S100B is a calcium sensing protein belonging to the S100 protein family with 
intracellular and extracellular roles. It is one of the EF hand homodimeric 
proteins, which is known to interact with various protein targets to regulate 
varied biological functions. Extracellular S100B has been recently reported to 
interact with FGF2 in a RAGE-independent manner. However, the recognition 
mechanism of S100B-FGF2 interaction at the molecular level remains unclear. In 
this study, the critical residues on S100B-FGF2 interface were mapped by 
combined information derived from NMR spectroscopy and site directed mutagenesis 
experiments. Utilizing NMR titration data, we generated the structural models of 
S100B-FGF2 complex from the computational docking program, HADDOCK which were 
further proved stable during 15ns unrestrained molecular dynamics (MD) 
simulations. Isothermal titration calorimetry studies indicated S100B 
interaction with FGF2 is an entropically favored process implying dominant role 
of hydrophobic contacts at the protein-protein interface. Residue level 
information of S100B interaction with FGF2 was useful to understand the varied 
target recognition ability of S100B and further explained its role in effecting 
extracellular signaling diversity. Mechanistic insights into the S100B-FGF2 
complex interface and cell-based assay studies involving mutants led us to 
conclude the novel role of S100B in FGF2 mediated FGFR1 receptor inactivation.