Structures of human folate receptors reveal biological trafficking states and diversity in folate and antifolate recognition

Abstract

Antifolates, folate analogs that inhibit vitamin B9 (folic acid)-using cellular enzymes, have been used over several decades for the treatment of cancer and inflammatory diseases. Cellular uptake of the antifolates in clinical use occurs primarily via widely expressed facilitative membrane transporters. More recently, human folate receptors (FRs), high affinity receptors that transport folate via endocytosis, have been proposed as targets for the specific delivery of new classes of antifolates or folate conjugates to tumors or sites of inflammation. The development of specific, FR-targeted antifolates would be accelerated if additional biophysical data, particularly structural models of the receptors, were available. Here we describe six distinct crystallographic models that provide insight into biological trafficking of FRs and distinct binding modes of folate and antifolates to these receptors. From comparison of the structures, we delineate discrete structural conformations representative of key stages in the endocytic trafficking of FRs and propose models for pH-dependent conformational changes. Additionally, we describe the molecular details of human FR in complex with three clinically prevalent antifolates, pemetrexed (also Alimta), aminopterin, and methotrexate. On the whole, our data form the basis for rapid design and implementation of unique, FR-targeted, folate-based drugs for the treatment of cancer and inflammatory diseases.

Keywords: isothermal titration calorimetry; targeted drug delivery.

Fig. 1.

Sequence alignment of human folate receptors α, β, and γ. Conserved cysteine and histidine residues are shown in red and green, respectively, and sites of N-glycan attachment are shown in blue. Secondary structure elements of the model representing the folate receptor in state I are presented above the sequences as cylinders for α-helices and arrows for β-strands. Regions that change conformations during proposed trafficking states are labeled with boundaries indicated above the secondary structures. Filled circles denote residues that interact directly with folate and all antifolates examined in this study. Residues that interact with a subset of ligands are shown as follows: PMX-specific, triangle; AMT- and MTX-specific, open circle; and FOL- and MTX-specific, star.

Fig. 2.

Model for trafficking of human folate receptors. (A) Schematic of ligand transport via endocytosis of hFR is depicted with three biological trafficking states. At the cell surface, the receptor at neutral to slightly basic pH is in an apo-FR conformation competent to bind ligand (state I). On ligand binding, structural transitions occur and lead to complex formation (state II). After endocytosis, ligand release occurs in the mildly acidic microenvironment of the recycling endosome. After ligand release and under acidic conditions, the receptor likely adopts a third distinct conformation (state III) before recycling to the cell surface. (B) Folic acid and the clinically prevalent anitfolate drugs examined in this study are shown.

Fig. 3.

Structures of folate receptors depicting states of biological trafficking. Cartoon models representing proposed states I, II, and III (A–C) of folate transport are represented by apo-hFRβ and the hFRβ/FOL complex at near neutral pH, and apo-hFRα at acidic pH, respectively. (A) Cartoon model of the apo-hFRβ structure is shown with conserved disulfides colored orange. (B) A cartoon depiction of the hFRβ/FOL complex shows the position of the ligand binding pocket with residues that interact with folate shown as sticks. (C) The apo-hFRα model illustrates global conformational differences in the structure of apo-hFR at pH 5.5 relative to the same at near neutral pH (cf. A and C). (D) Conformational differences between the three trafficking states are highlighted. Four regions of the folate receptors that undergo significant conformational changes are numbered with arrows to indicate the general direction of movements and colored as seen in A–C. Variable regions in each individual model are emphasized with darker shading of the same color.

Fig. 4.

Conformational changes in the hFR ligand binding pocket during states of biological trafficking. Residues that interact with folate in the complex structure are modeled to highlight the movements of conformationally variable loops (darker shading) between the open state at neutral pH (A), the folate complex (B), and the closed state at acidic pH (C). Polar interactions in the folate complex are designated with dashed lines. Tyr-76, which forms hydrophobic interactions benzoyl moiety of the folate ligand, is removed for clarity. Detailed interaction maps can be seen in Figs. S2 and S4–S6.

Fig. 5.

Histidine residues may promote or stabilize pH-dependent global conformational changes in hFR structures. (A) Conserved residues of hFRα and hFRβ form ionic contacts at low pH. A series of interactions facilitated by D51 and H54, residues from the anchor loop, which are solvent exposed at neutral pH, result in stabilization of Arg125 in a position that occludes the folate binding site. Electron density for the final 2m|F_o-DF_c| map contoured at 1.0 σ is shown with residue numbering based on hFRα. (B) Conserved histidines in hFRα and hFRβ may promote ligand dissociation and global changes in conformation based on changes in pH when moving from neutral (states I and II) to acidic (state III) conditions during trafficking. Residue numbers are indicated for hFRβ; equivalent numbering for hFRα would be incremented +6 relative to hFRβ (Fig. 1). Arrows serve as guides for the direction of movement for individual histidines. An asterisk indicates the H69 equivalent position in state III. This residue was not shown explicitly or modeled due to lack of electron density for the sidechain atoms. (C and D) The adaptive Poisson-Boltzmann solver (APBS) software was used to calculate electrostatic surface potentials for the hFRβ folate complex at pH 7.4 and 6.5 to assess possible differences in electrostatic potential experienced by hFRβ/FOL complex during trafficking.

Fig. 6.

Binding site interactions of antifolates with hFRβ. Interacting residues are highlighted to compare similarities and differences in the amino acid contacts in hFRβ complex structures with (A) pemetrexed, (B) aminopterin, and (C) methotrexate.