Nature Structural & Molecular Biology: doi: /nsmb Supplementary Figure 1

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1 Supplementary Figure 1 Model quality of C3b-miniFH-FI and C3b-miniFH crystal structures and overall structure comparisons. (a) Overall 2DF o -mf c electron density contoured at 1.0 for C3b-miniFH-FI (left) and C3b-miniFH (right). (b) Average B-factor and realspace correlation coefficient for and chain of C3b, minifh and FI for the two independent copies of the complex in the crystal. FI copy 1 exhibits higher real-space correlation and lower B-factor. (b) Missing electron densities for the poly-glycine linker between CCP4 and CCP19 of minifh in both copies C3b-miniFH-FI and C3b-miniFH structures. The distances between minifh CCP4 C-terminal and CCP19 N-terminal in the two copies of C3b-miniFH-FI and C3b-miniFH are Å. (d) Left-hand side: structural superposition of C3bminiFH-FI (grey and orange) with C3b-FH CCP1-4 (pdb: 2WII) (Wu, J. et al. Nature Immunology 10, , 2009) (blue and purple) and C3b-miniFH (cyan and black) shows similar binding of FH CCP1-4 to C3b. Right-hand side: structural superposition of TED domains in C3b-miniFH-FI (grey and orange) with C3b-miniFH (cyan and black) and C3d-FH CCP19-20 (pdb: 3OXU; yellow and green) (Morgan. H. P. et al. Nature SMB 18, , 2011) shows similar binding of FH CCP19-20 binding to C3b/C3d TED domains. (e) Cartoon representations of the main chains in the structure of free FI (purple) and of FI as observed in C3b-miniFH-FI (green); left side. Several loops could not be modeled in free FI due to disorder (Roversi, P. et al. PNAS 108, , 2011). In the right panel, electron densities (contoured at 1.0 level) for these loops and corresponding parts of the model as observed in C3b-miniFH-FI are shown. (f) Structural superposition of C3b CTC domains shows that the core of the CTC domain behaves as a rigid body. In both C3bminiFH (cyan) and C3b-miniFH-FI (grey) complex, CTC exhibits a -turn- configuration of the neck region, whereas in the C3b-FH CCP1-4 complex (blue) CTC exhibits a -helix in this region. Compared to C3b-miniFH-FI (grey) structure, the CTC domain in C3bminiFH (cyan) displays a 34 rotation due to a twisting of a hinge loop formed by residues E1515-K1526 (highlighted in purple). A simplified diagram in the right side illustrates this feature.

2 Supplementary Figure 2 Disease-related mutants at protein interfaces in C3b-FH-FI. (a) Cartoon representation of C3b-miniFH-FI showing disease-related mutation V1658A in C3b (indicated in sticks) located in the middle of the FI-binding site (with colors as defined in Fig. 1). (b) Structures of C3b-Bb-SCIN (pdb code: 2WIN) (Rooijakkers, S. H. M. et al. Nature Immunology 10, , 2009), C3b-FB (2XWJ) (Forneris, F. et al. Science 330, , 2010) and C3b-FB-FD (2XWB) (Forneris, F. et al. Science 330, , 2010) indicating that C3b V1658 is outside of FB-binding interface. C3b is shown in grey cartoon with V1658 shown in stick representation; FB and Bb are shown in cyan with either Mg 2+ or Ni 2+ occupying the metal-ion dependent adhesion site (MIDAS) position, as indicated by a sphere. (c) Disease-related residues variations on interaction interfaces of C3b-FI and minifh-fi. Red boxes indicate loss-of-function variations and the cyan box indicates a gain-of-function, suggesting that it is perhaps not involved in ahus (Nilsson, S. C. et al. Eur. J. Immunol. 39, , 2009). (c) Disease-related mutations on FH at the FH-FI interface. Residues on CCP1 possibly have an indirect effect on FI binding through their interactions with CCP2, since NMR chemical shift of the loop between Cys129 and Cys141 in FH CCP2 (indicated in blue) (Hocking, H. G. et al. JBC 283, , 2008).

3 Supplementary Figure 3 Stabilization of the FI SP domain (a) Cartoon representation of free FI (Roversi, P. et al. PNAS 108, , 2011) (cyan and purple for heavy and light chain resp.) superposed on FI in the C3b-miniFH-FI complex (blue and green for heavy and light chain resp.). The relative orientation of heavy and light chains differs by 11 between free FI and FI in the C3b-miniFH-FI complex. Residues at the heavy-light chain interface are shown in stick representation. (b) FH CCP 2-3 contact with loop , loop , loop and loop of FI SP domain. The contact regions in these loops are highlighted in purple. (c) Superposition FI SP with thrombin (Huntington, J. A. et al. Structure 11, , 2003; Lechtenberg, B. C. et al. PNAS 108, , 2011) (pdb: 1JOU) shows FI loop and loop correspond to thrombin 37 loop (exosite I) and 70 loop (exosite I) resp. and FI loop corresponds to thrombin loop.

4 Supplementary Figure 4 Flexibility of the scissile loops in C3b CUB. (a) Superposition of C3b CUB domains of C3b-miniFH-FI (cyan) and other C3b structures (Forneris, F. et al. EMBO J 10, , 2016) (colored in grey), C3b (pdb code: 5FO7), C3b-FH CCP1-4 (2WII), C3b-CR1 CCP15-17 (5FOB), C3b-MCP CCP1-4 (5FO8), C3b- DAF CCP2-4 (5FOA) and C3b-SPICE CCP1-4 (5FO9), shows conformational rearrangements in the first scissile loop. The first scissile bond Arg Ser1304 is indicated by sticks. (b) B-factor putty representations of the CUB domains with scissile bonds Arg1303- Ser1304, Arg1320-Ser1321 and Arg954-Glu955 are colored in yellow, red and blue resp., indicating increased flexibility for the loops containing the first and third scissile bonds in C3b prior to FI binding.

5 Supplementary Figure 5 Sequence and structure comparisons of DAF with cofactor regulators. (a) Structure-based sequence alignment of FH (CCP2-3), CR1 (CCP16-17), CR1 (CCP9-10), MCP (CCP2-3) and DAF (CCP3-4). Cysteines in CCP domains are indicated in bold characters. Residues of FH at the FI-binding interface indicated by red background; with, FI-interacting residues conserved among cofactor regulators indicated by red boxes. Red arrows indicate conserved FI-interacting residues not present in DAF. Residues buried in C3b-binding interfaces are indicted by blue background. (b) Overlay of FH CCP2-3 (orange) with DAF CCP3-4 (magenta). Interface residues that differ markedly between FH and DAF are shown in sticks. (c) Structural superposition of the complex of C3b-FH, C3b-CR1, C3b-MCP, C3b-SPICE and C3b-DAF on CCP ii and CCP iii. The orientation of the CUB domain in C3b-DAF crystal structure differs from the CUB position in the C3b complexes with regulators. Zoom-in on the righthand panel shows that CUB with regulator fragments positions the first scissile loop close to the catalytic center of FI SP, which is not the case for the arrangement observed in C3b-DAF.

6 Supplementary Figure 6 Charge variations in cofactors at FI interface. (a) Superposition of C3b-miniFH-FI with C3b-CR1 CCP15-17 (pdb: 5FOB) (Forneris, F. et al. EMBO J 10, , 2016), C3b-MCP CCP 1-4 (pdb: 5FO8) (Forneris, F. et al. EMBO J 10, , 2016), and C3b-SPICE CCP1-4 (pdb: 5FO9) (Forneris, F. et al. EMBO J 10, , 2016) shows FH contains an extra residue, i.e. E243 (highlighted by a purple box in sequence alignment), and a shorter loop between CCP2 Cys129 and Cys141 (stretch of missing residues highlighted by a cyan box in structure-based sequence alignment; with FH residue numbers indicated). FH D137 pointed to FI loop , which is rich in charge residues (indicated by sticks). At the corresponding position of FH D137, CR1, MCP and SPICE have a conserved lysine (indicated by a red box in sequence alignment). (b) Both the loop between CCP2 Cys129 and Cys141 of FH (indicated by red arrows) and its close by stretch loop of FI SP domain display high B-factors. (c) Superposition of SPICE CCP2 and CCP3 (pdb: 5FO9; grey) (Forneris, F. et al. EMBO J 10, , 2016) on FH CCP2 and CCP3 (orange) of C3b-miniFH-FI. Critical residues for SPICE cofactor activity are shown as sticks (Yadav, V. N. et al. Journal of Virology 82, , 2008). Y98 of SPICE docks into the hydrophobic pocket in FI, formed by W393, P402, L404 and I409. K107 and K120 are close to FI loop consisting of a series of positive and negative charge residues ( 435 xkkdxxkkdxexxr 448 ). Y103 of SPICE points away from FI-SP domain.

7 Supplementary Figure 7 Negative-stain EM and SAXS analysis of C3b, ic3b1 and ic3b. (a) C3b was incubated with VCP and FI for 2 hours at 37 C. Shown is chromatogram after separation on a monoq 15/50GL column. (b) SDS-PAGE of corresponding fractions. The red box indicates the sample fractions of ic3b1 used for EM and SAXS analyses. (c) Raw electron micrographs of purified C3b, ic3b1 and ic3b samples. (d) SAXS modeling. Ab-initio models of C3b (red), ic3b1 (green) and ic3b (blue). ic3b1 resembles C3b, whereas ic3b shows an elongated CUB-TED.

8 Supplementary Figure 8 Cofactor activities and relative binding affinities of regulator fragments. (a) SDS gel showing cleavage products from long-term cofactor assay (8-hours incubation at 37 C) showing FI with either minifh or FH CCP1-3 yield C3dg/C3d and C3c fragments corresponding to three cleavages, whereas FI with MCP only yields ic3b corresponding to two cleavages. (b) The left three columns show the processed SPR sensorgrams of the recombinant regulator segments (marked on the far left) to C3b (green), ic3b (blue), and C3c (red). C3 fragments were covalently coupled to individual sensor chip surfaces using amine coupling in a non-oriented manner. Regulators were injected for 60 s in a concentration range of 10 nm to 20 µm. Steady state responses were plotted against the regulator concentration, and all analyses were globally fit across all surfaces to a single binding site model (right column; shown as percentage of maximum binding capacity) to determine an apparent affinity value (K D app); R 2 values of the fits were all better than Please note that detailed quantitative interaction analyses of regulator molecules binding to C3b have previously been conducted by our group; these were based on oriented surface coupling of C3b via its thioester moiety in a close-to-physiological manner (Forneris, F. et al. EMBO J 10, , 2016). For the present study, and due to the absence of a thioester domain in C3c, all C3 fragments were immobilized in random orientation, leading to generally weaker affinity values that cannot be directly compared with previous reports regarding absolute numbers (hence the classification as apparent K D ). However, the immobilization of the three C3 fragments at closely matched surface density and using the same coupling method, and the simultaneous injection of regulators across all three surfaces ensures the qualitative/semi-quantitative assessment of relative binding activities, which was the main purpose of this experiment. Abbreviations: R max, maximum binding response; RU, resonance units.

9 Supplementary Figure 9 Disease-related mutants in FI. Classification of disease-related variations in FI (as listed in Supplement Table 1). Residues located in C3b-FI and FH-FI binding interfaces are indicated by purple spheres; mutations causing lower FI expression level in vivo indicated by blue spheres, mutations causing FI misfolding cyan, and mutations on the surface, but not at the binding interfaces, grey.

10 Supplementary Figure 10 Purification of FI. (a)western blot by anti-his antibody shows that purified recombinant human FI (with N-terminal His 6 tag) stored at 4 ºC is degraded slowly. (b) Chromatogram of gel filtration by Superdex /300 GL column. Sample fractions for crystallization are highlighted by red background.

11 Supplemental Notes and Table to: Regulator-dependent mechanisms of C3b processing by factor I allow for differentiation of immune responses Xiaoguang Xue 1, Jin Wu 1, Daniel Ricklin 2, Federico Forneris 1, Patrizia Di Crescenzio 2, Christoph Q. Schmidt 2,4, Joke Granneman 1, Thomas H. Sharp 3, John D. Lambris 2, Piet Gros Crystal and Structural Chemistry, Bijvoet Center for Biomolecular Research, Dept. Chemistry, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands Department of Pathology & Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA Section Electron Microscopy, Department of Molecular Cell Biology, Leiden University Medical Center, 2300 RC Leiden, the Netherlands Institute of Pharmacology of Natural Products and Clinical Pharmacology, Ulm University, Ulm, Germany Current addresses: FF: The Armenise-Harvard Laboratory of Structural Biology, Dept. Biology and Biotechnology, University of Pavia, Via Ferrata 9/A, Pavia, Italy, DR: Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland. Corresponding author: PG, p.gros@uu.nl ORCID: XG: FF: DR: PDC: THS: JDL: PG: Keywords: complement opsonins, regulators, cofactor activity, proteolysis, immune response

12 Mapping mutations in FI onto C3b-FH-FI A surprisingly large number of disease-related variations have been identified for FI 1-7. Supplemental Table 1 lists 89 identified mutations for a total of 525 residues that constitute FI (for references see Table 1). Mutations map to both the interior and the surface of FI (Supplemental Fig. 9). Seven mutations are known to reduce expression levels of FI in vivo. Thirty mutations with normal expression level concern hydrophobic residues and cysteines that upon mutation putatively destabilize the protein fold. Of the surface-exposed mutations, only nine map onto the binding interfaces of FI with C3b or FH. The remaining group of 43 residues concerns surface-exposed residues that are not located at a binding interface in C3b-miniFH-FI. Possibly, other interacting partners of FI, such as von Willebrand factor 8, may explain some of these surface mutations, which would imply that additional protein partners are involved in AMD or ahus. Alternatively, the high number of mutations is possibly related to the mechanism of induced activation of FI upon C3b-regulator binding, because mutations (either in the SP domain or the domains of the heavy chain) might hamper the direct or indirect stabilization of the SP domain that is required for its proteolytic activity. Supplemental Table 1 (next page). Disease related mutations in human FI. AMD and ahus related mutations are taken from 1-7. Mutations are grouped into separate categories. First, those reported to yield lower expression levels of FI. Second, those located at the interaction interfaces between FI heavy chain (FIM, SRCR, LA1, LA2, LNK) and light chain (SP) or between FI and C3b or FH. Third, residues buried within FI heavy chain and FI light chain, implicating potential folding problems upon mutation. Fourth, mutated residues exposed on the surface of FI, but not involved in known interaction interfaces.

13 Lower expression level Interaction interface Buried residues Surface exposed (not at know interaction sites) Mutants Domain Disease Mutants Domain Interface Disease Mutants Domain Disease Mutants Domain Disease P83Q FIM ahus P64L FIM S90N FIM ahus P83Q FIM G119R SRCR ahus R389H SP L131R SRCR AMD R406C SP FIM- C3b(CTC) FIM- C3b(CTC) SP- C3b(MG2) SP- FH(CCP2) ahus/amd H40R FIM AMD D27A FIM FI deficiency ahus C43F FIM ahus D44N FIM AMD AMD C54R FIM AMD P50A FIM ahus ahus C89Y FIM ahus T72S FIM ahus G188A SRCR AMD W456L SP SP-FIM ahus H118R SRCR ahus E109A FIM AMD A240G LA1 ahus/amd Y459S SP SP-FIM ahus/amd V127A SRCR AMD I416L SP ahus/amd D519N SP R575S SP SP- C3b(CUB) SP- C3b(CTC) M120I M120V SRCR ahus ahus V152M SRCR ahus/amd G125R SRCR AMD ahus V184M SRCR AMD N151S SRCR ahus R187Q SRCR AMD G162D SRCR AMD F198L SRCR AMD N177I SRCR ahus Y206N SRCR AMD H183R SRCR ahus C229R LA1 AMD R201S SRCR ahus V230M LA1 AMD R202I SRCR AMD C247G LA1 ahus T203I SRCR AMD G328R LNK AMD Q217H LA1 AMD V355M LNK AMD E224N LA1 AMD A356P SP AMD G243D LA1 ahus I357M SP ahus G248E LA1 ahus Y369S SP ahus A258T LA1 AMD W374C SP AMD Q260T LA2 AMD W399R SP ahus/amd G261D LA2 MPGN/AMD H418L SP AMD/FI deficiency G263V LA2 AMD A431T SP ahus G280D LA2 AMD I433T SP ahus G287R LA2 ahus/amd C467R SP AMD I306S LNK ahus G487C SP AMD D310E LNK AMD R502C SP AMD R317W R317Q LNK ahus V543A SP AMD G342E SP ahus W546L SP ahus G349R SP ahus I578T SP AMD G362A SP AMD D403N SP AMD G424D SP ahus Q462H SP AMD R474Q SP AMD D477H SP AMD I492L SP AMD G500R SP AMD G512S SP AMD K522T SP ahus N536K SP AMD E548G SP ahus P553S SP ahus E554V SP ahus

14 References 1. Bienaime, F. et al. Mutations in components of complement influence the outcome of Factor I- associated atypical hemolytic uremic syndrome. Kidney International 77, (2010). 2. Kavanagh, D. et al. Rare genetic variants in the CFI gene are associated with advanced agerelated macular degeneration and commonly result in reduced serum factor I levels. Hum. Mol. Genet. 24, (2015). 3. Fremeaux-Bacchi, V. et al. Complement factor I: a susceptibility gene for atypical haemolytic uraemic syndrome. J Med Genet 41, e84 e84 (2004). 4. Geelen, J. et al. A missense mutation in factor I (IF) predisposes to atypical haemolytic uraemic syndrome. Pediatr. Nephrol. 22, (2007). 5. van de Ven, J. P. H. et al. A functional variant in the CFI gene confers a high risk of age-related macular degeneration. Nature Genetics 45, (2013). 6. Nilsson, S. C. et al. A mutation in factor I that is associated with atypical hemolytic uremic syndrome does not affect the function of factor I in complement regulation. Mol. Immunol. 44, (2007). 7. Nilsson, S. C., Sim, R. B., Lea, S. M., Fremeaux-Bacchi, V. & Blom, A. M. Complement factor I in health and disease. Mol. Immunol. 48, (2011). 8. Feng, S., Liang, X., Kroll, M. H., Chung, D. W. & Afshar-Kharghan, V. von Willebrand factor is a cofactor in complement regulation. Blood 125, (2015).