Salt links dominate affinity of antibody HyHEL-5 for lysozyme through enthalpic contributions.
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).
Wibbenmeyer
J A
JA
Department of Biology and Biochemistry, University of Houston, Houston, Texas 77024-4792, USA.
Schuck
P
P
Smith-Gill
S J
SJ
Willson
R C
RC
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D000936:Antigen-Antibody Complex; D001120:Arginine / Q000378:metabolism; D001224:Aspartic Acid / Q000378:metabolism; D002645:Chickens; D003088:Colinus; D018360:Crystallography, X-Ray; D005973:Glutamine / Q000378:metabolism; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism; D008956:Models, Chemical; D009113:Muramidase / Q000276:immunology / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013329:Structure-Activity Relationship; D013696:Temperature
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D007140:Immunoglobulin Fab Fragments; D005973:Glutamine; D001224:Aspartic Acid; D001120:Arginine; D009113:Muramidase