Structure-function analyses of a stereotypic rheumatoid factor unravel the structural basis for germline-encoded antibody autoreactivity

Abstract

Rheumatoid factors (RFs) are autoantibodies against the fragment-crystallizable (Fc) region of IgG. In individuals with hematological diseases such as cryoglobulinemia and certain B cell lymphoma forms, the RFs derived from specific heavy- and light-chain germline pairs, so-called "stereotypic RFs," are frequently produced in copious amounts and form immune complexes with IgG in serum. Of note, many structural details of the antigen recognition mechanisms in RFs are unclear. Here we report the crystal structure of the RF YES8c derived from the IGHV1-69/IGKV3-20 germline pair, the most common of the stereotypic RFs, in complex with human IgG1-Fc at 2.8 Å resolution. We observed that YES8c binds to the CH2-CH3 elbow in the canonical antigen-binding manner involving a large antigen-antibody interface. On the basis of this observation, combined with mutational analyses, we propose a recognition mechanism common to IGHV1-69/IGKV3-20 RFs: (1) the interaction of the Leu⁴³²-His⁴³⁵ region of Fc enables the highly variable complementarity-determining region (CDR)-H3 to participate in the binding, (2) the hydrophobic tip in the CDR-H2 typical of IGHV1-69 antibodies recognizes the hydrophobic patch on Fc, and (3) the interaction of the highly conserved RF light chain with Fc is important for RF activity. These features may determine the putative epitope common to the IGHV1-69/IGKV3-20 RFs. We also showed that some mutations in the binding site of RF increase the affinity to Fc, which may aggravate hematological diseases. Our findings unravel the structural basis for germline-encoded antibody autoreactivity.

Keywords: IgG; autoimmunity; complex; crystal structure; structural biology.

Figure 1.

Binding specificity of YES8c to IgG subclasses. A, binding of the purified YES8c-Fab to the immobilized four human IgG subclasses (IgG1, IgG2, IgG3, and IgG4) was measured by ELISA. Data show mean ± S.D. (error bars) from triplicates from one representative experiment of two independent experiments with similar results. B, representative sensorgrams derived from injection of different concentrations of YES8c-Fab over immobilized human IgG1 by surface plasmon resonance analysis. The black horizontal bar indicates the region of the sensorgrams from which equilibrium binding responses are derived. RU, resonance unit. C, plot of the equilibrium binding response against the concentration of YES8c-Fab for the sensorgrams shown in B. The line represents a nonlinear fit of the 1:1 binding model. The K_d is 160 ± 30 μm (mean ± S.D.) from three independent experiments.

Figure 2.

Crystal structure of YES8c in complex with IgG1-Fc. A, overall structure of the YES8c–Fab–Fc complex in an asymmetric unit. In complex 1, the Fc and heavy and light chains are shown in magenta, cyan, and green, respectively, and in light pink, light blue, and light green in complex 2. The ovals show the missing constant region (CL and CH1). B, the YES8c–Fc complex and protein A–Fc complex (PDB code 1L6X) from the same viewpoint. C, comparison between the YES8c and RF-AN binding mode. In YES8c, the Fc and heavy and light chains are shown in magenta, cyan, and green, respectively, and in light pink, light blue, and light green in RF-AN. CDRs are shown in orange in the both structure.

Figure 3.

Fc recognition by the heavy chain of YES8c. A, stereo view of the protruding region in Leu⁴³²–His⁴³⁵ of Fc bound to YES8c. The heavy chain and the light chain of YES8c are shown as cyan and green surfaces, respectively, and the Fc is shown as magenta sticks. The CDR-H3 is shown in orange. The Leu⁴³²–His⁴³⁵ region is shown in yellow. A 2m|F_o| − D|F_c| map for the Leu⁴³²–His⁴³⁵ region is contoured at 1 σ (blue mesh). B, stereo view of the interaction between CDR-H3 (orange) and the Asn⁴³⁴ (yellow). Hydrogen bonds are shown as dashed lines. C, stereo view of the interaction between CDR-H3 and the CH2–CH3 cleft in Fc. The surfaces of CH2 and CH3 are shown in light pink and magenta, respectively. The Leu⁴³²–His⁴³⁵ region is shown in yellow. VH (excluding CDR-H3) and VL are shown as cyan and green surfaces, respectively. CDR-H3 is shown as orange sticks. A 2m|F_o| − D|F_c| map for the CDR-H3 is contoured at 1 σ (blue mesh). D, alignment of the amino acid residues in CDR-H3 of YES8c and the other IGHV1-69/IGKV3-20 RFs. The residues are numbered based on the Kabat numbering scheme. E, interactions between CDR-H2 of YES8c and Fc. The surface of Fc is shown with the hydrophobicity scale (48). A 2m|F_o| − D|F_c| map for the CDR-H2 is contoured at 1 σ (blue mesh). F, alignment of the amino acid residues in CDR-H2 of YES8c and the IGHV1-69/IGKV3-20 derived rheumatoid factors. G, binding of the mutants in the CDR-H2 of YES8c to the immobilized IgG1 by ELISA. Data show mean ± S.D. (error bars) from triplicates from one representative experiment of two independent experiments with similar results.

Figure 4.

Binding by the light chain of YES8c. A, interaction footprints in the light chain of YES8c to Fc (left panel) and the 90° rotated view (right panel). The contacting area of the light chain to Fc is shown in green. The other surface of the light chain is shown in pale yellow. Fc is shown as a magenta schematic model. B, binding of mutants in the light chain of YES8c to the immobilized IgG1 by ELISA. Data show mean ± S.D. (error bars) from triplicates from one representative experiment of two independent experiments with similar results.

Figure 5.

Binding of the germline-reverted mutant RFs to IgG1-Fc. A, binding of the germline-reverted mutants to IgG1-Fc was measured by ELISA. Data show mean ± S.D. (error bars) from triplicates from one representative experiment of two independent experiments with similar results. B, plot of equilibrium binding responses against concentration of YES8c-Fab mutants for the sensorgrams shown in Fig. S10. The solid line represents the nonlinear fit of the 1:1 binding model for the WT and the H-L53I mutant that gave K_d values of 130 μm and 84 μm, respectively. The responses for L-I28V and H-P33A could not be fit with the 1:1 binding model. RU, resonance unit.

Figure 6.

The YES8c sites relative to the increase in binding affinity for IgG1-Fc. A, the residues (H-Thr56 and H-Asn58) in CDR-H2 located near the acidic residues on Fc (Glu³⁴⁵ and Glu⁴³⁰). B, the residues (L-Gln27 and L-Ser27A) in CDR-L1. C, plot of equilibrium binding responses against concentration of YES8c-Fab mutants for the sensorgrams shown in Fig. S11, B–E. The solid line represents a nonlinear fit of the 1:1 binding model for H-T56K, H-N58K, L-Q27E, and L-S27AN. The K_d values are indicated. RU, resonance unit.