Structural basis for Na+ transport mechanism by a light-driven Na+ pump

Abstract

Krokinobacter eikastus rhodopsin 2 (KR2) is the first light-driven Na(+) pump discovered, and is viewed as a potential next-generation optogenetics tool. Since the positively charged Schiff base proton, located within the ion-conducting pathway of all light-driven ion pumps, was thought to prohibit the transport of a non-proton cation, the discovery of KR2 raised the question of how it achieves Na(+) transport. Here we present crystal structures of KR2 under neutral and acidic conditions, which represent the resting and M-like intermediate states, respectively. Structural and spectroscopic analyses revealed the gating mechanism, whereby the flipping of Asp116 sequesters the Schiff base proton from the conducting pathway to facilitate Na(+) transport. Together with the structure-based engineering of the first light-driven K(+) pumps, electrophysiological assays in mammalian neurons and behavioural assays in a nematode, our studies reveal the molecular basis for light-driven non-proton cation pumps and thus provide a framework that may advance the development of next-generation optogenetics.

Energetics and dynamics of a light-driven sodium-pumping rhodopsin

“…Furthermore, mechanistic studies of the Na + pumping in KR2 have important neurophysiological applications, as the protein can be used as an inhibitory optogenetic tool, which does not affect the pH balance of the organism (6,7,13,14). Despite several experimental studies (11,(15)(16)(17)(18), as well as the recently resolved X-ray structure (7,8), the exact molecular mechanism by which KR2 pumps Na + remains elusive.In this work, we show by classical atomistic molecular dynamics (MD) simulations and hybrid quantum mechanics/molecular mechanics (QM/MM) calculations how conformational, electrostatic, and hydration changes lead to transfer of the Na + ion across the membrane. Our calculations identify putative ion-binding sites that can stimulate site-directed mutagenesis experiments.…”

“…The rhodopsin family of proteins catalyzes such reactions by harnessing the energy from retinal photoisomerization and deprotonation reactions, followed by conformational changes that further trigger the pumping of ions across the membrane (5). All previously known ion-pumping rhodopsins function either as inward chloride or outward proton pumps, but the structure of the first Na + pumping rhodopsin, Krokinobacter eikastus rhodopsin 2 (KR2) (6), was recently resolved (7,8). In the absence of Na + ions, KR2 functions as an outward proton pump, similarly to bacteriorhodopsin (bR), but under physiological conditions, KR2 pumps Na + out of the cell (6, 9).…”

“…1A) (5,7,8). In contrast to bR, however, where a highly conserved Asp-Thr-Asp (DTD) motif is used to pump protons, KR2 employs a unique NDQ motif comprising residues Asn112, Asp116, and Gln123 (6).…”

“…Recently, the structure of the first light-activated Na + pump, Krokinobacter eikastus rhodopsin 2 (KR2), was resolved at atomic resolution [Kato HE, et al (2015) Nature 521: [48][49][50][51][52][53]. To elucidate its molecular mechanism for Na + pumping, we perform here extensive classical and quantum molecular dynamics (MD) simulations of transient photocycle states.…”

“…We also suggest putative structures for the transient photocycle intermediates, which we verify based on calculations of absorption spectra. Understanding the molecular mechanism of light-driven Na + transport by KR2 is essential for the rational design of novel light-driven ion pumps that hyperpolarize the membrane potential and could be used as inhibitory optogenetic tools (6,7,13,14). …”