Ko, Ling-NingLing-NingKoLim, Guo ZhenGuo ZhenLimChen, Jui-ChienJui-ChienChenKo, TaTaKoLi, Guan-YiGuan-YiLiCHII-SHEN YANG2025-06-162025-06-162025-05-14https://www.scopus.com/record/display.uri?eid=2-s2.0-105005116266&origin=resultslisthttps://scholars.lib.ntu.edu.tw/handle/123456789/730004The functionally unknown Middle rhodopsin (HwMR) is a microbial rhodopsin (mRho) identified in Haloquadratum walsbyi, an archaeon that thrives in a 2 M MgCl2 environment harmful to most other microorganisms. HwMR shares conserved and functionally critical residues with both bacteriorhodopsin (BR), a proton pump, and sensory rhodopsin II (SRII), which mediates phototaxis, even though HwMR exerts neither function. We previously reported HwMR as a unique mRho found to associate with Mg2+. Here, we show that HwMR can sense environmental Mg2+ concentration via the D84 residue according to characteristic maximum absorption wavelength shift, photocycle kinetics, and Mg2+ titration assay. X-ray crystallography of the wild-type HwMR and its D84N mutant produced two HwMR atomic structure models. Omit maps analysis of the wild-type HwMR model revealed D84 as a Mg2+ binding site. On the cytoplasmic side, omit maps also revealed Mg2+ association with T216. Both Mg2+ sites were absent in the D84N mutant. A cell-based light-driven conductivity assay provided evidence to propose that HwMR is an inward magnesium transporter, with D84 as the primary binding site and T216 as the transportation stabilizing site. A sequential model was proposed to illustrate Mg2+ transportation in HwMR.enjournal article10.1038/s41467-025-59795-y