Plants need to protect themselves from excess light, which causes photo-oxidative damage and lowers the efficiency of photosynthesis. Photosystem II subunit S (PsbS) is a pH sensor protein that plays a crucial role in plant photoprotection by detecting thylakoid lumen acidification in excess light conditions via two lumen-faced glutamates. However, how PsbS is activated under low-pH conditions is unknown. To reveal the molecular response of PsbS to low pH, here we perform an NMR, FTIR and 2DIR spectroscopic analysis of Physcomitrella patens PsbS and of the E176Q mutant in which an active glutamate has been replaced. The PsbS response mechanism at low pH involves the concerted action of repositioning of a short amphipathic helix containing E176 facing the lumen and folding of the luminal loop fragment adjacent to E71 to a 310-helix, providing clear evidence of a conformational pH switch. We propose that this concerted mechanism is a shared motif of proteins of the light-harvesting family that may control thylakoid inter-protein interactions driving photoregulatory responses.

The membrane protein Photosystem II subunit S (PsbS) is a pH sensor that plays an essential role in signaling light stress in plants to prevent photo oxidation and generation of detrimental reactive species. PsbS detects thylakoid lumen acidification in excess light conditions via two glutamates facing the lumen, however, its molecular mechanism for activation is elusive. We performed a spectroscopic analysis of wild type Physcomitrella patens PsbS and of mutants in which the active glutamates have been replaced (E69Q, E174Q and E69Q / E174Q). We discovered that E69 exerts allosteric control of PsbS dimerization, while E174 is essential for the secondary structural response to low pH. Based on our results, we propose a molecular pH response mechanism that involves a change in the amphiphatic short helix containing E174 facing the lumen, moving from the aqeuous phase into the hydrophobic membrane phase. This structural mechanism may be a shared motif of protein molecular switches of the light-harvesting family and its elucidation could open new routes for crops engineering to improve photosynthetic production of biomass.