鈣連蛋白S100P與RAGE蛋白V domain, Pentamidine,以 及Cromolyn sodium相互結合的研究

Abstract

S100P is a homodimeric protein that belongs to the S100 subfamily of EF hand of calcium binding proteins. Calcium bounded S100P is known to activate the RAGE receptor is known to stimulate both ERK and NF-kappa B signaling. RAGE receptor and S100P protein involved in a wide range of inflammation-related pathological states, such as vascular diseases, diabetes, neurodegeneration and cancer. As RAGE receptor and S100P play an important role in tumor formation, it is clear that preventing the formation of RAGE-S100P multi-protein complex is an effective strategy to inhibit various cancers. Despite having its importance, the detailed structural characterization of the S100P-RAGE complex has not yet been reported. In this study, we report model structure of S100P- V domain of RAGE complex and interactions of S100P with Pentamedine and cromolyn. In chapter I, we reported here the complete backbone and side chain NMR chemical shift assignment of S100P protein in calcium bound form. The assignment details reported here would be useful to identify the binding interface features of S100P with various its binding targets and its potential antagonists. In chapter II, we elucidated the structural interactions between S100P and RAGE V domain. We employed a variety of biophysical techniques, including isothermal titration calorimetry, fluorescence spectroscopy, multidimensional NMR spectroscopy, Docking (HADDOCK), mutagenesis study and functional assay to characterize the interactions between S100P and RAGE V domain. The binding constant was determined from isothermal titration calorimetry, fluorescence spectroscopy. The binding interfaces upon complex formation of S100P and RAGE V mapping from 15N-1H HSQC titrations. Further HADDOCK modeling and mutagenesis study and functional assay indicated the role of important residues of S100P protein for RAGE V domain protein interactions and suggest novel mode of S100P binding with other S100 proteins for RAGE receptor recognition. In this study, we also identified pentamidine as a small molecule that can bind to S100P and inhibit the interactions between S100P and the RAGE V domain, according to our HADDOCK binding model and functional assay studies. In chapter III, we focused on characterization of interactions between S100P and cromolyn. The binding between S100P and cromolyn characterized using a variety of biophysical methods, such as fluorescence spectroscopy and 15N-1H HSQC titrations. The binding constant (Kd) was determined by fluorescence spectroscopy and the data from 15N-1H HSQC titrations indicated that cromolyn binds to hydrophobic cavity of S100P protein, which located over loop-3, helix-4 of the helix-1 of the other monomer of the S100P dimer. Further conformational flexibility and stability of the S100P-cromolyn complex was studied by molecular dynamics study (GROMACS). Overall, the results indicated that designing hydrophobic ligands that can access the hydrophobic pocket that is located over the linker region, helix-4 from one monomer and helix-1 of another monomer of the S100P dimer can prevent RAGE interactions. The present study describes the binding properties of S100P-cromolyn and provides knowledge of the binding sites at the molecular level, which facilitate the design of better drugs to disrupt the S100P-RAGE pathway and treat various diseases, such as cancer, metastasis, and diabetes.