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

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

doi:10.1074/jbc.m116.732180

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.

Highlights

In earlier studies [4, 5], we provided isothermal titration calorimetry (ITC)4 data that suggested how Site 1 peptides might bind to the insulin receptor
C-terminal region of the insulin receptor ␣-chain; CR, cysteine-rich region; EH, endoglycosidase H; FnIII-1, -2, -3, the first, second, and third fibronectin type III domains; Fv, antibody variable domain; IR, insulin receptor; IR310.T, residues 1–310 of the human IR followed by seven residual linker and thrombin cleavage site residues; IR485, the L1-CR-L2 construct of IR comprising residues 1– 485; L1, first leucine-rich repeat domain; L1-␤2, the central ␤-sheet of the L1 domain; L2, second leucine-rich repeat domain; S519C16, the 16 C-terminal residues of the peptide S519; ␮IR, insulin microreceptor (the L1-CR module (IR310.T) of IR in complex with the IR ␣CT segment); TBSA, Tris-buffered saline plus azide; Variable Heavy (VH), variable heavy; Variable Light (VL), variable light
The dissociation constant, Kd, of the S519C16 peptide for the EH-treated IR310.T1⁄7Fv 83-7 complex was determined by ITC to be 2.5 Ϯ 0.6 nM (Fig. 2B; see “Experimental Procedures”), which is similar to that reported (Kd ϭ 2.6 Ϯ 0.7 nM) for S519C16 upon ITC against the IR485 construct [4], indicating that its relative affinity was not adversely affected by either removal of the L2 domain, partial glycosylation, or Fv 83-7 complexation

Summary

Results

Sample Characterization—SDS-PAGE analysis of the EH-treated IR310.T1⁄783-7 Fv revealed a small drop in molecular mass (ϳ3 kDa) compared with that of the untreated complex, slightly less than the drop in molecular mass (ϳ5 kDa) upon similar EH treatment of IR310.T in the absence of attached Fv (Fig. 2A) These data suggested that attachment of the Fv module resulted in a slight reduction in the accessibility of the endoglycosidase to the IR310.T N-linked glycan. IR310.T and 83-7 Fv moieties were left unmodeled due to poor or absent electron density (see “Experimental Procedures”) These residues all lay remote from the S519C16 binding site (Fig. 3) and corresponded to either (i) loop regions within IR310.T known to be of relatively high mobility (i.e. B-factor) in other structures of which IR310.T is a subset (e.g. Protein Data Bank code 2HR7 [8]) or (ii) polypeptide termini that arise as artificial truncations with respect to parent protein (i.e. human IR or 83-7 mAb).

No of reflections

Discussion

Experimental Procedures