It is well known that protein-protein interactions are fundamental to most of biological processes, including signal transduction, gene translation or transcription, enzyme activation or inhibition, and immune recognition. Contrast to the interaction between other normal protein-protein complexes, the binding between antibody and antigen is highly specific and stable . Previous studies have revealed that this specificity is dominantly determined by the contacting interface which is mainly composed of the variable domains of antibody [2, 3, 4, 5, 6]. It has been reported that with only 5% sequence change in the variable domains, antibodies can recognize specifically and bind tightly to 1010 different antigens . It is always interesting to study how antibody can recognize so large variety of antigens with so little change in sequence and thus deserve further investigation.
Characteristics of the binding interfaces of antibodies such as the size, shape, chemical, physical or structural complementation have been analyzed from different perspectives for a deeper understanding to antibody-antigen interactions [8, 9, 10]. Although the hydrophobic effect was considered as the major driving force for the general protein binding, the study of Tsai and co-workers indicated that hydrophobic amino acids were not the dominant part and a higher proportion of charged and polar residues could be found at the binding interfaces . Subsequent comparison between the interfaces of six antibody-antigen complexes and other protein-protein complexes reported that the residues composing the interface of antibody-antigen complexes were more polar, protruding and accessible . Currently, more and more results suggest that there are significant differences between the interfaces of immune and non-immune protein complexes. For instance, the interfaces of antigen-antibody complexes are particularly rich in Tyr, Arg, His, Phe and Trp [13, 14, 15, 16, 17]. Although further observations indicate that this enrichment ranking alters slightly with different data size, aromatic residues have always been found to occur more frequently at the binding sites of antibodies.
On the other hand, the contribution of enriched residues to the binding selectivity and specificity of antibody has aroused extensive interest . By the virtue of alanine scanning mutagenesis, the energetic contribution of respective residue to protein binding could be evaluated with the observed free energy variation derived from the introduced mutation [19, 20, 21, 22]. The results of mutations have frequently indicated that the affinity change of mutating certain interfacial residue is far more unpredictable which is considered as the hot spot residue at the binding interface . Some interfacial Tyr or Trp residues, but not all of them, have subsequently been identified as hot spots that contribute significantly to the high affinity of antibody-antigen interactions . Despite that the different conclusions have been derived from several individual experiments adopting different datasets and methodologies, the enrichment and important role of Tyr and Trp residues have been widely noticed at the binding interfaces of antibodies.
However, several questions are still open to be answered. Why are these aromatic residues enriched and preferred? How do they affect the affinity so largely and form the “hot spots”? Are there any special local environment existing around the Tyr or Trp residues to facilitate the interaction at the interface? … In order to answer these questions, an in-depth and large-scale analysis would be helpful focusing on the aromatic residues at the binding interfaces of antibody-antigen complexes. Here, we conducted a comprehensive analysis of 82 non-redundant interfaces of antibodies covering 509 immune complexes from the PDB database  and IMGT/3Dstructure-DB [26, 27]. The residue connection and spatial distribution were scanned for all interfacial Tyr and Trp residues following an enrichment analysis. Systematic study was further focused on the relationship between the distribution pattern and the energetic contribution of aromatic interfacial residues in order to reveal the function of the aromatic residues in the binding interfaces of antibodies.