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

@article{citeulike:5881830, 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 binding affinity by grafting the MAb3 epitope onto more distantly related scaffolds having a similar 4-helix bundle motif, such as human prolactin (23\% sequence identity) or granulocyte colony-stimulating factor, were unsuccessful. These studies show that only a small number of side chains on hGH confers tight binding affinity to MAb3 and that substituting or transplanting them onto another scaffold has narrow design tolerances.}, address = {Department of Protein Engineering, Genentech, Inc., South San Francisco, California 94080; Department of Pharmaceutical Chemistry, University of California at San Francisco, San Francisco, California 94143}, author = {Jin, Lei and Wells, James A.}, citeulike-article-id = {5881830}, citeulike-linkout-0 = {http://dx.doi.org/10.1002/pro.5560031219}, citeulike-linkout-1 = {http://www3.interscience.wiley.com/cgi-bin/abstract/121599374/ABSTRACT}, doi = {10.1002/pro.5560031219}, issn = {1469-896X}, journal = {Protein Science}, keywords = {antibody\_antigen, binding\_affinity, epitope, graft, vaccines}, number = {12}, pages = {2351--2357}, posted-at = {2009-10-03 17:56:41}, priority = {2}, title = {Dissecting the energetics of an antibody-antigen interface by alanine shaving and molecular grafting}, url = {http://dx.doi.org/10.1002/pro.5560031219}, volume = {3}, year = {1994} }

TY - JOUR ID - citeulike:5881830 L3 - citeulike-article-id:5881830 N2 - 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 binding affinity by grafting the MAb3 epitope onto more distantly related scaffolds having a similar 4-helix bundle motif, such as human prolactin (23% sequence identity) or granulocyte colony-stimulating factor, were unsuccessful. These studies show that only a small number of side chains on hGH confers tight binding affinity to MAb3 and that substituting or transplanting them onto another scaffold has narrow design tolerances. IS - 12 JF - Protein Science SN - 1469-896X AD - Department of Protein Engineering, Genentech, Inc., South San Francisco, California 94080; Department of Pharmaceutical Chemistry, University of California at San Francisco, San Francisco, California 94143 EP - 2357 TI - Dissecting the energetics of an antibody-antigen interface by alanine shaving and molecular grafting VL - 3 SP - 2351 KW - antibody_antigen KW - binding_affinity KW - epitope KW - graft KW - vaccines AU - Jin, Lei AU - Wells, James PY - 1994/// UR - http://dx.doi.org/10.1002/pro.5560031219 ER -