Figures and data
Structure of the complex between ISG65 and human C3b
a) Composite volume of locally refined regions determined using cryogenic electron microscopy for ISG65 bound to human C3b. ISG65 is coloured in different shades of blue and green, as indicated in the legend in the centre of the panel (loop1 and helix 2 are light blue, loop 2 and helix 3 are green and loop3 and helix 4 are dark blue). C3b is coloured in grey scale with the α-chain in light grey and the β-chain in dark grey. The TED domain is highlighted in orange and the CUB domain highlighted in yellow. b) Molecular model of the same complex with a colour scheme matching that of a). c) A schematic showing the features of ISG65, coloured as a). Regions resolved in the structure are indicated underneath the schematic using a green line and regions predicted to be disordered using AUCpreD45 are shown by the red line.
ISG65 forms two distinct interfaces with the TED and CUB domains of C3b
a) The ISG65-C3b model shown in transparent cryo-EM density. The top panel shows the interface between ISG65 and the TED domain (orange), with bottom panel showing the interface between loop L2 of ISG65 (green) and the CUB domain of C3b (yellow). In each case, the left-hand panel shows the intact structure, with a dotted box highlighting the region shown in an enlarged form in the right-hand panel. b) The ISG65 model superimposed onto a previously determined structure of C3 (PDB ID: 2A73)28 via the TED domain of the ISG65-C3b model. This is shown as a ribbon within a transparent surface representation. ISG65 can bind to C3 via the TED domain, via the same interface as previously identified for ISG65-C3d4. c) Surface plasmon resonance data showing responses from the injection of human serum (two-fold dilutions from a concentration of 8 mg/mL), C3 (two-fold dilutions from a concentration of 625 nM) and C3b (two-fold dilutions from a concentration of 500 nM, data previously shown4) over a flow cell conjugated with ISG65.
ISG65 does not block the formation of the C3 convertase
a) The structure of the ISG65-C3b complex docked into the electron microscopy-derived volumes obtained for the ISG65-C3b complex (left) and C3b alone (right). b) Composite models obtained by docking the C3b-ISG65 structure onto those of C3b bound to factors B and D (PDB ID: 2XWJ)29 or factor Bb (6RUR)10. c) An assay for C3 convertase formation in which C3b and factor D were each added at concentrations of 12 nM and C3 and factor B at concentrations of 600 nM. Samples were taken at different time points and were analysed by SDS-PAGE analysis with Coomassie straining. This was done in the absence (left-hand gel) and presence (right-hand gel) of 2 µM ISG65. The graphs show quantification by densitometry for factors B, Ba and C3a to assess convertase function. d) An equivalent assay to that shown in c), conducted in the absence of non-complement protein (left), or the presence of 2 µM ISG65 (central) or 2 µM BSA (right). The left-hand gel was run in non-reducing conditions while the right-hand gel was run in reducing conditions. e). An equivalent assay to that shown in c), conducted in presence of 2 µM ISG65 or of ISG65 variants lacking loop 1 (ΔL1), loop 2 (ΔL1), loop 3 (ΔL1) or the extended disordered C-terminal region (ΔC).
ISG65 overlaps the binding sites for complement receptors 2 and 3
Composite models obtained by docking the C3b-ISG65 structure onto those of C3b/d bound to factor H CCP19-20 (3OXU)31, CRIg (2ICF)32, CR1 CCP15-17 (5FO9)33, CR2 SCR1-2 (3OED)34 and CR3 I-domain (4M76)35. C3b/d is shown in a solid light grey surface, ISG65 is shown in a solid turquoise surface, and complement regulators are shown in transparent surface with ribbon in various colours.
Workflow of Cryo-EM data processing
Cryo-EM data collection and model building statistics.
