Immunological Optimization of a Generic Hydrophobic Pocket for High Affinity Hapten Binding and Diels–Alder Activity

Abstract

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.

Closely related antibody receptors exploit fundamentally different strategies for steroid recognition

“…Directional interactions, such as those provided by the polar atoms of the highly conserved Asn H35 , contribute significantly to catalysis and ligand recognition of 1E9 and DB3 (4,8,10). Antigen specificity of DB3 has been proposed to be focused on conserved interactions with the steroid D rings, such as hydrogen bond acceptor requirements at C17 and C20 of the steroids, and conserved van der Waals contacts with the buried steroid D ring (4,6).…”

“…1 A) to which it binds with nanomolar affinity. Structural analysis and theoretical calculations have attributed the catalytic efficiency of 1E9 to enthalpic stabilization of the reaction intermediate, near-perfect shape complementarity of the hydrophobic binding site for the transition state, and a strategically placed hydrogen bond (8)(9)(10).…”

“…Interestingly, some of the key combining site residues are identical; the hallmark residue of the VGAM3. 8 cases (4,10). Otherwise, for DB3, specific interactions between the cavity-lining residues and the steroid skeleton, particularly with the D ring, are essential (4, 5), whereas, for efficient catalysis by 1E9, shape complementarity combined with a few specific interactions are most important (8,9).…”

Switching Antibody Specificity through Minimal Mutation

“…3). The strategically positioned hydrogen bond between Asn H35 and one of the carbonyl groups of 6 stabilizes the complex and has been shown to be essential for catalytic activity, 20 while the indole ring of Trp H50 provides important π-stacking interactions with the succinimide group of the hapten. Two somatic mutations, Ser L89 Phe and Trp H47 Leu, which are unique to 1E9, are crucial for both the catalytic and binding properties of this antibody.…”

Sandwich Assay for Tacrolimus Using 2 Antitacrolimus Antibodies

“…This is because a bound hapten is mostly buried in the surface cavity of a binding protein (3)(4)(5)(6)(7) and tends to become inaccessible to a second binding event by another protein molecule due to steric hindrance. Therefore, most immunoassays for detection of haptens use a competitive format, in which only 1 antihapten antibody is used and the hapten to be detected competes with a labeled hapten for binding to the antibody.…”