A structural basis for the activity of retro-Diels–Alder catalytic antibodies: Evidence for a catalytic aromatic residue

Abstract

The nitroxyl synthase catalytic antibodies 10F11, 9D9, and 27C5 catalyze the release of nitroxyl from a bicyclic pro-drug by accelerating a retro-Diels-Alder reaction. The Fabs (antigen-binding fragments) of these three catalytic antibodies were cloned and sequenced. Fab 9D9 was crystallized in the apo-form and in complex with one transition state analogue of the reaction. Crystal structures of Fab 10F11 in complex with ligands mimicking substrate, transition state, and product have been determined at resolutions ranging from 1.8 to 2.3 Å. Antibodies 9D9 and 10F11 show increased shape complementarity (as quantified by the program SC) to the hapten and to a modeled transition state as compared with substrate and product. The shape complementarity is mediated to a large extent by an aromatic residue (tyrosine or tryptophan) at the bottom of the hydrophobic active pocket, which undergoes -stacking interactions with the aromatic rings of the ligands. Another factor contributing to the different reactivity of the regioisomers probably arises because of hydrogen-bonding interactions between the nitroxyl bridge and the backbone amide of PheH101 and possibly a conserved water molecule.

Structural contributions to multidrug recognition in the multidrug resistance (MDR) gene regulator, BmrR

“…The values calculated for BmrR are somewhat reduced relative to those found for ligand-specific ligand prototypes, which bury cognate ligands ca. 90% and exhibit Sc values >0.7 (19,20) (SI Appendix, Table S2). In agreement with X-ray structures of QacR and other MDR proteins, drug recognition by BmrR is dominated by apolar contacts involving aromatic and hydrophobic residues (SI Appendix, Fig.…”

Structural basis for Diels-Alder ribozyme-catalyzed carbon-carbon bond formation

“…6). The anthracene ring is stacked over a tryptophan, similar to the position of A3 in the ribozyme, with a few direct and water-mediated hydrogen bonds observed between heteroatoms on the bicyclic adduct and the amide backbone 41 .…”

“…Several such antibodies have been crystallized in complex with either products or transition state analogs 36,[39][40][41] . Common features observed in these structures are the formation of hydrophobic cores lined predominantly by the side chains of tyrosine, phenylalanine, valine and tryptophan residues, and the stacking of one reactant over an aromatic residue, preferably tryptophan.…”

“…Common features observed in these structures are the formation of hydrophobic cores lined predominantly by the side chains of tyrosine, phenylalanine, valine and tryptophan residues, and the stacking of one reactant over an aromatic residue, preferably tryptophan. Notably, the retro-Diels-Alderase catalytic antibody 10F11, the only antibody using anthracene as the diene 41 , forms a hydrophobic catalytic pocket with an overall shape similar to the Diels-Alder ribozyme pocket (Fig. 6).…”

Computational Enzyme Design

“…[48] Density functional theory (DFT) calculations were employed on models of the active site to study the interactions that can stabilize the transition state. [49] Kim et al reviewed and compared the noncovalent catalysis of Diels-Alder reactions by cyclodextrins, selfassembling capsules, antibodies, and RNAses, and concluded that-unlike enzyme catalysts-none of these hosts provide substantial specific binding of the transition states.…”