Dissecting the energetics of an antibody-antigen interface by alanine shaving and molecular grafting

Protein Science : a Publication of the Protein Society
L Jin, J A Wells

Abstract

Alanine-scanning mutagenesis on human growth hormone (hGH) identified 5 primary determinants (Arg 8, Asn 12, Arg 16, Asp 112, and Asp 116) for binding to a monoclonal antibody (MAb 3) (Jin L, Fendly BM, Wells JA, 1992, J Mol Biol 226:851-865). To further analyze the energetic importance of residues surrounding these five, we mutated all neighboring residues to alanine in groups of 7-16 (a procedure we call alanine shaving). Even the most extremely mutated variant, with 16 alanine substitutions, caused less than a 10-fold reduction in binding affinity to MAb3. By comparison, mutating any 1 of the 5 primary determinants to alanine 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 homologous residue (i.e., Arg to Lys or Asp to Glu) caused nearly as large a reduction in affinity as the corresponding alanine replacement. It was possible to graft the 5 primary binding determinants onto a nonbinding homologue of hGH, human placental lactogen (hPL), which has 86% sequence identity to hGH. The grafted hPL mutant bound 10-fold less tightly than hGH to MAb3 but bound as well as hGH when 2 additional framework mutations were introduced. Attempts to recover ...Continue Reading

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Oct 7, 1998·Protein Science : a Publication of the Protein Society·M G TaylorJ F Kirsch
Jun 16, 2010·Biochemistry·Joshua M GilmoreJeffrey L Urbauer
Jul 10, 2012·Journal of Chemical Information and Modeling·Brandon S ZerbeDima Kozakov
Nov 26, 2010·Nucleic Acids Research·Joan Segura, Narcis Fernandez-Fuentes
Mar 23, 2002·Protein Science : a Publication of the Protein Society·Olga BuczekJacek Otlewski
Nov 10, 2001·Annual Review of Biomedical Engineering·J Maynard, G Georgiou
Mar 3, 1999·Proceedings of the National Academy of Sciences of the United States of America·B M Brown, R T Sauer
Sep 18, 2014·BMC Bioinformatics·Prashant ShingateRamanathan Sowdhamini
Jul 28, 2004·Journal of Molecular Biology·Kun HuangMichael A Weiss
Sep 8, 2011·Protein Science : a Publication of the Protein Society·Yu ZhangBrendan P Orner
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Dec 29, 2005·Journal of Molecular Biology·Dagmar Hobson, Olke C Uhlenbeck
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