Higher-Resolution Structure of the Human Insulin Receptor Ectodomain: Multi-Modal Inclusion of the Insert Domain

Abstract

Insulin receptor (IR) signaling is critical to controlling nutrient uptake and metabolism. However, only a low-resolution (3.8 Å) structure currently exists for the IR ectodomain, with some segments ill-defined or unmodeled due to disorder. Here, we revise this structure using new diffraction data to 3.3 Å resolution that allow improved modeling of the N-linked glycans, the first and third fibronectin type III domains, and the insert domain. A novel haptic interactive molecular dynamics strategy was used to aid fitting to low-resolution electron density maps. The resulting model provides a foundation for investigation of structural transitions in IR upon ligand binding.

Figure 1. IR Ectodomain Structure

(A) Domain structure of the αβ monomer and of the disulfide-linked (αβ)₂ homodimer. Black lines denote inter-chain disulfide bonds. N-linked glycosylation sites are indicated for the monomer. (B) Inverted V-shaped arrangement of the domains within 3LOH. One monomer is in ribbon representation with domains labeled; the other is in molecular surface, apart from its ID which is in ribbon. (C and D) Revisions to FnIII-1 (C) and FnIII-3 (D). Regions of common residue register across 2DTG/ 3LOH and the revised structure are in gray; where the register differs, 2DTG/3LOH residues are in orange and those of the remodeled FnIII-1 in green, remodeled ID in black, and remodeled FnIII-3 in blue. Included in (D) is the ID segment 637–655 with the 647–860 intra-monomer α/β disulfide bond in purple rod representation. (E) Revised IR ectodomain homodimer, represented as in (B).

Figure 2. iMDFF Model of Residues Surrounding the Putative Inter-α-Chain Disulfide Bond at Cys683

Inter-chain disulfide bonds and the Asn671 glycan (in rod representation; labeled) are assigned to a large volume of a σ_A-weighted (F_obs-F_calc)-difference electron density (B_blur = 60 Ų), calculated prior to the inclusion of glycan and of the ID residues, in pink. The hairpin-like turn in the ID near Leu648 marked by a star.

Figure 3. Polypeptide Segments of IR Showing Altered H/D Exchange Profiles upon Ligand Binding and their Possible Burial upon Ligand Binding

(A) Asp496, Arg498, and Asp499 (within FnIII-1) lie adjacent to Glu453 and Arg454 (within L2 of the opposite monomer). (B) Residues 519–525 of the FnIII-1 CC′ loop lie adjacent to a conserved hydrophobic pocket on L2. (C) Residues 655–658 of the ID lie in proximity to the L1 surface. Residues described are in stick representation, while the molecular surface of amides of residues demonstrating altered H/D exchange profiles is in black and indicated by green arrows.