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. 2016 Jul 22;291(30):15473-81.
doi: 10.1074/jbc.M116.732180. Epub 2016 Jun 8.

Insulin Mimetic Peptide Disrupts the Primary Binding Site of the Insulin Receptor

Callum F Lawrence  1 , Mai B Margetts  1 , John G Menting  1 , Nicholas A Smith  2 , Brian J Smith  2 , Colin W Ward  1 , Michael C Lawrence  3
Affiliations

Insulin Mimetic Peptide Disrupts the Primary Binding Site of the Insulin Receptor

Callum F Lawrence et al. J Biol Chem. .

Abstract

Sets of synthetic peptides that interact with the insulin receptor ectodomain have been discovered by phage display and reported in the literature. These peptides were grouped into three classes termed Site 1, Site 2, and Site 3 based on their mutual competition of binding to the receptor. Further refinement has yielded, in particular, a 36-residue Site 2-Site 1 fusion peptide, S519, that binds the insulin receptor with subnanomolar affinity and exhibits agonist activity in both lipogenesis and glucose uptake assays. Here, we report three-dimensional crystallographic detail of the interaction of the C-terminal, 16-residue Site 1 component (S519C16) of S519 with the first leucine-rich repeat domain (L1) of the insulin receptor. Our structure shows that S519C16 binds to the same site on the L1 surface as that occupied by a critical component of the primary binding site, namely the helical C-terminal segment of the insulin receptor α-chain (termed αCT). In particular, the two phenylalanine residues within the FYXWF motif of S519C16 are seen to engage the insulin receptor L1 domain surface in a fashion almost identical to the respective αCT residues Phe(701) and Phe(705) The structure provides a platform for the further development of peptidic and/or small molecule agents directed toward the insulin receptor and/or the type 1 insulin-like growth factor receptor.

Keywords: Fv antibody fragment; insulin; insulin receptor; mimetic peptide; molecular modeling; peptides; protein structure; x-ray crystallography.

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Figures

FIGURE 1.
FIGURE 1.
Structures of the IR ectodomain and the insulin mimetic peptides. A, the folded over, Λ-shaped structure of the IR ectodomain. One monomer is in ribbon representation with domains labeled; the other is in molecular surface, apart from its insert domain (ID), which is in ribbon. B, the sequence similarity between IR αCT (residues 694–710) and S519C16 (residues 21–36) proposed to underlie their competition for binding the three-domain IR L1-CR-L2 construct IR485 (4, 8).
FIGURE 2.
FIGURE 2.
Sample characterization. A, representative non-reducing SDS-PAGE analysis of endoglycosidase H digestion of the IR310.T·83-7 Fv complex. Lanes 1 and 2, IR310.T; lane 3, endoglycosidase H-treated IR310.T·83-7 Fv; lanes 4 and 5, endoglycosidase H-treated IR310.T in the absence of bound 83-7 Fv. MW, molecular mass markers (kDa). B, representative isothermal titration calorimetry data for the titration of the S519C16 peptide against the IR310.T·83-7 Fv complex.
FIGURE 3.
FIGURE 3.
Overlay of ribbon representations of the structure determined here with the structure of the equivalent modules within that of the intact IR ectodomain in complex with two copies of both 83-7 Fab and 83-14 Fab (Protein Data Bank code 4ZXB). Light cyan, L1 domain of IR310.T; black, CR domain of IR310.T; orange, VL domain of 83-7 Fv bound to IR310.T; green, VH domain of 83-7 Fv bound to IR310.T; tan, S519C16 bound to IR310.T; white, L1-CR and 83-7 variable domains within the ectodomain complex. The overlay is based on the L1-CR modules. The polypeptide termini of the IR310.T·83-7 Fv complex that abut unmodeled residues of the complex are indicated by spheres (red for C termini and blue for N termini); these are remote from the S519C16 moiety. Also absent from the model are the N- and C-terminal glycine residues of S519C16.
FIGURE 4.
FIGURE 4.
Stereoimages of the (2mFobs − DFcalc) difference electron density associated with the S519C16 peptide within the IR310.T complex viewed parallel (A) and antiparallel (B) to the direction of the L1-β2 strands.
FIGURE 5.
FIGURE 5.
Interaction of the S519C16 helix with the IR L1-β2 surface. A, side-chain detail within the S519C16/IR L1-β2 interface. The backbone of IR L1-β2 and of S519C16 is shown in tube representation and colored light cyan and tan, respectively, with the surface of L1-β2 shown in transparent light cyan. B, comparison of the disposition of S519C16 on the surface of IR L1-β2 compared with that of IR αCT in its apo and insulin-complexed forms. C, comparison of the engagement of the side chains of S519C16 residues Phe27, Tyr28, Trp30, Phe31, and Gln34 with the IR L1-β2 surface compared with that of apo-IR αCT residues Phe701, Phe705, and Tyr708. The color scheme is as in A and B.
FIGURE 6.
FIGURE 6.
Representative models from the three respective clusters of putative locations for S519 in the context of the intact IR ectodomain. The ectodomain is shown in ribbon representation; the domain modules in one of the two legs of the Λ-shaped receptor is shown in light gray, whereas in the other leg, the L1 domain is shown in cyan and the FnIII domains are shown in yellow with the CR and L2 domains of that leg omitted for clarity. Sites of inter-α-chain disulfide are shown in green and labeled. The S519 peptide is shown in orange with it disulfide bond shown in green and labeled with an asterisk.
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References

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