How ligand binds to the type 1 insulin-like growth factor receptor

Abstract

Human type 1 insulin-like growth factor receptor is a homodimeric receptor tyrosine kinase that signals into pathways directing normal cellular growth, differentiation and proliferation, with aberrant signalling implicated in cancer. Insulin-like growth factor binding is understood to relax conformational restraints within the homodimer, initiating transphosphorylation of the tyrosine kinase domains. However, no three-dimensional structures exist for the receptor ectodomain to inform atomic-level understanding of these events. Here, we present crystal structures of the ectodomain in apo form and in complex with insulin-like growth factor I, the latter obtained by crystal soaking. These structures not only provide a wealth of detail of the growth factor interaction with the receptor's primary ligand-binding site but also indicate that ligand binding separates receptor domains by a mechanism of induced fit. Our findings are of importance to the design of agents targeting IGF-1R and its partner protein, the human insulin receptor.

Fig. 1

Structural biology of apo IRΔβ and insulin-bound μIR and the current model of ligand binding kinetics. a The Λ-shaped assembly of IRΔβ (PDB entry 4ZXB). Domain colours are L1 light blue, CR red, L2 orange, FnIII-1 green, FnIII-2 yellow, FnIII-3 dark blue, ID light magenta, αCT magenta. The foreground monomer is in ribbon representation, the background monomer in surface representation (apart from the ID element); dashed lines indicate disordered residues within the respective ID segments. b Human insulin (A chain grey, B chain black) bound to μIR (PDB entry 4OGA), coloured as in a. c Major pathway of ligand binding to IR and IGF-1R within the current kinetic model. S1, S2: site 1 and site 2 on one receptor monomer; S1΄, S2΄: site 1 and site 2 on the opposing receptor monomer. Red filled circle: ligand (i.e., IGF-I, IGF-II or insulin). d Steric overlap (asterisked) between insulin and the opposing fibronectin domain module of the structures depicted in a and b based on overlay of their common domain L1. αCT΄ is shown in both its apo conformation (thin magenta ribbon) and its insulin-complexed conformation (magenta ribbon) in order to illustrate its altered disposition upon insulin binding

Fig. 2

Stereo view of representative σ_A-weighted (2F_o−F_c) difference electron density. a The σ_A-weighted (2F_o−F_c) difference electron density in the vicinity of IGF-1RΔβ L1 domain residues 28–34 within the crystal of apo IGF-1RΔβ + Fv 24–60. The density is sharpened (B_sharp = −60 Ų) and displayed at a contour level of 1.7 σ (σ = root-mean-square deviation of the sharpened map). Density is shown only for volume within 2.0 Å of the atoms displayed. b The σ_A-weighted (2F_o−F_c) difference electron density in the vicinity of IGF-1RΔβ αCT residues 700–704 within the crystal of apo IGF-1RΔβ + Fv 24–60. The density is sharpened (B_sharp = −60 Ų) and displayed at a contour level of 0.33 σ (σ = root-mean-square deviation of the sharpened map). Density is shown only for volume within 2.5 Å of the atoms displayed. c σ_A-weighted (2F_o−F_c) difference electron density in the vicinity of IGF-I residues 11–18 within the crystal of the IGF-I-complexed IGF-1RΔβ + Fv 24–60. The density is sharpened (B_sharp = −60 Ų) and displayed at a contour level of 1.7 σ (where σ is the root-mean-square deviation of the sharpened map). Density is shown only for volume within 2.0 Å of the atoms displayed

Fig. 3

The crystal structure of apo IGF-1RΔβ. a The Π-shaped assembly of IGF-1RΔβ. Domain colours are L1 light blue, CR red, L2 orange, FnIII-1 green, FnIII-2 yellow, FnIII-3 dark blue, ID light magenta, αCT magenta. The foreground monomer is in ribbon representation, the background monomer in atomic sphere representation (apart from the ID element); dashed lines indicate disordered residues within the respective ID segments. b L1-CR-L2 module of IGF-1RΔβ (coloured as in a) overlaid onto that of IRΔβ (black) on the basis of corresponding residues within the L2 domain, showing the 26° difference in relative orientation of L1-CR and L2 in the two receptors. c L1/FnIII-2΄ pair of IGF-1RΔβ (coloured light blue and yellow, respectively) overlaid onto that of IRΔβ (black) on the basis of corresponding residues within domain FnIII-2΄, showing the 17° difference in relative orientation of L1 and FnIII-2΄ in the two receptors

Fig. 4

Configuration of αCT΄ and domains L1 and FnIII-2΄ of apo IGF-1RΔβ. a Ordering of the C-terminal region of the apo IGF-1RΔβ αCT΄ segment upon the surface of the adjacent domain FnIII-2΄. Inset below is a sequence alignment of residues at the C terminus of the respective α chains of IGF-1RΔβ and IRΔβ; residues in green are disordered in the crystal structure of the IRΔβ. Note that the αCT segment of IRΔβ is that of the A isoform of the receptor. b Association of the αCT΄ segment with domains L1 and FnIII-2΄ within the crystal structure of apo IGF-1RΔβ. c Association of the αCT΄ segment with domain L1 alone within the crystal structure of apo IRΔβ. Green dashed line represents the disordered C-terminal region of the α΄ chain of IRΔβ. The view direction in b and c is equivalent with respect to the domain L1. d Interaction between domains L1 and FnIII-2΄ within apo IGF-1RΔβ

Fig. 5

Mode of IGF-I binding to IGF-1RΔβ. a Bridge formed by IGF-I (black) between the site 1 components L1 and αCT΄ and FnIII-2΄, showing separation (asterisked) of the L1-CR module away from FnIII-2΄ and ID^N΄. b Overlay of one “leg” of the IGF-1RΔβ homodimer in its IGF-1-bound form (coloured ribbon) onto the corresponding domains of the apo IGF-1RΔβ homodimer (white ribbon). Alignment is based on domains FnIII-2΄ and FnIII-3΄. c Overlay (via L2) of the L1-CR-L2 module of IGF-1-bound IGF-1RΔβ (coloured ribbon) onto that of apo IGF-1RΔβ (white ribbon). Pro297, the hinge point, is in black and asterisked. d Interaction between the site-1-bound IGF-I (A domain black, B domain white) and FnIII-2΄

Fig. 6

Dissection of the interaction of IGF-I with binding site 1 of IGF-1R. a Conformation of L1-β₂ (light blue) and αCT΄ (magenta) in the apo IGF-1RΔβ structure, compared with b, its conformation in the IGF-I liganded IGF-1RΔβ structure. c Interaction between B domain of IGF-I (black) and the IGF-1R site 1 elements of L1-β₂ (light blue) and αCT΄ (magenta). The A domain of IGF-I (located in the foreground) is omitted for clarity. d Interaction between A domain of IGF-I (black) and the IGF-1R site 1 element αCT΄ (magenta); no interaction is observed between the A domain and L1-β₂ (light blue). The IGF-I B domain is in white

Fig. 7

Interaction of Fv 24–60 with IGF-1RΔβ. a Interaction of residues of domain CR of IGF-1RΔβ (red) with Fv 24–60 (variable heavy chain domain pink; variable light chain domain light grey). b Displacement by Fv 24–60 of the peptide loop formed by IGF-1RΔβ CR residues 254–265 towards the IGF-I binding site. The yellow ribbon is that of IGF-1R domain CR within the crystal structure of the isolated L1-CR-L2 fragment of IGF-1R obtained in the absence of attached Fv (PDB entry 1IGR), overlaid onto that of the IGF-I-bound IGF-1RΔβ structure (red) on the basis of common domain L1. Red sphere: IGF-I Asn26; blue sphere: IGF-I Pro39. The connecting IGF-I domain C residues 27–38 (indicated putatively by a blue dashed line) are disordered

Fig. 8

Induced fit binding of ligand to IGF-1R and IR. a Proposed kinetic scheme. S1, S2: site 1 and site 2 on one receptor monomer; S1΄, S2΄: site 1 and site 2 on the opposing receptor monomer. Black filled circle: hot ligand (i.e., IGF-I, IGF-II or insulin). Grey circle: cold ligand (i.e., IGF-I, IGF-II or insulin). a₁ and d₁: ligand association and dissociation rate constants for the high-affinity site. a₂ and d₂: ligand association and dissociation rate constants for doubly liganded, symmetrical receptor conformation. a₃ and d₃: association and dissociation rate constant for binding of a third insulin molecule (IR only, not applicable to IGF-1R). b Plot for accelerated dissociation of a pre-bound tracer-labelled ligand by cold (unlabelled) ligand. The dissociation time was 20 min. The experimental data were as described previously (reproduced in Supplementary Table 4) and are shown as blue triangles for IGF-1 and red diamonds for insulin, with the fit of the induced-fit model to these data shown as lines (IGF-I blue; insulin red)