Three‐dimensional structure of a fluorescein–Fab complex crystallized in 2‐methyl‐2,4‐pentanediol

Abstract

The crystal structure of a fluorescein-Fab (4-4-20) complex was determined at 2.7 A resolution by molecular replacement methods. The starting model was the refined 2.7 A structure of unliganded Fab from an autoantibody (BV04-01) with specificity for single-stranded DNA. In the 4-4-20 complex fluorescein fits tightly into a relatively deep slot formed by a network of tryptophan and tyrosine side chains. The planar xanthonyl ring of the hapten is accommodated at the bottom of the slot while the phenylcarboxyl group interfaces with solvent. Tyrosine 37 (light chain) and tryptophan 33 (heavy chain) flank the xanthonyl group and tryptophan 101 (light chain) provides the floor of the combining site. Tyrosine 103 (heavy chain) is situated near the phenyl ring of the hapten and tyrosine 102 (heavy chain) forms part of the boundary of the slot. Histidine 31 and arginine 39 of the light chain are located in positions adjacent to the two enolic groups at opposite ends of the xanthonyl ring, and thus account for neutralization of one of two negative charges in the haptenic dianion. Formation of an enol-arginine ion pair in a region of low dielectric constant may account for an incremental increase in affinity of 2-3 orders of magnitude in the 4-4-20 molecule relative to other members of an idiotypic family of monoclonal antifluorescyl antibodies. The phenyl carboxyl group of fluorescein appears to be hydrogen bonded to the phenolic hydroxyl group of tyrosine 37 of the light chain. A molecule of 2-methyl-2,4-pentanediol (MPD), trapped in the interface of the variable domains just below the fluorescein binding site, may be partly responsible for the decrease in affinity for the hapten in MPD.

Structural basis of peptide–carbohydrate mimicry in an antibody-combining site

“…All three residues are engaged in only two direct and two watermediated hydrogen bonds with the Fab. The hydrogen bond and other contacts between His P6 side chain and Lys L50 NZ, the first residue of the short loop CDR L2, are unusual in that this loop makes no contact with the saccharide in this or other complexes of antibody with carbohydrates (23,24) or haptens (25,26). The Ala P7 methyl side chain lies close to, but not completely inside, a small hydrophobic cavity (Tyr L32 and His L27D) (Fig.…”

Crystal Structure of a Recombinant Anti-estradiol Fab Fragment in Complex with 17β-Estradiol

“…Aromatic residues (particularly tyrosine and tryptophan) are abundant in the binding sites of antibodies (40,41) and seem to be especially common in the binding pockets of the antibodies recognizing small hydrophobic haptens (12,(42)(43)(44). These antibodies must generally be able to provide a nonpolar binding site in a polar aqueous environment, and the physico-chemical properties of the aromatic residues are apparently favorable for this purpose.…”

Macromolecular Accessibility of Fluorescent Taxoids Bound at a Paclitaxel Binding Site in the Microtubule Surface

“…8B). Because no complete structure of the IgG is available, the structure of the Fab fragment (4FAB (43,44)) was used. The modeling shows that in the best possible conformation of the hexanoyl linker, the antibody would have to interpenetrate the tubulin to bind to the fluorescein moiety of the bound Hexaflutax.…”