Defining the Interaction Between S100A13 and Annexin II Peptide: Insight into Non-Classical Secretion

Abstract

S100A13 is a calcium binding chaperone protein that is known to be involved in the non-classical export of signal peptide-less proteins, such as fibroblast growth factor (FGF-1) and interleukin-1α,. It has also been shown that the interaction of S100A13 with Annexin II, which exhibits an inducible flip-flop mechanism across the cell bilayer, helps the multiprotein release complex to traverse the membrane bilayer. The interaction of S100A13 and Annexin 2 has been characterized using various biophysical techniques including Isothermal Titration Calorimetry (ITC), Differential Scanning Calorimetry (DSC), and multidimensional NMR spectroscopy. Results of the Isothermal titration calorimetry (ITC) experiments show that holo-S100A13 exhibits preferential binding to Annexin II with high affinity in the low micro molar range compared to apo-S100A13. Equilibrium guanidine hydrochloride denaturation monitored by steady-state fluorescence and limited trypsin digestion analysis both reveal holo-S100A13 to be stabilized upon binding with the Annexin II peptide. ANS (8-anilino-1-napthalene sulfonate) binding experiments indicate that the presence of Annexin II peptide does not increase the solvent accessiblity of hydrophobic residues in holo-S100A13, which is a unique characteristic of S100A13. 1H-15N- HSQC NMR experiments reveal the binding site of the Annexin II peptide on holo-S100A13 to be distinctly different from other S100/Annexin interactions. In order to define the specificity of S100A13 for Annexin II specifically, the interaction of S100A13 with other Annexin peptides was characterized by ITC. The information gained from this study provides valuable information on the interaction between S100A13 and the Annexin II peptide and gives much needed insight into the mysterious pathway of non-classical release of the signal peptide-less proteins.