile, the 2 ml injection loop was washed with dH2O and equilibration buffer. When both equilibration and washing were completed, fragmented collection was started and the sample was injected on the amylose-resin column. At least 3 bed volumes equilibration buffer were loaded on the column until the UV280 absorbance has returned to baseline. After the complex was washed off, elution of EndoHf was initiated by loading elution buffer (20 mM HEPES pH 7.4, 150 mM NaCl and 10 mM maltose) on the column. Based on the elution profile, fractions were chosen to be analyzed according to Section 13.2.4.

12.6 Crystallization trials

12.6.1 Crystallization of hIL-34p180:hCSF-1RD1-D3 Crystallization trials of truncated IL-34 in complex with the first three extracellular domains of CSF-1R were performed using an in-house Mosquito® crystallization robot (TTP Labtech, Cambridge, USA). Several screens were set up, including Index, PEG Ion screen I & II, Crystal Screen Lite (Hampton Research, Aliso Viejo, CA, USA) and Proplex (Molecular Dimensions, Suffolk, UK). The screens were set up in a sitting drop vapor diffusion fashion using a 96-well plate (SwissCi, Zug, Switzerland) with a reservoir solution of 45 µl. All drops were 0.2 µl (0.1 µl protein with 0.1 µl reservoir solution) and crystallization was monitored under a light microscope.

12.6.2 Crystallization of deglycosylated hIL-34p180:hCSF-1RD1-D3

Several initial crystallization screens for the deglycosylated hIL-34p180:hCSF-1RD1-D3 complex were set up using the Mosquito® crystallization robot: Index, PEG Ion screen I & II, Crystal Screen Lite (Hampton Research, Aliso Viejo, CA, USA) and Proplex (Molecular Dimensions, Suffolk, UK). All screens were set up using a sitting drop vapor diffusion experiment in a 96- well plate (TTP Labtech, Cambridge, USA) with a 75 µl reservoir. All drops contained 0.1 µl protein and 0.1 µl reservoir solution. Needle-like crystals (50 µm x 10 µm) were observed In condition C11 (0.66 M ammonium sulfate, 0.066 M HEPES pH 7 and 0.33% PEG 8000) of the Index screen. By applying small changes to the crystallization conditions, important parameters such as the amount of nucleation and/or the growth rate of the crystals can be changed, hopefully leading to bigger and/or better diffracting crystals. Based on condition C11 of the Index screen, a grid screen was automatically set up in a sitting drop vapor diffusion experiment. In this crystallization screen, the pH and ammonium sulfate concentration were step-wise

61 Part 5: Material & Methods varied from pH 6 to pH 8 (steps of 0.5 pH units) and from 0.2 M to 0.9 M ammonium sulfate (steps of 0.1 M). The grid screen was performed in a 96-well plate with a 75 µl reservoir with 0.2 µl drops (1:1), and resulted in morphologically better crystals (birefringent under polarized light, more defined edges and a smoother surface) that were however still small in size (100 µm x 20 µm). In an attempt to yield even better crystals, the most promising condition F1 of the grid screen (0.7 M ammonium sulfate, 0.066 M HEPES pH 6 and 0.33 % PEG 8000) was used as a basis for the additive Silver Bullet™ screen (Hampton Research, Aliso Viejo, CA, USA). This screen contains a combinatorial library of more than 1090 chemicals, of which more ten 400 are unique. The screen was automatically set up using a sitting 0,2 µl (1 µl of the Silver Bullets™ screen + 1 µl protein in condition F1 of the grid screen) drop vapor diffusion experiment in a 96-well plate with a 75 µl reservoir. Several crystals could be detected in multiple conditions. Crystallization conditions B3 (0.25% w/v 5-Sulfoisophthalic acid monosodium salt, 0.25% w/v Cystathionine, 0.25% w/v Dithioerythritol, 0.25% w/v L- Citrullineand 0.02 M HEPES sodium pH 6,8) and B7 (0.33% w/v 1,4-Cyclohexanedicarboxylic acid, 0.33% w/v 2,2'-Thio-diglycolic acid, 0.33% w/v 5-Sulfoisophthalic acid monosodium salt and 0.33% w/v HEPES sodium pH 6.8) gave the largest crystals (respectively 50 µm x 50 µm x 80 µm and 30 µm x 30 µm x 200 µm). Crystallization in condition B7 was repeated 12 times in the same automated setup as above. The smallest crystals from two drops were harvested using a nylon loop and brought into a stabilizing buffer, containing reservoir solution with a 10 % higher concentration of precipitant. The solution was centrifuged at low speed for about 30 – 60 seconds and the supernatant was mixed with Laemmli buffer with 5% BME (“Wash 1” for drop 1 and drop 2). Again stabilizing buffer was added to the crystals followed by a centrifugation step and again the supernatant was collected and mixed with Laemmli buffer and 5% BME (“Wash 2” for drop 1 and drop 2). The remaining crystals were resuspended in Laemmli buffer with 5% BME. Next, crystals from a third drop were directly resuspended in Laemmli buffer with 5% BME without being washed. Finally, all samples were analyzed using an SDS-PAGE of which the proteins bands were silver-stained (Section 13.2.4). As a positive control, a sample from the deglycosylated complex before the start of the crystallization screening was used. All samples clearly indicated the presence of both hIL-34p180 and hCSF-1RD1-D3 in the crystals. The best looking crystals from condition B3 and from the repeated B7 conditions of the Silver Bullet™ screen were harvested from the drop using a nylon loop and brought in a cryoprotectant solution. This solution contained the stabilizing buffer of the corresponding condition with the cryoprotectant. Several cryoprotectants were tested, including 25% ethylene glycol, 25% glycerol, 25% glucose, 2 M sodium malonate and 3 M ammonium sulfate. For each cryoprotectant solution, the crystal was harvested from the solution and flash-frozen in liquid nitrogen for storage an travelling to the synchrotron facility. The protective effects of the cryoprotectant were estimated by the quality of the diffraction pattern, i.e. the appearance of ice rings, the diffraction resolution, the sharpness of spots and the spot separation. Crystals that were cryoprotected in stabilizing solution with 25% ethylene glycol appeared to give the best results.

62 Part 5: Material & Methods

12.6.3 Crystallization of hIL-34p180:hCSF-1RD2-D4

The first crystallization screen preformed on the hIL-34p180:hCSF-1RD2-D4 complex was a Crystal Screen Lite (Hampton Research, Aliso Viejo, CA, USA). The screen contained 48 conditions that were set up in a sitting drop vapor experiment with 0.2 µl (1 µl protein and 1 µl reservoir solution) drops. The screen was automatically set up in a 96-well plate with a 75 µl reservoir. Secondly, the Proplex screen (Molecular Dimensions, Suffolk, UK) was automatically set up in 96-well plate with a 75 µl reservoir. Also this crystallization experiment was in the sitting drop vapor diffusion geometry, but now with the use of 0.1 µl drops (50 nl protein + 50 nl reservoir solution). Crystals from condition C4 (0.2M lithium sulfate, 0.1M MES and 20% PEG 4000) were harvested from the drop using a nylon loop and brought into the cryoprotectant solution (15% PEG 400 added to reservoir solution of condition C10). Next, the crystals were harvested from cryoprotectant solution and flash-frozen in liquid nitrogen for storage an travel to the synchrotron facility.

12.7 Crystallographic experiments

12.7.1 Data collection from crystals containing deglycosylated hIL-34p180:hCSF-1RD1-D3 Data collection was performed at the I02 beamline of the Diamond Light Source (Harwell Science and Innovation Campus, Oxfordshire, UK). The focused X-ray beam was 80 µm x 20 µm, had a wavelength of 0.97949 Å and an flux of 3.0x1012 ph/sec. Crystals were automatically mounted in front of the beam and every dataset was recorded with an exposure time of 2 seconds in combination with a ADSC Q315r detector. An initial set of four images with an offset by 90° was recorded before the actual data collection of 180 images started. For this data collection, an oscillation range of 1° and a 0,79 m distance to the detector was used.

12.7.2 Data collection from hIL-34p180:hCSF-1RD2-D4 crystals Data collection was performed at the PXIII beamline of the Swiss Light Source (Paul Scherrer Institute, Villigen, Switzerland). The focused X-ray beam was 80 µm x 45 µm, had a wavelength of 1,00001 Å and an flux of 5x1011 ph/sec/400 mA. Crystals were automatically mounted in front of the beam and every dataset was recorded with a Pilatus 2M-F Detector. Prior to collection of data sets, an initial set of four images with an offset by 90° was recorded to estimate the optimal exposure time and evaluate centering of the crystal. Diffraction with exposure times of 0.01 seconds, 1 second, 2 seconds and 5 seconds were tested this way. At the end, datasets of 360° (oscillation range 0.1°; exposure time 0,09772 seconds), and 180° (oscillation range 1°, exposure time of 1,99772 seconds) were recorded with a 0,69999 m and 0.82 m distance to the detector respectively.

12.7.3 X-ray data analysis All recorded datasets were processed with the X-ray Detector Software (XDS) Program package (Kabsch, 2010)

63

Part 6 References

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69

Part 7 Appendices

Part 7: Appendices

Appendix 1. Preparing electrocompetent E. coli MC 1061 cells Note: All materials that are used during this protocol, must be ice-cooled for at least 20 minutes before use!

1. E. coli MC 1061 cells are inoculated in 2 ml LB (10 g/l bactotrypton, 5 g/l NaCl and 5 g/l yeast extract).

2. A preculture is prepared by bringing the 2 ml culture in 50 ml LB. The culture is grown overnight, shaking at 200 rmp and 37° C.

3. The preculture is used to inoculate 200 ml LB. The cells are grown at 37° C at the shaker until they reach an OD600 of 0.6.

4. The cell solution is decanted in 50 ml tubes and centrifuged at 4000 rmp in 4° C for 5 minutes.

5. The supernatants is discarded under sterile conditions.

6. De cell pellet is resuspended in 30 ml ice-cooled 10% (v/v) autoclaved glycerol and centrifuged at 4000 rmp in 4° C for 5 minutes.

7. The supernatant is discarded (carefully!) under sterile conditions.

8. The cell pellet is resuspended in 30 ml ice-cooled 10% (v/v) autoclaved glycerol for a second time, and centrifuged at 4000 rmp in 4° C for 5 minutes.

9. The supernatant is discarded (carefully!) under sterile conditions.

10. The cell pellet is resuspended in 30 ml ice-cooled 10% (v/v) autoclaved glycerol for the last time, and centrifuged at 4000 rmp in 4° C for 5 minutes.

11. Under sterile conditions, the supernatant is partially discarded, leaving 1 mm solution left above the cell pellet.

12. Without adding new glycerol, the cells are resuspended in the supernatant that was left over.

13. The cell solution is aliquoted in pre-cooled 0.2 ml epjes. In each epje, 60 µl is deposited.

14. Store the cells at -80° C till use.

71 Part 7: Appendices

Appendix 2. Transformation of electrocompetent E. coli MC 1061 cells and preparation of plasmid DNA NOTE: always work on ice and in sterile conditions!

1. The DNA stock solution is diluted or concentrated to 10 ng/µl.

2. Working in sterile conditions and on ice, 1 µl of the prepared DNA solution is added to 60 µl electrocompetent E. coli MC 1061 cells.

3. The cell solution with DNA is transferred to an ice-cooled electroporation cuvette.

4. The electroporation cuvette is placed in the E. coli Pulser™ (Bio-Rad, USA). The cells are electroporated at 2.5 kV, 25 µF and 200 Ohm.

5. After the pulse, 1 ml SOC medium (2% tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4 and 20 mM glucose) is quickly added to the cells. The cell solution is well mixed by pipetting several times up and down and placed at 37 ° C for 15 min.

6. After regeneration, de cell solution is transferred to a falcon and incubated for 1h at 37 °C while continuous shaking at 200 rmp.

7. The cell solution is plated on three selective carbenicillin LB plates (10 g/l bactotrypton, 5 g/l NaCl and 5 g/l yeast extract added with 100 µg/ml carbenicillin). For the first plate, 50 µl cell suspension is used, for the second 100 µl and for the third 200 µl. The plates are incubated overnight at 37° C.

8. Single positive colonies are picked up from the selective plate and used to inoculate 50 ml LB, containing 100 µg/ml carbenicillin. The cells in this preculture are grown in 37° C on a 200 rmp shaker to an OD600 of 2.5. The selective carbenicillin plates are stored at 4° C until further use.

9. Finally, the preculture is used to inoculate 10 l fresh LB medium.

10. Subsequent preparation of the plasmid DNA, the QIAGEN Plasmid Giga Kit (Qiagen, Venlo, The Netherlands) is used.

72 Part 7: Appendices

Appendix 3. Time course limited proteolysis of full-length hIL-34 in complex with hCSF-1RD1-D5 1. A 500 µg/ml and 50 µg/ml stock of elastase (Sigma-Aldrich, Bornem, Belgium) is prepared in 0.1 M Tris-HCl pH 8.

2. A 500 µg/ml and 50 µg/ml stock of chymotrypsin (Sigma-Aldrich, Bornem, Belgium) is prepared in 0.1 M Tris-HCl pH 8.

3. Eight solutions are prepaired:

a. 1% elastase + protein: i. 3 µl of 14 mg/ml hIL-34:hCSF-1RD1-D5 solution in 150 mM NaCl and 20 mM HEPES pH 7.4 ii. 2 µl of 500 µg/ml elastase stock iii. 5 µl of 1 M Tris-HCl pH 8 iv. 40 µl destilled dH2O b. Negative control: i. 2 µl of 500 µg/ml elastase stock ii. 5 µl of 1 M Tris-HCl pH 8 iii. 43 µl destilled dH2O

c. 0.1 % elastase + protein: i. 3 µl of 14 mg/ml hIL-34:hCSF-1RD1-D5 solution in 150 mM NaCl and 20 mM HEPES pH 7.4 ii. 2 µl of 50 µg/ml elastase stock iii. 5 µl of 1 M Tris-HCl pH 8 iv. 40 µl destilled dH2O d. Negative control: i. 2 µl of 50 µg/ml elastase stock ii. 5 µl of 1 M Tris-HCl pH 8 iii. 43 µl destilled dH2O

e. 1% chymotrypsin + protein: i. 3 µl of 14 mg/ml hIL-34:hCSF-1RD1-D5 solution in 150 mM NaCl and 20 mM HEPES pH 7.4 ii. 2 µl of 500 µg/ml chymotrypsin stock iii. 5 µl of 1 M Tris-HCl pH 8 iv. 40 µl destilled dH2O f. Negative control: i. 2 µl of 500 µg/ml chymotrypsin stock ii. 5 µl of 1 M Tris-HCl pH 8 iii. 43 µl destilled dH2O

g. 0.1% chymotrypsin + protein: i. 3 µl of 14 mg/ml hIL-34:hCSF-1RD1-D5 solution in 150 mM NaCl and 20 mM HEPES pH 7.4

73 Part 7: Appendices

ii. 2 µl of 50 µg/ml chymotrypsin stock iii. 5 µl of 1 M Tris-HCl pH 8 iv. 40 µl destilled dH2O h. Negative control: i. 2 µl of 50 µg/ml chymotrypsin stock ii. 5 µl of 1 M Tris-HCl pH 8 iii. 43 µl destilled dH2O

4. The samples are incubated at room temperature.

5. At time t = 1 min, t = 10 min, t = 30 min, t = 60 min and t = 150 min, 10 µl samples are taken, added with 10 µl Leammli buffer + 5% beta-mercaptoethanol and incubated for 10 minutes at 95° C. After incubation, the samples are stored at – 20°C until analysis by SDS-PAGE

74 Part 7: Appendices

Appendix 4. Calculated molecular masses and extinction coefficients of hIL-34 and hIL-34p180 alone and in complex with diverse hCSF-1R constructs.

Protein / protein Molecular weight (kDa) Amino acids ε (1000-1) complex

Full length hIL-34 26.11 221 42.78

hIL-34p180 21.9 180 34.3

hCSF-1RD1-D5 54.663 482 79.465

hCSF-1RD1-D3 31.5308 270 32.8

hCSF-1RD2-D4 32.886 293 35.660

FL hIL-34:hCSF-1RD1-D5 80.773 1406 122.245

FL hIL-34:hCSF-1RD1-D3 57.6408 838 75.58

FL hIL-34:hCSF-1RD2-D4 57,5388 514 78.44

hIL-34p180:hCSF-1RD1-D5 76.563 1324 113.765

hIL-34p180:hCSF-1RD1-D3 53.4308 450 67.1

hIL-34p180:hCSF-1RD2-D4 54.7886 473 69.960

Table A.2: Calculated molecular masses and extinction coefficients of hIL-34 and hIL-34p180 alone and in complex with diverse hCSF-1R constructs. All values are calculated using the ExPASy Protparam tool (http://web.expasy.org/protparam/) based on their sequences from the reviewed UniProtKB/Swiss-Prot database.

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