Understanding insulin and its receptor from their three-dimensional structures

Abstract

Background: Insulin's discovery 100 years ago and its ongoing use since that time to treat diabetes belies the molecular complexity of its structure and that of its receptor. Advances in single-particle cryo-electron microscopy have over the past three years revolutionized our understanding of the atomic detail of insulin-receptor interactions.

Scope of review: This review describes the three-dimensional structure of insulin and its receptor and details on how they interact. This review also highlights the current gaps in our structural understanding of the system.

Major conclusions: A near-complete picture has been obtained of the hormone receptor interactions, providing new insights into the kinetics of the interactions and necessitating a revision of the extant two-site cross-linking model of hormone receptor engagement. How insulin initially engages the receptor and the receptor's traversed trajectory as it undergoes conformational changes associated with activation remain areas for future investigation.

Keywords: Cryo-electron microscopy; Insulin; Insulin receptor; Protein structure; Receptor tyrosine kinase; X-ray crystallography.

Figure 1

Three-dimensional structure of insulin and its predicted receptor-binding surfaces. (A) Structure of the insulin monomer showing chains A (cyan) and B (magenta) together with two inter-chain disulfide bonds and one intra-chain disulfide bond. (B) Assembly of the insulin dimer and three dimers into the insulin hexamer. The two zinc atoms (green) lie on the hexamer axis. The three dimers are shown in varying representations (ribbon, ribbon plus transparent surface, and opaque surface, respectively). (C) Detail of the insulin dimer interface, with selected residues highlighted. (D) The so-called “classical” set of receptor-binding residues of insulin (stick representation); these overlap insulin's dimer-forming surface (transparent white). (E) Predicted additional set of receptor-binding residues of insulin (stick representation); these overlap insulin's hexamer-forming surface (transparent white). Panels are based on PDB 1MSO [32].

Figure 2

Three-dimensional structure of the human apo insulin-receptor (IR) ectodomain. (A) Domain layout of the disulfide-linked (αβ)₂ homodimer; close-up view shows the sequence detail of the α chain's C-terminal region. The domain nomenclature is defined in the main text. Interchain disulfide bonds are shown as green lines. (B) Bi-lobal structure of the IR TK domain (N-terminal lobe in blue, C-terminal lobe in yellow, and the activation loop in green). (C) Structure of the IR L1-CR-L2 module. (D) Structure of the αβ monomer showing its pairing with the (αβ)′ monomer to form the (αβ)₂ homodimer. The green disc (bottom right) represents the cell membrane. In the panel, the N and C termini of the α chain and β chain are denoted as α_N, α_C, β_N, and β_C, respectively. Dashed connectors indicate residue segments disordered in the crystal structure. (E) Detail of the engagement of the αCT′ helix with the L1-β₂ surface. Panels are based on PDB 1IRK [92], PDB 2HR7 [7] and PDB 4ZXB [95].

Figure 3

Insulin's engagement with its primary binding site on the receptor. (A) Schematic diagram illustrating both the folding out of the B-chain C-terminal region of insulin from the hormone's core (asterisk, top left), the reconfiguration upon insulin binding of the αCT′ helix on the L1-β₂ surface (asterisk, bottom left), and insulin's engagement with the L1+αCT′ tandem element (right). (B) Detail of the packing of αCT′ residues His710′ and Phe714′ into the hormone's core as well as the reconfiguration of the three aromatic residues PheB24, PheB25, and TyrB26 within insulin's B-chain C-terminal region. Panels are based on PDB 1MSO [32], PDB 4ZXB [95] and PDB 6VEP [16].

Figure 4

Insulin receptor (IR) constructs used in structures determined by cryoEM. (A) Holo-receptor IR construct employed by Uchikawa et al. [14]. (B) IR-A ectodomain constructs employed by Scapin et al. [12] and Gutmann et al. [15]. (C) Leucine-zippered ectodomain construct employed by Weis et al. [13] including the so-called Δβ modification that removes the highly glycosylated segment near the β chain's N terminus [8].

Figure 5

Three-dimensional structure of the single-insulin-bound insulin receptor ectodomain. (A) Overall domain configuration of the single-insulin-bound insulin receptor ectodomain determined in the context of the leucine-zippered construct. (B) Schematic demonstrating how the L1, CR, and L2 domains and the αCT′ segment relocate to the receptor “head” upon insulin binding. Domain names are circled for the insulin-free elements and boxed for the insulin-bound elements. Only a single receptor “leg” is shown (that is, domains L1, CR, and L2 from one monomer and domains FnIII-1, FnIII-2, FnIII-3, and αCT′ from the alternate monomer). (C) Detail of (A) showing engagement of insulin with domain FnIII-1′. (D) Detail of (A) showing the reconfiguration of αCT′ upon the L1 surface upon insulin binding. (E) Detail of (A) showing engagement of domains L2 and FnIII-1′ with the extended αCT′ helix and engagement of domain L1 with domain L2 upon insulin binding. (F) Detail of (A) showing how the domains FnIII-2, FnIII-2, FnIII-3, and FnIII-3′ fold inwards with respect to the apo receptor ectodomain structure, forming an interaction between domains FnIII-3 and FnIII-3′ while retaining an apo-like association of domain L1′ with domain FnIII-2. Panels are based on PDB 6HN5 [13], PDB 6HN4 [13] and PDB 4ZXB [95].

Figure 6

CryoEM structure of the two-insulin-bound receptor “head.” The left- and right-hand panels show orthogonal views of the isolated insulin receptor ectodomain prepared in complex with two insulins. Within this structure, domains FnIII-2, FnIII-2′, FnIII-3, and FnIII-3′ are disordered. The mode of interaction of the first insulin with the receptor domains L1, αCT′, and FnIII-1′ and of the second insulin with receptor domains L1′, αCT, and FnIII-1 are both effectively identical to that of the single insulin bound to the leucine-zippered construct shown in Figure 5. Panel is based on PDB 6CE9 [12].

Figure 7

Three-dimensional structure of the four-insulin-bound insulin receptor ectodomain. (A) Overall pseudo-symmetric structure of the four-insulin-bound insulin receptor ectodomain structure determined by Gutmann et al. [15]. The binding sites of the additional bound insulins are enclosed in dashed circles. (B) Detail of insulin's binding to the surface of domain FnIII-1′ in the structure, as seen in the structure determined by Uchikawa et al. [14]. (C) Interaction between domains FnIII-3 and FnIII-3′ in the structure determined by Gutmann et al. [15]. (D) Interaction between insert domain segments IDα and IDα′ in the structure determined by Uchikawa et al. [14]. Panels are based on PDB 6SOF [15] and PDB 6PXV [14].

Figure 8

Two-dimensional class averages obtained from negative-stain images of insulin-bound nanodisc-embedded holo insulin receptor. (A) Apo holo-receptor showing the characteristic Λ-shaped conformation assembled either into one or two nanodiscs (1U and 2U, respectively). (B) Insulin-complex holo-receptor assembled into nanodiscs after complexation with insulin. All display incorporation into a single nanodisc, suggesting that insulin binding permits or directs the combination of the ectodomain's membrane proximal elements. Arrows are the blobs putatively corresponding to the tyrosine kinase domain(s). The class averages reflect either a T-shaped conformation (1T) or a narrower conformation with two legs visible (II), the latter may in some instances be a side view of the 1T conformation. Based on Figure 3 in Gutmann et al. (2018) J Cell Biol 217, 1643-9 (https://doi.org/10.1083/jcb.201711047) and used under a Creative Commons License.

Figure 9

Transmembrane and cytoplasmic domains of the activated insulin receptor. (A) Putative association of the TM domains of the insulin-activated holo-receptor within a detergent micelle as detected by single-particle cryoEM [14]. The panel was extracted from Figure 1, Supplementary Figure 4 from Uchikawa et al. (2019) eLife 8, e48630 (https://doi.org/10.7554/eLife.48630) and used under a Creative Commons License. (B) Solution structure of the insulin receptor TM domain (backbone trace only, 20 structures superimposed [118]). The traces are rainbow-colored representations from blue (Met939) to red (Glu988). The kink at residues Gly960/Pro961 [118] is arrowed; the shaded box represents the membrane's approximate span. (C) Trans-association of upstream juxtamembrane segment with the respective TK domains (residues Tyr972, Tyr1158, Tyr1162, and Tyr1163 are phosphorylated) showing the interaction of the JM segment with the αC helix of the opposing TK domain [119]. Blue ribbons: N-terminal lobes of the respective TK domains; yellow ribbons: C-terminal lobes of the respective TK domains; green ribbons: phosphorylated activation loops; orange ribbons: respective upstream JM segments; gray surfaces: respective molecular surfaces of the TK domains. The labeled segments are from the same TK domain.