Molecular mechanism of light-driven sodium pumping

Kirill Kovalev ,Gleb Bourenkov ,Valentin Borshchevskiy ,Ernst Bamberg ,Alexey Alekseev ,Dmitrii Zabelskii ,Ivan Gushchin ,И И Сергеев ,Taras Balandin ,Egor V Zinovev ,Maksim Rulev ,Georg Büldt ,Egor Marin ,Dmytro Volkov ,Roman Astashkin ,Roeland Boer ,Antoine Royant ,Philippe Carpentier ,Nina Maliar ,Philipp Orekhov ,Christian Baeken ,X Carpena ,Svetlana Vaganova ,Valentin Gordeliy

doi:10.1038/s41467-020-16032-y

Abstract

The light-driven sodium-pumping rhodopsin KR2 from Krokinobacter eikastus is the only non-proton cation active transporter with demonstrated potential for optogenetics. However, the existing structural data on KR2 correspond exclusively to its ground state, and show no sodium inside the protein, which hampers the understanding of sodium-pumping mechanism. Here we present crystal structure of the O-intermediate of the physiologically relevant pentameric form of KR2 at the resolution of 2.1 Å, revealing a sodium ion near the retinal Schiff base, coordinated by N112 and D116 of the characteristic NDQ triad. We also obtained crystal structures of D116N and H30A variants, conducted metadynamics simulations and measured pumping activities of putative pathway mutants to demonstrate that sodium release likely proceeds alongside Q78 towards the structural sodium ion bound between KR2 protomers. Our findings highlight the importance of pentameric assembly for sodium pump function, and may be used for rational engineering of enhanced optogenetic tools.

Highlights

The light-driven sodium-pumping rhodopsin KR2 from Krokinobacter eikastus is the only nonproton cation active transporter with demonstrated potential for optogenetics
KR2 contains a characteristic for all known Na+-pumping rhodopsins (NaRs) set of N112, D116, Q123 residues in the helix C (NDQ motif)
As the difference electron density maps do not indicate even a low fraction of the ground state in the structure, show the Na+ bound near the RSB region, which is characteristic for the Ostate of KR2, and the O-state is a dominant intermediate of the KR2 photocycle, we consider the trapped intermediate as solely the O-state

Summary

Introduction

The light-driven sodium-pumping rhodopsin KR2 from Krokinobacter eikastus is the only nonproton cation active transporter with demonstrated potential for optogenetics. It was recently reported that in the ground state under physiological conditions (PDB ID: 6REW4) KR2 has a large water-filled cavity near the RSB (Schiff base cavity) This conformation of the protein was called ‘expanded’[4,12], and only occurs in the pentameric form of KR2. Pathways of proton in light-driven pumps cannot be the same as those in the cation pumps like NaRs. All history of studies of BR, halorhodopsin (HR) and sensory rhodopsin II (SRII) shows that high-resolution structures of the intermediate states of a rhodopsin are key for the understanding of the mechanisms. It is even more valid in the case of NaRs, as Na+ inside the protein is absent in the ground state of KR2, which provides a wide room for speculations on the mechanism of Na+ transport

Methods

Results

Conclusion