Crystal Structure of Two Anti-Porphyrin Antibodies with Peroxidase Activity

Abstract

We report the crystal structures at 2.05 and 2.45 Å resolution of two antibodies, 13G10 and 14H7, directed against an iron(III)-αααβ-carboxyphenylporphyrin, which display some peroxidase activity. Although these two antibodies differ by only one amino acid in their variable λ-light chain and display 86% sequence identity in their variable heavy chain, their complementary determining regions (CDR) CDRH1 and CDRH3 adopt very different conformations. The presence of Met or Leu residues at positions preceding residue H101 in CDRH3 in 13G10 and 14H7, respectively, yields to shallow combining sites pockets with different shapes that are mainly hydrophobic. The hapten and other carboxyphenyl-derivatized iron(III)-porphyrins have been modeled in the active sites of both antibodies using protein ligand docking with the program GOLD. The hapten is maintained in the antibody pockets of 13G10 and 14H7 by a strong network of hydrogen bonds with two or three carboxylates of the carboxyphenyl substituents of the porphyrin, respectively, as well as numerous stacking and van der Waals interactions with the very hydrophobic CDRH3. However, no amino acid residue was found to chelate the iron. Modeling also allows us to rationalize the recognition of alternative porphyrinic cofactors by the 13G10 and 14H7 antibodies and the effect of imidazole binding on the peroxidase activity of the 13G10/porphyrin complexes.

From “hemoabzymes” to “hemozymes”: towards new biocatalysts for selective oxidations

“…Then, to deliver the necessary electrons to reduce the intermediate iron-oxo species into the iron(III) resting state, the reducing cosubstrate interacts with the solvent exposed meso edge of the heme, but never enters the active site. Such a situation also occurs in the antibody-metalloporphyrin complexes since, as shown by the 3D structure of Fe III -MP9-7G12 94 and Fe III (ToCPP)13G10 complexes, 104 about two-thirds of the porphyrin are included in the binding site, leaving one-third of the porphyrin exposed to the solvent and then accessible to reducing co-substrates. It is thus not surprising that the anti-porphyrin antibody strategy leads to hemoabzymes with a good peroxidase activity.…”

“…4) using X-ray diffraction studies, associated with molecular modeling studies. 104 It appeared that the two l-light chain anti-porphyrin antibodies, 13G10 and 14H7, possess shallow hapten binding pockets compared with the other structurally characterized catalytic antibodies. The structural complementarity of a 3 b-Fe(ToCPP) to the hydrophobic binding pocket of antibodies 13G10 and 14H7 thus leads to a remarkable thermostability of the a 3 b-Fe(ToCPP)-antibody complexes.…”

Toward the Computational Design of Artificial Metalloenzymes: From Protein–Ligand Docking to Multiscale Approaches

“…Smaller cofactors are therefore expected to better fit inside the binding site of the enzyme and provide a wider asymmetric environment for enantioselective reactions. , A final scaffold we studied is a family of porphyrin-binding catalytic antibodies that are able to perform peroxidase activities. In conjunction with X-ray structures that were not conclusive on the geometry of the cofactor in the hapten recognition site, we could qualitatively rationalize both activity and binding …”

PyIgClassify: a database of antibody CDR structural classifications

“…Tentative associations of each cluster with gene locus (heavy, kappa and lambda) and species were provided. Recent databases of antibody CDR conformations have used our classification system ( 13 , 15 ) as a reference, and it has gained acceptance in the wider antibody literature ( 16 , 17 ) and in industry ( 18 – 20 ).…”