S100A5 is a calcium-binding protein of S100 family, which represents a major ligand to the receptor for advanced glycation end product (RAGE), a pattern recognition receptor engaged in diverse pathological processes. Here we have characterized calcium binding of S100A5 and the complex formation between S100A5 and RAGE using calorimetry and NMR spectroscopy. S100A5 binds to calcium ions in a sequential manner with the equilibrium dissociation constants (K-D) of 1.3 mu M and 3.5 mu M, which corresponds to the calcium-binding at the C-terminal and N-terminal EF-hands. Upon calcium binding, S100A5 interacts with the V domain of RAGE (RAGE-v) to form a heterotrimer (K-D similar to 5.9 mu M) that is distinct among the S100 family proteins. Chemical shift perturbation data from NMR titration experiments indicates that S100A5 employs the periphery of the dimer interface to interact with RAGE-v. Distinct binding mode and stoichiometry of RAGE against different S100 family proteins could be important to modulate diverse RAGE signaling. (C) 2016 Elsevier Inc. All rights reserved.