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 AUCpreD48 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)49 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 ISG65, ISG65ΔL2, and ISG65N188A,H189A,Y190A (two-fold serial dilutions from a concentration of 10 μM) over a flow cell coupled to biotin-C3b or biotin-C3d. Data is representative of three experimental repeats. Raw data available in Figure 2-Source Data 1.xlsx

ISG65 does not block the formation of the C3 convertase

a) The structure of the ISG65-C3b complex (without ISG65) 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)30 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 (ΔL2), loop 3 (ΔL3) or the extended disordered C-terminal region (ΔC-ter). Raw data available Figure 3-Source Data 1.

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)32, CRIg (2ICF)33, CR1 CCP15-17 (5FO9)34, CR2 SCR1-2 (3OED)35 and CR3 I-domain (4M76)36. 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.

Steps highlighted in blue were performed in SIMPLE, steps in green were performed in CryoSPARC, and steps in orange were performed in RELION.

surface plasmon resonance data

Two experimental replicates of the data shown in Figure 2c. Two-fold serial dilutions from a concentration range of 5000 to 312.5 nM. Data shown here is derived from two biological replicates of biotin-C3b, biotin-C3d, and ISG65, and one biological replicate of ISG65ΔL2, and ISG65N188A,H189A,Y190A. Raw data available in Figure 2-Figure Supplement 1-Source Data 1.xlsx and Figure 2-Figure Supplement 1-Source Data 2.xlsx.

Comparison of ISG65 sequences from T. brucei brucei (green labels), T. brucei rhodesiense (blue labels), and T. brucei gambiense (red labels).

Interacting residues from interface 1 are highlighted by green circles, and those from interface 2 by red circles. Raw data available in Figure 4-Figure Supplement 1-Source Data.

Cryo-EM data collection and model building statistics.

Kinetic parameters of ISG65 variants binding to biotinylated C3b and C3d, as measured by surface plasmon resonance.

Average values and standard deviation from three experimental repeats are reported.

Mass spectrometry analysis of ISG65-C3b conjugates observed in Figure 3.

The high molecular weight band observed in convertase assay in the presence of ISG65 was excised and analysed by mass spectrometry. The three most abundant proteins identified are presented.