Unidirectional ion transport mechanism of a light-driven chloride pump revealed using X-ray free electron lasers

Abstract

Light-driven chloride-pumping rhodopsins actively transport anions, including various halide ions, across cell membranes. Recent studies using time-resolved serial femtosecond crystallography (TR-SFX) have uncovered the structural changes and ion transfer mechanisms in light-driven cation-pumping rhodopsins. However, the mechanism by which the conformational changes pump an anion to achieve unidirectional ion transport, from the extracellular side to the cytoplasmic side, in anion-pumping rhodopsins remains enigmatic. We have collected TR-SFX data of Nonlabens marinus rhodopsin-3 (NM-R3), derived from a marine flavobacterium, at 10 µs and 1 ms time-points after photoexcitation. Our structural analysis reveals the conformational alterations during ion transfer and after ion release. Movements of the retinal chromophore initially displace a conserved tryptophan to the cytoplasmic side of NM-R3, accompanied with a slight shift of the halide ion bound to the retinal. After ion release, the inward movements of helix C and helix G and the lateral displacements of the retinal block access to the extracellular side of NM-R3. Anomalous signal data have also been obtained from NM-R3 crystals containing iodide ions. The anomalous density maps provide insight into the halide binding site for ion transfer in NM-R3.

A versatile approach to high-density microcrystals in lipidic cubic phase for room-temperature serial crystallography

“…Room-temperature SX at XFELs (SFX) and synchrotron sources (SSX) is applicable to membrane protein samples crystallized in lipidic cubic phase (LCP) (Caffrey & Cherezov, 2009;Liu et al, 2014). High-resolution structural data generated for integral membrane proteins (IMPs) (Axford et al, 2022;Johansson et al, 2019;Hosaka et al, 2022;Liu et al, 2013;Nango & Iwata, 2023;Nogly et al, 2015;Stauch et al, 2019;Zhang et al, 2017, to mention a few) are critical for understanding structure-function relationships, intermolecular interactions and protein dynamics, and for rational drug design (Hauser et al, 2018;Reis & Moraes, 2019). Structure determination of IMPs by traditional X-ray diffraction methods is challenging, since the crystals are usually extremely fragile and exceptionally sensitive to radiation damage.…”

“…SX of IMPs crystallized in LCP allows diffraction data to be acquired under near-physiological conditions, where the mesophase mimics the biological membrane. Furthermore, SX in LCP facilitates time-resolved studies, critical for generating mechanistic insights into the dynamic behaviour of IMPs (Hosaka et al, 2022;Mous et al, 2022;Nango et al, 2016;Nass Kovacs et al, 2019;Nogly et al, 2016Nogly et al, , 2018Oda et al, 2021;Skopintsev et al, 2020;Weinert et al, 2019;Yun et al, 2021).…”