A Thermodynamic Study of Ligand Binding to the First Three Domains of the Human Insulin Receptor: Relationship between the Receptor α-Chain C-Terminal Peptide and the Site 1 Insulin Mimetic Pepti

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• 作者：John G. Menting ; Colin W. Ward ; Mai B. Margetts ; Michael C. Lawrence
• 刊名：Biochemistry
• 出版年：2009
• 出版时间：June 16, 2009
• 年：2009
• 卷：48
• 期：23
• 页码：5492-5500
• 全文大小：810K
• 年卷期：v.48,no.23(June 16, 2009)
• ISSN：1520-4995

文摘

The C-terminal segment of the insulin receptor (IR) α-chain plays a critical role in insulin binding. This 16-residue peptide together with the central β-sheet of the receptor L1 domain forms one of the insulin binding surfaces of the IR monomer. Here we use isothermal titration calorimetry to assay directly the binding of the IR αCT peptide to an IR construct (IR485) consisting of the three N-terminal domains of the receptor monomer. Our measurements show further that the binding of the IR αCT peptide to IR485 competes with the binding of a prototypical “Site 1” insulin mimetic peptide to the same receptor fragment. The competitive nature of their binding appears to be reflected in a previously undetected sequence similarity between the IR αCT peptide and the Site 1 mimetic peptide. In contrast, a prototypical “Site 2” peptide has very limited affinity for IR485. Taken together, these results complement our recent observation that there is a possible structural relationship between these mimetic peptides and insulin itself. They also add support to the view that the segment of unexplained electron density lying on the surface of the central β-sheet of the L1 domain in the IR ectodomain crystal structure arises from the IR αCT peptide. Finally, we show that mutation of the critical IR αCT peptide residue Phe714 to alanine does not affect the peptide’s affinity for IR485 and conclude that the resultant loss of insulin binding with this mutation results from loss of interaction of the phenylalanine side chain with insulin.