Structural evidence for a programmed general base in the active site of a catalytic antibody

Abstract

The crystal structure of the complex of a catalytic antibody with its cationic hapten at 1.9-Å resolution demonstrates that the hapten amidinium group is stabilized through an ionic pair interaction with the carboxylate of a combining-site residue. The location of this carboxylate allows it to act as a general base in an allylic rearrangement. When compared with structures of other antibody complexes in which the positive moiety of the hapten is stabilized mostly by cation-interactions, this structure shows that the amidinium moiety is a useful candidate to elicit a carboxylate in an antibody combining site at a predetermined location with respect to the hapten. More generally, this structure highlights the advantage of a bidentate hapten for the programmed positioning of a chemically reactive residue in an antibody through charge complementarity to the hapten.A llylic rearrangements play a fundamental role in the biosynthesis of terpenes and steroid hormones and in the biodegradation of fatty acids (1). The enzymatic rearrangement of ␤-␥ unsaturated ketones requires a general base, usually a carboxylate, to abstract the ␣-proton of the ketone ( Fig. 1; refs. 1 and 2). This reaction leads to a dienol or dienolate high-energy intermediate 3 that does not possess a charge complementing that of the carboxylate. Therefore, antibodies elicited against a hapten that mimics the transition state or the dienol intermediate of allylic rearrangement would acquire a properly positioned general base carboxylate to catalyze this reaction only by serendipity. However, the extensive experience available on catalytic antibodies (3, 4) and the structures of catalytic antibodies elicited by transition state analogue haptens show that properly positioned chemically reactive residues are rarely present in the active site (5). An alternative approach that aims to generate functional residues, also termed ''bait and switch'' (6, 7), uses a charged hapten to induce the required complementary charged residue (8, 9). Haptens containing a positive charge have provided antibody catalysts for important reactions such as acyl transfer (7), elimination (9, 10), and phosphodiester hydrolysis (11). However, in the absence of structural data, it is not possible to establish unambiguously the nature and identity of the catalytic residue that has been induced and the relationship between the location of the haptenic charge and the position of the catalytic residue in the antibody combining site.Indeed, in the few cases in which the use of a hapten containing a positively charged moiety successfully induced catalytic antibodies and in which the structure of the hapten-antibody complex was determined, there was no negatively charged residue in the active site directly facing the positive charge, but stabilization of the haptenic charge was mediated mostly by cation-interactions (12-14). Herein, we report the structure, at 1.87-Å resolution, of the complex of an antibody catalyzing an allylic rearrangement with its cationic hapten. We provide direct ...

Structural origins of efficient proton abstraction from carbon by a catalytic antibody

“…However, Glu L34 has been estimated to be 10,000 times less effective as a base than Glu H50 in 34E4 (20). Although the carboxylate side chain of Glu L34 is well ordered and directed into the active site where it forms a salt bridge with the cationic hapten, the hydrophobic 4B2-binding pocket is more than three times larger than that of 34E4 (Ϸ8 Å ϫ 8 Å ϫ 12 Å) (44). As a consequence of its poor shape complementarity to planar 1, the probability of constraining the benzisoxazole substrate in a productive orientation relative to the catalytic base will be low.…”

“…Antibody 4B2, which was generated against compound 5 (43), also exploits a glutamate base [Glu L34 (44)] with an elevated pK a to promote the decomposition of 1. However, Glu L34 has been estimated to be 10,000 times less effective as a base than Glu H50 in 34E4 (20).…”

Bivalent Fv Antibody Fragments Obtained by Substituting the Constant Domains of a Fab Fragment with Heterotetrameric Molybdopterin Synthase

“…This catalytic antibody was chosen as a model, since it has been characterized structurally and expressed as an active catalytic scFv. [19][20][21] This approach demonstrates the ability of this original antibody format to drive the association of the Fv light and heavy chains through MPTS MoaD and MoaE dimerization and promote the antibody bi-valency through MPTS heterotetramerization.…”

Structural Basis for D-Amino Acid Transamination by the Pyridoxal 5′-Phosphate-dependent Catalytic Antibody 15A9

“…The lack of catalytic residues is one of the reasons why the rate enhancement brought about by catalytic antibodies only occasionally approaches that of enzymes. The "bait and switch" strategy, which uses a charged hapten to elicit a programmed chemically reactive charged residue (4,5), and the related procedure of "reactive immunization" (6) have been developed for obtaining catalytic antibodies endowed with reactive residues. Another possibility is to expand the catalytic scope of antibodies by incorporating a nonproteinaceous cofactor (7) that contributes to the catalytic efficacy while the antibody ensures substrate specificity and possibly reaction specificity.…”