TY - JOUR
T1 - Salt links dominate affinity of antibody HyHEL-5 for lysozyme through enthalpic contributions
AU - Wibbenmeyer, Jamie A.
AU - Schuck, Peter
AU - Smith-Gill, Sandra J.
AU - Willson, Richard C.
PY - 1999/9/17
Y1 - 1999/9/17
N2 - The binding of murine monoclonal antibody HyHEL-5 to lysozyme has been the subject of extensive crystallographic, computational, and experimental investigations. The complex of HyHEL-5 with hen egg lysozyme (HEL) features salt bridges between Fab heavy chain residue Glu50, and Arg45 and Arg68 of HEL. This interaction has been predicted to play a dominant role in the association on the basis of molecular electrostatics calculations. The association of aspartic acid and glutamine mutants at position 50(H) of the cloned HyHEL-5 Fab with HEL and bobwhite quail lysozyme (BQL), an avian variant bearing an Arg68 → Lys substitution in the epitope, was characterized by isothermal titration calorimetry and sedimentation equilibrium. Affinities for HEL were reduced by 400-fold (E50(H)D) and 40,000-fold (E50(H)Q) (ΔΔG°estimated at 4.0 and 6.4 kcal mol-1, respectively). The same mutations reduce affinity for BQL by only 7- and 55- fold, respectively, indicating a reduced specificity for HEL. The loss of affinity upon mutation is in each case primarily due to an unfavorable change in the enthalpy of the interaction; the entropic contribution is virtually unchanged. An enthalpy-entropy compensation exists for each interaction; ΔH°decreases, while ΔS°increases with temperature. The ΔCp for each mutant interaction is less negative than the wild-type. Mutant-cycle analysis suggests the mutations present in the HyHEL-5 Fab mutants are linked to those present in the BQL with coupling energies between 3 and 4 kcal mol-1.
AB - The binding of murine monoclonal antibody HyHEL-5 to lysozyme has been the subject of extensive crystallographic, computational, and experimental investigations. The complex of HyHEL-5 with hen egg lysozyme (HEL) features salt bridges between Fab heavy chain residue Glu50, and Arg45 and Arg68 of HEL. This interaction has been predicted to play a dominant role in the association on the basis of molecular electrostatics calculations. The association of aspartic acid and glutamine mutants at position 50(H) of the cloned HyHEL-5 Fab with HEL and bobwhite quail lysozyme (BQL), an avian variant bearing an Arg68 → Lys substitution in the epitope, was characterized by isothermal titration calorimetry and sedimentation equilibrium. Affinities for HEL were reduced by 400-fold (E50(H)D) and 40,000-fold (E50(H)Q) (ΔΔG°estimated at 4.0 and 6.4 kcal mol-1, respectively). The same mutations reduce affinity for BQL by only 7- and 55- fold, respectively, indicating a reduced specificity for HEL. The loss of affinity upon mutation is in each case primarily due to an unfavorable change in the enthalpy of the interaction; the entropic contribution is virtually unchanged. An enthalpy-entropy compensation exists for each interaction; ΔH°decreases, while ΔS°increases with temperature. The ΔCp for each mutant interaction is less negative than the wild-type. Mutant-cycle analysis suggests the mutations present in the HyHEL-5 Fab mutants are linked to those present in the BQL with coupling energies between 3 and 4 kcal mol-1.
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U2 - 10.1074/jbc.274.38.26838
DO - 10.1074/jbc.274.38.26838
M3 - Article
C2 - 10480891
AN - SCOPUS:0033578783
VL - 274
SP - 26838
EP - 26542
JO - The Journal of biological chemistry
JF - The Journal of biological chemistry
SN - 0021-9258
IS - 38
ER -