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      Contribution of TyrB26 to the Function and Stability of Insulin: STRUCTURE-ACTIVITY RELATIONSHIPS AT A CONSERVED HORMONE-RECEPTOR INTERFACE.

      The Journal of Biological Chemistry
      American Society for Biochemistry & Molecular Biology (ASBMB)
      receptor-tyrosine kinase, protein structure, non-standard mutagenesis, hormone, diabetes

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          Abstract

          Crystallographic studies of insulin bound to receptor domains have defined the primary hormone-receptor interface. We investigated the role of Tyr(B26), a conserved aromatic residue at this interface. To probe the evolutionary basis for such conservation, we constructed 18 variants at B26. Surprisingly, non-aromatic polar or charged side chains (such as Glu, Ser, or ornithine (Orn)) conferred high activity, whereas the weakest-binding analogs contained Val, Ile, and Leu substitutions. Modeling of variant complexes suggested that the B26 side chains pack within a shallow depression at the solvent-exposed periphery of the interface. This interface would disfavor large aliphatic side chains. The analogs with highest activity exhibited reduced thermodynamic stability and heightened susceptibility to fibrillation. Perturbed self-assembly was also demonstrated in studies of the charged variants (Orn and Glu); indeed, the Glu(B26) analog exhibited aberrant aggregation in either the presence or absence of zinc ions. Thus, although Tyr(B26) is part of insulin's receptor-binding surface, our results suggest that its conservation has been enjoined by the aromatic ring's contributions to native stability and self-assembly. We envisage that such classical structural relationships reflect the implicit threat of toxic misfolding (rather than hormonal function at the receptor level) as a general evolutionary determinant of extant protein sequences.

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          Most cited references69

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          Protein folding and misfolding.

          The manner in which a newly synthesized chain of amino acids transforms itself into a perfectly folded protein depends both on the intrinsic properties of the amino-acid sequence and on multiple contributing influences from the crowded cellular milieu. Folding and unfolding are crucial ways of regulating biological activity and targeting proteins to different cellular locations. Aggregation of misfolded proteins that escape the cellular quality-control mechanisms is a common feature of a wide range of highly debilitating and increasingly prevalent diseases.
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            Crystal structure of the activated insulin receptor tyrosine kinase in complex with peptide substrate and ATP analog.

            The crystal structure of the phosphorylated, activated form of the insulin receptor tyrosine kinase in complex with a peptide substrate and an ATP analog has been determined at 1.9 A resolution. The activation loop (A-loop) of the kinase undergoes a major conformational change upon autophosphorylation of Tyr1158, Tyr1162 and Tyr1163 within the loop, resulting in unrestricted access of ATP and protein substrates to the kinase active site. Phosphorylated Tyr1163 (pTyr1163) is the key phosphotyrosine in stabilizing the conformation of the tris-phosphorylated A-loop, whereas pTyr1158 is completely solvent-exposed, suggesting an availability for interaction with downstream signaling proteins. The YMXM-containing peptide substrate binds as a short anti-parallel beta-strand to the C-terminal end of the A-loop, with the methionine side chains occupying two hydrophobic pockets on the C-terminal lobe of the kinase. The structure thus reveals the molecular basis for insulin receptor activation via autophosphorylation, and provides insights into tyrosine kinase substrate specificity and the mechanism of phosphotransfer.
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              Structural biology of insulin and IGF1 receptors: implications for drug design.

              Type 2 diabetes mellitus -- in which the body produces insufficient amounts of insulin or the insulin that is produced does not function properly to control blood glucose -- is an increasingly common disorder. Prospective clinical studies have proven the benefits of tighter glucose control in reducing the frequency and severity of complications of the disease, leading to the advocation of earlier and more aggressive use of insulin therapy. Given the reluctance of patients with type 2 diabetes to inject themselves with insulin, orally active insulin mimetics would be a major therapeutic advance. Here, we discuss recent progress in understanding the structure-function relationships of the insulin and insulin-like growth factor 1 (IGF1) receptors, their mechanism of activation and their implications for the design of insulin-receptor agonists for diabetes therapy and IGF1-receptor antagonists for cancer therapy.
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                Author and article information

                Journal
                27129279
                4933216
                10.1074/jbc.M115.708347

                receptor-tyrosine kinase,protein structure,non-standard mutagenesis,hormone,diabetes

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