Immunological optimization of a generic hydrophobic pocket for high affinity hapten binding and Diels-Alder activity.
Antibody 1E9, which binds a tetrachloronorbornene derivative with subnanomolar affinity and catalyzes the Diels-Alder reaction between tetrachlorothiophene dioxide and N-ethylmaleimide with high efficiency, arose from a family of highly restricted germ-line immunoglobulins that bind diverse hydrophobic ligands. Two somatic mutations, one at position L89 in the light chain (SerL89Phe) and another at position H47 in the heavy chain (TrpH47Leu), have been postulated to be responsible for the unusually high degree of shape and chemical complementarity observed in the crystal structure of 1E9 complexed with its hapten. To test this hypothesis, the germ-line sequence at these two positions was restored by site-directed mutagenesis. The ensuing 160 to 3900-fold decrease in hapten affinity and the complete loss of catalytic activity support the hypothesis that these somatic mutations substantially remodel the antibody binding pocket. Mutation of the highly conserved hydrogen-bond donor AsnH35, which sits at the bottom of the active site and is a hallmark of this family of antibodies, is also catastrophic with respect to hapten binding and catalysis. In contrast, residues in the CDR H3 loop, which contributes a significant fraction of the hapten-contacting protein surface, have a more subtle influence on the properties of 1E9. Interestingly, while most changes in this loop have neutral or modestly deleterious effects, replacement of MetH100b at the floor of the pocket with phenylalanine leads to a significant sevenfold increase in catalytic activity. The latter result is surprising given the unusually close fit of the parent antibody to the transition-state analogue. Further fine-tuning of the interactions between 1E9 and its ligands by introducing mutations outside the active site could conceivably yield substantially more active catalysts.
Piatesi
Andrea
A
Laboratorium für Organische Chemie, Swiss Federal Institute of Technology ETH, ETH-Hönggerberg, 8093 Zürich, Switzerland.
Hilvert
Donald
D
D019943:Amino Acid Substitution / Q000235:genetics; D000906:Antibodies / Q000737:chemistry / Q000276:immunology; D001665:Binding Sites; D002384:Catalysis; D006241:Haptens / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D006860:Hydrogen Bonding; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D015394:Molecular Structure; D009154:Mutation / Q000235:genetics; D017434:Protein Structure, Tertiary
D000906:Antibodies; D006241:Haptens