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Molecular mechanism of light-driven sodium pumping.

Published on May 1, 2020in Nature Communications11.878
· DOI :10.1038/S41467-020-16032-Y
K. Kovalev7
Estimated H-index: 7
,
Roman Astashkin4
Estimated H-index: 4
(MIPT: Moscow Institute of Physics and Technology)
+ 21 AuthorsAntoine Royant22
Estimated H-index: 22
(CNRS: Centre national de la recherche scientifique)
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 A, 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. The Na+-pumping KR2 rhodopsin from Krokinobacter eikastus is a light-driven non-proton cation pump whose mechanism of pumping remains to be understood. Here authors solved crystal structures of the O-intermediate state of the pentameric form of KR2 and its D116N and H30A mutants, which sheds light on the mechanism of non-proton cation light-driven pumping.
  • References (52)
  • Citations (1)
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References52
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#1Akihiro Otomo (Osaka University)H-Index: 2
#2Misao Mizuno (Osaka University)H-Index: 12
Last. Yasuhisa Mizutani (Osaka University)H-Index: 24
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Krokinobacter rhodopsin 2 (KR2) serves as a light-driven sodium ion pump in the presence of sodium ion and works as a proton pump in the presence of larger monovalent cations such as potassium ion, rubidium ion, and cesium ion. Recent crystallographic studies revealed that KR2 forms a pentamer and possesses an ion binding site at the subunit interface. It is assumed that sodium ion bound at this binding site is not transported but contributes to thermal stability. Since KR2 can convert its funct...
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#1Tobias WeinertH-Index: 13
#2Petr SkopintsevH-Index: 8
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Conformational dynamics are essential for proteins to function. We adapted time-resolved serial crystallography developed at x-ray lasers to visualize protein motions using synchrotrons. We recorded the structural changes in the light-driven proton-pump bacteriorhodopsin over 200 milliseconds in time. The snapshot from the first 5 milliseconds after photoactivation shows structural changes associated with proton release at a quality comparable to that of previous x-ray laser experiments. From 10...
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#1Andreas Vogt (Humboldt University of Berlin)H-Index: 46
#2Arita Silapetere (Humboldt University of Berlin)H-Index: 3
Last. Peter Hegemann (Humboldt University of Berlin)H-Index: 60
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Abstract Light-driven sodium pumps (NaRs) are microbial rhodopsins that utilize light energy to actively transport sodium ions out of the cell. Here, we used targeted mutagenesis and electrophysiological methods in living cells to demonstrate that NaRs can be converted into light-activated cation channels by molecular engineering. Specifically, introduction of the R109Q mutation into the sodium ion pump of Dokdonia eikasta (KR2) results in passive ion conductance, with a high preference for pota...
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We conducted a comprehensive time-resolved resonance Raman spectroscopy study of the structures of the retinal chromophore during the photocycle of the sodium-ion pump Krokinobacter rhodopsin 2 (KR2). We succeeded in determining the structure of the chromophore in the unphotolyzed state and in the K, L, M, and O intermediates, by overcoming the problem that only a small fraction of the M intermediate is accumulated in the KR2 photocycle. The Schiff base in the retinal chromophore forms a strong ...
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Abstract Krokinobacter eikastus rhodopsin 2 (KR2) is a pentameric, light-driven ion pump, which selectively transports sodium or protons. The mechanism of ion selectivity and transfer is unknown. By using conventional as well as dynamic nuclear polarization (DNP)-enhanced solid-state NMR, we were able to analyse the retinal polyene chain between positions C10 and C15 as well as the Schiff base nitrogen in the KR2 resting state. In addition, 50% of the KR2 13C and 15N resonances could be assigned...
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#1K. KovalevH-Index: 7
#2Vitaly Polovinkin (CNRS: Centre national de la recherche scientifique)H-Index: 1
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Rhodopsins are the most universal biological light-energy transducers and abundant phototrophic mechanisms that evolved on Earth and have a remarkable diversity and potential for biotechnological applications. Recently, the first sodium-pumping rhodopsin KR2 from Krokinobacter eikastus was discovered and characterized. However, the existing structures of KR2 are contradictory, and the mechanism of Na + pumping is not yet understood. Here, we present a structure of the cationic (non H + ) light-d...
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#1Thomas A. WhiteH-Index: 1
This article provides a step-by-step guide to the use of the CrystFEL software for processing serial crystallography data from an X-ray free-electron laser or a synchrotron light source. Whereas previous papers have described the theory and algorithms and their rationale, this paper describes the steps to be performed from a user perspective, including command-line examples.
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#2Xuanxuan Li (THU: Tsinghua University)H-Index: 5
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: Non-cryogenic protein structures determined at ambient temperature may disclose significant information about protein activity. Chloride-pumping rhodopsin (ClR) exhibits a trend to hyperactivity induced by a change in the photoreaction rate because of a gradual decrease in temperature. Here, to track the structural changes that explain the differences in CIR activity resulting from these temperature changes, we used serial femtosecond crystallography (SFX) with an X-ray free electron laser (XF...
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Light-driven sodium ion pump rhodopsin (NaR) is a new functional class of microbial rhodopsin. A previous flash photolysis study of Krokinobacter eikastus rhodopsin 2 (KR2) revealed the presence of three kinetically distinct intermediates: K, L/M, and O. Previous low-temperature Fourier-transform infrared (FTIR) spectroscopy of KR2 showed that photoisomerization from the all-trans to the 13-cis form is the primary event of the Na+ pumping photocycle, but structural information on the subsequent ...
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#1Nina MaliarH-Index: 1
#2K. KovalevH-Index: 7
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The light-driven sodium pump KR2, found in 2013 in the marine bacteria Krokinobacter eikastus, serves as a model protein for the studies of the sodium-pumping microbial rhodopsins (NaRs). KR2 possesses a unique NDQ (N112, D116, and Q123) set of the amino acid residues in the functionally relevant positions, named the NDQ motif. The N112 was shown to determine the Na+/H+ selectivity and pumping efficiency of the protein. Thus, N112A mutation converts KR2 into an outward proton pump. However, no s...
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