Crystal structure of a natural light-gated anion channelrhodopsin

Li, Hai; Huang, Chia‐Ying; Govorunova, Elena G.; Schafer, Christopher T.; Sineshchekov, Oleg A.; Wang, Meitian; Zheng, Lei; Spudich, John L.

doi:10.7554/elife.41741

Cited by 36 publications

(70 citation statements)

References 43 publications

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“…15). Indeed, the CGs of the native cation channelrhodopsin-2 (CrChR2) 50 , chimeric cation channelrhodopsin (C1C2) 51 and also natural anion channelrhodopsin (GtACR1) 52,53 are composed of the S63-N258-E90, S102-N297-E129 and S43-N239-E68 triads ( Supplementary Fig. 15).…”

Section: Supplementary Textmentioning

confidence: 99%

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: Supplementary Textmentioning

confidence: 99%

Molecular mechanism of light-driven sodium pumping

Kovalev

Astashkin

Gushchin

et al. 2020

Preprint

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“…With respect to eukaryotic marine protists, proteorhodopsin-like genes have been identified in diatoms, haptophytes, cryptophytes, and dinoflagellates (e.g. Hovde and colleagues [7][8][9][10], and the references cited therein). Protein sequences of the proteorhodopsins of the primitive dinoflagellate Oxyrrhis marina are restricted to data given by Guo et al [11], Slamovits et al [12], and Zhang et al [13] and derived from translated expressed sequence tags (ESTs).…”

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confidence: 99%

A simple protocol for the isolation of proteorhodopsins of the dinoflagellate Oxyrrhis marina

et al. 2020

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For the first time, native proteorhodopsins of the marine dinoflagellate Oxyrrhis marina were isolated. Total cell membrane fractions were minced in a bead beater and solubilized with the detergent Triton X‐100. Subsequent sucrose density gradient centrifugation resulted in three or four red‐colored bands. Nonsolubilized, but still red colored, membranes sedimented at the bottom. For each of these bands, absorbance maxima were registered at approximately 514–516 nm with shoulders toward shorter wavelengths (470–490 nm). Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis revealed that the uppermost band represented free retinal chromophore, as it contained no protein. The other bands were almost pure proteorhodopsin fractions as the banding patterns showed one major protein of 25 kDa. Tryptic, in‐gel digestion of the 25 kDa proteins and of faint protein bands above and below 25 kDa was followed by mass spectrometry, confirming these protein bands to consist, nearly exclusively, proteorhodopsins. Only single peptides of few other proteins were detected. In total, at least seven predicted proteorhodopsin protein sequences were experimentally verified.

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“…Anion channelrhodopsins from the cryptophyte alga Guillardia theta ( Gt ACRs) are the most potent optogenetic inhibitors of neuronal firing and regulators of animal behavior currently available (Govorunova et al 2015; Mahn et al 2018; Messier et al 2018; Mohammad et al 2017; Wilson et al 2018). Recently we and others have obtained high-resolution X-ray structures of the dark (closed) state of G. theta anion channelrhodopsin 1 ( Gt ACR1) (Kim et al 2018; Li et al 2019). In contrast to available structures of cation channelrhodopsins (CCRs) from green algae (Kato et al 2012; Oda et al 2018; Volkov et al 2017), Gt ACR1 exhibits a narrow continuous intramolecular tunnel formed by helices 1-3 and 7 that connects the cytoplasmic and extracellular aqueous phases and presumably expands upon illumination to pass anions (Li et al 2019).…”

Section: Introductionmentioning

confidence: 99%

“…Recently we and others have obtained high-resolution X-ray structures of the dark (closed) state of G. theta anion channelrhodopsin 1 ( Gt ACR1) (Kim et al 2018; Li et al 2019). In contrast to available structures of cation channelrhodopsins (CCRs) from green algae (Kato et al 2012; Oda et al 2018; Volkov et al 2017), Gt ACR1 exhibits a narrow continuous intramolecular tunnel formed by helices 1-3 and 7 that connects the cytoplasmic and extracellular aqueous phases and presumably expands upon illumination to pass anions (Li et al 2019). However, no open channel structure is yet available, and many questions regarding its architecture remain unanswered.…”

Section: Introductionmentioning

confidence: 99%

“… ABSTRACT The crystal structure of Gt ACR1 from Guillardia theta revealed an intramolecular tunnel predicted to expand to form the anion-conducting channel upon photoactivation (Li et al 2019). The location of the retinylidene photoactive site within the tunnel raised the question of whether, in addition to triggering channel opening by photoisomerization, the site also participates in later channel processes.…”

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