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 A. 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 pi-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.

Figure 1

Reaction scheme and haptens used in this study. The retro-Diels–Alder reaction of pro-drug 1a (k_un = 9.8 10⁻⁶ s⁻¹ conditions: 31°C in PBS pH 7.4, 10% vol/vol dimethylformamide) releases nitroxyl and anthracene 2a. The reaction is catalyzed by antibodies 9D9 (anti-3), 10F11 (anti-5), and 27C5 (anti-3,4). The reaction with the regioisomeric substrate 1b (k_un = 7.1 10⁻⁶ s⁻¹) is also catalyzed weakly by the antibodies. The unreactive substrate analog 6 and product analogue 2b were used for crystallization. Substrate analogue 1c and product analog 2c and transition state model 7 were used for docking studies.

Figure 2

Sequence alignment and comparison of the CDR H (A) and CDR L (B) corresponding to the three nitroxyl-synthase catalytic antibodies 9D9, 10F11, and 27C5, the Diels–Alderase antibodies 1E9 and 39-A11, and the noncatalytic antibody Mrk-16. Identical residues are shown in red, similar residues are shown in blue, and different residues are shown in black. Black stars represent residues in close contact with the hapten.

Figure 3

Active site of the abzyme. (Left) 10F11 in complex with OXY (compound 6). The hydrogen bonding network is shown as dotted lines. (Right) Surface representation of the binding pocket of Fab 10F11 with the transition state analogue BCN (compound 4) of the retro-Diels–Alder reaction. View is from the top of the binding site showing the fit of the ligand BCN. The light pink transparent surface represents V_L and the light blue transparent surface represents V_H. Note the presence of water molecules inside the pocket. Figure prepared with dino (http://www.dino3d.org).

Figure 4

(Left) Overlay of 10F11⋅6 (green)“OXY,” 10F11⋅4 (yellow) “BCN,” and 10F11⋅2b (magenta) “ANT.” The small movement of the flap H99–H102 is clearly visible at Ser-H100. Water molecules S1 and S26 are shown as small spheres, and water molecule S127, which is only present in the 10.F11⋅6 structure, is shown as large cyan sphere. (Right) Overlay of 10F11 with 9D9. 9D9 is shown in orange, 10F11 is shown in cyan. Amino acids labeled in black represent the conserved residues, and red labels indicate the exchanges of amino acids at equivalent positions.