STRUCTURAL STUDIES OF PAS DOMAIN SIGNALING MECHANISMS

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Description (applicant's description): PAS domains mediate protein/protein interactions among proteins that regulate a wide range of biological functions, including gene expression. The protein-binding capabilities of several PAS domains are modulated by small organic ligands, providing a mechanism for these compounds to regulate complex biological activities. Misregulation of these pathways is deleterious, as demonstrated by the central role of PAS/toxin complexes in developing the carcinogenic effects of several environmental toxins. The proposed research will characterize how PAS domains specifically interact with ligands and other proteins and how these binding events are integrated within a small (100-120 residue) domain. To achieve this, NMR-based structural studies will be conducted in three general areas: 1). Studies of single PAS domains from four eukaryotic proteins, using novel methods to rapidly identify soluble fragments of these domains for NMR studies. Structural information has been obtained using standard methods on two such domains (12 and 20kDa) and suggests that ligand- and protein-binding sites are flexible in the absence of interacting partners. 2). Studies of PAS/ligand complexes. Starting with the information obtained on ligand-free domains, small molecule libraries will be screened by NMR-based methods to identify potential ligands. Preliminary data from these studies demonstrate that several compounds bind to one of the PAS domains under investigation, rigidifying several flexible regions observed in the ligand-free form. These conformational changes will be studied in more detail by examining the structures and dynamics of PAS/ligand complexes. 3). Studies of PAS/protein complexes. Most of these are formed by PAS/PAS heterodimerization, facilitating the use of isotopic labeling to simplify the NMR spectra of these large (about 30kDa) complexes. A mixed resolution approach will be used, generating models of the complex by docking by high resolution structures of the individual domains using multiple low-resolution restraints acquired on the intact complex. These views of PAS domains in different conformations will provide insight into the mechanism of their regulation and possibly open new approaches to the design of therapeutic agents that could assemble/disassemble PAS-mediated complexes.