Association energetics of cross-reactive and specific antibodies


HyHEL-8, HyHEL-10, and HyHEL-26 (HH8, HH10, and HH26, respectively) are murine monoclonal IgG1 antibodies which share over 90% variable-region amino acid sequence identity and recognize identical structurally characterized epitopes on hen egg white lysozyme (HEL). Previous immunochemical and surface plasmon resonance-based studies have shown that these antibodies differ widely in their tolerance of mutations in the epitope. While HH8 is the most cross-reactive, HH26 is rigidified by a more extensive network of intramolecular salt links and is highly specific, with both association and dissociation rates strongly affected by epitope mutations. HH10 is of intermediate specificity, and epitope mutations produce changes primarily in the dissociation rate. Calorimetric characterization of the association energetics of these three antibodies with the native antigen HEL and with Japanese quail egg white lysozyme (JQL), a naturally occurring avian variant, shows that the energetics of interaction correlate with cross-reactivity and specificity. These results suggest that the greater cross-reactivity of HH8 may be mediated by a combination of conformational flexibility and less specific intermolecular interactions. Thermodynamic calculations suggest that upon association HH8 incurs the largest configurational entropic penalty and also the smallest loss of enthalpic driving force with variant antigen. Much smaller structural perturbations are expected in the formation of the less flexible HH26 complex, and the large loss of enthalpic driving force observed with variant antigen reflects its specificity. The observed thermodynamic parameters correlate well with the observed functional behavior of the antibodies and illustrate fundamental differences in thermodynamic characteristics between cross-reactive and specific molecular recognition.



Protein-protein recognition, antibody-antigen complexes, cross-reactivity and specificity, titration calorimetry, affinity maturation, thermodynamics


Copyright 2009 Biochemistry. This is a post-print of a published paper that is available at: Recommended citation: Mohan, S., Katerina Kourentzi, Kari A. Schick, Christian Uehara, Claudia A. Lipschultz, Mauro Acchione, Morgan E. DeSantis, Sandra J. Smith-Gill, and Richard C. Willson. "Association energetics of cross-reactive and specific antibodies." Biochemistry 48, no. 6 (2009): 1390-1398. DOI: 10.1021/bi801901d. This item has been deposited in accordance with publisher copyright and licensing terms and with the author's permission.