Revisiting the role of glycosylation in the structure of human IgG Fc

Abstract

Binding of the Fc domain of Immunoglobulin G (IgG) to Fcγ receptors on leukocytes can initiate a series of signaling events resulting in antibody-dependent cell-mediated cytotoxicity (ADCC) and other important immune responses. Fc domains lacking glycosylation at N297 have greatly diminished Fcγ receptor binding and lack the ability to initiate a robust ADCC response. Earlier structural studies of Fc domains with either full length or truncated N297 glycans led to the proposal that these glycans can stabilize an "open" Fc conformation recognized by Fcγ receptors. We determined the structure of an E. coli expressed, aglycosylated human Fc domain at 3.1 Å resolution and observed significant disorder in the C'E loop, a region critical for Fcγ receptor binding, as well as a decrease in distance between the C(H)2 domains relative to glycosylated Fc structures. However, comparison of the aglycosylated human Fc structure with enzymatically deglycosylated Fc structures revealed large differences in the relative orientations and distances between C(H)2 domains. To provide a better appreciation of the physiologically relevant conformation of the Fc domain in solution, we determined Radii of Gyration (R(g)) by small-angle X-ray scattering (SAXS) and found that the aglycosylated Fc displays a larger R(g) than glycosylated Fc, suggesting a more open C(H)2 orientation under these conditions. Moreover, the R(g) of aglycosylated Fc was reduced by mutations at the C(H)2-C(H)3 interface (E382V/M428I), which confer highly selective binding to FcγRI and novel biological activities.

Figure 1

Overall structure of aglycosylated IgG Fc domain solved at 3.1 Å resolution. The range of B-factors of all Cαs are represented by rainbow-colors ranging from blue (low) to red (high). Two homodimers A,B and C,D are present in the asymmetric unit. The location of the C′E loop is noted in the C,D dimer.

Figure 2

Comparison of C′E loops of glycosylated and aglycosylated C_H2 domains. (a) The A chain C_H2 domain of the glycosylated Fc 3AVE (grey) is aligned with the A chain of our aglycosylated C_H2 domain (3S7G, red). Differences in the C′E loop orientation can be seen in the ribbon diagram. Y296, N297 and S298 orientations at the apex of the C′E loop differ significantly between glycosylated and aglycosylated C_H2 domains. (b) The C′E loops of glycosylated 3AVE (grey) are shown aligned with three previously solved C_H2 domains lacking glycosylation, 2DJ9 (yellow), 3DNK (blue), 3S7G A chain (red) and 3S7G B chain (green). Y296 side chain orientations differ significantly from the glycosylated structure and each other.

Figure 3

Comparison of overall opening/closing of C_H2 domains among glycosylated and deglycosylated structures. (a) Glycosylated Fc 3AVE (grey) is compared with the A,B dimer of aglycosylated 3S7G (red). Pro 329 distances (measured from Cαs) are shown as dotted lines. P329 distances for 3AVE and 3S7G are 25.1 Å and 18.5 Å respectively. E382 and M428 are represented as sticks (cyan) to highlight the location of the Fc5 mutation (b) The A,B dimer of aglycosylated 3S7G (red) is compared with deglycosylated human Fc, 3DNK (blue) and the deglycosylated mouse Fc, 3HKF (green). P329 distances for 3DNK and 3HKF are 27.6 Å and 11.6 Å respectively.

Figure 4

SAXS analysis of glycosylated and aglycosylated Fcs. (a) Scattering profiles from 0.5 second exposure are shown for glycosylated Fc (yellow) aglycosylated Fc (red) and aglycosylated Fc5 (blue). These profiles were obtained by extrapolating curves at different concentrations to the zero concentration. The logarithm of scattering intensity is shown as a function of the reciprocal vector (S). (b) The distance distribution functions (P(r)) of glycosylated Fc (yellow) aglcyosylated Fc (red) and aglycosylated Fc5 (blue) are shown. Curves were calculated from the SAXS scattering data in (a).