Structural Analysis of Mutants of High-Affinity and Low-Affinity p -Azophenylarsonate-Specific Antibodies Generated by Alanine Scanning of Heavy Chain Complementarity-Determining Region 2

Abstract

Alanine scanning was used to determine the affinity contributions of 10 side chain amino acids (residues at position 50–60 inclusive) of H chain complementarity-determining region 2 (HCDR2) of the somatically mutated high-affinity anti-p-azophenylarsonate Ab, 36–71. Each mutated H chain gene was expressed in the context of mutated (36–71L) and the unmutated (36–65L) L chains to also assess the contribution of L chain mutations to affinity. Combined data from fluorescence quenching, direct binding, inhibition, and capture assays indicated that mutating H:Tyr50 and H:Tyr57 to Ala in the 36–71 H chain results in significant loss of binding with both mutated (36–71L) or unmutated (36–65L) L chain, although the decrease was more pronounced when unmutated L chain was used. All other HCDR2 mutations in 36–71 had minimal effect on Ab affinity when expressed with 36–71 L chain. However, in the context of unmutated L chain, of H:Gly54 to Ala resulted in significant loss of binding, while Abs containing Asn52 to Ala, Pro53 to Ala, or Ile58 to Ala mutation exhibited 4.3- to 7.1-fold reduced affinities. When alanine scanning was performed instead on certain HCDR2 residues of the germline-encoded (unmutated) 36–65 Ab and expressed with unmutated L chain as Fab in bacteria, these mutants exhibited affinities similar to or slightly higher than the wild-type 36–65. These findings indicate an important role of certain HCDR2 side chain residues on Ab affinity and the constraints imposed by L chain mutations in maintaining Ag binding.