Salt links dominate affinity of antibody HyHEL-5 for lysozyme through enthalpic contributions

J Biol Chem. 1999 Sep 17;274(38):26838-42. doi: 10.1074/jbc.274.38.26838.


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 Glu(50), and Arg(45) and Arg(68) 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 Arg(68) --> 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) (DeltaDeltaG degrees 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; DeltaH degrees decreases, while DeltaS degrees increases with temperature. The DeltaCp 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).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Monoclonal / metabolism*
  • Antigen-Antibody Complex*
  • Arginine / metabolism
  • Aspartic Acid / metabolism
  • Chickens
  • Colinus
  • Crystallography, X-Ray
  • Glutamine / metabolism
  • Immunoglobulin Fab Fragments / metabolism
  • Models, Chemical
  • Muramidase / immunology
  • Muramidase / metabolism*
  • Protein Binding
  • Protein Conformation
  • Protein Denaturation
  • Protein Folding
  • Structure-Activity Relationship
  • Temperature


  • Antibodies, Monoclonal
  • Antigen-Antibody Complex
  • Immunoglobulin Fab Fragments
  • Glutamine
  • Aspartic Acid
  • Arginine
  • Muramidase