The gaseous plant hormone ethylene influences many physiological processes in plant growth and development. Plant ethylene responses are mediated by a family of ethylene receptors, in which the N-terminal transmembrane domains are responsible for ethylene binding and membrane localization. Until now, little structural information was available on the molecular mechanism of ethylene responses by the transmembrane binding domain of ethylene receptors. Here, we screened different constructs, fusion tags, detergents, and purification methods of the transmembrane sensor domain of ethylene receptors. However, due to their highly hydrophobic transmembrane domain (TMD), only a KSI-fused LeETR21–131from tomato yielded a good-quality nuclear magnetic resonance (NMR) spectrum in the organic solvent. Interestingly, a dimer model of LeETR21–131built by the AlphaFold2 algorithm showed greatly converged structures. The interaction analysis of ethylene and LeETR21–131using molecular docking and molecular dynamics (MD) simulations demonstrated the potential binding sites of ethylene in LeETR21–131. Our exploration provides valuable knowledge for further understanding of the ethylene-perception process in ethylene receptors.Keywords:LeETR2;AlphaFold2;membrane protein expression;solution NMR;organic solvent;molecular docking;molecular dynamics simulation
Han Liu,Lanjing Li,Ting Liang,Ru Huan,Bruno Hagenbuch,Chunshan Gui
Human organic anion transporting polypeptide (OATP) 1B1 and 1B3 are two highly homologous liver-specific uptake transporters. However, 2’,7’-dichlorofluorescein (DCF) is preferably transported by OATP1B1. In the present study, the molecular mechanisms for the selective transport of DCF by OATP1B1 were investigated by constructing and characterizing an array of OATP1B1/1B3 chimeras and site-directed mutagenesis. Our results show that transmembrane domain (TM) 10 is crucial for the surface expression and function of OATP1B1, in which Q541 and L545 play the most important roles in DCF transport. Replacement of TM10 in OATP1B1 with its OATP1B3 counterpart led to OATP1B1’s complete intracellular retention. Q541 and L545 may interact with DCF directly via hydrogen bonding and hydrophobic interactions. The decrease of DCF uptake by Q541A and L545S was due to their reduced binding affinity for DCF as compared with OATP1B1. In addition, Q541 and L545 are also crucial for the transport of estradiol-17β-glucuronide (E17βG) but not for the transport of estrone-3-sulfate (E3S), indicating different interaction modes between DCF/E17βG and E3S in OATP1B1. Taken together, Q541 and L545 in TM10 are critical for OATP1B1-mediated DCF uptake, but their effect is substrate-dependent.
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