Antibody Recognition of Disordered Antigens
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This paper examines the molecular basis for antibody recognition of disordered antigens, providing important insights into how the immune system interacts with intrinsically disordered proteins. The authors challenge the prevailing notion that disordered antigens are poor targets for antibody responses, demonstrating instead that disordered epitopes are frequently recognized by high-affinity antibodies.
The study begins by analyzing a large dataset of protein antigens extracted from the Immune Epitope Database (IEDB). Using the IUPred algorithm to predict intrinsic disorder, the authors find that over 27% of residues in these antigens are predicted to be disordered - a level comparable to many eukaryotic proteomes. Importantly, they observe that antibody binding assays involving disordered epitopes actually report positive results more frequently than those for ordered epitopes. This suggests that conformational disorder does not impede antibody recognition, contrary to previous speculation.
To further explore the relationship between disorder and antigenicity, the authors examined how the frequency of positive antibody binding results varied with IUPred score (a measure of predicted disorder). They found that antigenicity generally increased with increasing IUPred score over most of the range. However, this trend closely tracked the relationship between IUPred score and solvent accessibility, suggesting that the increased antigenicity of disordered regions may be largely due to their greater exposure to solvent and thus to antibodies.
A key question addressed by the study is whether disordered antigens can elicit high-affinity antibody responses. It has been suggested that the entropic cost of inducing order in a disordered epitope upon antibody binding would significantly reduce affinity. However, by analyzing experimentally determined binding affinities reported in the IEDB, the authors find only a modest 5-fold difference in median affinity between ordered and disordered epitopes. This difference is much smaller than might be expected based on theoretical considerations of the entropic penalty. The authors note that this affinity difference equates to only about 0.1 kcal/mol per residue for a typical 9-residue disordered epitope - far less than recent estimates of the entropic cost of protein folding (1.0-1.5 kcal/mol per residue).
To investigate the structural basis for this unexpectedly high affinity, MacRaild et al. analyzed a dataset of 872 antibody-antigen complex structures from the Protein Data Bank. They found that disordered epitopes tend to be substantially smaller than ordered epitopes, both in terms of the number of residues contacting the antibody and the total buried surface area. A typical disordered epitope comprises just 9 residues burying 695 Å2 on the antigen, compared to 21 residues burying 847 Å2 for ordered epitopes.
Interestingly, despite their smaller size, disordered epitopes appear to interact more intimately with their cognate antibodies. The authors observe that disordered epitopes bury a larger fraction of their surface area upon binding and show greater shape complementarity with the antibody paratope. The paratopes that recognize disordered epitopes tend to be more concave, effectively enveloping the smaller epitope. This is reflected in differences in the lengths of the antibody complementarity-determining regions (CDRs), with antibodies to disordered epitopes having shorter central CDR H3 loops but longer flanking CDR H2 and L1 loops.
The paper also examines the density and distribution of key interactions at the antibody-antigen interface. While the absolute number of hydrogen bonds and salt bridges is lower for disordered epitope complexes (due to the smaller interface size), the density of these polar contacts is actually higher. Residues in disordered epitopes are more than 50% more likely to form salt bridges and twice as likely to form hydrogen bonds with the antibody compared to residues in ordered epitopes.
These structural features - smaller but more intimately contacted epitopes with a higher density of polar interactions - help explain how antibodies to disordered antigens can achieve high affinity despite the entropic penalty of binding. By limiting the number of residues involved, the entropic cost is minimized, while maximizing the extent and complementarity of interactions for those residues that do engage the antibody optimizes the compensating enthalpic component of binding.
The authors also investigated whether antigen disorder affects the process of affinity maturation - the mechanism by which antibodies develop increased affinity through somatic mutation during the immune response. By comparing antibody sequences to germline genes, they found no significant difference in the number of somatic mutations between antibodies recognizing ordered versus disordered epitopes. This suggests that affinity maturation is not impaired by antigen disorder.
An important implication of the study relates to antibody specificity. The authors used computational alanine scanning to predict the energetic effects of mutating individual residues at the antibody-antigen interface. They found that mutations within disordered epitopes were much more likely to significantly impact binding affinity compared to mutations in ordered epitopes. Disordered epitopes showed a markedly higher density of energetic "hot spot" residues - those whose mutation is predicted to strongly destabilize the complex. This suggests that antibody recognition of disordered epitopes may be particularly sensitive to sequence variation in the antigen.
Interestingly, the distribution of hot spots shows a striking asymmetry between antibody and antigen. While disordered epitopes are enriched in hot spot residues compared to ordered epitopes, their cognate antibody paratopes actually show a depletion of hot spots. The authors note that similar asymmetry has been observed in other protein-protein interactions involving disordered proteins. This implies that the effects of disorder on binding specificity may depend on which partner in the interaction is varied - mutations in the disordered partner (antigen) are more likely to disrupt binding, while the interaction may be more robust to variation in the ordered partner (antibody).
The findings of this study have important implications for understanding immune responses to pathogens. Many pathogenic organisms, particularly parasites, have proteomes enriched in disordered proteins. The ability of these disordered antigens to elicit high-affinity, specific antibody responses suggests they could be viable vaccine targets. However, the heightened sensitivity to epitope sequence variation may also make it easier for pathogens to evade antibody recognition through mutation of disordered epitopes.
In conclusion, this comprehensive analysis demonstrates that disordered epitopes are bona fide targets of high-affinity and specific antibody responses. The paper reveals key structural features that allow antibodies to efficiently recognize disordered antigens despite the entropic challenges involved. It also highlights important differences in the nature of antibody recognition between ordered and disordered epitopes, particularly in terms of the distribution of energetically critical residues. These insights advance our understanding of protein-protein interactions involving disordered proteins and have significant implications for vaccine development and our broader conception of how conformational disorder impacts molecular recognition.
The study's findings challenge prevailing assumptions about the interplay between protein disorder and immune recognition. By demonstrating that disorder does not preclude high-affinity antibody binding, and may even enhance certain aspects of recognition, the work opens up new avenues for exploring disordered proteins as therapeutic targets. At the same time, the observed sensitivity of these interactions to epitope sequence variation provides a new perspective on how pathogens may exploit disorder in their antigenic proteins to evade immune responses. Overall, this paper makes a significant contribution to our understanding of both immunology and the functional roles of intrinsically disordered proteins.
https://www.cell.com/structure/pdf/S0969-2126(15)00470-0.pdf
