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Abstract
Antibodies protect organisms from a huge variety of foreign antigens. Antibody diversity originates from both genetic and structural levels. Antigen recognition relies on complementarity between antigen-antibody interfaces. Recent methodological advances in structural biology and the accompanying rapid increase of the number of crystal structures of proteins have enabled atomic-level manipulation of protein structures to effect alterations in function. In this study, we explored the designability of electrostatic complementarity at an antigen-antibody interface on the basis of a crystal structure of the complex. We designed several variants with altered charged residues at the interface and characterized the designed variants by surface plasmon resonance, circular dichroism, differential scanning calorimetry, and molecular dynamics simulations. Both successes and failures of the structure-based design are discussed. The variants that compensate electrostatic interactions can restore the interface complementarity, enabling the cognate antigen-antibody binding. Retrospectively, we also show that these mutational effects could be predicted by the simulations. Our study demonstrates the importance of charged residues on the physical properties of this antigen-antibody interaction and suggests that computational approaches can facilitate design of antibodies that recognize a weakly immunogenic antigen.
Highlights
Antigens far outnumber antibody genes, sufficient antibody diversity results from genetic and structural variation[1]
Miklos et al subsequently designed thermostable antibodies based on a supercharging strategy[14]; they demonstrated that the designed antibodies had better thermal stability and binding affinity than the parent antibodies even though the altered positions were not in the complementarity determining region (CDR) but in the framework regions (FRs)
These analyses indicate that Molecular dynamics (MD) simulations can reveal dynamic influences that cannot be detected by analysis of a static crystal structure and can be used to predict the effects of protein manipulations on function
Summary
Antigens far outnumber antibody genes, sufficient antibody diversity results from genetic and structural variation[1]. Fukunaga and Tsumoto improved the affinity of an anti-human cardiac troponin I antibody by substitution of multiple amino acids in FRs with charged residues[16] These studies indicate that manipulating electrostatic interactions can improve physicochemical properties of antigen-antibody interactions. Clark et al attempted direct calculation of relative antigen-antibody binding affinities using a free energy perturbation protocol and replica exchange solute tempering method and was able to predict the effect of alanine mutations on the relative binding affinities[19] These analyses indicate that MD simulations can reveal dynamic influences that cannot be detected by analysis of a static crystal structure and can be used to predict the effects of protein manipulations on function. We attempted to predict designability of the charged residues solely using a computational approach and discuss a possible strategy to efficiently elicit antibodies against weakly immunogenic antigens by surface charge manipulations
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