A Crystallographic Study Unveils a Novel Magnesium Transporting Rhodopsin

Abstract The functionally unknown Middle rhodopsin (HwMR) is microbial rhodopsin (mRho) identified in Haloquadratum walsbyi, a microbe that thrives in 2 M MgCl₂ environment harmed to most microorganisms. HwMR shares conserved and functionally critical residues with both bacteriorhodopsin (BR), a proton pump, and sensory rhodopsin (SR) that function in phototaxis, even though HwMR exerts neither function. We previously reported that it uniquely associates with Mg²⁺. Here, we showed that HwMR was an inward magnesium regulator, evidenced by solving the atomic structures of wild-type and D84N mutant and a cell-based light-driven conductivity assay. HwMR can sense environmental Mg²⁺ concentration via the D84 residue according to maximum absorbance (Abs-max) and photocycle kinetics results. Furthermore, two main Mg²⁺ binding sites were identified in HwMR wild-type structure but not in D84N mutant. Structural analysis demonstrated that D84N induced changes in the BC-loop on the extracellular side, causing a lower hydrophobicity and disturbing resistance against external solvents. D84N also altered the local environment in solvent-excluded areas and Coulombic electrostatic potentials around the Mg²⁺ binding sites. On the cytoplasmic side, T216 was found to stabilize Mg²⁺ directly, a phenomenon also supported by optical properties experiments. Finally, a sequential model was proposed to illustrate Mg²⁺ transportation in HwMR.