Long-distance perturbation on Schiff base–counterion interactions by His30 and the extracellular Na<sup>+</sup>-binding site in <i>Krokinobacter</i> rhodopsin 2

Shigeta, Arisu; Itô, Shota; Kaneko, Ryuji; Tomida, Sahoko; Inoue, Kazuhide; Kandori, Hideki; Kawamura, Izuru

doi:10.1039/c8cp00626a

Cited by 17 publications

(21 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It means that the rearrangements on the surface of the KR2 occur not directly in response to the retinal isomerization upon photon absorption, but rather due to the redistribution of charges in the central inner part of the protein protomer. Such long-distance interactions between the RSB-counterion and pentamer surface were already studied for the WT and H30A variant of KR2 14,25 . Recently, it was also shown that there is an allosteric communication between the interprotomer Na + -binding site and the RSB hydrogen bond already in the ground state 26 .…”

Section: Resultsmentioning

confidence: 97%

Molecular mechanism of light-driven sodium pumping

et al. 2020

View full text Add to dashboard Cite

The light-driven sodium-pumping rhodopsin KR2 from Krokinobacter eikastus is the only nonproton 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.

show abstract

Section: Resultsmentioning

confidence: 97%

Molecular mechanism of light-driven sodium pumping

et al. 2020

View full text Add to dashboard Cite

show abstract

“…As structure of KR2 depends dramatically on the oligomeric state, we decided to mutate the oligomerization interface to destroy the pentameric assembly and simultaneously check the functional activity of the protein variants. Recently, it was shown that the alterations at the oligomerization interface around His 30 affect the oligomeric assembly and change protein selectivity and Schiff base–counterion interaction ( 8 , 20 , 28 ). Consequently, we generated H30L, H30K, and Y154F mutants of KR2, which were aimed at breaking the His 30 -Tyr 154 hydrogen bond, located in the middle of the oligomerization interface and therefore stabilizing the pentamer.…”

Section: Resultsmentioning

confidence: 99%

Structure and mechanisms of sodium-pumping KR2 rhodopsin

et al. 2019

View full text Add to dashboard Cite

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.

show abstract

“…There is also no clear effect on the 15 N resonance of the protonated Schiff base (not shown). An additional shielding was only reported for KR2 H30A (Shigeta et al, 2018). Furthermore, no chemical shift changes could be detected for T83 (data not shown), which is directly involved in coordinating Na + binding (Fig.…”

Section: Effect Of Na +mentioning

confidence: 87%

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

View full text Add to dashboard Cite

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.

show abstract

Long-distance perturbation on Schiff base–counterion interactions by His30 and the extracellular Na⁺-binding site in Krokinobacter rhodopsin 2

Abstract: Proton pumping ability of a light-driven Na+ pumping Krokinobacter rhodopsin 2 (KR2) is inhibited by H30A and lack of bound Na+ at extracellular site.

Cited by 17 publications

References 29 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

Contact Info

Product

Resources

About

Long-distance perturbation on Schiff base–counterion interactions by His30 and the extracellular Na+-binding site in Krokinobacter rhodopsin 2

Abstract: Proton pumping ability of a light-driven Na+ pumping Krokinobacter rhodopsin 2 (KR2) is inhibited by H30A and lack of bound Na+ at extracellular site.

Cited by 17 publications

References 29 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

Contact Info

Product

Resources

About

Long-distance perturbation on Schiff base–counterion interactions by His30 and the extracellular Na⁺-binding site in Krokinobacter rhodopsin 2