Fig 1.
Mouse IgG2c Fc is comparable to other IgGs but shows differences in crucial features.
A. A cartoon model of mouse IgG2c Fc solved by x-ray crystallography. Domain and secondary structure element labels (B.) are noted. C. An overlay of two mouse IgG2c Fc models with different N-glycan composition. D. A cartoon schematic showing the glycoforms studied here, individual carbohydrate residues are indicated by colored shapes according to the SNFG system [2]. The colors of individual Fcs will be used as indicated throughout the text to denote sequence and glycan variants. E. Sequence and secondary structure arrangement of the Fc Cγ2 domains.
Fig 2.
2d HSQC spectra of the Fc N-glycan (1)GlcNAc 1H1-13C1 reveals orthologous Fcs provide different N-glycan environments.
The spectra of human IgG1 Fc were previously reported [20].
Fig 3.
Mouse IgG2c Fc and 2b Fc show a high degree of similarity with a complex-type N-glycan, however, differences in loop residues impact conformation.
A. Ribbon diagram of the mIgG2c Fc model shows clear Van der Waals contacts between Y296 and (0)Fuc. B. The ribbon diagram for mIgG2b Fc shows a different organization of the DE loop. C. A comparison of the 2b Fc and 2c Fc models shows the effect of residue 300 in DE loop conformation and D. sequence differences in the FG loop.
Table 1.
Binding of mIgG2c Fc and mIgG2b Fc to mFcγRIV measured with surface plasmon resonance.
Table 2.
Summary of crystallographic data.
Fig 4.
Truncating the mIgG2c Fc N-glycan alters the DE loop conformation.
A-B) Comparison of mIgG2c Fc models with a complex-type N-glycan (orange ribbon) with a truncated (1)GlcNAc N-glycan (green ribbon). C-D) Electron density of the DE loops show significant displacements in the loop conformation (contoured to 1.5 σ). E-F) Electron density of the N-glycans show the usual contacts in the case of full length glycan, and a 180° rotation of the (1)GlcNAc residue in the truncated glycoform (contoured to 1.5 σ).
Fig 5.
1H-13C HSQCs of anomeric (1)GlcNAc correlations from mouse IgG2b and mouse IgG2c Fc show near interconversion with amino acid substitution.
Fig 6.
Mouse IgG2c and hIgG1 Fc show different strand and loop interactions.
A. Mouse and human Fc fragments show a high degree of global similarity with an rmsd of 0.7 Å. B. P291 of hIgG1 disrupts a beta sheet formation in the C' strand, and shifts the register by one residue that is relieved by a bulge at mIgG2c D295. Differences and similarities are identified in residues that mediate loop interactions in mIgG2c Fc (C) and hIgG1 Fc (D).