Closely related antibody receptors exploit fundamentally different strategies for steroid recognition

Abstract

Molecular recognition by the adaptive immune system relies on specific high-affinity antibody receptors that are generated from a restricted set of starting sequences through homologous recombination and somatic mutation. The steroid binding antibody DB3 and the catalytic Diels-Alderase antibody 1E9 derive from the same germ line sequences but exhibit very distinct specificities and functions. However, mutation of only two of the 36 sequence differences in the variable domains, Leu H47 Trp and Arg H100 Trp, converts 1E9 into a high-affinity steroid receptor with a ligand recognition profile similar to DB3. To understand how these changes switch binding specificity and function, we determined the crystal structures of the 1E9 Leu H47 Trp/Arg H100 Trp double mutant (1E9dm) as an unliganded Fab at 2.05 Å resolution and in complex with two configurationally distinct steroids at 2.40 and 2.85 Å. Surprisingly, despite the functional mimicry of DB3, 1E9dm employs a distinct steroid binding mechanism. Extensive structural rearrangements occur in the combining site, where residue H47 acts as a specificity switch and H100 adapts to different ligands. Unlike DB3, 1E9dm does not use alternative binding pockets or different sets of hydrogen-bonding interactions to bind configurationally distinct steroids. Rather, the different steroids are inserted more deeply into the 1E9dm combining site, creating more hydrophobic contacts that energetically compensate for the lack of hydrogen bonds. These findings demonstrate how subtle mutations within an existing molecular scaffold can dramatically modulate the function of immune receptors by inducing unanticipated, but compensating, mechanisms of ligand interaction.antibody-antigen complex ͉ modulation of receptor specificity ͉ molecular recognition ͉ protein engineering ͉ x-ray crystallography

Prediction of the binding interface between monoclonal antibody m102.4 and Nipah attachment glycoprotein using structure-guided alanine scanning and computational docking

“…The remaining residue position (31 of VH) has Asn in the sequence published by Xu et al but Lys in the sequence published by the patent. To probe the involvement of residues, which are alanine in WT epitope or paratope, we mutated alanine to tryptophan as the large aromatic side chain of tryptophan is expected to have the highest disruptive potential to the binding interface 42 . Importantly, 18/19 identified paratope residues are from the heavy chain, which is logical given that the interaction between m102.3 and GP is dominated by the heavy chain.…”

High-Affinity Nucleic-Acid-Based Receptors for Steroids

“…On the other hand, the unique advantages of oligonucleotide-based receptors over other types of receptors is that they can be readily isolated by streamlined in vitro selection and amplification procedures and tailored by rational adjustments in stringencies and focused counter-selections. , However, we were concerned that aptamer receptors defined by shorter sequences, e.g. , <40 nucleotides, would have only limited affinities for purely hydrophobic targets, because the free energy benefits of hydrophobic interactions are directly proportional to contact surface areas. , For hydrophilic steroids, such as those containing hydroxyl groups, selectivity may be easier to achieve due to more oriented polar interactions, but these may interfere with our goal of achieving high-affinity receptors with limited size. We were further concerned that concomitant increases in binding pocket complexity would make aptamers even less likely to be successfully isolated in selections.…”

The testosterone binding mechanism of an antibody derived from a naïve human scFv library

“…Two mutations (Leu52HTrp and Arg113HTrp) directed to the binding site residues of 1E9 Fab were sufficient to improve the steroid binding affinity of 1E9 Fab. The crystal structures of the 1E9 double mutant Fab (1E9dm) in complex with progesterone and 5b-androstane-3,17-dione showed the major role of hydrophobic interactions in the complex formation (Verdino et al, 2008). In both 1E9dm Fab complex structures, the hapten molecule is bound in a sandwich between the amino acid residues Trp55H and Trp113H.…”

“…The three-dimensional structures have been determined for three anti-estradiol antibodies [57-2, 17E12 and 10G6 (Lamminmäki and Kankare, 2001;Monnet et al, 2002)], an anti-progesterone antibody DB3 (Arevalo et al, 1994), two anti-digoxin antibodies (26-10 and 40-50 (Jeffrey et al, 1993;Jeffrey et al, 1995), a glucuronide binding antibody Fv4155 fragment (Trinh et al, 1997) and a mutated catalytic Diels-Alderase antibody Fab fragment 1E9dm (Verdino et al, 2008). The relative orientation of the bound steroid molecule, number of hydrogen bonds between the hapten and antibody and the contribution of the antibody heavy chain to steroid binding vary among these structures.…”