Structural resolution of a tandem hormone-binding element in the insulin receptor and its implications for design of peptide agonists

Abstract

The C-terminal segment of the human insulin receptor α-chain (designated αCT) is critical to insulin binding as has been previously demonstrated by alanine scanning mutagenesis and photo-crosslinking. To date no information regarding the structure of this segment within the receptor has been available. We employ here the technique of thermal-factor sharpening to enhance the interpretability of the electron-density maps associated with the earlier crystal structure of the human insulin receptor ectodomain. The αCT segment is now resolved as being engaged with the central β-sheet of the first leucine-rich repeat (L1) domain of the receptor. The segment is α-helical in conformation and extends 11 residues N-terminal of the classical αCTsegment boundary originally defined by peptide mapping. This tandem structural element (αCT-L1) thus defines the intact primary insulin-binding surface of the apo-receptor. The structure, together with isothermal titration calorimetry data of mutant αCT peptides binding to an insulin minireceptor, leads to the conclusion that putative "insulin-mimetic" peptides in the literature act at least in part as mimics of the αCT segment as well as of insulin. Photo-cross-linking by novel bifunctional insulin derivatives demonstrates that the interaction of insulin with the αCT segment and the L1 domain occurs in trans, i.e., these components of the primary binding site are contributed by alternate α-chains within the insulin receptor homodimer. The tandem structural element defines a new target for the design of insulin agonists for the treatment of diabetes mellitus.inding of insulin to the insulin receptor initiates a signaling cascade in target tissues as the first step in the regulation of metabolic homeostasis. However, a molecular description of how insulin binds and activates its receptor remains elusive. Whereas determination of the structure of insulin (Fig. 1A) represented an early triumph of protein crystallography (2), the structure of the much larger receptor ectodomain homodimer (in apo form) has only recently been crystallographically analyzed (3). The latter structure and its implications for the nature of the hormone-binding sites have been extensively reviewed (4-7). Briefly, the insulin receptor is a disulfide-linked dimer wherein each proreceptor monomer is proteolytically cleaved into an N-terminal α-chain and a C-terminal β-chain (Fig. 1B). A single disulfide bond links the α-and β-chains within each monomer. The extracellular portion of the insulin receptor includes both α-chains as well as 194 residues (Ser724-Lys917) of each β-chain. Each receptor monomer consists of several structural domains (Fig. 1B), including a leucine-rich repeat domain L1 (residues 1-157), a cysteine-rich region (CR, residues 158-310), a second leucine-rich repeat domain L2 (residues 311-470), and three fibronectin type-III domains: FnIII-1 (residues 471-595), FnIII-2 (residues 596-808), and FnIII-3 (residues 809-906). FnIII-2 contains a ∼120-residue insert domain (ID, residues 638-756) that co...

Structure of the insulin receptor–insulin complex by single-particle cryo-EM analysis

“…The insulin and the α-CT helix were positioned by overlaying the microreceptor structure from PDB entry 3W11 9 onto the L1 domain of the complex and rigid body fitting them into the available density. The α-CT helix was manually extended, while the full insulin was built using as reference one of the monomers from PDB entry 1ZNI.…”

“…Structures of the individual sub-domains comprising the ECD (L1, CR, L2, and fibronectin type III domains FnIII-1, 2 and 3) are known, and the crystal structure of the apo IR ECD 8 provides one view of their quaternary organization. In the apo IR ECD structure, density for the insertion domain (120 residues located within FnIII-2, containing the cleavage site that generates the α and β chains, and the subunit α C-terminal helix, α-CT) was poor, and only a portion of α-CT helix was visible 9,10 . While only one (αβ) heterodimer is present in the asymmetric unit of the IR crystal structure, a symmetry generated tetramer ((αβ) 2 ) was proposed to represent the biologically relevant form of the ECD 8 .…”

Photocyclic behavior of rhodopsin induced by an atypical isomerization mechanism

“…4A). Thermal factor sharpening of 2F o -F c electron-density maps was performed at B sharp below the negative Wilson B value of the diffraction data to upweight the higher-resolution terms (31)(32)(33)(34)(35)(36)(37). In addition to the Rh6mr crystal structure, bovine opsin lacking the 6mr chromophore was crystallized at 2.7 Å. Crystallization of bovine opsin was performed under the same conditions as Rh6mr except for the exclusion of 6mr.…”

Protective hinge in insulin opens to enable its receptor engagement

“…Involvement of the two α-subunits is asymmetric: the primary insulin-binding site (site 1*) comprises the central β-sheet (L1-β 2 ) of the first leucine-rich repeat domain (L1) of one α-subunit and the partially helical Cterminal segment (αCT) of the other α-subunit (Fig. 1A) (10). Such binding initiates conformational changes leading to transphosphorylation of the β-subunits' intracellular tyrosine kinase (TK) domains.…”