Probing the combining site of an anti-carbohydrate antibody by saturation-mutagenesis: role of the heavy-chain CDR3 residues.
The carbohydrate-binding site in Fab fragments of an antibody specific for Salmonella serogroup B O-polysaccharide has been probed by site-directed mutagenesis using an Escherichia coli expression system. Of the six hypervariable loops, the CDR3 of the heavy chain was selected for exhaustive study because of its significant contribution to binding-site topography. A total of 90 mutants were produced and screened by an affinity electrophoresis/Western blotting method. Those of particular interest were further characterized by enzyme immunoassay, and on this basis seven of the mutant Fabs were selected for thermodynamic characterization by titration microcalorimetry. With regard to residues that hydrogen bond to ligand through backbone interactions, Gly102H could not be substituted, while several side chains could be introduced at Gly100H and Tyr103H with relatively little effect on antigen binding. There was, however, a preference for nonpolar side chains at position 103H. Substitution of His101H with carboxylate and amide side chains gave mutants with binding affinities approaching that of the wild type; complete side-chain removal by mutation to Gly was tolerated with a 10-fold reduction in binding constant. Analysis of binding by titration microcalorimetry revealed some dramatic thermodynamic changes hidden by the similarity of the binding constants. Similar effects were observed with residue changes in an Arg-Asp salt-bridge at the base of the loop. These results indicate that alterations to higher affinity anti-carbohydrate antibodies are characterized by an enthalpy-entropy compensation factor which allows for fundamental changes in the nature of the binding interactions but impedes engineering for increases in affinity.
Brummell
D A
DA
Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario.
Sharma
V P
VP
Anand
N N
NN
Bilous
D
D
Dubuc
G
G
Michniewicz
J
J
MacKenzie
C R
CR
Sadowska
J
J
Sigurskjold
B W
BW
Sinnott
B
B
D000595:Amino Acid Sequence; D000907:Antibodies, Bacterial / Q000737:chemistry / Q000235:genetics; D001483:Base Sequence; D001666:Binding Sites, Antibody / Q000235:genetics; D015153:Blotting, Western; D002151:Calorimetry; D050260:Carbohydrate Metabolism; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000276:immunology*; D004269:DNA, Bacterial; D006860:Hydrogen Bonding; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry* / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000276:immunology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012475:Salmonella / Q000276:immunology
D000907:Antibodies, Bacterial; D002241:Carbohydrates; D004269:DNA, Bacterial; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region