Local and global anatomy of antibody-protein antigen recognition

Abstract

Deciphering antibody-protein antigen recognition is of fundamental and practical significance. We constructed an antibody structural dataset, partitioned it into human and murine subgroups, and compared it with nonantibody protein-protein complexes. We investigated the physicochemical properties of regions on and away from the antibody-antigen interfaces, including net charge, overall antibody charge distributions, and their potential role in antigen interaction. We observed that amino acid preference in antibody-protein antigen recognition is entropy driven, with residues having low side-chain entropy appearing to compensate for the high backbone entropy in interaction with protein antigens. Antibodies prefer charged and polar antigen residues and bridging water molecules. They also prefer positive net charge, presumably to promote interaction with negatively charged protein antigens, which are common in proteomes. Antibody-antigen interfaces have large percentages of Tyr, Ser, and Asp, but little Lys. Electrostatic and hydrophobic interactions in the Ag binding sites might be coupled with Fab domains through organized charge and residue distributions away from the binding interfaces. Here we describe some features of antibody-antigen interfaces and of Fab domains as compared with nonantibody protein-protein interactions. The distributions of interface residues in human and murine antibodies do not differ significantly. Overall, our results provide not only a local but also a global anatomy of antibody structures.

Keywords: allosteric regulation; antibody-antigen recognition; binding epitopes; mAb drugs; protein-protein interaction.

Figure 1

Antibodies use less charged residue and less hydrophobic residues in protein-protein interaction. Amino acid interaction propensities at different cutoff distances for protein-protein interactions. A. 5 Å; B. 3.5 Å; and C. 3.0 Å. Blue bar: Non-antibody proteins-protein interactions, red bar: Antibody amino acids in antibody-protein antigen interactions.

Figure 2

Antibodies use Tyr and Ser, instead of Lys to interact with Asp/Glu on antigen interface. (A)–(D). Amino acid propensities on antibody interface to recognize Arg, Lys, Asp, and Glu residues on antigen interface, respectively.

Figure 3

Antibodies minimize side-chain entropy in antigen recognition. (A) Side-chain entropies for the residues within different distances to antigen heavy atoms. (B) Density distributions of sidechain entropy for the amino-acids in antibody proteins mostly lower than non-antibody proteins, except the junction region between variable and constant regions.

Figure 4

Tyr, Ser, and Asp residues are water bridge hot spots in antibody-protein interactions. (A) antibody amino acid propensities to have water bridged interaction with antigen at 3.5Å cutoff distance. (B) and (C) Change of amino acid frequencies between water bridged interaction and direct interaction for antibody (B) and antigen (C).

Figure 5

Human antibodies use less tyrosine and more serine than murine antibody in antigen recognition. Green bar: Non-antibody proteins-protein interactions; red bar: Human antibodies; and blue bar: murine antibodies. (A) and (B) Comparison of human and murine amino acid propensities on antibody (A) and antigen (B) interfaces. (C) Antibody and antigen have opposite residue propensity in comparison with non-antibody related protein-protein interaction. X-axis: P_i (Antibody) - P_i (nonantibody receptor), Y-axis: P_i (Antigen) - P_i (nonantibody ligand). (D) Total amino acid frequencies on the antibody-antigen interfaces follow the same trend of general protein-protein interaction. X-axis: (P_i (Antibody) + P_i (antigen))/2, Y-axis: (P_i (protein receptor) + P_i (protein ligand))/2.

Figure 6

Antibodies have organized charge distributions based on the radial distance away from antigen heavy atoms. Antigen is represented as orange ribbon. The contact layer within 5 Å to antigen is highly negative, followed by positively charged layer. The non-antibody proteins have different distribution pattern.

Figure 7

Global distribution of antibody residues based on their distances from antigen C_α-carbon underlies structural aspect of allosteric communication across Fab domain. (A–C): residue distribution density away from antigen for ionizable residues for human antibody, mouse/rat antibody, and non-antibody proteins, respectively. (D–F) The corresponding charge distributions residues for human antibody, mouse/rat antibody, and non-antibody proteins, respectively.

Figure 8

Germline VDJ recombination of antibody indicates that the antibodies are mostly positively charged.

Figure 9

Protein net-charge distributions for antibody-antigen complex and non-antibody protein complex indicate that human antibody have more charge interaction correlation.