How Does Thiosulfate-Binding Protein CysP Efficiently Transport S2O3²ˉ

Abstract

Sulfur, one of major elements, constitutes lots of functional organic molecules in vivo[1]. Its content is strictly controlled by many transport systems. In Gram-negative bacteria, sulfur/thiosulfate can be efficiently transferred into cell by sulfate/thiosulfate transport system which consists of ATP dependent membrane protein CysU/CysW, inner member binding protein CysA and system unique protein (thiosulfatebinding protein CysP or sulfate-binding protein Sbp).Although function overlapping, the two systems show obvious substrate specificity. Thiosulfate transport system has stronger binding affinity with thiosulfate than sulfate. Its unique protein CysP shows highly sequence conservation (>90%) among lots of pathogenic gram-negative bacteria. Thus, CysP has been suggested to be a good candidate of vaccine antigen. Here, we elaborate its exact maturing progress. It presents as a dimer in cytoplasm with a signal peptide and then shows as the trimer in periplasmic space once its signal peptide is deleted. Interestingly, our thermal shift data indicate that, its native substrate S2O3 2-, unlike SO4 2-, doesn’t show obvious effect on strengthening the thermal stability of immature CysP although both S2O3 2- and SO4 2- can enhance mature CysP thermal stability, which may come from different binding affinity as revealed from SPR data which show that the deletion leads to the enhancement in its substrate specificity, i.e. the binding affinity (Kd) is enhanced about 8 times (3.9 and 32.6 nM) between mature CysP and its potential substrate ions, S2O32- or SO4 2-, instead of comparable binding (17.43 and 11.1 nM) when the signal peptide is still located at the Nterminal of CysP. Furthermore, we show that the native CysP bind 8-fold S2O3 2-, whereas without obvious binding to Mo and Se. Our crystallography data further support these arguments, that substrate ion S2O3 2- is compactly filled into a 7.6Å cavity by interaction with Y12, D13, S48, N67, S134, N136, and F196. This work is supported by a grant from Research Grants Council of Hong Kong (170461P).