All Publications


  • Ultrafast structural changes direct the first molecular events of vision NATURE Gruhl, T., Weinert, T., Rodrigues, M. J., Milne, C. J., Ortolani, G., Nass, K., Nango, E., Sen, S., Johnson, P. M., Cirelli, C., Furrer, A., Mous, S., Skopintsev, P., James, D., Dworkowski, F., Bath, P., Kekilli, D., Ozerov, D., Tanaka, R., Glover, H., Bacellar, C., Brunle, S., Casadei, C. M., Diethelm, A. D., Gashi, D., Gotthard, G., Guixa-Gonzalez, R., Joti, Y., Kabanova, V., Knopp, G., Lesca, E., Ma, P., Martiel, I., Muhle, J., Owada, S., Pamula, F., Sarabi, D., Tejero, O., Tsai, C., Varma, N., Wach, A., Boutet, S., Tono, K., Nogly, P., Deupi, X., Iwata, S., Neutze, R., Standfuss, J., Schertler, G., Panneels, V. 2023; 615 (7954): 939-+

    Abstract

    Vision is initiated by the rhodopsin family of light-sensitive G protein-coupled receptors (GPCRs)1. A photon is absorbed by the 11-cis retinal chromophore of rhodopsin, which isomerizes within 200 femtoseconds to the all-trans conformation2, thereby initiating the cellular signal transduction processes that ultimately lead to vision. However, the intramolecular mechanism by which the photoactivated retinal induces the activation events inside rhodopsin remains experimentally unclear. Here we use ultrafast time-resolved crystallography at room temperature3 to determine how an isomerized twisted all-trans retinal stores the photon energy that is required to initiate the protein conformational changes associated with the formation of the G protein-binding signalling state. The distorted retinal at a 1-ps time delay after photoactivation has pulled away from half of its numerous interactions with its binding pocket, and the excess of the photon energy is released through an anisotropic protein breathing motion in the direction of the extracellular space. Notably, the very early structural motions in the protein side chains of rhodopsin appear in regions that are involved in later stages of the conserved class A GPCR activation mechanism. Our study sheds light on the earliest stages of vision in vertebrates and points to fundamental aspects of the molecular mechanisms of agonist-mediated GPCR activation.

    View details for DOI 10.1038/s41586-023-05863-6

    View details for Web of Science ID 000985863500011

    View details for PubMedID 36949205

    View details for PubMedCentralID PMC10060157

  • Dynamics and mechanism of a light-driven chloride pump SCIENCE Mous, S., Gotthard, G., Ehrenberg, D., Sen, S., Weinert, T., Johnson, P. M., James, D., Nass, K., Furrer, A., Kekilli, D., Ma, P., Bruenle, S., Casadei, C., Martiel, I., Dworkowski, F., Gashi, D., Skopintsev, P., Wranik, M., Knopp, G., Panepucci, E., Panneels, V., Cirelli, C., Ozerov, D., Schertler, G. X., Wang, M., Milne, C., Standfuss, J., Schapiro, I., Heberle, J., Nogly, P. 2022; 375 (6583): 845-+

    Abstract

    Chloride transport by microbial rhodopsins is an essential process for which molecular details such as the mechanisms that convert light energy to drive ion pumping and ensure the unidirectionality of the transport have remained elusive. We combined time-resolved serial crystallography with time-resolved spectroscopy and multiscale simulations to elucidate the molecular mechanism of a chloride-pumping rhodopsin and the structural dynamics throughout the transport cycle. We traced transient anion-binding sites, obtained evidence for how light energy is used in the pumping mechanism, and identified steric and electrostatic molecular gates ensuring unidirectional transport. An interaction with the π-electron system of the retinal supports transient chloride ion binding across a major bottleneck in the transport pathway. These results allow us to propose key mechanistic features enabling finely controlled chloride transport across the cell membrane in this light-powered chloride ion pump.

    View details for DOI 10.1126/science.abj6663

    View details for Web of Science ID 000764232800039

    View details for PubMedID 35113649

  • Femtosecond-to-millisecond structural changes in a light-driven sodium pump NATURE Skopintsev, P., Ehrenberg, D., Weinert, T., James, D., Kar, R. K., Johnson, P. 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., Bruenle, S., Deupi, X., Schertler, G. X., Benoit, R. M., Panneels, V., Nogly, P., Schapiro, I., Milne, C., Heberle, J., Standfuss, J. 2020; 583 (7815): 314-+

    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.

    View details for DOI 10.1038/s41586-020-2307-8

    View details for Web of Science ID 000535225300003

    View details for PubMedID 32499654

    View details for PubMedCentralID 5364028

  • Proton uptake mechanism in bacteriorhodopsin captured by serial synchrotron crystallography SCIENCE Weinert, T., Skopintsev, P., James, D., Dworkowski, F., Panepucci, E., Kekilli, D., Furrer, A., Brunle, S., Mous, S., Ozerov, D., Nogly, P., Wang, M., Standfuss, J. 2019; 365 (6448): 61-+

    Abstract

    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 to 15 milliseconds onwards, we observe large additional structural rearrangements up to 9 angstroms on the cytoplasmic side. Rotation of leucine-93 and phenylalanine-219 opens a hydrophobic barrier, leading to the formation of a water chain connecting the intracellular aspartic acid-96 with the retinal Schiff base. The formation of this proton wire recharges the membrane pump with a proton for the next cycle.

    View details for DOI 10.1126/science.aaw8634

    View details for Web of Science ID 000474432700032

    View details for PubMedID 31273117

  • Functional and structural characterization of an ECF-type ABC transporter for vitamin B12 ELIFE Santos, J. A., Rempel, S., Mous, S. M., Pereira, C. T., ter Beek, J., de Gier, J., Guskov, A., Slotboom, D. J. 2018; 7

    Abstract

    Vitamin B12 (cobalamin) is the most complex B-type vitamin and is synthetized exclusively in a limited number of prokaryotes. Its biologically active variants contain rare organometallic bonds, which are used by enzymes in a variety of central metabolic pathways such as L-methionine synthesis and ribonucleotide reduction. Although its biosynthesis and role as co-factor are well understood, knowledge about uptake of cobalamin by prokaryotic auxotrophs is scarce. Here, we characterize a cobalamin-specific ECF-type ABC transporter from Lactobacillus delbrueckii, ECF-CbrT, and demonstrate that it mediates the specific, ATP-dependent uptake of cobalamin. We solved the crystal structure of ECF-CbrT in an apo conformation to 3.4 Å resolution. Comparison with the ECF transporter for folate (ECF-FolT2) from the same organism, reveals how the identical ECF module adjusts to interact with the different substrate binding proteins FolT2 and CbrT. ECF-CbrT is unrelated to the well-characterized B12 transporter BtuCDF, but their biochemical features indicate functional convergence.

    View details for DOI 10.7554/eLife.35828

    View details for Web of Science ID 000435002300001

    View details for PubMedID 29809140

    View details for PubMedCentralID PMC5997447