Accepted Manuscript A Type III Complement Factor D Deficiency: Structural insights for inhibition of the alternative pathway Christopher C.T. Sng, MB BChir, Sorcha O’Byrne, BSc, Daniil M. Prigozhin, PhD, Matthias R. Bauer, PhD, Jennifer C. Harvey, BSc, Michelle Ruhle, BBiomedSc, Ben G. Challis, PhD, Sara Lear, MBBS, Lee D. Roberts, PhD, Sarita Workman, RN MSc, Tobias Janowitz, PhD, Lukasz Magiera, PhD, Rainer Doffinger, PhD FRCPath, Matthew S. Buckland, PhD FRCPath, Duncan J. Jodrell, DM MSc FRCP, Robert K. Semple, MB PhD, Timothy J. Wilson, PhD, Yorgo Modis, PhD, James E.D. Thaventhiran, PhD FRCPath PII: S0091-6749(18)30327-0 DOI: 10.1016/j.jaci.2018.01.048 Reference: YMAI 13343 To appear in: Journal of Allergy and Clinical Immunology Received Date: 24 October 2017 Revised Date: 2 January 2018 Accepted Date: 16 January 2018 Please cite this article as: Sng CCT, O’Byrne S, Prigozhin DM, Bauer MR, Harvey JC, Ruhle M, Challis BG, Lear S, Roberts LD, Workman S, Janowitz T, Magiera L, Doffinger R, Buckland MS, Jodrell DJ, Semple RK, Wilson TJ, Modis Y, Thaventhiran JED, A Type III Complement Factor D Deficiency: Structural insights for inhibition of the alternative pathway, Journal of Allergy and Clinical Immunology (2018), doi: 10.1016/j.jaci.2018.01.048. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. M AN US CR IP T AC CE PT ED ACCEPTED MANUSCRIPT Sng et al. 1 TITLE PAGE 1 2 Letter to the Editor 3 4 A Type III Complement Factor D Deficiency: Structural insights for inhibition of the 5 alternative pathway. 6 7 Christopher C. T. Sng, MB BChir,1* Sorcha O’Byrne, BSc,2* Daniil M. Prigozhin, PhD3*, 8 Matthias R. Bauer, PhD,4* Jennifer C. Harvey, BSc,5 Michelle Ruhle, BBiomedSc,6 Ben G. 9 Challis, PhD,7 Sara Lear, MBBS,2 Lee D. Roberts, PhD,9 Sarita Workman, RN MSc,5 Tobias 10 Janowitz, PhD,1 Lukasz Magiera, PhD,1 Rainer Doffinger, PhD FRCPath,2 Matthew S. 11 Buckland, PhD FRCPath,5 Duncan J. Jodrell, DM MSc FRCP,1 Robert K. Semple, MB PhD,7 12 8 Timothy J. Wilson, PhD,10 Yorgo Modis, PhD,3 James E. D. Thaventhiran, PhD FRCPath.1 13 2 11 12 14 15 1Cancer Research UK Cambridge Institute, Robinson Way, Cambridge, CB2 0RE U.K. 16 2Department of Clinical Immunology, Cambridge University Hospitals NHS Trust, 17 Addenbrooke’s Hospital, Cambridge, CB2 0QQ U.K. 18 3Molecular Immunity Unit, Department of Medicine, MRC Laboratory of Molecular Biology, 19 Cambridge Biomedical Campus, Francis Crick Ave, Cambridge CB2 0QH, U.K. 20 4 MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Francis Crick 21 Ave, Cambridge CB2 0QH, U.K. 22 5Department of Immunology, Royal Free London NHS Foundation Trust, Pond Street, 23 Hampstead, London, NW3 2QG U.K. 24 M AN US CR IP T AC CE PT ED ACCEPTED MANUSCRIPT Sng et al. 2 6The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, 25 Victoria, Australia 3058 26 7Wellcome Trust-MRC Institute of Metabolic Science, Addenbrooke’s Hospital, Cambridge, 27 CB2 0QQ U.K. 28 8University of Edinburgh Centre for Cardiovascular Sciences, Queen's Medical Research 29 Institute, Little France Crescent, Edinburgh EH16 4TJ. 30 9Leeds Institute of Cardiovascular and Metabolic Medicine, LIGHT Laboratories 31 University of Leeds, LS2 9JT U.K. 32 10Department of Microbiology, Miami University, 68 Pearson Hall, 700 E. High Street, 33 Oxford, OH 45056 U.S.A. 34 11Department of Medicine, University of Cambridge, Addenbrooke’s Hospital, Cambridge, 35 CB2 0QQ U.K. 36 12MRC Toxicology Unit, Hodgkin Building, University of Leicester, LE1 9HN U.K. 37 * These authors contributed equally to this work. 38 39 Corresponding author 40 James E. D. Thaventhiran, PhD 41 Department of Medicine 42 Addenbrooke’s Hospital 43 Box 157 44 Hills Rd 45 Cambridge 46 CB2 0QQ U.K. 47 48 Telephone: +44 7740 703599 49 M AN US CR IP T AC CE PT ED ACCEPTED MANUSCRIPT Sng et al. 3 Fax: +44 1223 336846 50 E-mail: jedt2@cam.ac.uk 51 52 Declaration of all sources of funding: JEDT is supported by an MRC Clinician Scientist 53 Fellowship (MR/L006197/1). This work was funded by BRC III PPG funding and a 54 Wellcome Trust Senior Research Fellowship to Y.M. (101908/Z/13/Z). RKS is funded by the 55 Wellcome Trust [grant number WT098498 and strategic award 100574/Z/12/Z], the United 56 Kingdom Medical Research Council [MRC_MC_UU_12012/5] and the United Kingdom 57 National Institute for Health Research (NIHR) Cambridge Biomedical Research Centre. TJ is 58 funded by Cancer Research UK (Clinician Scientist Fellowship C42738/A24868). BGC is 59 now a full-time employee of Astra-Zeneca. 60 61 Word count: 1012 62 63 M AN US CR IP T AC CE PT ED ACCEPTED MANUSCRIPT Sng et al. 4 Capsule summary 64 We fully characterise the first reported functional deficiency of complement factor D in a 65 patient. The structural analysis yielded a novel approach by which this key enzyme could be 66 inhibited to treat inflammatory diseases. 67 Key words 68 Adipsin, age-related macular degeneration, alternative pathway, complement deficiency, 69 complement serine protease, drug development, factor D, glucose homeostasis, single-70 nucleotide variant, type III deficiency 71 72 Abbreviations used 73 AMD: age-related macular degeneration 74 AP: alternative complement pathway 75 AP50: alternative pathway haemolytic activity 76 CH50: classical pathway haemolytic activity 77 FB: complement factor B 78 FD: complement factor D 79 MD: molecular dynamics 80 WT: wild-type 81 M AN US CR IP T AC CE PT ED ACCEPTED MANUSCRIPT Sng et al. 5 To the Editor: 82 We investigated an alternative complement pathway (AP) deficiency in a patient with absent 83 alternative pathway haemolytic activity (AP50) but normal classical pathway haemolytic 84 activity recovering from invasive meningococcal infection (for patient and sibling details, see 85 Appendix A in this article’s Online Repository). Serum reconstitution with proximal AP 86 components suggested a Factor D (FD) deficiency (Fig 1A). Sanger sequencing of CFD 87 identified a rare homozygous missense mutation (c.602G>C) in exon 4 in the patient (II-1) 88 and sibling (II-2), resulting in an arginine to proline substitution (p. R176P) (see Fig E1, A, in 89 this article’s Online Repository). This genotype co-segregated with an AP50-null phenotype, 90 as the parents, both heterozygotes, had normal AP50 (Fig 1B). In contrast to previous 91 confirmed FD deficiencies,1-3 all members of the pedigree had normal levels of circulating 92 FD, as corroborated by western blot (see Fig E1, B). Meanwhile, identical circular dichroism 93 spectra and melting curves of recombinant wild-type (WT) and R176P FD precluded gross 94 changes in FD structure or stability, suggesting a functional deficiency (Fig 1C and see Fig 95 E1, C). We assessed the cleavage of C3b-bound Factor B (FB) by recombinant WT and 96 mutant FD (R176P, R176A, R176Q). WT FD could cleave C3b-bound FB to produce 97 fragments Bb and Ba. Conversely, R176P FD demonstrated diminished in vitro catalytic 98 activity at all concentrations, and had negligible activity at physiological concentration (0.04 99 µM) (Fig 1D and see E1, D). Reconstitution of FD-depleted serum with R176P FD also 100 demonstrated impaired AP mediated haemolysis (see Fig E1, E). 101 102 FD’s serine protease activity depends on obligatory binding to the C3bB complex via four 103 exosite loops (residues 132-135, 155-159, 173-176, 203-209). This leads to rearrangement of 104 the self-inhibitory loop (199-202), allowing realignment of His41 and Asp89 with Ser183 to 105 form the active catalytic triad (see Fig E2, A and B, in this article’s Online Repository).4, 5 106 M AN US CR IP T AC CE PT ED ACCEPTED MANUSCRIPT Sng et al. 6 Mutation R176P lies outside the active site, within one of the FB-binding exosite loops. We 107 used molecular dynamics (MD) stimulations to study how the R176P mutation affects the FD 108 protein fold (see Fig E2, C). In mutant FD, we observed a rearrangement of the exosite loop 109 155-161 within 50 ns of simulation (Fig 2A). This was unexpected because loop 155-161 was 110 not in direct contact with residue 176. Average structures generated from the final 50 ns of 111 simulation for WT and mutant FD (R176P and R176A) demonstrated that key FB-binding 112 residues Asp161 and Arg157 were shifted by 4.3 Å and 1.9 Å respectively (Cα average 113 position) (Fig 2B). Superimposing these MD average structures onto the crystal structure of 114 the C3bB-D complex revealed that Asp161 and Arg157 assumed a conformation that no 115 longer supported binding due to loss of shape and charge complementarity to the FB surface 116 (Fig 2C). The other three exosite loops retained their binding-competent conformations. After 117 assuming the new conformation, exosite loop 155-161 demonstrated higher conformational 118 mobility (root mean square fluctuation) relative to WT (Fig E2, D and E). In contrast, the 119 mobility of loops containing catalytic residues His41 and Asp89 decreased in the mutants. 120 Using the distance between His41 and Ser183 during MD simulations as a proxy for the 121 active site conformation, we observed that WT could sample the short distance necessary for 122 a catalytically active conformation (Fig 2D). Conversely, in both mutant simulations, the 123 distance remained larger, consistent with His41 pointing away from the active site. Therefore, 124 in addition to disruption of key FB-binding residues, mutations R176P and R176A appear to 125 stabilise the self-inhibited conformation of free FD. 126 127 To assess the binding of FD to C3bB, we used surface plasmon resonance. Co-injection of 128 catalytically inactive FD (WT/S183A) with FB demonstrated a dose-dependent increase in 129 binding to C3b and complex formation (Fig 2E). In contrast, R176P/S183A FD lacked any 130 detectable binding (Fig 2F). Consistent with the stochastic transitions of free WT FD to the 131 M AN US CR IP T AC CE PT ED ACCEPTED MANUSCRIPT Sng et al. 7 active conformation observed in the MD simulation, FD has a low level of esterolytic activity 132 towards a small synthetic substrate, Z-Lys-SBzl (Fig 2D). Surprisingly, R176P FD 133 demonstrated a loss of esterolytic activity similar to the active site mutant, S183A (see Fig 134 2G). 135 136 Deficiency of properdin, the most common AP deficiency, can result from absent (type I), 137 low (type II) or normal but non-functioning (type III) protein levels (for reference, see 138 Reference E10 in this article’s Online Repository). Meanwhile, previously confirmed 139 deficiencies of activating complement serine proteases have all resulted in low or absent gene 140 product. We have identified a unique deficiency: R176P FD is fully expressed and stable, but 141 enzymatically inert, constituting a functional or Type III deficiency. Recent preclinical 142 evidence6 that FD deficient mice are susceptible to diabetes prompted metabolic assessment 143 in the FD deficient patients. No abnormality was detected (for details, see Appendix B, Fig 144 E3 and Table E1 in this article’s Online Repository). 145 146 Over-activation of AP is implicated in numerous inflammatory disorders, including age-147 related macular degeneration (AMD). Therefore, blockade of the AP by targeting the rate-148 limiting enzyme, FD, is an attractive approach to controlling disease progression. An anti-FD 149 Fab fragment targeting the two distal exosite loops has shown some benefit in phase II 150 clinical trials for treatment of dry AMD.7 In vitro studies indicate that it inhibits binding to 151 the C3bB complex but increases esterolytic activity towards small-molecule substrates.8 This 152 may result in unwanted clinical effects due to non-specific activity or limit its efficacy in 153 vivo. In the case of R176P FD, both FB-binding and esterolytic activity are abrogated 154 through exosite hindrance and stabilisation of the self-inhibited state. Loop 173-176 is thus a 155 promising target for allosteric inhibitors of FD that stabilise the inhibitory loop in addition to 156 M AN US CR IP T AC CE PT ED ACCEPTED MANUSCRIPT Sng et al. 8 binding-blockade. A structure-based design approach to targeting FD has recently succeeded 157 in identifying candidate FD inhibitors where high-throughput screens had failed,9 158 highlighting the benefits of integrating structural information into candidate drug screens. 159 Comprehensive definition of the structural and molecular determinants of in vivo FD activity 160 is critical for this. This study of the R176P mutation demonstrates how in-depth mechanistic 161 analysis of rare complement deficiencies can deliver such insight validated clinically by in 162 vivo human evidence of AP blockade. 163 164 Our acknowledgements can be found in this article’s Online Repository. 165 166 Christopher C. T. Sng, MB BChir1* 167 Sorcha O’Byrne, BSc2* 168 Daniil M. Prigozhin, PhD3* 169 Matthias R. Bauer, PhD4* 170 Jennifer C. Harvey, BSc5 171 Michelle Ruhle, BBiomedSc6 172 Ben G. Challis, PhD7 173 Sara Lear, MBBS2 174 Lee D. Roberts, PhD9 175 Sarita Workman, RN MSc5 176 Tobias Janowitz, PhD1 177 Lukasz Magiera, PhD1 178 Rainer Doffinger, PhD FRCPath2 179 Matthew S. Buckland, PhD FRCPath5 180 Duncan J. Jodrell, DM MSc FRCP1 181 M AN US CR IP T AC CE PT ED ACCEPTED MANUSCRIPT Sng et al. 9 Robert K. Semple, MB PhD7 8 182 Timothy J. Wilson, PhD10 183 Yorgo Modis, PhD3 184 James E. D. Thaventhiran, PhD FRCPath1 2 11 12 185 186 From 1Cancer Research UK Cambridge Institute, Cambridge, United Kingdom; 2Department 187 of Clinical Immunology, Cambridge University Hospitals NHS Trust, Addenbrooke’s 188 Hospital, Cambridge, United Kingdom; 3Molecular Immunity Unit, Department of Medicine, 189 MRC Laboratory of Molecular Biology, Cambridge, United Kingdom; 4MRC Laboratory of 190 Molecular Biology, Cambridge, United Kingdom; 5Department of Immunology, Royal Free 191 London NHS Foundation Trust, London, United Kingdom; 6The Walter and Eliza Hall 192 Institute of Medical Research, Parkville, Victoria, Australia; 7Wellcome Trust-MRC Institute 193 of Metabolic Science, Addenbrooke’s Hospital, Cambridge, United Kingdom; 8University of 194 Edinburgh Centre for Cardiovascular Sciences, Queen's Medical Research Institute, Little 195 France Crescent, Edinburgh EH16 4TJ; 9Leeds Institute of Cardiovascular and Metabolic 196 Medicine, LIGHT Laboratories, University of Leeds, Leeds, United Kingdom; 10Department 197 of Microbiology, Miami University, Oxford, United States of America; 11Department of 198 Medicine, University of Cambridge, Addenbrooke’s Hospital, Cambridge, United Kingdom; 199 and the 12MRC Toxicology Unit, Hodgkin Building, University of Leicester, LE1 9HN 200 United Kingdom. 201 * These authors contributed equally to this work. 202 203 204 M AN US CR IP T AC CE PT ED ACCEPTED MANUSCRIPT Sng et al. 10 References 205 1. Hiemstra PS, Langeler E, Compier B, Keepers Y, Leijh PC, van den Barselaar MT, et 206 al. Complete and partial deficiencies of complement factor D in a Dutch family. J 207 Clin Invest 1989; 84:1957-61. 208 2. Biesma DH, Hannema AJ, van Velzen-Blad H, Mulder L, van Zwieten R, Kluijt I, et 209 al. A family with complement factor D deficiency. J Clin Invest 2001; 108:233-40. 210 3. Sprong T, Roos D, Weemaes C, Neeleman C, Geesing CL, Mollnes TE, et al. 211 Deficient alternative complement pathway activation due to factor D deficiency by 2 212 novel mutations in the complement factor D gene in a family with meningococcal 213 infections. Blood 2006; 107:4865-70. 214 4. Narayana SV, Carson M, el-Kabbani O, Kilpatrick JM, Moore D, Chen X, et al. 215 Structure of human factor D. A complement system protein at 2.0 A resolution. J Mol 216 Biol 1994; 235:695-708. 217 5. Forneris F, Ricklin D, Wu J, Tzekou A, Wallace RS, Lambris JD, et al. Structures of 218 C3b in complex with factors B and D give insight into complement convertase 219 formation. Science 2010; 330:1816-20. 220 6. Lo JC, Ljubicic S, Leibiger B, Kern M, Leibiger IB, Moede T, et al. Adipsin is an 221 adipokine that improves beta cell function in diabetes. Cell 2014; 158:41-53. 222 7. Yaspan BL, Williams DF, Holz FG, Regillo CD, Li Z, Dressen A, et al. Targeting 223 factor D of the alternative complement pathway reduces geographic atrophy 224 progression secondary to age-related macular degeneration. Sci Transl Med 2017; 9. 225 8. Katschke KJ, Jr., Wu P, Ganesan R, Kelley RF, Mathieu MA, Hass PE, et al. 226 Inhibiting alternative pathway complement activation by targeting the factor D 227 exosite. J Biol Chem 2012; 287:12886-92. 228 M AN US CR IP T AC CE PT ED ACCEPTED MANUSCRIPT Sng et al. 11 9. Maibaum J, Liao SM, Vulpetti A, Ostermann N, Randl S, Rudisser S, et al. Small-229 molecule factor D inhibitors targeting the alternative complement pathway. Nat Chem 230 Biol 2016; 12:1105-10. 231 232 233 M AN US CR IP T AC CE PT ED ACCEPTED MANUSCRIPT Sng et al. 12 Figure legends 234 Figure 1: Assessing the contribution of mutation R176P to AP dysfunction. 235 (A) AP50 assay assessing patient serum supplemented with properdin (P), factor B (FB) or 236 factor D (FD). 237 (B) The immediate family pedigree of the patient with the CFD genotype, serum AP50 and 238 serum FD concentrations displayed. D, WT allele. d, mutant allele (c.602G>C). 239 (C) Thermal shift assay of WT and R176P FD. 240 (D) Serial dilutions of recombinant WT or R176P FD were incubated with C3b and FB. The 241 SDS-PAGE gel, stained with AcquaStain, shows the individual proteins and resultant 242 products. 243 244 Figure 2: Defining the effects of the R176P mutation on FD function. 245 (A and B) FB-binding exosite loop 155-167 assumes a new conformation in mutant FD 246 simulation. Arrows highlight average Cα position shifts of two residues that bind C3bB in the 247 R176P FD simulation. 248 (C) Loss of shape complementarity at the FD-C3bB interface. FD exosite loops from 249 published co-crystal structures (white, PDB ID: 2XWB) overlaid with the simulated loops of 250 WT and mutant FD. 251 (D) Distance sampled between the active site Nε2 nitrogen of His41 and Oγ of Ser183 during 252 each simulation. The shorter distance is necessary for catalytic activity. 253 (E and F) SPR binding measurement of enzymatically inactive recombinant FD 254 (R176P/S183A or WT/S183A) to C3bB complex. 255 (G) Steady state kinetics for Z-Lys-SBzl cleavage by WT, R176P, R176A and catalytically 256 inactive control S183A FD. 257 M AN US CR IP T AC CE PT ED ACCEPTED MANUSCRIPTCo ntr ol ser um Pa tie nt ser um Pa tie nt ser um + P Pa tie nt ser um + F B Pa tie nt ser um + F D FD on ly A I II D/d 121% 2.5 D/d 125% 2.3 d/d 0% 2.3 d/d 0% 2.0 P CFD genotype: AP50: Serum FD [mg/L]: CFD genotype: AP50: Serum FD [mg/L]: 1 2B C Temperature (°C) WT R176P 0.0 0.5 1.0 1.5 40 60 80 100 + - - - + + + + + + + + - + - - + + + + + + + + - - 2.5 - 2.5 - 1 - 0.2 - 0.04 - - - - 2.5 - 2.5 - 1 - 0.2 - 0.04 250 kDa C3b FB Bb Ba FD 150 kDa 100 kDa 75 kDa 50 kDa 37 kDa 25 kDa 20 kDa C3bD FB R176P FD [μM] WT FD [μM] M AN US CR IP T AC CE PT ED ACCEPTED MANUSCRIPT 0 0.5 1 1.5 2 2.5 0 20 40 60 80 100 WT R176P R176A A FD Loop 155-167 Backbone RM SD (Å ) Time (ns) B Exosite Repacking WT R176P R176A Loop 155-167 Arg157 Cα 1.9 ÅAsp161 Cα 4.3 Å 0 2 4 6 8 10 12 14 0 20 40 60 80 100 WT R176P R176A D Active Site His41-Ser183 Distance (Å ) Time (ns) C FB Surface Binding Arg157 Asp161 Thr158 Lys208 Arg207 Arg175 Asn132 His133Asn206 1.9 Å 4.3 Å FB + WT/S183A FD FB FB FB + R176P/S183A FD E F G 0 200 400 600 800 0 200 400 600 800 Time (s) SP R re sp on se (R U) 0 200 400 600 800 0 200 400 600 800 Time (s) 0 1 2 3 4 0 1×10-5 2×10-5 3×10-5 Substrate [mM] kc at (a pp )( s- 1 ) WT R176P S183A R176A M AN US CR IP T AC CE PT ED ACCEPTED MANUSCRIPT Sng et al. Supplementary material 1 APPENDIX A 1 Patient details 2 A 19-year-old, South Asian female presented with a 24-hour history of high fever, rigors, 3 delirium and diarrhoea. On clinical examination, she was febrile with a purpuric rash and a 4 reduced level of consciousness (Glasgow Coma scale score: 9/15). Intravenous antibiotic 5 therapy was initiated for provisionally diagnosed meningococcal septicaemia. She was 6 intubated and transferred to the intensive care unit where she developed disseminated 7 intravascular coagulation, for which she received treatment. Results from blood cultures 8 drawn at the time of admission confirmed an infection with Neisseria meningitides serogroup 9 Y. Her clinical condition improved with intensive care support and antimicrobial therapy. 10 She was discharged after two weeks with minimal sequelae including bilateral leg scarring, a 11 sacral pressure sore and mild bilateral hearing loss. 12 13 At the age of 5 years, she had received bilateral tympanostomy tubes for recurrent ear 14 infections and otitis media with effusion but had no other unusual infections as a child. She 15 received the full course of childhood vaccinations as per the national immunisation schedule. 16 17 On screening for immunodeficiency, laboratory measurement demonstrated a normal full 18 blood count with normal counts of lymphoid cells. The titres of C3, C4, mannose-binding 19 lectin and C1q were within normal range, but there was undetectable alternative 20 pathwayhaemolytic activity (AP50) in conjunction with normal classical pathway haemolytic 21 activity. In view of her complement deficiency, she was prescribed lifelong 22 phenoxymethylpenicillin as antimicrobial prophylaxis. She was also vaccinated for 23 meningitis ACWY, meningitis C, pneumococcus and haemophilus influenza B to which she 24 developed high antibody titre responses. 25 M AN US CR IP T AC CE PT ED ACCEPTED MANUSCRIPT Sng et al. Supplementary material 2 26 Her sole sibling, a younger male, who was homozygous for the same mutation, leading to an 27 identical pattern on immunodeficiency screening, was healthy at assessment. He reported no 28 excess of infections in the past. Of note, he reported having been treated empirically for 29 suspected meningitis, aged 11, whilst travelling in Mauritius from which he recovered with 30 no sequalae after a standard course of antibiotics. 31 32 APPENDIX B 33 Functional Factor D deficiency does not result in impaired oral glucose tolerance 34 Recent pre-clinical evidenceE1 that FD regulates insulin secretion prompted metabolic 35 assessment of the patient and her sibling. They had a BMI of 19.3 kg/m2 and 23.1 kg/m2, 36 respectively. Fasting venous plasma glucose (5.2-5.4 mmol/L) and insulin (29-39 pmol/L) 37 levels were normal in both subjects (Fig E3). Similarly, plasma glucose excursions were 38 normal in response to an oral glucose (75g) challenge. At 120 minutes following glucose 39 administration, glucose levels remained normal (4.0 mmol/L). Furthermore, circulating 40 concentrations of leptin and adiponectin, adipokines which regulate insulin sensitivity, were 41 normal, as were fasting lipid profiles in both subjects. Thus, glucose homeostasis is not 42 impaired in the context of genetic, and therefore lifelong, FD deficiency. 43 44 These results are consistent with the finding that FD knock-out mice developed impaired 45 glucose tolerance only on a long-term diabetogenic diet. This suggests that FD may 46 contribute little to glucose homeostasis in the absence of prolonged metabolic stress. 47 Alternatively, the role of FD in glucose homeostasis could be independent of binding to 48 C3bB or independent of its serine protease activity and, by extension, independent of its 49 downstream effects on the complement cascade. While congenital deficiency of FD alone 50 M AN US CR IP T AC CE PT ED ACCEPTED MANUSCRIPT Sng et al. Supplementary material 3 may not lead to insulin insufficiency, Lo et al.’s findings warrant observation of oral glucose 51 tolerance in such FD deficient patients under extreme metabolic stress and at older age. 52 Further research will be required to understand the role of FD in glucose homeostasis and 53 FD-deficient family pedigrees offer a useful clinical insight to this question. 54 M AN US CR IP T AC CE PT ED ACCEPTED MANUSCRIPT Sng et al. Supplementary material 4 METHODS 55 Informed consent statement 56 All study participants gave their informed consent as appropriate under approved protocols 57 from local institutional review boards. The research was conducted at University College 58 London and the University of Cambridge under approved protocols (#04/Q0501/119 for 59 affected individuals, #07/H0720/182 for family members). 60 61 Alternative pathway haemolytic activity measurement 62 AP100 RC003.1 Kit (Binding Site) agar-chicken erythrocyte plates were prepared according 63 to the manufacturer’s instructions, with kit control and calibration solutions added. 5µl 64 aliquots of test serum were added to individual wells on the plates over ice. The loaded plates 65 were then stored at 4°C for 18 hours to allow radial diffusion of serum components, followed 66 by incubation at 37°C for 90 minutes to develop zones of lysis. The plates were then digitally 67 scanned at high-resolution, and the diameters of zones of lysis were measured using ImageJ 68 1.x computer software. Representative plates were selected for figures. The diameter of lysis 69 correlates with alternative pathway activity (AP50) and is expressed out of 100% relative to 70 kit control. Purified human Factor D (FD), Factor B and properdin for reconstitution assays 71 were purchased from Complement Tech, Inc. 72 73 Sanger sequencing 74 Genomic DNA was isolated from blood samples with QIAamp Kits (QIAGEN). The CFD 75 gene polymerase chain reaction was performed with primers annealing to intron sequences 76 close to each exon as described previouslyE2. Specifically, regarding the R176P mutation, a 77 258-bp genomic fragment comprising exon 4 was amplified by PCR with the primers 5'-78 CTGGGGCATAGTCAACCAC-3' and 5'-TGGGCCCTGTTCCTACTTG-3'. The cDNA 79 M AN US CR IP T AC CE PT ED ACCEPTED MANUSCRIPT Sng et al. Supplementary material 5 numbering for the CFD variant identified is based on transcript NCBI Ref Seq accession no. 80 NM_001928/Ensembl accession no. ENST00000327726.6, beginning at the ATG start 81 codon. The genomic coordinates refer to the GRCh37 genome build. 82 83 Western blot analysis 84 Pooled control and the patient serum were diluted to 1:40 in tris-buffered saline and resolved 85 by SDS-PAGE on NuPAGE 4-12% Bis-Tris Gels, then blotted to nitrocellulose membranes. 86 FD was detected using goat anti-human FD (AF1824; R&D) and donkey anti-goat-IgG 87 IRDye 680CW (LI-COR Biosciences, Lincoln, NE, USA) secondary antibodies. The 88 membranes were imaged using the Odyssey Infrared Imaging System (LI-COR Biosciences, 89 Nebraska, USA) 90 91 Recombinant CFD expression and purification 92 Lentiviral transfer plasmid, envelope plasmid (pMD2.G; gift from Didier Trono; AddGene 93 plasmid #12260) and packaging plasmid (psPAX2; AddGene; gift from Didier Trono; 94 AddGene plasmid #12259) were used to transfect HEK293T cells to produce lentiviral 95 particles. The transfer vector (modified pLenti-CMV-GFP-Puro; gift of Eric Campeau – 96 Addgene 17448) included human FD cDNA (WT, R176P, R176A, S183A) with C-terminus 97 hexahistidine tag upstream of an IRES-Thy1.1 and a puromycin resistance gene (PuroR). 98 Transfection was carried out using Lipofectamine 3000 and, after 24hrs, the media containing 99 the lentiviral particles was used immediately to stably transduce newly plated HEK293T 100 cells. After puromycin selection, stably transduced 293T cells were incubated with FreeStyle 101 media (Gibco) supplemented with 6X Glutamax and 2mM valproic acid. After 7-14 days, 102 secreted recombinant CFD was purified from this media using cobalt immobilised metal 103 affinity chromatography. CFD was eluted in 150mM imidazole in PBS and buffer exchanged 104 M AN US CR IP T AC CE PT ED ACCEPTED MANUSCRIPT Sng et al. Supplementary material 6 by centrifugal concentration (Vivaspin® 20; 10,000Da pore size; Sartorius). Purity of the 105 sample was confirmed on SDS-PAGE and mass spectrometry. 106 107 Measuring in vitro catalytic activity of recombinant FD 108 Purified human C3b and FB were purchased from Complement Technology, Inc. 109 Recombinant WT, R176P or R176A FD were mixed in varying concentrations (1.0 µM, 0.2 110 µM, 0.04 µM) with C3b (1.0 µM) and FB (1.0 µM) in veronal buffer (Lonza) with 10 mM 111 MgCl2 to a final volume of 20 µL. Reaction tubes were incubated for 10 minutes at 37°C 112 before the addition of sample loading buffer (NuPAGE® LDS Sample Buffer) to terminate 113 the reaction. The samples were then heated to 70°C for 10 minutes and resolved by SDS-114 PAGE on a Novex NuPAGE 4-12% Bis-Tris Gel. The gels were developed overnight with 115 AcquaStain (Bulldog Bio), washed for 1 hour with distilled water, dried and digitally scanned 116 at high-resolution. Analysis of percentage cleavage of FB was calculated by densitometry 117 analysis using ImageJ 1.x computer software. Statistical comparisons between WT and 118 R176P FD activity were performed at each concentration, from 4 independent experiments 119 using the Kruskall-Wallis non-parametric t-test. 120 121 Circular dichroism spectroscopy and thermal shift assay 122 WT and R176P catalytically inactive (S183A) proteins were purified by size exclusion 123 chromatography in chloride-free 0.1M sodium phosphate pH 7.0, diluted to a concentration 124 of 2 mg/mL (72.4 µM), and loaded into a 0.1 mm quartz sample cell. Circular dichroism 125 spectra were recorded at 20°C on a Jasco J-810 spectropolarimeter equipped with a Jasco 126 PTC-348WI temperature controller. Spectra were acquired from 190-260 nm with 0.1 nm 127 resolution and 1 nm bandwidth. Final spectra are the sum of 20 scans acquired at 50 128 nm/minute. Thermal shift assay. 2 µg of protein was mixed with SYPRO Orange in PBS 129 M AN US CR IP T AC CE PT ED ACCEPTED MANUSCRIPT Sng et al. Supplementary material 7 with 25mM HEPES and fluorescence data acquired on a ViiA 7 real-time PCR system with 130 thermal denaturation over increasing temperatures observed using 1°C intervals. 131 132 Molecular dynamics (MD) simulation of mutant FD 133 Starting models were derived from crystal structures of S183A FD (PDB ID 2XW9, 1.2 Å 134 resolution) reported previously.E3 The catalytic residue was reverted to serine during the MD 135 setup. CootE4 was used to place the Pro176 side chain in the Arg176 experimental density 136 while minimizing clashes with surrounding atoms and aiming to achieve a favourable initial 137 geometry. The resulting structures were further adjusted in UCSF Chimera.E5 The 138 GROMACS packageE6 was used to set up and run MD simulations. The AMBER99SB-ILDN 139 force fieldE7 and TIP3P water model were used and the structures placed in dodecahedral 140 boxes with 10 Å padding and surrounded with solvent including water and 150 mM NaCl. 141 Following steepest gradient energy minimization, a modified Berendsen thermostat (two 142 groups, time constant 0.1 ps, temperature 310 K) followed by a Berendsen barostat (isotropic, 143 coupling constant 0.5 ps, reference pressure 1 bar) were coupled to the system over 100 ps. 144 100 ns runs of unrestrained MD trajectories were produced. Following removal of periodic 145 boundary condition artefacts, MD runs were visualised and analysed in Chimera and bulk 146 statistics extracted using GROMACS analysis routines. 147 148 Surface plasmon resonance 149 Binding experiments were carried out based on established protocol using a Biacore T200 150 instrument.E8 FB and FD were buffer exchanged by gel filtration into veronal buffer with 151 10mM MgCl2. C3b was immobilised on the CM5 chip by amine coupling to achieve 8000 152 resonance units. A dual injection programme was designed where 0.1 µM or 1 µM FB was 153 injected at a flow rate of 30uL/min for 3 minutes, followed by a second injection of a mix of 154 M AN US CR IP T AC CE PT ED ACCEPTED MANUSCRIPT Sng et al. Supplementary material 8 0.1 µM or 1 µM FB and FD at 30uL/min for 4 minutes. After 5 minutes for dissociation, the 155 chip was regenerated by three 5-minute washes in 40mM acetate + 3M NaCl (pH 5.5). The 156 chip was re-equilibrated in assay buffer for 5 minutes. Catalytically inactive FD (S183A) or 157 double mutant R176P/S183A were used to emulate the binding response of wild-type or 158 R176P respectively while preventing cleavage of FB and subsequent dissociation of the 159 complex. 160 161 Esterolytic activity of FD 162 Z-Lys-SBzl was purchased from Sigma-Aldrich in powder form and reconstituted to 100 mM 163 in 70% DMSO. The assay buffer consisted of 50 mM HEPES (pH7.5), 220mM NaCl and 2 164 mM of Ellman's reagent (5,5-dithio-bis-(2-nitrobenzoic acid) [DTNB]; Sigma-Aldrich). Each 165 reaction mixture contained FD (80nM), variable Z-Lys-SBzl concentrations (0.2-3.2 mM) 166 and 8% v/v of DMSO in a final volume of 200 µl. Solutions were pre-warmed to 37°C before 167 addition of substrate to initiate the reaction. Hydrolysis of Z-lys-SBzl was measured using 168 CLARIOstar FS microplate reader through equimolar formation of chromophore 2-nitro-5-169 thiobenzoate at 405 nm every 30 seconds for 90 minutes (ε = 13,600 M-1cm-1). The rate of 170 hydrolysis was determined from linear slopes of the reaction curves. Reaction velocities, 171 expressed in apparent turnover values were plotted against substrate concentration. 172 M AN US CR IP T AC CE PT ED ACCEPTED MANUSCRIPT Sng et al. Supplementary material 9 Table E1 173 Analyte Proband Sibling Leptin (ng/ml) 10.6 11.3 Adiponectin (µg/ml) 9.7 6.5 NEFA (µmol/L) 391 212 Cholesterol (mmol/L) 4.2 4 HDL (mmol/L) 1.53 1.26 LDL (mmol/L) 2.3 2.3 Triglycerides (mmol/L) 0.8 0.9 HbA1c (mmol/mol) 36 35 174 NEFA, non-esterified fatty acids. HDL, high-density lipoprotein. LDL, low-density 175 lipoprotein. 176 M AN US CR IP T AC CE PT ED ACCEPTED MANUSCRIPT Sng et al. Supplementary material 10 References 177 E1. Lo JC, Ljubicic S, Leibiger B, Kern M, Leibiger IB, Moede T, et al. Adipsin is an 178 adipokine that improves beta cell function in diabetes. Cell 2014; 158:41-53. 179 E2. Sprong T, Roos D, Weemaes C, Neeleman C, Geesing CL, Mollnes TE, et al. 180 Deficient alternative complement pathway activation due to factor D deficiency by 2 181 novel mutations in the complement factor D gene in a family with meningococcal 182 infections. Blood 2006; 107:4865-70. 183 E3. Forneris F, Ricklin D, Wu J, Tzekou A, Wallace RS, Lambris JD, et al. Structures of 184 C3b in complex with factors B and D give insight into complement convertase 185 formation. Science 2010; 330:1816-20. 186 E4. Emsley P, Lohkamp B, Scott WG, Cowtan K. Features and development of Coot. 187 Acta Crystallogr D Biol Crystallogr 2010; 66:486-501. 188 E5. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, et al. 189 UCSF Chimera--a visualization system for exploratory research and analysis. J 190 Comput Chem 2004; 25:1605-12. 191 E6. Abraham MJ, Murtola T, Schulz R, Pall S, Smith JC, Hess B, et al. GROMACS: High 192 performance molecular simulations through multi-level parallelism from laptops to 193 supercomputers. SoftwareX 2015; 1-2:19-25. 194 E7. Lindorff-Larsen K, Piana S, Palmo K, Maragakis P, Klepeis JL, Dror RO, et al. 195 Improved side-chain torsion potentials for the Amber ff99SB protein force field. 196 Proteins 2010; 78:1950-8. 197 E8. Katschke KJ, Jr., Wu P, Ganesan R, Kelley RF, Mathieu MA, Hass PE, et al. 198 Inhibiting alternative pathway complement activation by targeting the factor D 199 exosite. J Biol Chem 2012; 287:12886-92. 200 M AN US CR IP T AC CE PT ED ACCEPTED MANUSCRIPT Sng et al. Supplementary material 11 E9. Lek M, Karczewski KJ, Minikel EV, Samocha KE, Banks E, Fennell T, et al. 201 Analysis of protein-coding genetic variation in 60,706 humans. Nature 2016; 202 536:285-91. 203 E10. Fredrikson GN, Westberg J, Kuijper EJ, Tijssen CC, Sjoholm AG, Uhlen M, et al. 204 Molecular characterization of properdin deficiency type III: dysfunction produced by 205 a single point mutation in exon 9 of the structural gene causing a tyrosine to aspartic 206 acid interchange. J Immunol 1996; 157:3666-71. 207 208 M AN US CR IP T AC CE PT ED ACCEPTED MANUSCRIPT Sng et al. Supplementary material 12 Figure legends 209 Figure E1: Mutation R176P results in a type III FD deficiency. 210 (A) Chromatograms for the DNA sequence adjacent to position c.602 are shown for each 211 member of the pedigree. The identified variant is rare: the EXAC database reports mutation 212 R176P (variant 19:861943 G/C) at an allele frequency of 1.049x10-4, with no homozygotes.E9 213 (B) Western blot analysis of FD in serum from the patient and healthy control. 214 (C) Secondary structural compositions of WT and R176P FD were evaluated using circular 215 dichroism spectroscopy. 216 (D) Comparison of in vitro catalytic activity of recombinant WT, R176P, R176Q and R176A 217 FD in terms of FB cleavage. (***, p<.001; ****, p<.0001). 218 (E) Recombinant WT and R176P FD were tested for the ability to reconstitute alternative 219 pathway haemolytic activity when added to FD-depleted serum. 220 221 Figure E2: Mutation R176P stabilises the self-inhibited state of FD. 222 (A) Structure of free FDE3 (PDB ID: 2XW9) showing the catalytic triad (Ser183-His41-223 Asp89) in an inactive conformation stabilised by the self-inhibitory loop 199-202 (red) and 224 an ion bridge between Asp177 and Agr202. The exosite loops are shown in yellow. 225 (B) Structure of C3bB-bound FDE3 (PDB ID: 2XWB) omitting the C3b and FB components. 226 FD exosite loops retain a conformation similar to that of unbound FD. 227 (C) WT, R176P and R176A structures were stable over 100 ns of unrestrained molecular 228 dynamics simulation with explicit solvent. RMSD, root mean square deviation. 229 (D) Root mean square fluctuation (RMSF) in WT and mutant FD over the second half of the 230 trajectory. 231 M AN US CR IP T AC CE PT ED ACCEPTED MANUSCRIPT Sng et al. Supplementary material 13 (E) Differences in WT versus R176 RMSF mapped to the FD structured. MD predicted 232 increased mobility in exosite loops, notably 155-167, and decreased mobility in loops 233 carrying the catalytic His41 and Asp89 residues. 234 235 Fig E3: Assessment of glucose tolerance in patients with functional FD deficiency. 236 Patient and sibling were given 75g of oral glucose at 0 minutes and blood glucose was 237 measured at regular intervals between 0 - 120 minutes. The error bars indicate the range of 238 plasma glucose concentrations between the patient and sibling. 239 M AN US CR IP T AC CE PT ED ACCEPTED MANUSCRIPT Control I-1 I-2 II-1 II-2 CC 25kDa 20kDa Control Patient Blank WT R176P 180 200 220 240 260 280 -30 -20 -10 0 10 Wavelength (nm) El ip tic ity (m de g) 0.01 0.1 1 10 0 50 100 WT R176P R176Q R176A **** **** *** *** p < 0.001 **** p < 0.0001FB c le av ag e (% ) Factor D [µM] 2.5 wild-type R176P 1.0 0.2 0.04FD [μM] A B C D E M AN US CR IP T AC CE PT ED ACCEPTED MANUSCRIPTA B C D Free FD (Inactive) C3bB-bound FD (Active) Arg176 Ser183 Asp89 His41 Arg202 Asp177 132-135 173-176 199-202 Inhibitory Loop 203-209 155-159 Exosite E Ser183 His41 R176P Less mobile in R176P More mobile in R176P 1 Å -1 Å WT - R176P Cα RMSF 0 1 2 3 0 25 50 75 100 125 150 175 200 225 WT R176P R176A Cα Fluctuation 50 - 100 ns C α R M S F (Å ) Residue Number 0 0.5 1 1.5 2 2.5 0 20 40 60 80 100 WT R176P R176A FD Backbone RM SD (Å ) Time (ns) M AN US CR IP T AC CE PT ED ACCEPTED MANUSCRIPT 0 20 40 60 80 100 120 0 2 4 6 8 10 0 200 400 600 800 Time (min) G lu co se (m m ol /L ) Insulin (pm ol/L) Glucose (mmol/L) Insulin (pmol/L)