Cryo-EM structures elucidate the multiligand receptor nature of megalin

Abstract

Megalin (low-density lipoprotein receptor-related protein 2) is a giant glycoprotein of about 600 kDa, mediating the endocytosis of more than 60 ligands, including those of proteins, peptides, and drug compounds [S. Goto, M. Hosojima, H. Kabasawa, A. Saito, Int. J. Biochem. Cell Biol. 157, 106393 (2023)]. It is expressed predominantly in renal proximal tubule epithelial cells, as well as in the brain, lungs, eyes, inner ear, thyroid gland, and placenta. Megalin is also known to mediate the endocytosis of toxic compounds, particularly those that cause renal and hearing disorders [Y. Hori et al., J. Am. Soc. Nephrol. 28, 1783-1791 (2017)]. Genetic megalin deficiency causes Donnai-Barrow syndrome/facio-oculo-acoustico-renal syndrome in humans. However, it is not known how megalin interacts with such a wide variety of ligands and plays pathological roles in various organs. In this study, we elucidated the dimeric architecture of megalin, purified from rat kidneys, using cryoelectron microscopy. The maps revealed the densities of endogenous ligands bound to various regions throughout the dimer, elucidating the multiligand receptor nature of megalin. We also determined the structure of megalin in complex with receptor-associated protein, a molecular chaperone for megalin. The results will facilitate further studies on the pathophysiology of megalin-dependent multiligand endocytic pathways in multiple organs and will also be useful for the development of megalin-targeted drugs for renal and hearing disorders, Alzheimer's disease [B. V. Zlokovic et al., Proc. Natl. Acad. Sci. U.S.A. 93, 4229-4234 (1996)], and other illnesses.

Keywords: cryoelectron microscopy; endocytosis; ligand binding; megalin; proximal tubule.

Fig. 1.

Structure of rat megalin. (A) Cryo-EM density of overall rat megalin, viewed parallel to the membrane plane. Domains are indicated within the panel. (B) Cartoon representation of the domain composition of rat megalin. CR, EGF-type repeat, and YWTD-containing β-propeller are indicated by ovals, wide pentagons, and hexagons, respectively. (C) Overall structure of rat megalin shown as a ribbon model. One of the protomers is shown in rainbow colors, while the other is shown in gray or is transparent.

Fig. 2.

Ligand-binding sites of megalin. (A) Unsharpened cryo-EM density of rat megalin viewed parallel to the membrane plane (Left) and a cutaway surface of the density at the dotted line viewed from the cytoplasmic side (Right). Density is shown for the low- (transparent) and high-contoured (colored) areas in the Left panel, and the low-contoured map shows the micelle density of the TM domain (TMD). The colors correspond to those in Fig. 1, with O- and N-glycans highlighted in pink. Extra densities of endogenous ligands are indicated by red arrowheads. The ligand densities are observed at the binding sites indicated in the panels, except for LBD I, whose density is rather weak and disordered in the middle of the CRs. (B) Fo-Fc densities of ligand-binding sites are highlighted in purple, showing the extra strong density of endogenous ligands. Rat megalin is shown as a ribbon model. (C–E) Surface representations are shown for the head domain (C), β-basket (D), and β-pocket (E). β-propellers and CRs are indicated. Positive and negative Fo-Fc density maps are shown in purple and red, respectively. A probable architecture of protein ligands is shown as purple sticks. Two N-glycans on the CRs of β-pocket are indicated in E. (F–H) Ribbon models are shown for the head domain (F), β-basket (G), and β-pocket (H). Ca²⁺ ions are indicated by red spheres. O-glycans are predominantly present on the spacer regions of CRs but are on the opposite sides in the β-basket (G) and β-pocket (H). The Insets show the zoomed-in region of (I and J), with the viewpoints indicated by the eye symbols. (I–L) Zoomed-in views showing the interactions within the ligand-binding sites for the head domain (I and J), β-basket (K), and β-pocket (L). Hydrogen-bond interactions are indicated by yellow dotted lines.

Fig. 3.

Binding of peptide ligands in β-propellers. (A–D) Zoomed-in views of the β-propellers are shown for the β-basket (A), β-pocket (B), the leg domain (C), and the head domain (D). β-propeller numbers (β1–8) are shown in the Lower Left corner, and blade numbers are indicated in the Upper Left panel (β1). Colors are the same as those in Fig. 1. Possible architectures of peptide ligands are shown as light purple sticks. β-propeller residues important for ligand recognition are shown as sticks. A pair of Asp/Glu–Arg residues, on the third and fourth blades, respectively, are conserved in β1–3, 6, and 7, whereas β4 and β5 have different motifs for ligand recognition. Amino acid residues of the ligands are assigned based on the density shapes and the similarity to the previous ligand-bound structures of the YWTD-containing β-propellers (SI Appendix, Extended Data Fig. 5 A–C), while densities with fewer features are assigned as Ala. The residue number of the ligands is defined such that the residues recognized by the conserved motifs (e.g., Asn in β2) are zero.

Fig. 4.

Structure of rat megalin in complex with RAP. (A) Overall structure of rat megalin–RAP complex shown as ribbon models. The megalin color codes are the same as those in Fig. 1, while RAP is shown in red. Two protomers of RAP bind to the megalin dimer, with each protomer bridging two protomers of megalin. Insets indicate the zoomed-in regions in (B and C). (B–D) Zoomed-in views of the megalin–RAP complex of rat RAP D1 (B) and D3 (C) in the present structure, and human RAP D3 in complex with CRs (LA3–4) of LDLR (PDB: 2FCW) (D). Residues involved in the interactions are shown as sticks (Left). Right panels show the surface electrostatic potentials of the RAP domains, viewed from a perpendicular angle relative to the Left panels. Positively charged patches are highlighted as blue dotted circles. Complex formation is mediated through different patches of RAP in the two structures. Residue number is not consistent between human and rat RAP (SI Appendix, Extended Data Fig. 8A). (E) Surface plasmon resonance (SPR) response curves are shown for the interaction of full-length as well as wild-type forms of D1, D2, and D3 of RAP with megalin immobilized on a sensor. Line colors indicate the concentration of the analyte proteins (a twofold dilution series starting at 200 nM [gray], with seven points of concentration change [red: lowest]). The average value of the dissociation constant (K_D) with the SD is shown in each panel. (F) SPR response curves are shown for the interaction of wild-type as well as patch mutant forms of each domain with megalin immobilized on a sensor. The concentration of the analyte proteins was 200 nM. Representative sensorgrams are shown from multiple experiments (n = 4; E and F).

Fig. 5.

Schematic model of ligand binding in megalin. Proposed model of the ligand interaction is shown for the β-pocket and β-basket, LBD I and III, and the β-cage. The ligand-binding sites consist of CRs and β-propellers, and ligand preference is likely dependent on the constituent modules. Ligand size may be limited by the space of each ligand-binding mode.