Complement is activated by elevated IgG3 hexameric platforms and deposits C4b onto distinct antibody domains

Abstract

IgG3 is unique among the IgG subclasses due to its extended hinge, allotypic diversity and enhanced effector functions, including highly efficient pathogen neutralisation and complement activation. It is also underrepresented as an immunotherapeutic candidate, partly due to a lack of structural information. Here, we use cryoEM to solve structures of antigen-bound IgG3 alone and in complex with complement components. These structures reveal a propensity for IgG3-Fab clustering, which is possible due to the IgG3-specific flexible upper hinge region and may maximise pathogen neutralisation by forming high-density antibody arrays. IgG3 forms elevated hexameric Fc platforms that extend above the protein corona to maximise binding to receptors and the complement C1 complex, which here adopts a unique protease conformation that may precede C1 activation. Mass spectrometry reveals that C1 deposits C4b directly onto specific IgG3 residues proximal to the Fab domains. Structural analysis shows this to be caused by the height of the C1-IgG3 complex. Together, these data provide structural insights into the role of the unique IgG3 extended hinge, which will aid the development and design of upcoming immunotherapeutics based on IgG3.

Fig. 1. Characterisation of antigen-bound IgG1 and IgG3.

a, b Slices 10 nm thick through tomograms of antigen-bound IgG1 (a) and IgG3 (b). Magnified regions (bottom) with membrane-to-Fc platform measurements. Scale bars represent 100 nm (top) and 50 nm (bottom). c Slice 5 nm thick through a subtomogram average of the ordered Fab domains viewed from the side. Two Fab domains are coloured violet and green, and membrane denoted as cyan. Repeating distance of 3.8 nm and 7 nm height of striations are shown. d Possible model of the Fab array from the same orientation as (b and c). Blue and yellow represent the heavy chain and the light chain of IgG3 Fab domains, respectively. e Slice 5 nm thick through a subtomogram average of aligned Fab domains observed at the apex of liposomes (Supplementary Fig. 5c, d). Two Fab domains are coloured violet and green, and repeating distances of 3.8 nm and 5.25 nm shown. f Possible model of the Fab array from the same orientation as (e), effectively rotated 90° from (b and c). Heavy and light chains are blue and yellow, respectively.

Fig. 2. A model of antigen-bound hexameric IgG3.

a Hexameric IgG3-Fc platform C6-symmetric map (grey) and model (coloured ribbons). b Complete model of IgG3 hexamer viewed from three different directions. Light chains are grey, each heavy chain pair is a separate colour. c Proposed IgG1 hexamer showing monovalent Fab binding. The second Fab is oriented away from the surface (arrows). d Proposed IgG3 hexameric models; the longer upper hinge region allows either monovalent (left) or divalent (middle) antigen binding. Divalent Fabs associate (middle) and form an array, causing the Fc platform to become elevated (right). For (c and d), the heavy chain is blue and the light chain is yellow.

Fig. 3. Structures of IgG3-C1-C4b on antigenic liposomes.

a, b C1 complexes (black arrowheads) on IgG1-coated liposomes (a) and IgG3-coated liposomes (b). Scale bars represent 100 nm (top) and 50 nm (bottom). c, d Denoised tomographic slices 10 nm thick of opsonized liposomes coated with IgG1 (c) and IgG3 (d) (top). Schematic diagrams (bottom) show the location of antibody platforms (blue lines) on the liposomes (grey). Pink and green represent 12 nm and 24 nm-thick heights of the protein corona. Scale bars represent 100 nm. e, f Subtomogram map (left) and fitted model (right) of the complete IgG3-C1-C4b complex. C1q, blue; C1r, purple; C1s, pink; C4b, orange; IgG3, green; liposome, grey. g Focussed refinement of the C1 complex showing empty density near the Fc domain (white arrow) and adjacent to one of the C1r SP domains (black arrowhead). h Model built into the subtomogram map showing the C1s arm adopting a conformation close to the C1r SP domain, annotated with measured distances.

Fig. 4. CryoEM and MS characterisation of C4b binding to IgG3.

a Density corresponding to C4b (orange) reveals a tilted molecule bound to IgG3 Fab domains (green). gC1q are displayed in blue, C1s is displayed in pink and the liposome surface is displayed in grey. b Example of a Coomassie-stained gel showing purified IgG3 (green arrow), C4b (orange arrow), and mixtures with liposomes (Lipos) and human serum (NHS). A new band appeared upon mixture of all required components (blue arrow). Gel was repeated three times. c Model of C4b bound to IgG3 showing sites for covalent attachment as determined using MS. Sites of attachment are indicated with the following colours: STSGGTAALGCLVK, pink; SCDTPPPCPR, cyan; VSNKALPAPIEK, red. Residues identified by MS that attach to C4b are denoted as spheres. The C4b-TED reactive glutamine residue is shown as a blue sphere. d MS analysis of tryptic peptides of C4b (GCGEQTmIYLAPTLAASR, orange; m, oxidised methionine) crosslinked to IgG1 (upper, red) and IgG3 (lower, green). For both samples, extracted ion chromatograms show traces for the C4b peptide (orange) attached to VSNKALPAPIEK (32 min). Only IgG3 has C4b conjugated to other sites. Crosslinked residues are denoted in bold and underlined in the colour corresponding to (c).

Fig. 5. Functional consequences of ordered IgG3-Fab and elevated IgG3-Fc domains.

a Neutralisation by IgG1 (left, red) and IgG3 (right, green). IgG3 can pack much closer together than IgG1 by forming a Fab array and cover more of the pathogen surface (blue), potentially enhancing pathogen neutralisation. b The higher IgG3 (green) platform is more exposed than IgG1 (red) on opsonized membranes, allowing greater FcγR access. c Height comparisons of various complement components with IgG3 and IgG1 on a surface. C4, orange (PDB code 4XAM); C3, cyan (PDB code 2I07); IgG1, red; IgG3, green; MAC, purple (PDB code 6H03).

Fig. 6. Insights into C1s activation and C4b deposition by IgG3-C1 complexes.

a Cross activation by C1 complexes. The C1r arm (blue) of one complex can react with the C1r’ arm (blue) of the adjacent complex. The conformation of C1s (pink) on IgG3 platforms also enables activation by the C1r’ protease (purple) of an adjacent complex. b Crystal structures showing the large, 9 nm, rearrangement of domains after cleavage of C4 (PDB code 5JPM) to form C4b (PDB code 4XAM). Domains are coloured as follows: α-chain, red; β chain, orange; γ chain, yellow. c The direct route from C4 to C4b (blue arrow) would orient the reactive thioester (blue dot) towards the β chain, potentially enhancing intramolecular conjugation (blue cross), and formation of an inactive C4b molecule. d Instead, if an indirect route is taken, the thioester would first be oriented away from the rest of C4b (blue arrow), before formation of the final C4b structure. This would also orient the thioester towards IgG3-Fc, resulting in crosslinking to lysine K326 (blue cross), as seen by MS.