Specific zinc binding to heliorhodopsin

Abstract

Heliorhodopsins (HeRs), a recently discovered family of rhodopsins, have an inverted membrane topology compared to animal and microbial rhodopsins. The slow photocycle of HeRs suggests a light-sensor function, although the actual function remains unknown. Although HeRs exhibit no specific binding of monovalent cations or anions, recent ATR-FTIR spectroscopy studies have demonstrated the binding of Zn²⁺ to HeR from Thermoplasmatales archaeon (TaHeR) and 48C12. Even though ion-specific FTIR spectra were observed for many divalent cations, only helical structural perturbations were observed for Zn²⁺-binding, suggesting a possible modification of the HeR function by Zn²⁺. The present study shows that Zn²⁺-binding lowers the thermal stability of TaHeR, and slows back proton transfer to the retinal Schiff base (M decay) during its photocycle. Zn²⁺-binding was similarly observed for a TaHeR opsin that lacks the retinal chromophore. We then studied the Zn²⁺-binding site by means of the ATR-FTIR spectroscopy of site-directed mutants. Among five and four mutants of His and Asp/Glu, respectively, only E150Q exhibited a completely different spectral feature of the α-helix (amide-I) in ATR-FTIR spectroscopy, suggesting that E150 is responsible for Zn²⁺-binding. Molecular dynamics (MD) simulations built a coordination structure of Zn²⁺-bound TaHeR, where E150 and protein bound water molecules participate in direct coordination. It was concluded that the specific binding site of Zn²⁺ is located at the cytoplasmic side of TaHeR, and that Zn²⁺-binding affects the structure and structural dynamics, possibly modifying the unknown function of TaHeR.