How insulin binds to and triggers the insulin receptor (IR) represents a fundamental problem at the intersection of basic science and clinical medicine. This application, building on a long-term program of research by a team of investigators, promises to provide a new foundation for analysis of structure-function relationships. Prior progress in the field was limited by the absence of a crystal structure of the hormone-IR complex. In accordance with the Aims of the last renewal, collaborative crystal structures have been obtained of (i) the primary hormone-binding surface of the receptor ectodomain ("Site 1";Smith, B.J. et al. Proc. Natl. Acad. Sci. USA 107, 6771-6 (2010)) and (ii) insulin bound to Site 1 (the "micro-receptor" complex;Menting, J.G. et al. Nature, 493, 241-5 (2013)). The physiological significance of these structures have been validated in the context of the holo-receptor (a) through site-specific photo-cross-linking studies (Xu, B., et al. J. Biol. Chem. 284, 14597-08 (2009)) "Paper of the Week" and (b) by mutagenesis with application to monogenic syndromes of diabetes mellitus associated with mutations in insulin or its receptor (Whittaker, J., et al. Prc. Natl. Acad. Sci. USA 109, 11166 (2012)). We request continued NIH support to address the following Specific Aims.
Aim 1. Functional Mapping of the Functional Surfaces of Insulin. We will employ mutagenesis and residue-specific photo-cross-linking to map the cognate Site-1 and Site-2 binding surfaces of insulin and the IR.
Aim 2 Structure of a Model Hormone-Receptor Complex. We seek to improve the resolution and completeness of the micro-receptor structure through synthesis of heavy-atom derivatives of insulin and design of ultra-stable insulin analogs. Inferred structure-function relationships will be tested by non-standard protein design.
Aim 3. Biophysical Probes of the Micro-Receptor and Successive Complexes. Two biophysical probes of structure and dynamics will be employed: (a) TROSY-based heteronuclear NMR studies of the micro-receptor complex at 900 MHz and (b) synchrotron-based hydroxyl-radical footprinting (with MS-MS detection) of the micro-receptor and larger receptor fragments, culminating in comparative studies of the holoreceptor.
Aim 4. Toward the Structure of a Hormone-Ectodomain Complex. As a long-term goal, we seek to extend our collaboration with M. Lawrence (Melbourne, AU) to a crystal structure of a hormone-ectodomain complex. Together, these Aims represent the culmination of a 25-year program of research that has sought to combine basic science and its potential translation. Insulin and the insulin receptor represent an ancestral signaling system in metazoans of central importance to human health. My colleagues and I are grateful to the Study Section and NIDDK for making this continuing adventure possible.

Public Health Relevance

How insulin binds to and triggers the insulin receptor represents a major unsolved problem in cell biology and medicine with broad implications for the treatment of diabetes mellitus. This program of research seeks both to decipher the molecular mechanisms by which such binding and signaling occurs. An interdisciplinary approach is proposed combining biochemical, biophysical and structural techniques.

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Molecular and Cellular Endocrinology Study Section (MCE)
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Sechi, Salvatore
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Case Western Reserve University
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Avital-Shmilovici, Michal; Mandal, Kalyaneswar; Gates, Zachary P et al. (2013) Fully convergent chemical synthesis of ester insulin: determination of the high resolution X-ray structure by racemic protein crystallography. J Am Chem Soc 135:3173-85
Weiss, Michael A (2013) Diabetes mellitus due to the toxic misfolding of proinsulin variants. FEBS Lett 587:1942-50
Phillips, Nelson B; Whittaker, Jonathan; Ismail-Beigi, Faramarz et al. (2012) Insulin fibrillation and protein design: topological resistance of single-chain analogs to thermal degradation with application to a pump reservoir. J Diabetes Sci Technol 6:277-88
Kiselar, Janna G; Datt, Manish; Chance, Mark R et al. (2011) Structural analysis of proinsulin hexamer assembly by hydroxyl radical footprinting and computational modeling. J Biol Chem 286:43710-6
Sohma, Youhei; Hua, Qing-Xin; Whittaker, Jonathan et al. (2010) Design and folding of [GluA4(ObetaThrB30)]insulin ("ester insulin"): a minimal proinsulin surrogate that can be chemically converted into human insulin. Angew Chem Int Ed Engl 49:5489-93
Smith, Brian J; Huang, Kun; Kong, Geoffrey et al. (2010) Structural resolution of a tandem hormone-binding element in the insulin receptor and its implications for design of peptide agonists. Proc Natl Acad Sci U S A 107:6771-6
Yang, Yanwu; Hua, Qing-Xin; Liu, Jin et al. (2010) Solution structure of proinsulin: connecting domain flexibility and prohormone processing. J Biol Chem 285:7847-51
Sohma, Youhei; Hua, Qing-xin; Liu, Ming et al. (2010) Contribution of residue B5 to the folding and function of insulin and IGF-I: constraints and fine-tuning in the evolution of a protein family. J Biol Chem 285:5040-55
Hua, Qing-xin; Xu, Bin; Huang, Kun et al. (2009) Enhancing the activity of a protein by stereospecific unfolding: conformational life cycle of insulin and its evolutionary origins. J Biol Chem 284:14586-96
Xu, Bin; Huang, Kun; Chu, Ying-Chi et al. (2009) Decoding the cryptic active conformation of a protein by synthetic photoscanning: insulin inserts a detachable arm between receptor domains. J Biol Chem 284:14597-608

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