Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology
Marshae Lashley - Fisk University
Co-Author(s): Isis Christopher, Kofi Amoah, Emmanuel Jackson, Kinara Byrd, Dana Franklin, and Steven Damo, Fisk University, Nashville, TN
S100 proteins are members of the EF-hand class of the S100 family of calcium binding proteins that regulate a diverse set of biological functions. Several members have been shown to activate the receptor for advanced glycation end products (RAGE), a critical receptor in the inflammation response. Increased expression of S100 proteins and RAGE are implicated in a number of inflammatory disorders. Therefore, characterization of S100-RAGE protein-protein interactions are important for understanding the molecular basis of the inflammatory response. Models of S100A12 and S100A8/S100A9 complexes with RAGE were calculated using ZDOCK, a web-based interface for protein-protein docking. Analysis of these models identified a conserved positively charged binding surface of RAGE comprised of lysine 39 and lysine 107. This surface was validated experimentally by creating charged reversal mutations in RAGE that were expressed recombinantly and characterized using a fluorescence based binding assay. These mutant RAGE constructs showed decreased binding affinity for S100A8/S100A9 by a factor of four, suggesting both lysine 39 and lysine 107 residues for binding S100 proteins. To identify inhibitors of S100-RAGE binding, a virtual screen was conducted of a collated library of one thousand small molecules. Future research will involve validation of the virtual screen and x-ray crystallographic studies of S100-RAGE complexes.
Models of S100A12 and S100A8/S100A9 complexes with RAGE were calculated using ZDOCK, a web-based interface for protein-protein docking. Analysis of these models identified a conserved positively charged binding surface of RAGE comprised of lysine 39 and lysine 107. This surface was validated experimentally by creating charged reversal mutations in RAGE that were expressed recombinantly and characterized using a fluorescence based binding assay. These mutant RAGE constructs showed decreased binding affinity for S100A8/S100A9 by a factor of four, suggesting both lysine 39 and lysine 107 residues for binding S100 proteins. To identify inhibitors of S100-RAGE binding, a virtual screen was conducted of a collated library of one thousand small molecules. Future research will involve validation of the virtual screen and x-ray crystallographic studies of S100-RAGE complexes.
Funder Acknowledgement(s): This work was supported by the NSF Award #s 1332491, 1400969; NIH R25MD010396.
Faculty Advisor: Steven Damo, sdamo@fisk.edu
Role: I worked with a team of students to express and purify the S100 proteins used in this study and the RAGE protein. Also I conducted computational analysis to study protein-protein interactions and assisted with analyzing all the results.