Femtosecond-to-millisecond structural changes in a light-driven sodium pump

Petr Skopintsev; David Ehrenberg; Tobias Weinert; Daniel James; Rajiv K. Kar; Philip J.M. Johnson; Dmitry Ozerov; Antonia Furrer; Isabelle Martiel; Florian Dworkowski; Karol Nass; Gregor Knopp; Claudio Cirelli; Christopher Arrell; Dardan Gashi; Sandra Mous; Maximilian Wranik; Thomas Gruhl; Demet Kekilli; Steffen Brünle; Xavier Deupi; Gebhard F.X. Schertler; Roger M. Benoit; Valerie Panneels; Przemyslaw Nogly; Igor Schapiro; Christopher Milne; Joachim Heberle; Jörg Standfuss

doi:10.1038/s41586-020-2307-8

Femtosecond-to-millisecond structural changes in a light-driven sodium pump

Petr Skopintsev, David Ehrenberg, Tobias Weinert, Daniel James, Rajiv K. Kar, Philip J.M. Johnson, Dmitry Ozerov, Antonia Furrer, Isabelle Martiel, Florian Dworkowski, Karol Nass, Gregor Knopp, Claudio Cirelli, Christopher Arrell, Dardan Gashi, Sandra Mous, Maximilian Wranik, Thomas Gruhl, Demet Kekilli, Steffen BrünleXavier Deupi, Gebhard F.X. Schertler, Roger M. Benoit, Valerie Panneels, Przemyslaw Nogly, Igor Schapiro, Christopher Milne, Joachim Heberle, Jörg Standfuss^*

^*Corresponding author for this work

Institute of Chemistry

Research output: Contribution to journal › Article › peer-review

92 Scopus citations

Abstract

Light-driven sodium pumps actively transport small cations across cellular membranes¹. These pumps are used by microorganisms to convert light into membrane potential and have become useful optogenetic tools with applications in neuroscience. Although the resting state structures of the prototypical sodium pump Krokinobacter eikastus rhodopsin 2 (KR2) have been solved^2,3, it is unclear how structural alterations over time allow sodium to be translocated against a concentration gradient. Here, using the Swiss X-ray Free Electron Laser⁴, we have collected serial crystallographic data at ten pump–probe delays from femtoseconds to milliseconds. High-resolution structural snapshots throughout the KR2 photocycle show how retinal isomerization is completed on the femtosecond timescale and changes the local structure of the binding pocket in the early nanoseconds. Subsequent rearrangements and deprotonation of the retinal Schiff base open an electrostatic gate in microseconds. Structural and spectroscopic data, in combination with quantum chemical calculations, indicate that a sodium ion binds transiently close to the retinal within one millisecond. In the last structural intermediate, at 20 milliseconds after activation, we identified a potential second sodium-binding site close to the extracellular exit. These results provide direct molecular insight into the dynamics of active cation transport across biological membranes.

Original language	American English
Pages (from-to)	314-318
Number of pages	5
Journal	Nature
Volume	583
Issue number	7815
DOIs	https://doi.org/10.1038/s41586-020-2307-8
State	Published - 9 Jul 2020

Bibliographical note

Funding Information:
Acknowledgements This project was funded by the following agencies: The Swiss National Science Foundation project grants 31003A_141235 and 31003A_159558 (to J.S.), PZ00P3_174169 (to P.N.), 310030_192780 (to X.D.) and 310030B_173335 (to G.F.X.S.). We further acknowledge support by the NCCR:MUST (to C.M. and J.S.). The German Research Foundation supported the work through SFB-1078, project B3 and SPP-1926, HE 2063/6-1 (to J.H). This project has received funding from the European Union’s Horizon 2020 research and innovation program under Marie-Sklodowska-Curie grant agreements 701646 and 701647. I.S. acknowledges funding by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant no. 678169 ‘PhotoMutant’). I.S. thanks the SFB 1078 ‘Protonation Dynamics in Protein Function’ for the Mercator fellowship. R.K.K. acknowledges support from the Lady Davis Trust for the Arskin postdoctoral fellowship. We thank N. Varma for discussions on TR-SFX sample jetting and the Macromolecular Crystallography group for support during testing of crystals at the Swiss Light Source. We further thank everybody involved in ensuring the smooth operation of the Swiss X-ray Free Electron Laser during our experiments.

Publisher Copyright:
© 2020, The Author(s), under exclusive licence to Springer Nature Limited.

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Skopintsev, P., Ehrenberg, D., Weinert, T., James, D., Kar, R. K., Johnson, P. J. M., Ozerov, D., Furrer, A., Martiel, I., Dworkowski, F., Nass, K., Knopp, G., Cirelli, C., Arrell, C., Gashi, D., Mous, S., Wranik, M., Gruhl, T., Kekilli, D., ... Standfuss, J. (2020). Femtosecond-to-millisecond structural changes in a light-driven sodium pump. Nature, 583(7815), 314-318. https://doi.org/10.1038/s41586-020-2307-8

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title = "Femtosecond-to-millisecond structural changes in a light-driven sodium pump",

abstract = "Light-driven sodium pumps actively transport small cations across cellular membranes1. These pumps are used by microorganisms to convert light into membrane potential and have become useful optogenetic tools with applications in neuroscience. Although the resting state structures of the prototypical sodium pump Krokinobacter eikastus rhodopsin 2 (KR2) have been solved2,3, it is unclear how structural alterations over time allow sodium to be translocated against a concentration gradient. Here, using the Swiss X-ray Free Electron Laser4, we have collected serial crystallographic data at ten pump–probe delays from femtoseconds to milliseconds. High-resolution structural snapshots throughout the KR2 photocycle show how retinal isomerization is completed on the femtosecond timescale and changes the local structure of the binding pocket in the early nanoseconds. Subsequent rearrangements and deprotonation of the retinal Schiff base open an electrostatic gate in microseconds. Structural and spectroscopic data, in combination with quantum chemical calculations, indicate that a sodium ion binds transiently close to the retinal within one millisecond. In the last structural intermediate, at 20 milliseconds after activation, we identified a potential second sodium-binding site close to the extracellular exit. These results provide direct molecular insight into the dynamics of active cation transport across biological membranes.",

author = "Petr Skopintsev and David Ehrenberg and Tobias Weinert and Daniel James and Kar, {Rajiv K.} and Johnson, {Philip J.M.} and Dmitry Ozerov and Antonia Furrer and Isabelle Martiel and Florian Dworkowski and Karol Nass and Gregor Knopp and Claudio Cirelli and Christopher Arrell and Dardan Gashi and Sandra Mous and Maximilian Wranik and Thomas Gruhl and Demet Kekilli and Steffen Br{\"u}nle and Xavier Deupi and Schertler, {Gebhard F.X.} and Benoit, {Roger M.} and Valerie Panneels and Przemyslaw Nogly and Igor Schapiro and Christopher Milne and Joachim Heberle and J{\"o}rg Standfuss",

note = "Funding Information: Acknowledgements This project was funded by the following agencies: The Swiss National Science Foundation project grants 31003A_141235 and 31003A_159558 (to J.S.), PZ00P3_174169 (to P.N.), 310030_192780 (to X.D.) and 310030B_173335 (to G.F.X.S.). We further acknowledge support by the NCCR:MUST (to C.M. and J.S.). The German Research Foundation supported the work through SFB-1078, project B3 and SPP-1926, HE 2063/6-1 (to J.H). This project has received funding from the European Union{\textquoteright}s Horizon 2020 research and innovation program under Marie-Sklodowska-Curie grant agreements 701646 and 701647. I.S. acknowledges funding by the European Research Council (ERC) under the European Union{\textquoteright}s Horizon 2020 research and innovation program (grant no. 678169 {\textquoteleft}PhotoMutant{\textquoteright}). I.S. thanks the SFB 1078 {\textquoteleft}Protonation Dynamics in Protein Function{\textquoteright} for the Mercator fellowship. R.K.K. acknowledges support from the Lady Davis Trust for the Arskin postdoctoral fellowship. We thank N. Varma for discussions on TR-SFX sample jetting and the Macromolecular Crystallography group for support during testing of crystals at the Swiss Light Source. We further thank everybody involved in ensuring the smooth operation of the Swiss X-ray Free Electron Laser during our experiments. Publisher Copyright: {\textcopyright} 2020, The Author(s), under exclusive licence to Springer Nature Limited.",

year = "2020",

month = jul,

day = "9",

doi = "10.1038/s41586-020-2307-8",

language = "American English",

volume = "583",

pages = "314--318",

journal = "Nature",

issn = "0028-0836",

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Skopintsev, P, Ehrenberg, D, Weinert, T, James, D, Kar, RK, Johnson, PJM, Ozerov, D, Furrer, A, Martiel, I, Dworkowski, F, Nass, K, Knopp, G, Cirelli, C, Arrell, C, Gashi, D, Mous, S, Wranik, M, Gruhl, T, Kekilli, D, Brünle, S, Deupi, X, Schertler, GFX, Benoit, RM, Panneels, V, Nogly, P, Schapiro, I, Milne, C, Heberle, J & Standfuss, J 2020, 'Femtosecond-to-millisecond structural changes in a light-driven sodium pump', Nature, vol. 583, no. 7815, pp. 314-318. https://doi.org/10.1038/s41586-020-2307-8

TY - JOUR

T1 - Femtosecond-to-millisecond structural changes in a light-driven sodium pump

AU - Skopintsev, Petr

AU - Ehrenberg, David

AU - Weinert, Tobias

AU - James, Daniel

AU - Kar, Rajiv K.

AU - Johnson, Philip J.M.

AU - Ozerov, Dmitry

AU - Furrer, Antonia

AU - Martiel, Isabelle

AU - Dworkowski, Florian

AU - Nass, Karol

AU - Knopp, Gregor

AU - Cirelli, Claudio

AU - Arrell, Christopher

AU - Gashi, Dardan

AU - Mous, Sandra

AU - Wranik, Maximilian

AU - Gruhl, Thomas

AU - Kekilli, Demet

AU - Brünle, Steffen

AU - Deupi, Xavier

AU - Schertler, Gebhard F.X.

AU - Benoit, Roger M.

AU - Panneels, Valerie

AU - Nogly, Przemyslaw

AU - Schapiro, Igor

AU - Milne, Christopher

AU - Heberle, Joachim

AU - Standfuss, Jörg

N1 - Funding Information: Acknowledgements This project was funded by the following agencies: The Swiss National Science Foundation project grants 31003A_141235 and 31003A_159558 (to J.S.), PZ00P3_174169 (to P.N.), 310030_192780 (to X.D.) and 310030B_173335 (to G.F.X.S.). We further acknowledge support by the NCCR:MUST (to C.M. and J.S.). The German Research Foundation supported the work through SFB-1078, project B3 and SPP-1926, HE 2063/6-1 (to J.H). This project has received funding from the European Union’s Horizon 2020 research and innovation program under Marie-Sklodowska-Curie grant agreements 701646 and 701647. I.S. acknowledges funding by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant no. 678169 ‘PhotoMutant’). I.S. thanks the SFB 1078 ‘Protonation Dynamics in Protein Function’ for the Mercator fellowship. R.K.K. acknowledges support from the Lady Davis Trust for the Arskin postdoctoral fellowship. We thank N. Varma for discussions on TR-SFX sample jetting and the Macromolecular Crystallography group for support during testing of crystals at the Swiss Light Source. We further thank everybody involved in ensuring the smooth operation of the Swiss X-ray Free Electron Laser during our experiments. Publisher Copyright: © 2020, The Author(s), under exclusive licence to Springer Nature Limited.

PY - 2020/7/9

Y1 - 2020/7/9

N2 - Light-driven sodium pumps actively transport small cations across cellular membranes1. These pumps are used by microorganisms to convert light into membrane potential and have become useful optogenetic tools with applications in neuroscience. Although the resting state structures of the prototypical sodium pump Krokinobacter eikastus rhodopsin 2 (KR2) have been solved2,3, it is unclear how structural alterations over time allow sodium to be translocated against a concentration gradient. Here, using the Swiss X-ray Free Electron Laser4, we have collected serial crystallographic data at ten pump–probe delays from femtoseconds to milliseconds. High-resolution structural snapshots throughout the KR2 photocycle show how retinal isomerization is completed on the femtosecond timescale and changes the local structure of the binding pocket in the early nanoseconds. Subsequent rearrangements and deprotonation of the retinal Schiff base open an electrostatic gate in microseconds. Structural and spectroscopic data, in combination with quantum chemical calculations, indicate that a sodium ion binds transiently close to the retinal within one millisecond. In the last structural intermediate, at 20 milliseconds after activation, we identified a potential second sodium-binding site close to the extracellular exit. These results provide direct molecular insight into the dynamics of active cation transport across biological membranes.

AB - Light-driven sodium pumps actively transport small cations across cellular membranes1. These pumps are used by microorganisms to convert light into membrane potential and have become useful optogenetic tools with applications in neuroscience. Although the resting state structures of the prototypical sodium pump Krokinobacter eikastus rhodopsin 2 (KR2) have been solved2,3, it is unclear how structural alterations over time allow sodium to be translocated against a concentration gradient. Here, using the Swiss X-ray Free Electron Laser4, we have collected serial crystallographic data at ten pump–probe delays from femtoseconds to milliseconds. High-resolution structural snapshots throughout the KR2 photocycle show how retinal isomerization is completed on the femtosecond timescale and changes the local structure of the binding pocket in the early nanoseconds. Subsequent rearrangements and deprotonation of the retinal Schiff base open an electrostatic gate in microseconds. Structural and spectroscopic data, in combination with quantum chemical calculations, indicate that a sodium ion binds transiently close to the retinal within one millisecond. In the last structural intermediate, at 20 milliseconds after activation, we identified a potential second sodium-binding site close to the extracellular exit. These results provide direct molecular insight into the dynamics of active cation transport across biological membranes.

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DO - 10.1038/s41586-020-2307-8

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JF - Nature

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ER -

Femtosecond-to-millisecond structural changes in a light-driven sodium pump

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