FcαRI binding at the IgA1 CH2-CH3 interface induces long-range conformational changes that are transmitted to the hinge region

Abstract

IgA effector functions include proinflammatory immune responses triggered upon clustering of the IgA-specific receptor, FcαRI, by IgA immune complexes. FcαRI binds to the IgA1-Fc domain (Fcα) at the C_H2-C_H3 junction and, except for C_H2 L257 and L258, all side-chain contacts are contributed by the C_H3 domain. In this study, we used experimental and computational approaches to elucidate energetic and conformational aspects of FcαRI binding to IgA. The energetic contribution of each IgA residue in the binding interface was assessed by alanine-scanning mutagenesis and equilibrium surface plasmon resonance (SPR). As expected, hydrophobic residues central to the binding site have strong energetic contributions to the FcαRI:Fcα interaction. Surprisingly, individual mutation of C_H2 residues L257 and L258, found at the periphery of the FcαRI binding site, dramatically reduced binding affinity. Comparison of antibody:receptor complexes involving IgA or its precursor IgY revealed a conserved receptor binding site at the C_H2-C_H3 junction (or its equivalent). Given the importance of residues near the C_H2-C_H3 junction, we used coarse-grained Langevin dynamics simulations to understand the functional dynamics in Fcα. Our simulations indicate that FcαRI binding, either in an asymmetric (1:1) or symmetric (2:1) complex with Fcα, propagated long-range conformational changes across the Fc domains, potentially impacting the hinge and Fab regions. Subsequent SPR experiments confirmed that FcαRI binding to the Fcα C_H2-C_H3 junction altered the kinetics of HAA lectin binding at the IgA1 hinge. Receptor-induced long-distance conformational transitions have important implications for the interaction of aberrantly glycosylated IgA1 with anti-glycan autoantibodies in IgA nephropathy.

Keywords: IgA1 antibody; binding energetics; molecular-dynamics simulations; principal-component analysis; surface plasmon resonance.

Fig. 1.

FcαRI binds IgA1 at a hydrophobic region of the Cα2–Cα3 junction. (A) Model of the 2:1 complex between FcαRI (blue) and IgA1 (green/orange) (3, 6) based on the crystal structure of the FcαRI:Fcα complex (PDB ID code 1OW0) and the solution structure of full-length IgA1 (PDB ID code 1IGA). (B and C) Characteristics of FcαRI binding site on Fcα: amino acid properties (hydrophobic, yellow; positively charged, blue; negatively charged, red; polar, green) (B); percent contribution of each Fcα residue with side chain contacts to the binding surface (C).

Fig. 2.

SPR analysis of FcαRI binding to Fcα variants identifies energetic hot-spot residues. (A) Coplotted SPR binding isotherms of wild-type Fcα and mutants binding to FcαRI. Fcα variants were coupled to the SPR chip and soluble FcαRI was flowed over. Fcα L257A, M433A, F443A, and L258A mutations resulted in the largest decreases in binding affinity. (B) Plotting of the experimentally determined ΔΔG values on Fcα. (C) Location of the Cα of all mutated residues, colored according to the ΔΔG values for each alanine mutant.

Fig. 3.

Comparison of Fcα and Fcυ crystal structures reveals variability in the C_H2 domain position, indicating the Cα2–Cα3 junction acts as a hinge point. (A) Structure of the Fcα heavy chain, showing the location of L257 and L258 at the bottom of the Cα2 domain (highlighted with transparent spheres). (B) Overlay of Fcα from the FcαRI-bound complex (blue) with unbound IgY Cυ3–Cυ4 (magenta) revealed a 14.4-Å shift between the top of the Cα2 and Cυ3 domains. (C) The coarse-grained model of Fcα is shown in a bead representation, with each amino acid represented using two beads. First bead (blue), representing the backbone, is located at C_α position, and the second bead (red), representing the side chain, is located at the center of mass of the amino acid’s side chain.

Fig. 4.

Backbone flexibility in distinct Fcα complexes. (A) The root-mean-square fluctuations (RMSFs) of Cα atoms of Fcα amino acids in unliganded Fcα (black); 1:1 FcαRI–Fcα (red) and 2:1 FcαRI–Fcα (green) complexes are shown for the cis (trans) heavy chain in the Upper (Lower) panel. In the asymmetric 1:1 FcαRI–Fcα complex, the receptor is bound to the cis Fcα heavy chain. (B) Amino acids (green) corresponding to the R1, R2, and R3 regions in A, located primarily near the linker between the Cα2 and Cα3 domains, experience the strongest RMSF dampening upon receptor binding. The yellow spheres with labels indicate the amino acid positions in this set, which are highlighted as important for receptor binding in mutagenesis studies. Six structural regions (orange and green loop in the Cα3 domain) with the largest flexibility (RMSF > 1.5 Å) primarily include loops involved in the intersubunit interface.

Fig. 5.

Principal-component analysis (PCA) of MD trajectories of distinct Fcα complexes. (A) The largest 20 eigenvalues of the PC modes for different Fcα complexes: Fcα (black), 1:1 FcαRI–Fcα (red), and 2:1 FcαRI–Fcα (green). (B–D) Fcα motions associated with mode 1 (largest eigenvalue) in the three systems studied. The red vectors illustrate the amplitude and the direction of residue motion for (B) Fcα alone, (C) 1:1 FcαRI–Fcα, and (D) 2:1 FcαRI–Fcα.

Fig. 6.

Fcα motions associated with the highest ranked PC modes. The five panels indicate the mode motions for modes 1–5 for unliganded Fcα. The red vectors indicate the direction of residue motion, and the vector length indicates the relative amplitude of the residue motion in each mode.

Fig. 7.

Maps of directional correlation coefficients of all amino acid pairs in the unliganded Fcα for the five highest ranked PC modes. Correlation of amino acid pairs in modes 1–5. Strong correlation of a given pair of residues is indicated in red, and strong anticorrelation is shown in blue.

Fig. 8.

Long-distance allosteric interactions between intersubunit pairs involving FcαRI binding sites. (A) Intersubunit residue pairs that switch from weak directional correlation in unliganded Fcα to strong correlation or anticorrelation upon receptor binding. Amino acid pairs, highlighted by distinct colors, include experimentally identified FcαRI-binding sites L258 (purple, cis), S387 (blue, cis), E437 (cyan, cis), L441 (pink, cis), A442 (magenta, trans), and F443 (white, cis; black, trans). (B) Intersubunit residue pairs that switch from strongly correlated (anticorrelated) motions in the unliganded Fcα to strongly anticorrelated (correlated) motions in FcαRI:Fcα complexes. Shown are pairs that include FcαRI binding sites L441 (pink, cis), A442 (red, cis), F443 (white, cis), R382 (silver, trans), S387 (yellow, trans), E389 (gray, trans), L441 (orange, trans), L442 (magenta, trans), and F443 (black, trans).

Fig. 9.

FcαRI binding influences HAA binding at the IgA1 hinge. (A) Comparison of SPR curves under steady-state conditions for mIgA1κ binding to the lectin HAA in the presence or absence of FcαRI indicates differences in kinetics of binding. (B) Kinetic binding data comparing HAA binding to mIgA1κ in the presence and absence of FcαRI binding revealed faster on rates of binding in the presence of FcαRI. Kinetic data and fits are shown in detail in SI Appendix, Fig. S7. (C) Model for scissor-like action of Fcα and hinge when FcαRI binds. FcαRI binding at the Cα2–Cα3 junction induces long-range conformational changes that are transmitted up into the hinge and Fab regions. The change in relative proximity of hinge O-glycans increases the rate of binding by the lectin HAA. For clarity, the conformation of the hinge and Fab in the unbound form are shown on the left side, and the proposed conformational change is illustrated on the right side.