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

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 leucinerich 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 ␣-chain (termed ␣CT). 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 ␣CT 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.In 2002, sets of synthetic peptides were identified that could compete for insulin binding to the human insulin receptor extracellular region and that had affinities in the high nanomolar to low micromolar range (1). Based on competition studies, the putative epitopes of these peptides were grouped into three non-overlapping sites, termed Sites 1, 2, and 3.3 Some Site 1 peptides were able to activate the receptor tyrosine kinase and act as agonists in an insulin-dependent fat cell assay, whereas Site 2 and Site 3 peptides were found to act as antagonists both in phosphorylation and fat cell assays. The highest affinity Site 1 peptides contained the motif FYXWF, whereas Site 2 peptides were characterized by either a short or long disulfide loop (1).Optimized versions of the Site 1 and Site 2 peptides were subsequently described (2), in particular those that were linked in tandem in various ways. Several of these linked peptides were found to function as either potent antagonists (e.g. peptide S661, a Site 1-Site 2 combination) or as potent agonists (e.g. peptide S597, a Site 2-Site 1 combination), depending on the order in which the individual Site 1 and Site 2 peptides were linked (2, 3). 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. The insulin receptor (IR) itself is a disulfide-linked (␣␤) 2 homodimer; each ␣␤ monomer comprises, from its N terminus, two homologous leucine-rich repeat domains (L1 and L2) separated by a cysteine-rich region (CR) comprising eight disulfide-linked modules (6). These domains are followed by three fibronectin type III d...

Face-time with TAR: Portraits of an HIV-1 RNA with diverse modes of effector recognition relevant for drug discovery

“…In terms of the buried surface area and shape complementarity of the TAR-TBP6.7 interface, a total of 718 Å 2 of TAR is sequestered, wherein 384 Å 2 is attributable to the ␤2-␤3 loop. Recognition of TAR by TBP6.7 gives an S c value of 0.79 (77), which is comparable with S c values of peptides selected by phage display to bind the insulin receptor ectodomain (136). Overall, these properties closely resemble comparable metrics for the BIV and HIV TAR-Tat complexes (Fig.…”

Insulin mimetic peptide S371 folds into a helical structure

“…Given steric constraints on α CT and its displacement on insulin binding, our models outlined above collectively suggest that in the IR ectodomain S371 binds to L1 in a conformation similar to that of α CT in its apo or displaced positions. These conclusions are consistent with a recently reported crystallographic structure of the mimetic peptide S519C16 (the C‐terminal 16‐residue Site 1 component of S519 that is identical to S371) in complex with a fragment of the IR ectodomain that contains the L1 domain . We show a snapshot of the S519C16/L1 complex in Fig.…”

“…As S371 is added to the C‐terminus of the affinity‐optimized agonist S519, we speculate that the Site 2 part of S519 likely folds into short helical motifs similar to the A‐chain of insulin, particularly given that the Site 2 sequence also contains a disulfide bond . Some tentative modeling of the Site 2 sequence in a recent study supports this suggestion . Conversely, S371 is added at the N‐terminus of antagonist S661 and in case it occupies the position of the insulin B‐chain (as shown in Fig.…”

The signalling conformation of the insulin receptor ectodomain

“…IRΔβ-zip (Supplementary Table 1 ) was purified by insulin elution from an insulin-affinity column, then complexed with the variable domain module (Fv) of the anti-IR antibody 83-7 12 , and finally subjected to mild endoglycosidase H treatment—the latter two steps initially intended to aid crystallographic study of the complex 13 . Insulin remained bound to IRΔβ-zip throughout these steps (see Methods and Supplementary Fig.…”

“…Fv 83-7 was prepared using a Brevibacillus expression system using protocols identical to those described previously 13 . Briefly, codon- and expression-optimised DNA corresponding to murine monoclonal antibody 83-7 variable heavy (VH) chain residues 1-118 5 , 6 , 12 followed by the sequence SLVPRGSSSEQKLISEEDLN (thrombin cleavage site + c-myc tag) was synthesized and then cloned into the vector pCDNA3.1 by DNA2.0 (USA).…”