Switching antibody specificity through minimal mutation.
Antibody 1E9, which was elicited with a hexachloronorbornene derivative and catalyzes the Diels-Alder reaction between tetrachlorothiophene dioxide and N-ethylmaleimide with high efficiency, was successfully reengineered to bind a range of structurally diverse steroids with nanomolar affinities. Remarkably, two mutations (Leu(H47)Trp/Arg(H100)Trp) out of 36 total sequence differences suffice to switch the selectivity of 1E9 to that of the progesterone-binding antibody DB3. In contrast to the double mutant, which tightly binds multiple steroids with differently configured A-B ring junctions, the individual Leu(H47)Trp and Arg(H100)Trp single mutants both exhibit significantly greater specificity than DB3, preferentially binding 5alpha-pregnan-3beta-ol-20-one (K(d) approximately 5 nM) over other steroids. These findings illustrate how easily differently shaped binding pockets can be created through subtle changes to the same primordial germ line template.
Piatesi
Andrea
A
Laboratory of Organic Chemistry, ETH Zürich, Hönggerberg HCI F 339, CH-8093 Zürich, Switzerland.
Aldag
Caroline
C
Hilvert
Donald
D
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000378:metabolism*; D000918:Antibody Specificity; D001666:Binding Sites, Antibody / Q000235:genetics; D003429:Cross Reactions / Q000235:genetics*; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000379:methods; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000502:physiology; D011374:Progesterone / Q000276:immunology* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013256:Steroids / Q000276:immunology / Q000378:metabolism
D000911:Antibodies, Monoclonal; D050505:Mutant Proteins; D013256:Steroids; D011374:Progesterone