19 18 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 1 / 2 2 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 1 / 2 2 Macromolecular Crystallography Group Beamlines I24 and B23 High-resolution photoreceptor structures offer a blueprint for newmethods and applications Related publication: Bada Juarez, J. F., Judge, P. J., Adam, S., Axford, D., Vinals, J., Birch, J., Kwan, T. O. C., Hoi, K. K., Yen, H.-Y., Vial, A., Milhiet, P.-E., Robinson, C. V., Schapiro, I., Moraes, I., &Watts, A. Structures of the archaerhodopsin-3 transporter reveal that disordering of internal water networks underpins receptor sensitization. Nature Communications , 12 (1), 629. (2021). DOI: 10.1038/s41467-020-20596-0 Publication keywords : Archaerhodopsin; Optogenetics; Receptor Sensitisation; Ion transport; Lipidic cubic phase A rchaerhodopsin-3 (AR3) is a light-sensitive protein expressed by Halorubrum sodomense , an organism that grows in the Dead Sea. Mutants of the protein are routinely used in neuroscience experiments to selectively silence individual nerve cells and detect changes in transmembrane voltage. However, these mutants are designed without knowledge of the protein’s structure. An international team of researchers visualised the photoreceptor at unprecedented resolution using the I24 and B23 beamlines and reported the first ever structure of the ground state of AR3. In this state, the protein is configured to transport one H + ion across the cell membrane for each photon absorbed. The teamwas also able to crystallise the photoreceptor in a second conformation, a desensitised state that AR3 adopts in the prolonged absence of light. The superb resolution achieved for these AR3 structures is among the highest for a wild-type membrane protein deposited in the Protein Data Bank. The high-resolution crystal structures were essential for understanding the workings of the protein. Obtaining such high-quality diffraction data would not have been possible without the state-of-the-art microfocus I24 beamline at Diamond. Circular Dichroism (CD) measurements from the B23 Beamline allowed the team to quantify the alpha-helical content of AR3 and correlate it with other biophysical information. This data provides structural biologists and protein engineerswith the ‘blueprints’ to AR3, opening theway for the development of newtools andmethodologies in the fields of neuroscience, cell biology and beyond. Archaerhodopsin-3 (AR3) is a light-sensitive protein produced by the archaebacterium Halorubrum sodomense, which grows in the Dead Sea. The protein converts energy from sunlight to a transmembrane electrical gradient, which is used by the organism to power growth and reproduction. In the laboratory, AR3 can also be expressed recombinantly in mammalian cells and is used extensively in neurobiology experiments. When light is shone on nerve cells containing AR3, the protein prevents them from transmitting nerve impulses. This selective silencing of nerves (a methodology known as optogenetics 1 ) is used by neuroscientists to understand the roles of individual neurons in brain function. The photoreceptor is also used to measure electrical voltages across cell membranes in vivo 2 . Although AR3 is widely used in several types of laboratory experiment, the lack of structural data for the photoreceptor has hindered the development of new methods. The data produced in this research provide the detailed blueprints for the protein and pave the way for new applications in neurobiology and biotechnology. Like many light-sensitive receptor proteins, AR3 is found in the membrane of the archaebacterial cell. It absorbs light using a chromophore, retinal, which is better known as one of the forms of Vitamin A. When light shines on the retinal, it changes shape, transitioning from an extended all- trans isomer to a bent 13- cis isomer. The protein can also adopt a desensitised state, in which it does not respond to light, but until now the process of inactivation has not been understood. The high-resolution structural data obtained from crystals of AR3 (Fig. 1a) on the I24microfocus beamline at Diamond Light Source, have enabled the molecular mechanisms of AR3 desensitisation to be explained, and these insights have implications for our understanding of other receptor proteins. Wild-type AR3 was expressed in the native organism and purified, unusually, without the use of detergent. It was crystallised in lipidic cubic phase and diffraction data were acquired on the I24 beamline at cryo temperatures. One subset of the crystals was grown in the absence of light and diffraction data collected from these samples were used to generate the dark-adapted, desensitised structure, which was solved to 1.3 Å resolution. The second subset of crystals was grown under illumination and the data collected gave rise to the resting state structure. At 1.07 Å resolution, the light-adapted, resting-state structure is one of the highest resolution membrane protein structures solved to date (Fig. 1b). The structures show that AR3 undergoes a series of previously unobserved post-translational modifications during its maturation, including the cleavage of the first six amino acids at the N-terminus, the cyclisation of Gln7 to form a pyroglutamyl group and the covalent conjugation of retinal to Lys226 via a Schiff base (Fig. 1c) 3 . There are key differences between the resting and desensitised states of the protein in the chromophore and the surrounding region of the protein. In thedesensitised state (PDB: 6GUX), 13- cis andall-trans retinal isomers are present in a calculated occupancy ratio of 70% and 30%, respectively (Fig. 2). In the resting state (PDB:6S6C), the chromophore is resolved as two different conformations of the same all- trans isomer (Fig. 2), with relative occupancies of 75% and 25%. The structures also suggest that the amino acids and water molecules inside the protein are more mobile in the resting state than in the desensitised state. The energy barriers to isomerisation of the retinal were calculated using the crystal structures as a starting point. In the desensitised form, the 13- cis isomer of retinal was more stable than the all- trans isomer, (ΔGcis-trans=−1.9 kcal/ mol). The relatively small difference in energies is consistent with the presence of both isomers in the crystal structure. In contrast, the energy difference between the isomers in the resting state is larger (ΔGcis-trans=10.9 kcal/ mol) and the all-trans form is more stable (Fig. 3). The energetic profiles are determined by the environment of the retinal 5 in each state, which is in turn dependent on the order and position of the internal water molecules and the stability of the networks of non-covalent bonds surrounding the chromophore. Our structures suggest that the greater internal disorder of the resting state heavily influences the response of the protein to visible light. In contrast, the lower internal mobility in the desensitised state may prevent AR3 from being activated. These AR3 structures are amajor advance for the study of structure-function relationships of integral membrane proteins. The resolution obtained (1.07 Å), using diffraction data from the I24microfocus beamline, is currently unmatched for this group of proteins. The structural information generatedwill nowprovide the details required to design improved protein variants for use in optogenetics experiments, and for applications in biotechnology. Calculations, using the structures as a starting point, reveal how small differences in the electronic environment of the chromophore binding site can determine the behaviour of the resting and desensitised states of the protein. Finally, the differences revealed in the order of internal water molecules and the associated networks of non-covalent bonds between the two states, may help us to unlock the more complex mechanisms of activation and desensitisation observed inmammalian receptor proteins. References: 1. Pastrana, E. Optogenetics: controlling cell function with light. Nature Methods . 8 (1), 24–25 (2011). DOI: 10.1038/nmeth.f.323 2. Chow, B. Y. et al. High-performance genetically targetable optical neural silencing by light-driven proton pumps. Nature . 463 (7277), 98–102 (2010). DOI: 10.1038/nature08652 3. Hoi, K. et al. Detergent-free Lipodisq Nanoparticles Facilitate High- Resolution Mass Spectrometry of Folded Integral Membrane Proteins. Nano Letters , 21 (7), 2824–2831 (2021). DOI: 10.1021/acs.nanolett.0c04911 4. Ihara, K. et al . Met-145 is a key residue in the dark adaptation of bacteriorhodopsin homologs. Biophysical Journal . 67 (3), 1187–1191 (1994) DOI: 10.1016/S0006-3495(94)80587-9 5. Baudry, J. et al. Simulation Analysis of the Retinal Conformational Equilibrium in Dark-Adapted Bacteriorhodopsin. Biophysical Journal . 76 (4), 1909–1917 (1999). DOI: 10.1016/S0006-3495(99)77349-2 Funding acknowledgement: This work was supported by the BBSRC (BB/N006011/1), DSTL (DSTLX-1000099768),Wellcome Trust (20289/Z/16/Z) and the DFG (SFB 1078). Additional funding is acknowledged from BEIS and the ERC. Corresponding authors: Dr Isabel Moraes, National Physical Laboratory,
[email protected] Prof. AnthonyWatts, Oxford University,
[email protected] Figure 1: (a) Crystals of AR3 visualised under circularly polarised light; (b) Overlay of the structures of the resting state of AR3 (white) and the related protein, bacteriorhodopsin (purple). Secondary structure elements are shown in ribbon representation. The chromophore is shown in stick representation. The approximate positions of the intracellular (IC) and extracellular (EC) sides of the membrane are indicated with dotted lines; (c) The pyroglutamyl group at the N-terminus of AR3 is generated by the removal of the signal peptide and the modification of Gln7. The 2Fobs– Fcalc electron density map (blue mesh) is contoured at 1.2σ. Figure 2: Comparison of the internal structures of the desensitised (PDB: 6GUX) and resting state (PDB: 6S6C) structures of AR3. (Top) In the desensitised state (left) the C13 = C14 retinal bond has been modelled with 70% cis and 30% trans isomers (dark and light pink respectively). In the LA state (right) retinal is modelled in the all-trans state only, but as two different conformers. (Bottom) Structures of the internal H-bond networks close to the retinal chromophore. Predicted H-bonds are indicated by yellow dashes. The 2Fobs–Fcalc electron density maps (blue mesh) are contoured at 1.2σ. Figure 3: Calculated potentials of mean force (PMF) for the isomerisation of the C12– C13 = C14–C15 dihedral of retinal in dark-adapted (blue) and light-adapted (red) AR3. The PMF was computed by sampling the retinal isomerisation from all-trans to 13-cis and vice versa. Each point on the curve is generated from two independent 0.5 ns QM(SCC-DFTB)/MM MD trajectories, initiated from two separated equilibrated starting structures. The protein backbone was fixed in place, but all other atoms (including those in the chromophore and amino acid sidechains) were allowed to move.