Biophysical Characterization of PDX1 interactions with DNA and other transcriptio

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DESCRIPTION (provided by applicant): This project aims to biophysically characterize the interactions of pancreas duodenum homeobox 1 (PDX1) with promoter sequences and other transcription factors. The structure of PDX1 is that of a globular homeodomain (HD) embedded within tails that lack secondary structure and that are generally under-studied. The overall hypothesis of this project is that the disordered tails serve a central function in the mechanism o transcriptional regulation by PDX1; establishing the role of these regions will define the molecular origins of diabetic phenotypes originating from PDX1 mutants harbored within them. PDX1 is a HD-containing transcription factor found to be at the core of regulating insulin transcription, and that is also involved in the regulation of various ¿ -cell specific genes. The disordered tails are involved in mediating protein-protein interactions; however, 1,2 their direct roles in gene regulation are indeterminate. Eleven mutants of PDX1 result in a form of type 2 3-8 diabetes; 9 are located within the disordered tails. These regions have been reasonably characterized at the cell biology level and through promoter activation assays; the direct roles of these regions or the molecular details of PDX1 mutants that result in diabetes remain undefined. Nuclear Magnetic Resonance (NMR) spectroscopy will be used to the atomistically characterize the structure of all regions in PDX1 and mutants. NMR is the technique of choice due to the highly dynamic character of its disordered regions. Simultaneously, Isothermal Titration Calorimetry (ITC) will provide sensitive and complete thermodynamic information defining PDX1 interactions with DNA and other proteins. We have complete 1 backbone assignments of the unbound and bound forms of the HD, which were generated using traditional H 15 9-12 13 and N methods. Novel C detection methods will be employed to characterize the disordered regions. 15 Conformational dynamics of all variants and mutants will be studied by measuring the N spin relaxation and relaxation dispersion. NMR will also be utilized to study the interactions of PDX1 variants and mutants with DNA or other proteins. The thermodynamics of PDX1 interactions with 11 gene promoter elements from 5 genes and interactions with other proteins will be studied by ITC. Based on all ITC data, a catalogue of the binding affinity, stoichiometry, entropy of binding, free energy of the reaction, change in heat capacity and apparent binding enthalpy will be generated. Preliminary binding studies of the PDX1-HD titrated into a sequence containing the core site, 5'-TAAT-3', and the insulin promoter element A3 demonstrate a 1:1 binding stoichiometry at near 14,15 physiological pH and Kd values consistent with in vitro studies. Preliminary NMR revealed a high quality spectrum of the bound form of the HD and ITC data of the HD yielded quantitative binding parameters. The comprehensive structural and binding studies proposed here are needed to more fully characterize the impact of the disordered regions on PDX1 function and the conclusions drawn should be readily generalized for application to other transcription factors containing HD embedded in disordered tails. PUBLIC HEALTH RELEVANCE: The protein, PDX1 serves a vital role in maintaining proper function of the pancreas and glucose sensing necessary for proper metabolism. Mutations to this protein result in malfunctions that lead to type 2 diabetes. Thus, determining the fundamental properties of PDX1 through the proposed work will provide insight into the mechanisms that contribute to malfunction, while providing a framework for future biomedical and therapeutic programs.