HwMR is a novel magnesium-associated protein

Microbial rhodopsins (MRho) are vital proteins in Haloarchaea for solar light sensing in extreme living environments. Among them, Haloquadratum walsbyi (Hw) is a species known to survive high MgCl₂ concentrations, with a total of three MRhos identified, including a high-acid-tolerance light-driven proton outward pump, HwBR, a chloride-insensitive chloride pump, HwHR, and a functionally unknown HwMR. Here, we showed that HwMR is the sole magnesium-sensitive MRho among all tested MRho proteins from Haloarchaea. We identified at least D84 as one of the key residues mediating such magnesium ion association in HwMR. Sequence analysis and molecular modeling suggested HwMR to have an extra H8 helix in the cytosolic region like those in signal-transduction-type MRho of deltarhodopsin-3 (dR-3) and Anabaena sensory rhodopsin (ASR). Further, HwMR showed a distinctly prolonged M-state formation under a high concentration of Mg²⁺. On the other hand, an H8 helix truncated mutant preserved photocycle kinetics like the wild type, but it led to missing M-state structure. Our findings clearly suggested not only that HwMR is a novel Mg²⁺-associated protein but that the association with both Mg²⁺ and the H8 domain stabilizes M-state formation in HwMR. We conclude that Mg²⁺ association and H8 are crucial in stabilizing HwMR M state, which is a well-known photoreceptor signaling state.