A single amino acid distorts the Fc γ receptor IIIb/CD16b structure upon binding immunoglobulin G1 and reduces affinity relative to CD16a

Abstract

Therapeutic mAbs engage Fc γ receptor III (CD16) to elicit a protective cell-mediated response and destroy the target tissue. Newer drugs designed to bind CD16a with increased affinity surprisingly also elicit protective CD16b-mediated responses. However, it is unclear why IgG binds CD16a with more than 10-fold higher affinity than CD16b even though these receptors share more than 97% identity. Here we identified one residue, Gly-129, that contributes to the greater IgG binding affinity of CD16a. The CD16b variant D129G bound IgG1 Fc with 2-fold higher affinity than CD16a and with 90-fold higher affinity than the WT. Conversely, the binding affinity of CD16a-G129D was decreased 128-fold relative to WT CD16a and comparably to that of WT CD16b. The interaction of IgG1 Fc with CD16a, but not with CD16b, is known to be sensitive to the composition of the asparagine-linked carbohydrates (N-glycans) attached to the receptor. CD16a and CD16b-D129G displaying minimally processed oligomannose N-glycans bound to IgG1 Fc with about 5.2-fold increased affinity compared with variants with highly processed complex-type N-glycans. CD16b and the CD16a-G129D variant exhibited a smaller 1.9-fold affinity increase with oligomannose N-glycans. A model of glycosylated CD16b bound to IgG1 Fc determined to 2.2 Å resolution combined with a 250-ns all-atom molecular dynamics simulation showed that the larger Asp-129 residue deformed the Fc-binding surface. These results reveal how Asp-129 in CD16b affects its binding affinity for IgG1 Fc and suggest that antibodies engineered to engage CD16b with high affinity must accommodate the Asp-129 side chain.

Keywords: IgG; N-linked glycosylation; X-ray crystallography; fragment crystallizable region; immunity; molecular dynamics; monoclonal antibody; posttranslational modification (PTM); protein–protein interaction.

Figure 1.

FcγR III/CD16 is expressed in two forms from two distinct genes. A and B, FcγRIIIb/CD16b is highly expressed on neutrophils, and FcγRIIIa/CD16a is the predominant FcγR expressed on NK cells. B, these proteins differ by only four amino acid residues (highlighted in yellow) in the mature proteins. The N-terminal signal peptide is indicated in red and is not found in the fully processed proteins. The C-terminal residues of CD16b shown in orange are cleaved prior to addition of the glycosylphosphatidylinositol anchor at the newly uncovered C-terminal serine. N-glycosylation sites are indicated with gray shading. Both proteins are cleaved by ADAM17 and released from the cell surface following cell activation. Resolved N-glycan residues are shown as spheres.

Figure 2.

Total ion current of procainamide-derived CD16b N-glycans expressed in two distinct glycoforms and analyzed by HILIC-MS. Top, CD16b was expressed to display Man5 N-glycans. Bottom, CD16b with CT N-glycans.

Figure 3.

The binding affinity and binding kinetics of the CD16b D129G protein are comparable with the high-affinity CD16a protein. Shown are representative binding sensorgrams for the CD16 variants binding to IgG1 Fc (G0 glycoform). Dissociation constants were determined using equilibrium intensity values, with the exception of K_D values marked with an asterisk, which were calculated by fitting kinetic data. Errors indicate uncertainties from the curve fitting procedures.

Figure 4.

A glycine at position 129 of CD16 is essential for high-affinity IgG1 Fc binding. Dissociation constants indicate the impact of the IgG1 Fc N-glycan, CD16 in N-glycan, and the residue at position 129 on binding affinity. Error bars indicate the error of fit for the interactions.

Figure 5.

CD16a binds IgG1 Fc with greater affinity and makes a closer approach to IgG1 Fc through the loop containing residue 129. A and B, the overall complexes formed by CD16a (A) and CD16b (B) with IgG1 Fc are highly similar with a root mean square deviation of 0.499 Å. The protein backbone is shown as ribbons, and N-glycans are shown as sticks. C, the electron density of the CD16b region surrounding Asp-129 is well-resolved at 1.5 σ. D and E, the Gly-129 amide nitrogen atom of CD16a (D) is located 1.3 Å closer to the carbonyl carbon of the IgG1 Fc Asn-297 than the amide nitrogen of Asp-129 from CD16B (E).

Figure 6.

Asp-129 in CD16b decreases the distance between β strands and distorts the interface formed between the Asn-162-glycan and Arg-155 compared with Gly-129 and CD16a. A, the CD16a complex with glycosylated CD16a is shown in red (PDB code 5VU0 (20)) and superimposed on the unliganded (unlig.) CD16b (PDB code 1E4J (39)), shown in gray. Note that the position of the Asp-129 side chain clashes with the IgG1 Fc C'E loop. B, glycosylated CD16b in complex with IgG1 Fc is shown in orange and superimposed on the unliganded CD16b, shown in gray. IgG1 Fc binding distorts the conformation surrounding Asp-129, shortening the distance from Asp-129 to Arg-155. C and E, two views of CD16a, with the polypeptide surface shown in gray and relevant residues as red sticks. D and F, two poses of CD16b, with the polypeptide surface shown in gray and relevant residues as orange sticks. This Asp-129–mediated strand distortion perturbs the interface formed between the (1)GlcNAc residue of the CD16 Asn-162 glycan and Arg-155.

Figure 7.

Internuclear distances observed in crystallography are maintained during 250 ns all-atom molecular dynamics simulations of the CD16-IgG1 Fc complexes. Left panels, a 10-frame average of each distance over the course of the simulation. Right panels, histograms reporting the number of frames that correspond to each distance. Vertical arrows indicate the distance measured from structures determined by X-ray crystallography (CD16a-IgG1 Fc G0 (PDB code 5VU0 (20)); CD16b-IgG1 Fc G0, this work). Distances correspond to those measured in Figs. 5 and 6. X129 refers to residue Asp-129 of CD16b and Gly-129 of CD16a.