Differences in Electrostatic Properties at Antibody–Antigen Binding Sites: Implications for Specificity and Cross-Reactivity

Abstract

Antibodies HyHEL8, HyHEL10, and HyHEL26 (HH8, HH10, and HH26, respectively) recognize highly overlapping epitopes on hen egg-white lysozyme (HEL) with similar affinities, but with different specificities. HH8 binding to HEL is least sensitive toward mutations in the epitope and thus is most cross-reactive, HH26 is most sensitive, whereas the sensitivity of HH10 lies in between HH8 and HH26. Here we have investigated intra- and intermolecular interactions in three antibody-protein complexes: theoretical models of HH8-HEL and HH26-HEL complexes, and the x-ray crystal structure of HH10-HEL complex. Our results show that HH8-HEL has the lowest number and HH26-HEL has the highest number of intra- and intermolecular hydrogen bonds. The number of salt bridges is lowest in HH8-HEL and highest in HH26-HEL. The binding site salt bridges in HH8-HEL are not networked, and are weak, whereas, in HH26-HEL, an intramolecular salt-bridge triad at the binding site is networked to an intermolecular triad to form a pentad. The pentad and each salt bridge of this pentad are exceptionally stabilizing. The number of binding-site salt bridges and their strengths are intermediate in HH10-HEL, with an intramolecular triad. Our further calculations show that the electrostatic component contributes the most to binding energy of HH26-HEL, whereas the hydrophobic component contributes the most in the case of HH8-HEL. A "hot-spot" epitope residue Lys-97 forms an intermolecular salt bridge in HH8-HEL, and participates in the intermolecular pentad in the HH26-HEL complex. Mutant modeling and surface plasmon resonance (SPR) studies show that this hot-spot epitope residue contributes significantly more to the binding than an adjacent epitope residue, Lys-96, which does not form a salt bridge in any of the three HH-HEL complexes. Furthermore, the effect of mutating Lys-97 is most severe in HH26-HEL. Lys-96, being a charged residue, also contributes the most in HH26-HEL among the three complexes. The SPR results on these mutants also highlight that the apparent "electrostatic steering" on net on rates actually act at post-collision level stabilization of the complex. The significance of this work is the observed variations in electrostatic interactions among the three complexes. Our work demonstrates that higher electrostatics, both as a number of short-range electrostatic interactions and their contributions, leads to higher binding specificity. Strong salt bridges, their networking, and electrostatically driven binding, limit flexibilities through geometric constrains. In contrast, hydrophobic driven binding and low levels of electrostatic interactions are associated with conformational flexibility and cross-reactivity.

Antibodies Use Heme as a Cofactor to Extend Their Pathogen Elimination Activity and to Acquire New Effector Functions

“…Indeed, the salt concentration dependence of the kinetic rate constants indicated that the native antibody used electrostatic interactions, whereas the heme-exposed antibody relied mainly on nonpolar forces for binding to the same antigen. It has been shown that antibodies that use the hydrophobic effect for antigen binding are usually polyreactive (33)(34)(35)(36). Our observation on the enlargement of the antibody repertoire upon heme exposure is in full agreement with these findings.…”

Internal Point Mutations of the Capsid Modify the Serotype of Rice Yellow Mottle Virus

“…Moreover, the absence of response with this MAb is associated with substitutions that modify the nature of the residues (in particular, the change at position 115 from polar to hydrophobic). The role of the electrostatic interactions involving, for example, alanine or threonine at the antigen-antibody binding site was previously demonstrated (24). We concluded that the residues 114 and 115 probably correspond to the epitope recognized by MAb D.…”

Mimotope mapping as a complementary strategy to define allergen IgE-epitopes: Peach Pru p 3 allergen as a model☆

“…2C), it seems also reasonable to predict a weak ionic activity associated to these IgE-binding surfaces. Since it has been proposed that strong electrostatic interactions should be expected in high-affinity antibody-antigen complexes whereas weaker electrostatic activity inherent to less specific contacts should be indicative of cross-reactivity (Sinha et al, 2002), one can speculate that these surfaces should preferably exhibit cross-reactive behaviour.…”