PROTEIN STRUCTURE AND FOLDING
Complementary Combining Site Contact Residue Mutations of the Anti-digoxin Fab 26–10 Permit High Affinity Wild-type Binding*

https://doi.org/10.1074/jbc.M110444200Get rights and content
Under a Creative Commons license
open access

Antibody 26–10, obtained in a secondary immune response, binds digoxin with high affinity (K a = 1.3 × 1010m−1) because of extensive shape complementarity. We demonstrated previously that mutations of the hapten contact residue HTrp-100 to Arg (where H refers to the heavy chain) resulted in increased specificity for digoxin analogs substituted at the cardenolide 16 position. However, mutagenesis of H:CDR1 did not result in such a specificity change despite the proximity of the H:CDR1 hapten contact residue Asn-35 to the cardenolide 16 position. Here we constructed a bacteriophage-displayed library containing randomized mutations at H chain residues 30–35 in a 26–10 mutant containing Arg-100 (26–10-RRALD). Phage were selected by panning against digoxin, gitoxin (16-OH), and 16-acetylgitoxin coupled to bovine serum albumin. Clones that retained wild-type Asn at position 35 showed preferred binding to gitoxin, like the 26–10-RRALD parent. In contrast, clones containing Val-35 selected mainly on digoxin-bovine serum albumin demonstrated a shift back to wild-type specificity. Several clones containing Val-35 bound digoxin with increased affinity, approaching that of the wild type in a few instances, in contrast to the mutation Val-35 in the wild-type 26–10 background, which reduces affinity for digoxin 90-fold. It has therefore proven possible to reorder the 26–10 binding site by mutations including two major contact residues on opposite sides of the site and yet to retain high affinity for binding for digoxin. Thus, even among antibodies that have undergone affinity maturation in vivo, different structural solutions to high affinity binding may be revealed.

Cited by (0)

Published, JBC Papers in Press, February 19, 2002, DOI 10.1074/jbc.M110444200

*

This work was supported by Grant HL47415 from the National Institutes of Health (to M.N.M.) and Grant CA24432 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

Present address: Dept. of Hematology/Oncology, Children's Hospital Medical Center, 3333 Burnet Ave., Cincinnati, OH 45229.

Both authors contributed equally to this work.

Present address: Dept. of Respiratory Diseases, Wyeth Research, 200 Cambridge Park Dr., Cambridge, MA 02140.