Interaction of a designed interleukin-10 epitope mimic with an antibody studied by isothermal titration microcalorimetry.
The mechanism of recognition of proteins and peptides by antibodies and the factors determining binding affinity and specificity are mediated by essentially the same features. However, additional effects of the usually unfolded and flexible solution structure of peptide ligands have to be considered. In an earlier study we designed and optimized six peptides (pepI to pepVI) mimicking the discontinuous binding site of interleukin-10 for the anti-interleukin-10 monoclonal antibody (mab) CB/RS/1. Three of them were selected for analysis of their solution conformation by circular dichroism measurements. The peptides differ in the content of alpha-helices and in the inducibility of helical secondary structures by trifluoroethanol. These properties, however, do not correlate with the binding affinity. PepVI, a 32-mer cyclic epitope mimic, has the highest affinity to mab CB/RS/1 identified to date. CD difference spectroscopy suggests an increase of the alpha-helix content of pepVI with complex formation. Binding of pepVI to mab CB/RS/1 is characterized by a large negative, favorable binding enthalpy and a smaller unfavorable loss of entropy (DeltaH degrees = -16.4 kcal x mol(-1), TDeltaS degrees = -6.9 kcal x mol(-1)) resulting in DeltaG degrees = -9.5 kcal x mol(-1) at 25 degrees C as determined by isothermal titration calorimetry. Binding of pepVI is enthalpically driven over the entire temperature range studied (10-35 degrees C). Complex formation is not accompanied by proton uptake or release. A negative heat capacity change DeltaC(p) of -0.354 kcal x mol(-1) x K(-1) was determined from the temperature dependence of DeltaH degrees. The selection of protein mimics with the observed thermodynamic properties is promoted by the applied identification and iterative optimization procedure.
Welfle
K
K
Max-Delbrück-Centrum für Molekulare Medizin, Berlin, Germany. welfle@mdc-berlin.de
Misselwitz
R
R
Sabat
R
R
Volk
H D
HD
Schneider-Mergener
J
J
Reineke
U
U
Welfle
H
H
D000937:Antigen-Antibody Reactions; D001666:Binding Sites, Antibody; D001667:Binding, Competitive / Q000276:immunology; D002151:Calorimetry; D002942:Circular Dichroism; D018985:Epitopes, B-Lymphocyte / Q000276:immunology*; D016753:Interleukin-10 / Q000276:immunology*; D018716:Molecular Mimicry / Q000276:immunology; D019151:Peptide Library
D018985:Epitopes, B-Lymphocyte; D019151:Peptide Library; D016753:Interleukin-10