Complement Factor H: Solution Structures and Interactions with Ligands Thesis Presented for the Degree of Doctor of Philosphy by Azubuike Ifeanyi Okemefuna Institute of Structural and Molecular Biology, University College London June 2009 I, Azubuike Ifeanyi Okemefuna, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. Abstract Factor H (FH) is a plasma glycoprotein that plays a central role in regulating the alternative pathway of complement. It is composed of 20 short complement regulator (SCR) domains. The SCR-1/5 fragment is required for decay acceleration and cofactor activity, while the SCR-16/20 fragment possesses binding sites for complement C3d and host cell-surface polyanionic ligands. C3d is a 35-kDa fragment of C3b, the activated form of the central complement protein C3. C- reactive protein (CRP) is an acute-phase reactant that activates complement through the classical pathway but inhibits the alternative pathway. In this thesis, X-ray scattering, analytical ultracentrifugation (AUC) and constrained modelling were used to determine solution structures for the SCR-1/5 and SCR-16/20 fragments, as well as for intact FH. Surface plasmon resonance (SPR) was used in conjunction with these methods to investigate the interactions of FH with C3d and CRP in the fluid and solid phases. Structural studies revealed that at physiological concentrations, SCR-1/5 is monomeric and SCR-16/20 exists in a weak monomer-dimer equilibrium. In the best-fit models, both FH fragments adopt a partially folded-back orientation in solution, and the SCR-16/20 dimer is formed by association of two SCR-20 domains. FH exists in a partially folded-back orientation in solution and forms oligomers. Here, these FH oligomers were shown to exist under different buffer conditions of NaCl concentration and pH, and these two factors also influence the conformation of FH. New, significantly improved molecular structures showed that FH orientation is maintained by short-to-middle distance interdomain interactions. There was no evidence of long distance interactions between the N- and C-terminals of FH. Studies on the interaction of FH with C3d identified a number of multimeric complexes formed between C3d and both SCR-16/20 and native FH. Binding to C3d did not significantly increase the overall length of FH, and this interaction is NaCl concentration-dependent. CRP, another ligand of FH, is known to exist physiologically as a pentamer. Here, CRP is shown to exist in a pentamer-decamer equilibrium under physiological buffer conditions. This equilibrium is NaCl concentration dependent and occurs in both fluid and solid phases. SPR binding studies showed that interaction with CRP inhibits FH oligomerisation, and further identified a novel CRP-binding site within SCR-16/20 of FH. Thus ionic strength- dependence may be a general feature of FH interaction with its ligands. These results provide insight into the complement regulatory activity of FH. i Acknowledgements I would like to thank my supervisor Professor S. J. Perkins for his guidance and encouragement throughout the period of my work with him. I am also grateful to the other two members of my thesis committee, Dr. Andrew Martin and Professor Martin Crompton, for their advice and support. I would like to thank my collaborators, including Professor David L. Gordon, Dr Rebecca Ormsby, Dr. Tania Sadlon and Ms. Kim Griggs for their expertise and for supplying the proteins and expression systems used in my studies. I am very grateful to the UCL graduate school and Overseas Research Fund for a postgraduate scholarship. My thanks also go to Mr. Jayesh Gor for his invaluable help with experiments and lab work. I thank Dr. Patricia Furtado for her helpful and gentle advice, and my colleagues at UCL for their untiring help, understanding and patience. And lastly, to my parents and family - thank you all for believing. ii Contents Page Chapter One The Complement Cascade 1 1.1 Introduction to complement 2 1.2 Complement activation 3 1.2.1 C3 in complement activation 5 1.2.2 The classical pathway 7 1.2.3 The alternative pathway 9 1.2.4 The lectin pathway 11 1.2.5 The membrane attack complex 12 1.3 Regulation of complement activation 12 1.4 Biosynthesis of complement proteins 16 1.5 Complement in disease 16 Chapter Two Factor H of Complement 19 2.1 Factor H expression and biosynthesis 20 2.1.1 The Factor H gene family 20 2.1.2 Factor H expression and sequence 20 2.1.3 Biosynthesis of Factor H 21 2.2 Structure of Factor H 21 2.2.1 The SCR domain structure 21 2.2.2 Structures of Factor H SCR domains 24 2.2.3 Factor H inter-domain arrangements 27 2.3 Function of Factor H in complement 28 2.3.1 Regulation of complement activation 28 2.3.2 Factor H binding to C3b 30 2.3.3 Factor H binding to CRP 31 2.3.3.1 Introduction to human CRP 31 2.3.3.2 Factor H interaction with CRP as a means of complement 33 regulation 2.3.3.3 The role of the interaction between Factor H 34 and CRP in the molecular mechanism of age-related macular degeneration iii 2.3.4 Factor H binding to other ligands 35 2.4 Factor H mutations and disease 35 Chapter Three Protein Structure and Interaction Measurement 38 3.1 Introduction to protein structure determination 39 3.2 Analytical ultracentrifugation 41 3.2.1 Introduction to the analytical ultracentrifuge 41 3.2.2 Uses of analytical ultracentrifugation 42 3.2.3. Theory of sedimentation 42 3.2.4. Instrumentation 46 3.2.4.1. The rotor 46 3.2.4.2. The cells 46 3.2.4.3. Absorbance optics 48 3.2.4.4. Interference optics 48 3.2.5. Applications of analytical ultracentrifugation 50 3.2.5.1. Sedimentation velocity 50 3.2.5.2. Sedimentation equilibrium 51 3.2.5.3 Analytical ultracentrifugation data analyses 51 3.3 Solution scattering 55 3.3.1 Introduction to solution scattering 55 3.3.2 Types of solution scattering 56 3.3.3 Theory of solution scattering 56 3.3.4 Properties of X-ray scattering experiments 58 3.3.5 Instrumentation 61 3.3.6 X-ray sources 61 3.3.7 Data interpretation 65 3.3.7.1 Guinier analyses 65 3.3.7.2 Distance distribution function analyses 66 3.4 Biomolecular modelling 69 3.4.1 Scattering curve modelling 69 3.4.1.1 Analysis of protein composition 69 3.4.1.2 Creating atomic models 70 3.4.1.3 Molecular model searches 70 iv 3.4.1.4 Sphere modeling 71 3.4.1.5 Debye scattering curve calculation 72 3.4.2 Identification of best-fit models 72 3.4.3 Calculation of hydrodynamic properties 73 3.5 Introduction to protein interaction measurement 73 3.5.1 Surface plasmon resonance 75 3.5.2 Experiments on the Biacore X100 77 3.5.3 Applications of Biacore systems 79 3.5.3.1 Binding analyses 80 3.5.3.2 Equilibrium analyses 80 3.5.3.3 Concentration assays 81 Chapter Four The Regulatory SCR-1/5 and Cell-surface-binding SCR-16/20 87 Fragments of Factor H Reveal Partially Folded-back Solution Structures and Different Self-associative Properties 4.1 Introduction 88 4.2 Results and Discussion 89 4.2.1 X-ray scattering data for SCR-1/5 and SCR-16/20 89 4.2.2 Analytical ultracentrifugation data for SCR-1/5 and SCR-16/20 97 4.2.3 Constrained scattering modelling of SCR-1/5 and 105 monomeric SCR-16/20 4.2.4 Trial models for dimeric SCR-16/20 114 4.3 Conclusions 117 4.4 Materials and Methods 125 4.4.1 Expression and preparation of SCR-1/5 and SCR-16/20 125 4.4.2 X-ray scattering data collection and analysis 126 4.4.3. Sedimentation velocity and equilibrium data 128 for SCR-1/5 and SCR-16/20 4.4.4. Modelling of the SCR-1/5 and SCR-16/20 solution structures 129 4.4.5 Debye scattering and sedimentation coefficient modelling 130 4.4.6 Protein data bank accession number 131 v Chapter Five Electrostatic Interactions Contribute to the Folded-back 132 Conformation of Wild-type Human Factor H 5.1 Introduction 133 5.2 Results and Discussion 134 5.2.1 Analytical ultracentrifugation of Factor H 134 5.2.2 X-ray scattering data for Factor H 146 5.2.3 Constrained scattering modelling of monomeric Factor H 150 5.3 Conclusions 158 5.4 Materials and Methods 167 5.4.1 Purification of Factor H 167 5.4.2 Sedimentation velocity data for Factor H 169 5.4.3 X-ray scattering data collection and analysis 170 5.4.4 Modelling of the Factor H solution structures 171 5.4.5 Debye scattering and sedimentation coefficient modelling 172 5.4.6 Protein Data Bank accession number 173 Chapter Six Multimeric Interactions Between Complement Factor H and its C3d 174 Ligand Provide New Insight on Complement Regulation 6.1 Introduction 175 6.2 Results and Discussion 176 6.2.1 Solution structural data for the Factor H-C3d complex 176 6.2.2 Analytical ultracentrifugation of C3d 179 6.2.2.1 Sedimentation velocity data for C3d 179 6.2.2.2 Sedimentation equilibrium data for C3d 181 6.2.3 Analytical ultracentrifugation of the SCR-16/20 and Factor H 183 complexes with C3d 6.2.3.1 Sedimentation velocity data for the SCR-16/20 complex 183 with C3d 6.2.3.2 Sedimentation velocity data for the complex 188 of native Factor H with C3d 6.2.4 Surface plasmon resonance studies of the complexes of Factor H 190 and SCR-16/20 with C3d vi 6.2.5 X-ray scattering data of the SCR-16/20 complex with C3d 197 6.2.6 Molecular modelling of SCR-16/20 complexes with C3d 201 6.3 Conclusions 205 6.4 Materials and methods 211 6.4.1 Protein expression and purification 211 6.4.2 Analytical ultracentrifugation data and analysis 215 6.4.2.1 Sedimentation velocity data for C3d and its complexes 215 with Factor H and SCR-16/20 6.4.2.2 Sedimentation equilibrium data for C3d 216 6.4.3