Structural basis for molecular recognition of folic acid by folate receptors

Abstract

Folate receptors (FRα, FRβ and FRγ) are cysteine-rich cell-surface glycoproteins that bind folate with high affinity to mediate cellular uptake of folate. Although expressed at very low levels in most tissues, folate receptors, especially FRα, are expressed at high levels in numerous cancers to meet the folate demand of rapidly dividing cells under low folate conditions. The folate dependency of many tumours has been therapeutically and diagnostically exploited by administration of anti-FRα antibodies, high-affinity antifolates, folate-based imaging agents and folate-conjugated drugs and toxins. To understand how folate binds its receptors, we determined the crystal structure of human FRα in complex with folic acid at 2.8 Å resolution. FRα has a globular structure stabilized by eight disulphide bonds and contains a deep open folate-binding pocket comprised of residues that are conserved in all receptor subtypes. The folate pteroate moiety is buried inside the receptor, whereas its glutamate moiety is solvent-exposed and sticks out of the pocket entrance, allowing it to be conjugated to drugs without adversely affecting FRα binding. The extensive interactions between the receptor and ligand readily explain the high folate-binding affinity of folate receptors and provide a template for designing more specific drugs targeting the folate receptor system.

Figure 1. Structure of FRα bound to folic acid

a, Two views of the complex, with FRα in green, folic acid in grey, NAG in orange and the disulphide bonds depicted as yellow sticks. The N and C termini are labelled. b, Ribbon diagram of FRα, with folic acid and NAG in green stick presentations, overlaid with the semi-transparent receptor surface. c, Charge distribution surface of FRα with a close-up view of the ligand-binding pocket entrance. Folic acid carbon atoms are coloured grey, nitrogen atoms blue, and oxygen atoms red. A colour-code bar (bottom) shows an electrostatic scale from −3 to +3 eV.

Figure 2. Structural and biochemical analysis of FRα–folic acid interactions

a, The σA-weighted 2F_o – F_c electron density map for folic acid, shown as a grey mesh. b, The internal charge distribution surface of the binding pocket is shown using the same colour code as in Fig. 1c, with folic acid shown in stick presentation. c, Folic acid-binding network with close-ups of the folic acid head and tail groups. Residues that line the binding pocket are shown in green and folic acid is shown in grey. Hydrogen bonds are indicated by dashed lines.

Figure 3. Folic acid affinities of FRα ligand-binding-pocket mutants

a, Interaction map of folic acid with ligand-binding-pocket residues. The folic acid chemical structure is shown in magenta, pocket residues in black and hydrogen bonds as green dashed lines with bond distances (Å) indicated. Hydrophobic interactions are presented as curved red lines. The pteroate and glutamate moieties of folic acid are indicated above the map. b, Folic acid affinities of wild-type and mutant FRα proteins as measured by [³H]-folic acid binding assay (see Supplementary Figs 7 and 8 for binding isotherms). The numbers on top of the bars indicate the fold decrease in affinity (increase in K_d) relative to wild-type FRα. Error bars indicate s.d. (n –2).