Abstract 1382: Use of polarized light spectroscopy (CD) to study STAT3 folding and STAT3:ligand interactions

Abstract

Abstract STAT3 is a homodimeric transcription factor that is over-expressed in variety of solid tumours. It is known to trigger transcription of a range of genes associated with tumour growth and metastasis. In order to use the STAT3/STAT3 dimer for drug discovery, the factors affecting dimerisation of the monomeric protein and the interaction with ligands must be understood. In this study, we have demonstrated that Circular Dichroism (CD) can be used as a tool to study these processes. We have found that formation of the dimeric STAT3/STAT3 complex results in an induced CD signal which is absent in monomeric protein. Thus, CD can be used to optimize conditions for the production and storage of un-phosphorylated (un-P-) and phosphorylated (P-) STAT3. A similar induced signal was observed when a small molecule (or peptide ligand) interacted with either un-P- or P-STAT3 due to chiral perturbation of the protein structure or electron rearrangements. Crucially, the intensity of the induced CD signal was found to depend on the strength and geometry of the interaction of the binding partner. CD was also used to determine the secondary structure of un-P- and P-STAT3. HIS- and FLAG-tagged STAT3 were examined using the far-UV spectral region (190-250 nm), and the regular folded environment of the peptidic bonds was used to determine the alpha-helix, beta-sheet, and random coil content of different batches of STAT3 protein. For batches of P-STAT3, the composition was found to be 28.3% α-helix, 22.3% β-sheet and 49.4% random coils. For un-P-STAT3, the β-sheet content was slightly higher (23.2%) while the α-helix content was lower (26.8%). Next, protein:ligand interactions which alter the conformation of the protein were studied. Titration of un-P-STAT monomer with different small-molecule inhibitors produced a significant change in the CD spectrum consistent with modification of secondary structure due to altered folding. In particular, significant effects on the secondary structures of both un-P- and P-STAT3 were observed upon binding of the natural peptidic ligands LKTKF and LPQTV. Crucially, the different complex geometries adopted by the proteins upon interaction with ligands could be used to determine the stoichiometry of interaction and the orientation of the ligands in the binding pocket of the protein. These results demonstrate that CD spectroscopy can be used to evaluate STAT3 structure and ligand interactions at the molecular level, and thus represents a useful analytical tool in the development of small molecule inhibitors for potential therapeutic use. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1382. doi:10.1158/1538-7445.AM2011-1382