Abstract
Retinal bound light-driven proton pumps are widespread in eukaryotic and prokaryotic organisms. Among these pumps, bacteriorhodopsin (BR) proteins cooperate with ATP synthase to convert captured solar energy into a biologically consumable form, ATP. In an acidic environment or when pumped-out protons accumulate in the extracellular region, the maximum absorbance of BR proteins shifts markedly to the longer wavelengths. These conditions affect the light-driven proton pumping functional exertion as well. In this study, wild-type crystal structure of a BR with optical stability under wide pH range from a square halophilic archaeon, Haloquadratum walsbyi (HwBR), was solved in two crystal forms. One crystal form, refined to 1.85 Å resolution, contains a trimer in the asymmetric unit, whereas another contains an antiparallel dimer was refined at 2.58 Å. HwBR could not be classified into any existing subgroup of archaeal BR proteins based on the protein sequence phylogenetic tree, and it showed unique absorption spectral stability when exposed to low pH values. All structures showed a unique hydrogen-bonding network between Arg(82) and Thr(201), linking the BC and FG loops to shield the retinal-binding pocket in the interior from the extracellular environment. This result was supported by R82E mutation that attenuated the optical stability. The negatively charged cytoplasmic side and the Arg(82)-Thr(201) hydrogen bond may play an important role in the proton translocation trend in HwBR under acidic conditions. Our findings have unveiled a strategy adopted by BR proteins to solidify their defenses against unfavorable environments and maintain their optical properties associated with proton pumping.
Highlights
Most bacteriorhodopsins demonstrate red-shifted spectrum in acidic condition
Our findings have unveiled a strategy adopted by BR proteins to solidify their defenses against unfavorable environments and maintain their optical properties associated with proton pumping
The amino acid sequence of HwBR from a quadrateshaped bacterium, H. walsbyi, constitutes a novel group with HmBRII according to the phylogenetic tree analysis (Fig. 1B)
Summary
Most bacteriorhodopsins demonstrate red-shifted spectrum in acidic condition. Results: Structures of Haloquadratum walsbyi bacteriorhodopsin explain stable action spectra from pH 2 to 8. All structures showed a unique hydrogen-bonding network between Arg and Thr201, linking the BC and FG loops to shield the retinal-binding pocket in the interior from the extracellular environment HmBRII showed high optical stability in acidic conditions even down to pH of 1.6 and maintained its light-driven proton pumping activity at pH of 4.0 [19]. The crystal structures of HwBR revealed that a unique arginine residue stabilizes the extracellular loop region by forming hydrogen bonds with a threonine residue located in the membrane edge of extracellular region The importance of this local structure, which shields the interior environment of HwBR from the low pH extracellular area, was further validated by the mutagenesis approach
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