Structural Analysis of Mutants of High-Affinity and Low-Affinityp-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.

Probing a protein–protein interaction by in vitro evolution

“…Homologue scanning, an alternative method that involves replacement of wild-type residues with amino acids having similar side-chain chemistries, is thought to further define a subset of paratope residues that are an absolute requirement for antigen binding (9). In the majority of cases, it has been concluded that residues within the functional paratope should not be randomized for improved potency, because they are likely to be intolerant of any amino acid substitutions (11,12). Alanine and homologue scanning are therefore reliable methods to determine which residues to avoid mutating during the process of antibody optimization.…”

Detect, correct, retract: How to manage incorrect structural models

“…A register-shift error in the complementarity determining region (CDR) L2 residues L51–L57 was also corrected. This register shift is not found in the search model (1jfq; Fab 36–71 with anti- p -azophenylarsonate; [68]) used for molecular replacement. However, this error could easily be propagated into any structures using 2a6i as a molecular replacement probe.…”