Origins of specificity and affinity in antibody-protein interactions

Abstract

Natural antibodies are frequently elicited to recognize diverse protein surfaces, where the sequence features of the epitopes are frequently indistinguishable from those of nonepitope protein surfaces. It is not clearly understood how the paratopes are able to recognize sequence-wise featureless epitopes and how a natural antibody repertoire with limited variants can recognize seemingly unlimited protein antigens foreign to the host immune system. In this work, computational methods were used to predict the functional paratopes with the 3D antibody variable domain structure as input. The predicted functional paratopes were reasonably validated by the hot spot residues known from experimental alanine scanning measurements. The functional paratope (hot spot) predictions on a set of 111 antibody-antigen complex structures indicate that aromatic, mostly tyrosyl, side chains constitute the major part of the predicted functional paratopes, with short-chain hydrophilic residues forming the minor portion of the predicted functional paratopes. These aromatic side chains interact mostly with the epitope main chain atoms and side-chain carbons. The functional paratopes are surrounded by favorable polar atomistic contacts in the structural paratope-epitope interfaces; more that 80% these polar contacts are electrostatically favorable and about 40% of these polar contacts form direct hydrogen bonds across the interfaces. These results indicate that a limited repertoire of antibodies bearing paratopes with diverse structural contours enriched with aromatic side chains among short-chain hydrophilic residues can recognize all sorts of protein surfaces, because the determinants for antibody recognition are common physicochemical features ubiquitously distributed over all protein surfaces.

Keywords: epitope prediction; functional epitope; interface hot spot; paratope prediction; protein antigenic site.

Fig. 1.

Prediction benchmarks of antibody/antigen binding sites on antibody/antigen surfaces. (A) Correlations of PPI_CL of antibody surface atoms to atomic burial in antibody–antigen interfaces. Atom-based PPI_CL range (shown in the x axis of the panel) for antibody surface atoms is correlated to the averaged burial level (measured by dSASA) of the subgroup of atoms in the antibody–antigen complexes predicted within the confidence level range. The correlation is shown by the square symbols, corresponding to the y axis on the right of the panel. The distribution of the atom-based predictions as shown by the diamond symbols, corresponding to the y axis on the left of the panel, is plotted against the PPI_CL range in the x axis. The data were derived from the independent test with the ISMBLab-PPI predictors on the antibodies in the S111 dataset. (B) The same as in A for the antigens in the S111 dataset. Additional benchmarks are shown in Fig. S1. The prediction algorithm and parameters follow the optimal settings without modification as previously described (30).

Fig. 2.

Comparison of the functional paratopes with the predicted hot spot residues on the antibody structures. (A) The residues of the functional paratopes on antibody FvD1.3 (PDB code: 1VFB) are highlighted with carbon atoms colored in yellow, green, and cyan. The residues with carbon atoms colored in cyan and green are the hot spot residues (∆∆G > 1 kcal/mol) interacting with lysozyme. The residues with carbon atoms colored in yellow and green are the hot spot residues (∆∆G > 1 kcal/mol) interacting with FvE5.2. (B) The residues of the functional paratopes on antibody bH1 (PDB code: 3BDY) are highlighted in color. The residues with carbon atoms colored in cyan and green are the hot spot residues (∆∆G > 1 kcal/mol) interacting with VEGF. The residues with carbon atoms colored in yellow and green are the hot spot residues (∆∆G > 1 kcal/mol) interacting with HER2. (C) The residues of the functional paratope of the anti-lysozym antibody HyHEL-63 (PDB code: 1DQJ) (∆∆G > 1 kcal/mol) are highlighted in cyan. (D–F) The carbon atoms of the residues in the potential functional paratope (PFP)(PPI) (PPI_CL ≥ 0.45) are colored in orange and pink. The carbon atoms of the residues in the PFP(proABC) (proABC_CP ≥ 80%) are colored in orange and magenta. Oxygen atoms are colored in red, and nitrogen atoms are colored in blue in A–F. The prediction results are compared with the actual hot spots in the table below the structures, where TP (true positive), FP (false positive), TN (true negative), FN (false negative), PRE (precision), ACC (accuracy), SEN (sensitivity), MCC (Matthews correlation coefficient), SPC (specificity), and F1 (F-score) are defined in Eqs. S4–S9.

Fig. 3.

Two-class classification prediction benchmarks for the representative antibody–antigen complex structures from the S111 dataset. The distributions of the prediction precisions and sensitivities for the test set antibody structures are shown in A and B, respectively. The red histograms (related to the left side y axis of the panels) and the purple cumulative curves (related to the right side y axis of the panels) show the results for the ISMBLab-PPI predictors with PPI_CL ≥ 0.45; the blue histograms (related to the left side y axis of the panels) and the green cumulative curves (related to the right side y axis of the panels) show the results for the proABC predictor with proABC_CP ≥ 80%. The detailed benchmark results for the ISMBLab-PPI predictions are shown in Table S1. The computational methods are described in SI Materials and Methods.

Fig. 4.

Amino acid type preferences and protein atom type preferences in the paratope–epitope interfaces. (A) The amino acid type preferences on the paratopes from the antibody–protein complex structures in the S111 dataset are shown in the histograms colored in blue, green, cyan, and red for SP(0), SP(0.2), PFP(proABC), and PFP(PPI), respectively. The background amino acid type preferences on average protein solvent accessible surface areas are shown in the purple histogram, calculated with the protein structures in the P9468 dataset (SI Materials and Methods). The y axis shows the fraction of the amino acid type in the x axis. (B) The amino acid type preferences on the epitopes from the antibody–protein complex structures in the S111 dataset are shown in the histograms colored in blue, green, cyan, and red for SE(0), SE(0.2), PFE(proABC), and PFE(PPI), respectively. The background amino acid type preferences on average protein solvent accessible surface areas are shown in the purple histogram. (C) The protein atom type preferences on the paratopes from the antibody–protein complex structures in S111 are shown in the histograms colored in blue, green, cyan, and red for the SP(0), SP(0.2), PFP(proABC), and PFP(PPI) respectively. The protein atom type preferences, calculated with protein structures from the P9468 dataset, on average protein solvent accessible surface areas are shown in the purple histogram. The y axis shows the fraction of the protein atom type in the x axis. (D) The protein atom type preferences on the epitopes from the antibody–protein complex structures in the S111 dataset are shown in the histograms colored in blue, green, cyan, and red for the SE(0), SE(0.2), PFE(proABC), and PFE(PPI), respectively. The background protein atom type preferences on average protein solvent accessible surface areas are shown in the purple histogram.

Fig. 5.

Distributions of pairwise atomistic contacts in antibody–protein interactions and in nonantibody protein–protein interaction interfaces. (A) Pairwise atomistic contacts are sorted in five groups as shown in the y axis (more details are discussed in the main text). The atom type groups are defined in the first column of Table 1. (Left) Results for antibody–protein interfaces from the S111 dataset, calculated with PFP(proABC)–PFE(proABC) interfaces. (Center) Results for antibody–protein interfaces from the S111 dataset, calculated with PFP(PPI)–PFE(PPI) interfaces. (Right) Results for PPI interfaces from the S430 dataset. The x axis shows the number of pairwise atomistic contacts per interface. The histograms are cumulative: the blue part of the histogram in the Left and Center shows the distributions for the PFP(proABC)–PFE(proABC) and PFP(PPI)–PFE(PPI) interfaces, respectively, in antibody–protein interactions; the blue+red histogram shows the distributions for the SP(0.2)–SE(0.2) interfaces; the blue+red+green histogram shows the distributions for the SP(0)–SE(0) interfaces. (Right) The same presentation method is used for the PPI interfaces defined by PPI_CL ≥ 0.45&dSASA ≥ 0.2, dSASA ≥ 0.2, and dSASA > 0, respectively. (B) Pairwise atomistic contacts involving paratope aromatic atoms are sorted into five groups (main text) as shown in the y axis. The x axis shows the number of atomistic contact pairs per interface. Other specifications are the same as in A. (C) (Left) The y axis shows the fraction of antibody–protein complexes in the S111 dataset with the number of A:B + A:C atomistic contact pairs (x axis) per interface; the purple, blue, red, and green curves are calculated with PFP(proABC)–PFE(proABC), PFP(PPI)–PFE(PPI), SP(0.2)–SE(0.2), and SP(0)/SE(0) interfaces, respectively. (Right) The y axis shows the fraction of protein–protein complexes in the S430 dataset with the number of A:B + A:C atomistic contact pairs (x axis) per interface; the blue, red, and green curves are calculated with PPI_CL ≥ 0.45&dSASA ≥ 0.2, dSASA ≥ 0.2, and dSASA > 0 interfaces, respectively. (D) Polar pairwise atomistic contacts are sorted into six groups as shown in the y axis (more details are discussed in the main text). The polar atom groups are defined in the second column of Table 1. The x axis shows the number of contacts per interface. Other specifications are the same as in A. (E) Direct H-bonded polar pairwise atomistic contacts per interface for the six polar contact groups are shown. Other specifications are the same as in A.

Fig. 6.

Propensity for tyrosine side-chain interaction on protein surface residues. (A) The residues of the lysozyme are color coded according to the tyrosyl side-chain interacting propensity (from blue to white to red representing low, medium, and high interacting propensity for tyrosyl side chain). The stick models of tyrosyl side chains are from three known anti-HEL antibodies: yellow, HyHEL-5 (PDB code: 1YQV); green, HyHEL-10 (PDB code: 3HFM); cyan, FvD1.3 (PDB code: 1VFB). The quantitative propensities are shown in Fig. S2, which shows the residue-based tyrosyl side-chain interacting propensity of HEL, plotted against the residue number of the antigen protein HEL. The tyrosyl side-chain interacting propensities are calculated with the scoring matrix shown in Table S8 (main text and SI Materials and Methods). (B) Tyrosyl side-chain interacting propensity score distributions calculated with epitope atoms (blue) and nonepitope atoms (red) in the protein antigens of the S111 dataset are compared. The t test P value for the two distributions is close to 1.