Solid-State Nuclear Magnetic Resonance Structural Study of the Retinal-Binding Pocket in Sodium Ion Pump Rhodopsin

Shigeta, Arisu; Itô, Shota; Inoue, Kazuhide; Okitsu, Takashi; Wada, Akimori; Kawamura, Izuru

doi:10.1021/acs.biochem.6b00999

Cited by 30 publications

(82 citation statements)

References 66 publications

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“…Indeed, it was shown, that the hydrogen bond between the RSBH + and D116exists in only a fraction of this KR2 variant. Moreover, as it was demonstrated by the NMR studies, the RSBH + is in a multiple conformations in D116N 56 . Therefore, we refined our crystallographic data on the mutant with the both alternative RSBH + orientations in the final model ( Supplementary Fig.…”

Section: Double Conformation Of the Rsbh + In D116n Mutantmentioning

confidence: 51%

Molecular mechanism of light-driven sodium pumping

Kovalev

Astashkin

Gushchin

et al. 2020

Preprint

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ABSTRACTMicrobial rhodopsins appeared to be the most abundant light-harvesting proteins on the Earth and are the major contributes to the solar energy captured in the sea. They possess highly diverse biological functions. Explosion of research on microbial rhodopsins led to breakthroughs in their applications, in particular, in neuroscience.An unexpected new discovery was a Na+-pumping KR2 rhodopsin from Krokinobacter eikastus, the first light-driven non-proton cation pump. A fundamental difference between proton and other cation pumps is that non-proton pumps cannot use tunneling or Grotthuss mechanism for the ion translocation and, therefore, Na+ pumping cannot be understood in the framework of classical proton pump, like bacteriorhodopsin. Extensive studies on the molecular mechanism of KR2 failed to reveal mechanism of pumping. The existing high-resolution structures relate only to the ground state of the protein and revealed no Na+ inside the protein, which is unusual for active ion transporters.KR2 is only known non proton cation transporter with demonstrated remarkable potential for optogenetic applications and, therefore, elucidation of the mechanism of cation transport is important. To understand conception of cation pumping we solved crystal structures of the functionally key O-intermediate state of physiologically relevant pentameric form of KR2 and its D116N and H30A key mutants at high resolution and performed additional functional studies.The structure of the O-state reveals a sodium ion near the retinal Schiff base coordinated by N112 and D116 residues of the characteristic (for the whole family) NDQ triad. The structural and functional data show that cation uptake and release are driven by a switching mechanism. Surprisely, Na+ pathway in KR2 is lined with the chain of polar pores/cavities, similarly to the channelrhodopsin-2. Using Parinello fast molecular dynamics approach we obtained a molecular movie of a probable ion release.Our data provides insight into the mechanism of a non-proton cation light-driven pumping, strongly suggest close relation of sodium pumps to channel rhodopsins and, we believe, expand the present knowledge of rhodopsin world. Certainly they might be used for engineering of cation pumps and ion channels for optogenetics.

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Section: Double Conformation Of the Rsbh + In D116n Mutantmentioning

confidence: 51%

Molecular mechanism of light-driven sodium pumping

Kovalev

Astashkin

Gushchin

et al. 2020

Preprint

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“…The distance between the Asp 116 oxygen and the Schiff base nitrogen is increased to 3.2 Å, and at pH 8.0, it is only 2.8 Å. The weakening of Asp 116 interactions with RSB around pH 5.0 was also observed with the solid-state NMR study of KR2 retinal-binding pocket and was assigned to the protonation of Asp 116 ( 19 ). The change in protonation of Asp 116 can also be indicated by the shift of absorption maximum of KR2 in crystals from 528 to 550 nm.…”

Section: Resultsmentioning

confidence: 72%

“…As the protein acts as the H + pump at acidic pH even at physiological concentration of sodium, we define the low pH KR2 form as the H + -pumping form. In addition, optical properties of the protein are altered with the decrease of pH ( 8 ), and structural rearrangements in the retinal-binding pocket were recently reported ( 19 ). Structural insights into the nature of transitions in KR2 with the pH change may help to better understand the mechanism of Na + pumping.…”

Section: Resultsmentioning

confidence: 99%

Structure and mechanisms of sodium-pumping KR2 rhodopsin

et al. 2019

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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 fromKrokinobacter eikastuswas 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-driven pump at physiological pH in its pentameric form. We also present 13 atomic structures and functional data on the KR2 and its mutants, including potassium pumps, which show that oligomerization of the microbial rhodopsin is obligatory for its biological function. The studies reveal the structure of KR2 at nonphysiological low pH where it acts as a proton pump. The structure provides new insights into the mechanisms of microbial rhodopsins and opens the way to a rational design of novel cation pumps for optogenetics.

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“…on the 13 C chemical shifts of C14 and C20 (Shigeta et al, 2017). The C20 chemical shift reported by Shigeta et al is a very sensitive reporter for the all-trans-retinal configuration.…”

Section: Resultsmentioning

confidence: 89%

Solid-state NMR analysis of the sodium pump Krokinobacter rhodopsin 2 and its H30A mutant

Kaur

Kriebel

Eberhardt

et al. 2019

Journal of Structural Biology

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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 C andN resonances could be assigned by multidimensional high-field solid-state NMR experiments. Assigned residues include part of the NDQ motif as well as sodium binding sites. Based on these data, the structural effects of the H30A mutation, which seems to shift the ion selectivity of KR2 primarily to Na, could be analysed. Our data show that it causes long-range effects within the retinal binding pocket and at the extracellular Na binding site, which can be explained by perturbations of interactions across the protomer interfaces within the KR2 complex. This study is complemented by data from time-resolved optical spectroscopy.

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Solid-State Nuclear Magnetic Resonance Structural Study of the Retinal-Binding Pocket in Sodium Ion Pump Rhodopsin

Cited by 30 publications

References 66 publications

Molecular mechanism of light-driven sodium pumping

Molecular mechanism of light-driven sodium pumping

Structure and mechanisms of sodium-pumping KR2 rhodopsin

Solid-state NMR analysis of the sodium pump Krokinobacter rhodopsin 2 and its H30A mutant

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