X-ray Crystallographic Structure and Oligomerization of Gloeobacter Rhodopsin

Takefumi Morizumi,Oliver P Ernst,Leonid S Brown,Wei-Lin Ou,Hideki Kandori,Ned Van Eps,Keiichi Inoue

doi:10.1038/s41598-019-47445-5

Takefumi Morizumi, Oliver P Ernst + Show 5 more

Open Access

https://doi.org/10.1038/s41598-019-47445-5

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Abstract

Gloeobacter rhodopsin (GR) is a cyanobacterial proton pump which can be potentially applied to optogenetics. We solved the crystal structure of GR and found that it has overall similarity to the homologous proton pump from Salinibacter ruber, xanthorhodopsin (XR). We identified distinct structural characteristics of GR’s hydrogen bonding network in the transmembrane domain as well as the displacement of extracellular sides of the transmembrane helices relative to those of XR. Employing Raman spectroscopy and flash-photolysis, we found that GR in the crystals exists in a state which displays retinal conformation and photochemical cycle similar to the functional form observed in lipids. Based on the crystal structure of GR, we selected a site for spin labeling to determine GR’s oligomerization state using double electron–electron resonance (DEER) spectroscopy and demonstrated the pH-dependent pentamer formation of GR. Determination of the structure of GR as well as its pentamerizing propensity enabled us to reveal the role of structural motifs (extended helices, 3-omega motif and flipped B-C loop) commonly found among light-driven bacterial pumps in oligomer formation. Here we propose a new concept to classify these pumps based on the relationship between their oligomerization propensities and these structural determinants.

Highlights

Many organisms utilize light-driven transporter proteins to capture light for energy conversion or cell signaling
We identify pentameric oligomerization of Gloeobacter rhodopsin (GR) in detergent micelles using double electron–electron resonance (DEER) spectroscopy[27], which we demonstrate to be a powerful tool for determining oligomeric assemblies of microbial rhodopsins
Similar to other microbial rhodopsins, the seven transmembrane helices are bundled in a clockwise fashion when observed from the cytoplasmic side, and the all-trans-retinal chromophore is covalently bound to the opsin protein moiety via a Schiff base linkage with the Lys[257] side chain

Summary

Introduction

Many organisms utilize light-driven transporter proteins to capture light for energy conversion or cell signaling. Electron paramagnetic resonance (EPR) spectroscopy combined with site-directed spin-labeling is a powerful tool for characterizing conformational changes in microbial rhodopsins, and for intermolecular distance measurements between protomers providing structural insight into oligomer formation of these 7-TM proteins[19,20].

Results

Conclusion