DISSECTING THE ENERGETICS OF AN ANTIBODY-ANTIGEN INTERFACE BY ALANINESHAVING AND MOLECULAR GRAFTING

Alanine-scanning mutagenesis on human growth hormone (hGH) identified 5 primary determinants (Arg 8, Asn 12, Arg 16, Asp 112, and Asp 116) f or binding to a monoclonal antibody (MAb 3) (Jin L, Fendly BM, Wells J A, 1992, Mol Biol 226:851-865). To further analyze the energetic impor tance of residues surrounding these five, we mutated all neighboring r esidues to alanine in groups of 7-16 (a procedure we call alanine shav ing). Even the most extremely mutated variant, with 16 alanine substit utions, caused less than a 10-fold reduction in binding affinity to MA b3, By comparison, mutating any 1 of the 5 primary determinants to ala nine caused a 6- to >500-fold reduction in affinity. Replacing any of the 4 charged residues (Arg 8, Arg 16, Asp 112, and Asp 116) with a ho mologous residue (i.e., Arg to Lys or Asp to Glu) caused nearly as lar ge a reduction in affinity as the corresponding alanine replacement. I t was possible to graft the 5 primary binding determinants onto a nonb inding homologue of hGH, human placental lactogen (hPL), which has 86% sequence identity to hGH. The grafted hPL mutant bound 10-fold less t ightly than hGH to MAb3 but bound as well as hGH when 2 additional fra mework mutations were introduced. Attempts to recover binding affinity by grafting the MAb3 epitope onto more distantly related scaffolds ha ving a similar 4-helix bundle motif, such as human prolactin (23% sequ ence identity) or granulocyte colony-stimulating factor, were unsucces sful. These studies show that only a small number of side chains on hG H confers tight binding affinity to MAb3 and that substituting or tran splanting them onto another scaffold has narrow design tolerances.