Ultrafast structural changes direct the first molecular events of vision

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.

Main

Rhodopsin, the vertebrate receptor for low-light vision, is concentrated within the disk membranes of rod cells in the retina. Rhodopsin transforms the absorption of light into a physiological signal through conformational changes that activate the intracellular G protein transducin—a member of the Gi/o/t family—initiating a signalling cascade, resulting in electrical impulses sent to the brain and ultimately leading to visual perception. The structure of rhodopsin consists of seven transmembrane (TM) α-helices with an 11-cis retinal chromophore covalently bound through a protonated Schiff base (PSB) to Lys296^7.43 of TM7 (the superscript values on amino acids containing the TM domain location refer to the Ballesteros–Weinstein scheme, explained in the ‘Residue numbering’ section of the Methods). This buried ligand is located within the TM bundle towards the extracellular side, like in many class A GPCRs. Retinal also contacts extracellular loop 2 (ECL2), which forms a lid over the chromophore and contains a highly conserved disulfide bridge (Cys110^3.25–Cys187^ECL2) connecting to the central helix TM3 (see box 1 of ref. ¹). Glu113^3.28 provides a negatively charged counterion⁴ that forms a salt bridge with the PSB (Fig. 1a–c) and thereby participates in the stabilization of the receptor resting state⁵. From our understanding of the evolution of visual pigments^6,7, we know that, originally, Glu181^ECL2 was the only residue able to neutralize the positive charge of the Schiff base. This ‘ancestral counterion’⁸, which still functions as a complex counterion in invertebrates⁶, remains connected through a water-mediated hydrogen bond network to the PSB of vertebrate rhodopsins (Fig. 1b). The second, main counterion Glu113^3.28 appeared during evolution and both residues are important for the activation mechanism. Structures of light-activated rhodopsin trapped at low temperature^9,10, structures of the late Meta II active state^11,12,13, and copious computational¹⁴, biochemical and spectroscopic studies^15,16,17 have provided important insights into the mechanism of signal transduction in rhodopsin. However, methods that provide both a high spatial and temporal resolution are required to obtain a complete experimentally derived picture of the activation mechanism at the atomic scale from femtoseconds to milliseconds.

In recent years, time-resolved crystallography^3,18 at X-ray free-electron lasers (XFEL) has been used to reveal ultrafast structural changes in myoglobin¹⁹, photoactive yellow protein^20,21, fluorescent proteins²², bacterial phytochromes²³, microbial proton^24,25, sodium²⁶ and chloride²⁷ pumps, a bacterial photosynthetic reaction centre²⁸ and also with non-photosensory proteins with exogenously added non-natural photocaged ligands^29,30. In time-resolved serial femtosecond crystallography (TR-SFX), the protein molecules in the crystals are photoactivated with an optical laser pulse and the structure is probed with an X-ray pulse from an XFEL after a specified time delay. As each crystal generates one diffraction pattern, the experiment is carried out in a serial manner: the frames are collected from tens of thousands of randomly oriented crystals. The TR-SFX method is complementary to spectroscopy methods, revealing structural detail at the atomic level in the femtosecond domain, without directly resolving charge effects, hydrogen bond interactions and electronic changes. The atomic resolution comes at the cost of less clearly defined illumination conditions³¹, which are a matter of discussion^24,25,32, and systematic studies investigating these in more detail are underway³³.

Here we used TR-SFX at room temperature to follow the light-induced conversion of the inverse agonist 11-cis retinal to an agonist all-trans in a vertebrate opsin. Our observations reveal how this translates into early structural changes within the protein. After 1 ps, we observe a twisted retinal that stores energy while structural motions in the protein radiate as an anisotropic propagation away from the retinal chromophore. The rhodopsin structure, 100 ps later, reveals a slightly more relaxed conformation.

Room temperature structure of rhodopsin

The room-temperature SFX structure of rhodopsin in the inactive dark state was obtained at a resolution of 1.8 Å (Extended Data Table 1 (dark state)) from microcrystals grown in a lipidic cubic phase (LCP). As with most membrane protein structures determined from LCP-grown crystals, these crystals display a type I lattice³⁴ forming stacks of protein two-dimensional layers built through hydrophobic interactions (Extended Data Fig. 1a–c). Potentially physiologically relevant dimers of rhodopsin molecules³⁵ form contacts between the TM1 and helix 8 (H8) segments of each monomer and are assembled in a head-to-tail manner generating the asymmetric unit. By close inspection of the diffraction data and the resulting electron density maps, the presence of translation-related crystal domains was detected. Measured intensities were corrected to account for this, globally improving the quality and interpretability of the maps³⁶ (Methods, Extended Data Fig. 1d–i and Extended Data Table 1). Overall, the SFX structure in the inactive dark state of rhodopsin (Fig. 1a) is very similar to other crystal structures collected at cryogenic temperatures (for example, Protein Data Bank (PDB) 1GZM; root mean square deviation = 0.33 Å on C_α atoms)³⁷. In contrast to earlier structures solved in cryogenic conditions, the present room temperature structure reveals electron density for all of the previously described functional and structural water molecules. These include the water-mediated cluster around the ancestral counterion Glu181 and its polar tyrosine cage (Fig. 1b), which have a role later in the photoactivation process^38,39. Moreover, a new ordered water molecule was resolved near the Schiff base connecting the proximal counterion Glu113^3.28 to Met86^2.53 (Fig. 1c) and Ala117^3.32. This interaction has a central position at the TM2–TM3–TM7 interface in the TM bundle.

A picosecond light-induced bent retinal

The first metastable intermediate of rhodopsin (bathorhodopsin, Batho-Rh)^2,15,40 arises 200 fs after photoactivation. It is fully populated by ∆t = 1 ps (refs. ^14,41) and persists for tens of nanoseconds^42,43. To characterize the structure of Batho-Rh, we collected TR-SFX data at the Swiss and Japanese XFELs (Extended Data Fig. 2) from LCP-grown microcrystals of rhodopsin photoactivated under a regime in which laser-induced heating is low (Extended Data Fig. 3, Methods) and in which we recovered high-quality difference electron density maps below the maximum activation (Extended Data Fig. 3) using a femtosecond-pump laser with a 480-nm wavelength for three time delays of photoactivation of ∆t = 1 ps, 10 ps and 100 ps. High-quality TR-SFX data (Extended Data Table 1) are represented as difference Fourier electron density maps in Fig. 2a,b and Extended Data Fig. 4. For the shorter time delay, changes in electron density are distributed in a highly anisotropic manner, clustering in the immediate vicinity of the buried retinal chromophore and propagating towards the cytoplasmic side of the protein through the TM5 and TM6 helices (Extended Data Fig. 5) at a minimum speed of 18 Å ps⁻¹ measured along TM6 (1,800 m s⁻¹, slightly above the speed of sound in water and in accordance with the speed of sound in ribonuclease A crystals⁴⁴). This structural anisotropy had completely decayed by Δt = 100 ps….