| Summary: | 博士 === 國立清華大學 === 生物資訊與結構生物研究所 === 104 === Membrane-embedded pyrophosphatase (M-PPase) hydrolyzes PPi to drive the transportation of H+ or/and Na+ across plasma/vacuolar membranes. M-PPase is composed of 16 transmembrane helices (TMs) with the ion translocation pathway constructed by core TMs 5/6/11/12/15/16. The key residues R242/D294/K742/E301 in TMs 5/6/16 in proton translocation pathway of Vigna radiata H+-pumping M-PPase (VrH+-PPase) have been identified. However, the detailed ion transport mechanism is still unclear. In this study, the crystal structures of the VrH+-PPase-2Pi and VrH+-PPase-Pi complexes were determined. It suggests that opening and closing of the PPi-binding pocket are mediated by motions in inner TMs 5/6 and outer TMs 13/14. From the mutagenesis study of T228D in TM5 and L555K in TM12 at the hydrophobic gate of VrH+-PPase, water molecules were observed at the hydrophobic gates of T228D and L555K mutants’ structures. It suggests that the hydrophobic gate of VrH+-PPase could potentially be hydrated to allow the proton passing through the exit channel. Furthermore, the mutagenesis study of E225 in TM5 at the exit channel of VrH+-PPase shows that E225S and E225H mutants’ structures preserve the essential interaction of E225 with R562 in TM12 at the end of the exit channel to the lumen. Overall, the essential proton carrier R242 in TM5 accepts the proton from D287/D731 in TMs 6/16 at the entrance of proton transport, and the salt-bridge of E225-R562 is located at the exit of proton release. It proposes that R242 and R562 might act as the initiator and terminator of proton translocation, respectively.
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