Rhodopsin from Haloquadratum walsbyi is a light-driven magnesium transporter

The 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.