Adam L. Corper

Structure of human IgM rheumatoid factor Fab bound to its autoantigen IgG Fc reveals a novel topology of antibody-antigen interaction.

Rheumatoid factors are the characteristic autoantibodies of rheumatoid arthritis, which bind to the Fc regions of IgG molecules. Here we report the crystal structure of the Fab fragment of a patient-derived IgM rheumatoid factor (RF-AN) complexed with human lgG4 Fc, at 3.2 Å resolution. This is the first structure of an autoantibody–autoantigen complex. The epitope recognised in IgG Fc includes the Cγ2/Cγ3 cleft region, and overlaps the binding sites of bacterial Fc-binding proteins. The antibody residues involved in autorecognition are all located at the edge of the conventional combining site surface, leaving much of the latter available, potentially, for recognition of a different antigen. Since an important contact residue is a somatic mutation, the structure implicates antigen-driven selection, following somatic mutation of germline genes, in the production of pathogenic rheumatoid factors.

The I-Ag7 MHC Class II Molecule Linked to Murine Diabetes Is a Promiscuous Peptide Binder

Susceptibility to insulin-dependent diabetes mellitus is linked to MHC class II genes. The only MHC class II molecule expressed by nonobese diabetic (NOD) mice, I-Ag7, shares a common α-chain with I-Ad but has a peculiar β-chain. As with most β-chain alleles linked to diabetes susceptibility, I-Ag7 contains a nonaspartic residue at position β57. We have produced large amounts of empty I-Ag7 molecules using a fly expression system to characterize its biochemical properties and peptide binding by phage-displayed peptide libraries. The identification of a specific binding peptide derived from glutamic acid decarboxylase (GAD65) has allowed us to crystallize and obtain the three-dimensional structure of I-Ag7. Structural information was critical in evaluating the binding studies. I-Ag7, like I-Ad, appears to be very promiscuous in terms of peptide binding. Their binding motifs are degenerate and contain small and/or small hydrophobic residues at P4 and P6 of the peptide, a motif frequently found in most globular proteins. The degree of promiscuity is increased for I-Ag7 over I-Ad as a consequence of a larger P9 pocket that can specifically accommodate negatively charged residues, as well as possibly residues with bulky side chains. So, although I-Ad and I-Ag7 are structurally closely related, stable molecules and good peptide binders, they differ functionally in their ability to bind significantly different peptide repertoires that are heavily influenced by the presence or the absence of a negatively charged residue at position 57 of the β-chain. These characteristics link I-Ag7 with autoimmune diseases, such as insulin-dependent diabetes mellitus.

The structure and origin of rheumatoid factors

Abstract The recently determined X-ray crystal structure of a human rheumatoid factor Fab bound to IgG Fc provides the basis of a new hypothesis for the origin of these autoantibodies in rheumatoid arthritis. The observation that Fc is bound outside the conventional antigen combining site suggests a novel form of crossreactivity with simultaneous binding of another antigen, potentiated by somatic mutation. This article discusses the implications for the induction of these autoantibodies.

Crystal Structure Of MHC Class II I-Ab in Complex with a Human CLIP Peptide: Prediction of an I-Ab Peptide-binding Motif

Association between the class II major histocompatibility complex (MHC) and the class II invariant chain-associated peptide (CLIP) occurs naturally as an intermediate step in the MHC class II processing pathway. Here, we report the crystal structure of the murine class II MHC molecule I-A(b) in complex with human CLIP at 2.15A resolution. The structure of I-A(b) accounts, via the peptide-binding grooves unique physicochemistry, for the distinct peptide repertoire bound by this allele. CLIP adopts a similar conformation to peptides bound by other I-A alleles, reinforcing the notion that CLIP is presented as a conventional peptide antigen. When compared to the related HLA-DR3/CLIP complex structure, the CLIP peptide displays a slightly different conformation and distinct interaction pattern with residues in I-A(b). In addition, after examining the published sequences of peptides presented by I-A(b), we discuss the possibility of predicting peptide alignment in the I-A(b) binding groove using a simple scoring matrix.

Soluble CD36 ectodomain binds negatively charged diacylglycerol ligands and acts as a co-receptor for TLR2.

Background Cluster of differentiation 36 (CD36) is a transmembrane glycoprotein involved in many biological processes, such as platelet biology, angiogenesis and in the aetiopathology of atherosclerosis and cardiovascular diseases. Toll-like receptors (TLRs) are one of the most important receptors of the innate immune system. Their main function is the recognition of conserved structure of microorganisms. This recognition triggers signaling pathways that activate transcription of cytokines and co-stimulatory molecules which participate in the generation of an immune response against microbes. In particular, TLR2 has been shown to recognize a broad range of ligands. Recently, we showed that CD36 serves as a co-receptor for TLR2 and enhances recognition of specific diacylglycerides derived from bacteria. Methodology/ Principal Findings Here, we investigate the mechanism by which CD36 contributes to ligand recognition and activation of TLR2 signaling pathway. We show that the ectodomain of murine CD36 (mCD36ED) directly interacts with negatively charged diacylglycerol ligands, which explains the specificity and selectivity of CD36 as a TLR2 co-receptor. We also show that mCD36ED amplifies the pro-inflammatory response to lipoteichoic acid in macrophages of wild-type mice and restores the pro-inflammatory response of macrophages from mice deficient in CD36 (oblivious), but not from mice deficient in cluster of differentiation 14 (CD14) (heedless). Conclusion/ Significance These data indicate that the CD36 ectodomain is the only relevant domain for activation of TLR2 signaling pathway and that CD36 and CD14 have a non-redundant role for loading ligands onto TLR2 in the plasma-membrane. The pro-inflammatory role of soluble CD36 can be relevant in the activation of the immune response against pathogens, as well as in the progression of chronic diseases. Therefore, an increased level of soluble forms of CD36, which has been reported to be increased in type II diabetic patients, could accelerate atherosclerosis by increasing the pro-inflammatory response to diacylglycerol ligands.

Crystal structure of a non-canonical low-affinity peptide complexed with MHC class I: a new approach for vaccine design.

Peptides bind with high affinity to MHC class I molecules by anchoring certain side-chains (anchors) into specificity pockets in the MHC peptide-binding groove. Peptides that do not contain these canonical anchor residues normally have low affinity, resulting in impaired pMHC stability and loss of immunogenicity. Here, we report the crystal structure at 1.6 A resolution of an immunogenic, low-affinity peptide from the tumor-associated antigen MUC1, bound to H-2Kb. Stable binding is still achieved despite small, non-canonical residues in the C and F anchor pockets. This structure reveals how low-affinity peptides can be utilized in the design of novel peptide-based tumor vaccines. The molecular interactions elucidated in this non-canonical low-affinity peptide MHC complex should help uncover additional immunogenic peptides from primary protein sequences and aid in the design of alternative approaches for T-cell vaccines.

New Design of MHC Class II Tetramers to Accommodate Fundamental Principles of Antigen Presentation

Direct identification and isolation of Ag-specific T cells became possible with the development of MHC tetramers, based on fluorescent avidins displaying biotinylated peptide-MHC complexes. This approach, extensively used for MHC class I-restricted T cells, has met very limited success with class II peptide-MHC complex tetramers (pMHCT-2) for the detection of CD4+-specific T cells. In addition, a very large number of these reagents, although capable of specifically activating T cells after being coated on solid support, is still unable to stain. To try to understand this puzzle and design usable tetramers, we examined each parameter critical for the production of pMHCT-2 using the I-Ad-OVA system as a model. Through this process, the geometry of peptide-MHC display by avidin tetramers was examined, as well as the stability of rMHC molecules. However, we discovered that the most important factor limiting the reactivity of pMHCT-2 was the display of peptides. Indeed, long peptides, as presented by MHC class II molecules, can be bound to I-A/HLA-DQ molecules in more than one register, as suggested by structural studies. This mode of anchorless peptide binding allows the selection of a broader repertoire on single peptides and should favor anti-infectious immune responses. Thus, beyond the technical improvements that we propose, the redesign of pMHCT-2 will give us the tools to evaluate the real size of the CD4 T cell repertoire and help us in the production and testing of new vaccines.

Crystallization of a complex between the Fab fragment of a human immunoglobulin M (IgM) rheumatoid factor (RF-AN) and the Fc fragment of human IgG4.

Rheumatoid factors (RF) are the characteristic autoantibodies found in patients with rheumatoid arthritis. They recognize epitopes in the Fc region of immunoglobulin G (IgG) and are often of the IgM isotype. In order to analyse the nature of RF–Fc interactions, we have crystallized a complex between the Fab fragment of a human monoclonal IgM rheumatoid factor (RF–AN) and the Fc fragment of human IgG4. The stoichiometry of the complex within the crystals was found to be 2 : 1 Fab : Fc. The crystals diffracted X‐rays to 0.3 nm resolution, and the space group was C2, with cell dimensions a = 16.03 nm, b = 8.19 nm, c = 6.42 nm, β = 98.3°. We have also determined the sequence of the variable region of the RF–AN light chain, not hitherto reported. This belongs to the VλIII‐a subgroup and is closely related to the germline gene Humlv318, from which it differs in three amino acid residues. This is the first reported crystallized complex between a human autoantibody and its autoantigen.

The diabetogenic mouse MHC class II molecule I-Ag7 is endowed with a switch that modulates TCR affinity.

Genetic susceptibility to autoimmunity is frequently associated with specific MHC alleles. Diabetogenic MHC class II molecules, such as human HLA-DQ8 and mouse I-Ag7, typically have a small, uncharged amino acid residue at position 57 of their beta chain (beta57); this results in the absence of a salt bridge between beta57 and Argalpha76, which is adjacent to the P9 pocket of the peptide-binding groove. However, the influence of Argalpha76 on the selection of the TCR repertoire remains unknown, particularly when the MHC molecule binds a peptide with a neutral amino acid residue at position P9. Here, we have shown that diabetogenic MHC class II molecules bound to a peptide with a neutral P9 residue primarily selected and expanded cells expressing TCRs bearing a negatively charged residue in the first segment of their complementarity determining region 3beta. The crystal structure of one such TCR in complex with I-Ag7 bound to a peptide containing a neutral P9 residue revealed that a network of favorable long-range (greater than 4 A) electrostatic interactions existed among Argalpha76, the neutral P9 residue, and TCR, which supported the substantially increased TCR/peptide-MHC affinity. This network could be modulated or switched to a lower affinity interaction by the introduction of a negative charge at position P9 of the peptide. Our results support the existence of a switch at residue beta57 of the I-Ag7 and HLA-DQ8 class II molecules and potentially link normal thymic TCR selection with abnormal peripheral behavior.

The Structure of a Human Rheumatoid Factor Bound to IgG Fc

This is the first crystal structure analysis of a complex between an autoantibody and its autoantigen, and it reveals a mode of interaction never before seen in an antibody-antigen complex. Not only are there relatively few antibody contact residues, contributing perhaps to its very low affinity, but these residues are to be found on only one side of the potential combining site surface. Indeed, so many CDR residues are not involved in Fc binding, including those in the central region of the combining site, that it is easy to envisage that this RF may have another, entirely different, specificity. The antibody may therefore have originated in response to another, as yet unidentified, antigen, and the reactivity with IgG Fc may be an unfortunate cross-reactivity. Certainly some of the CDR residues which do interact with IgG Fc are germline encoded, but significantly one of only two residues in the light chain, Pro56, which makes many contacts with Fc, is a somatic mutation. Since this mutation would appear to make a significant contribution to the binding affinity, it is therefore evidence for an antigen driven response to the IgG Fc in the generation of this autoantibody. The Fc epitope recognised by RF-AN is strikingly similar to the binding sites for the bacterial binding proteins A and G, but the significance of this is not clear. What is clear however is that the epitope does not include any part of the Fc carbohydrate residues, although the structure of the complex does reveal that there is an alteration in the carbohydrate conformation when the galactose residues are absent. Loss of the interaction between the terminal galactose residue on the alpha (1-6) linked branch and the C gamma 2 domain appears to allow the carbohydrate chains to become mobile, at the same time exposing a predominantly hydrophobic patch on the C gamma 2 surface. Accessibility to either the agalactosyl carbohydrate chains or the newly exposed residues may account for the enhanced reactivity for G0-IgG that has been reported for certain RFs, and such an epitope need not be very different to that recognised by RF-AN. In order to understand more completely the effect of the presence or absence of the terminal galactose residue, the fully galactosylated glycoform of Fc must be studied for comparison; this work is underway. It is also important now to study a RF which is known to sense this difference in oligosaccharide composition, and also to study RFs of higher affinity, of the IgG class, and from the synovium. RF-AN was the first RF to be immortalised as a cell line, and in many ways it is a typical RF (in terms of specificity, relationship to germline sequence and affinity), but we must now establish whether the novel structural features revealed in this analysis are indeed typical of other RFs. Only when comparisons can be made between RFs of different origin and with contrasting functional properties will we begin to understand what constitutes a pathogenic RF, and the mechanism by which such auto-reactive antibodies are generated.