- Prof. Gebhard F.X. Schertler
Structural Biology ETH Zürich D-BIOL
Head of Biology and Chemistry
Department
Paul Scherrer Institut
Laboratory of Biomolecular Research,
LBR
OFLC 109
CH-5232 Villigen PSI
gebhard.schertler@psi.ch
phone +41 56 310 4265
http://bio.web.psi.ch - +41 56 310 4265
Gebhard F X Schertler
Paul Scherrer Institute, Biomolecular Research, Faculty Member
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Swiss Federal Institute of Technology (ETH), D-BIOL, Faculty Member add
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Structure and Dynamics of Membrane Proteinsedit
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Manfred Schweiger, Dieter Oesterhelt, Hartmuth Michel, Richard Hendersonedit
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Chemistry, Crystallography, Medicine, Multidisciplinary, Macromolecular X-Ray Crystallography, and 10 moreProtein Stability, Humans, G protein-coupled receptors, Salt Bridge, Protein Secondary Structure Prediction, Receptor, Protein Conformation, Protein Binding, Rhodopsin, and G Protein Coupled Receptors
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Chemistry, Crystallography, Medicine, Multidisciplinary, Protein Engineering, and 15 moreAntibodies, Humans, Secondary, Protein structure, Protein Domains, Receptors, Cryo Electron Microscopy, G protein, Protein Binding, Nature Communications, Gtp Binding Proteins, Rhodopsin, Nucleotides, Monoclonal, and binding sites
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Significance G protein–coupled receptors (GPCRs) are important pharmaceutical targets for the treatment of a broad spectrum of diseases. Upon ligand binding, GPCRs initiate intracellular signaling pathways by interacting with partner... more
Significance G protein–coupled receptors (GPCRs) are important pharmaceutical targets for the treatment of a broad spectrum of diseases. Upon ligand binding, GPCRs initiate intracellular signaling pathways by interacting with partner proteins. Assays that quantify the interplay between ligand binding and initiation of downstream signaling cascades are critical in the early stages of drug development. We have developed a high-throughput mass spectrometry method to unravel GPCR–protein complex interplay and demonstrated its use with three GPCRs to provide quantitative information about ligand-modulated coupling selectivity. This method provides insights into the molecular details of GPCR interactions and could serve as an approach for discovery of drugs that initiate specific cell-signaling pathways.
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Ptychographic hard X-ray computed tomography (PXCT) is a recent method allowing imaging with quantitative electron-density contrast. Here, we imaged, at cryogenic temperature and without sectioning, cellular and subcellular structures of... more
Ptychographic hard X-ray computed tomography (PXCT) is a recent method allowing imaging with quantitative electron-density contrast. Here, we imaged, at cryogenic temperature and without sectioning, cellular and subcellular structures of a chemically fixed and stained wild-type mouse retina, including axons and synapses, with complete isotropic 3D information over tens of microns. Comparison with tomograms of degenerative retina from a mouse model of retinitis pigmentosa illustrates the potential of this method for analyzing disease processes like neurodegeneration at sub-200 nm resolution. As a non-destructive imaging method, PXCT is very suitable for correlative imaging. Within the outer plexiform layer containing the photoreceptor synapses, we identified somatic synapses. We used a small region inside the X-ray-imaged sample for further high-resolution focused ion beam/scanning electron microscope tomography. The subcellular structures of synapses obtained with the X-ray techniqu...
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Light-sensitive G protein-coupled receptors (GPCRs)—rhodopsins—absorb photons to isomerize their covalently bound retinal, triggering conformational changes that result in downstream signaling cascades. Monostable rhodopsins release... more
Light-sensitive G protein-coupled receptors (GPCRs)—rhodopsins—absorb photons to isomerize their covalently bound retinal, triggering conformational changes that result in downstream signaling cascades. Monostable rhodopsins release retinal upon isomerization as opposed to the retinal in bistable rhodopsins that “reisomerize” upon absorption of a second photon. Understanding the mechanistic differences between these light-sensitive GPCRs has been hindered by the scarcity of recombinant models of the latter. Here, we reveal the high-resolution crystal structure of a recombinant bistable rhodopsin, jumping spider rhodopsin-1, bound to the inverse agonist 9- cis retinal. We observe a water-mediated network around the ligand hinting toward the basis of their bistable nature. In contrast to bovine rhodopsin (monostable), the transmembrane bundle of jumping spider rhodopsin-1 as well that of the bistable squid rhodopsin adopts a more “activation-ready” conformation often observed in other...
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Animals sense light using photosensitive proteins—rhodopsins—containing a chromophore—retinal—that intrinsically absorbs in the ultraviolet. Visible light-sensitivity depends primarily on protonation of the retinylidene Schiff base (SB),... more
Animals sense light using photosensitive proteins—rhodopsins—containing a chromophore—retinal—that intrinsically absorbs in the ultraviolet. Visible light-sensitivity depends primarily on protonation of the retinylidene Schiff base (SB), which requires a negatively-charged amino acid residue—counterion—for stabilization. Little is known about how the most common counterion among varied rhodopsins, Glu181, functions. Here, we demonstrate that in a spider visual rhodopsin, orthologue of mammal melanopsins relevant to circadian rhythms, the Glu181 counterion functions likely by forming a hydrogen-bonding network, where Ser186 is a key mediator of the Glu181–SB interaction. We also suggest that upon light activation, the Glu181–SB interaction rearranges while Ser186 changes its contribution. This is in contrast to how the counterion of vertebrate visual rhodopsins, Glu113, functions, which forms a salt bridge with the SB. Our results shed light on the molecular mechanisms of visible lig...
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The structure of the rhodopsin/mini-G o complex reveals new insights on G protein selectivity.
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G protein coupled receptors (GPCRs) are a key family of membrane proteins in all eukaryotes and also very important drug targets for medical intervention. The extensively studied visual pigment rhodopsin is a prime example of a family A... more
G protein coupled receptors (GPCRs) are a key family of membrane proteins in all eukaryotes and also very important drug targets for medical intervention. The extensively studied visual pigment rhodopsin is a prime example of a family A GPCR. Its chromophore ligand retinal is covalently linked to a lysine in helix seven forming a protonated Schiff base. Interestingly, this is the same situation in other-non-GPCR-retinal proteins, like the prototype light-driven microbial proton pump bacteriorhodopsin, albeit there is no (or only a very remote) phylogenetical link. Close to the retinal ligand, several water molecules help to organise a functionally important hydrogen bond network that undergoes significant changes during photo-activation. Such water-mediated networks are also critical for ligand binding of other GPCRs and they are becoming increasingly important in drug discovery. GPCRs also contain a partially conserved water mediated hydrogen bond network that stabilises the ground...
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Ultrafast isomerization of retinal is the primary step in photoresponsive biological functions including vision in humans and ion-transport across bacterial membranes. We studied the sub-picosecond structural dynamics of retinal... more
Ultrafast isomerization of retinal is the primary step in photoresponsive biological functions including vision in humans and ion-transport across bacterial membranes. We studied the sub-picosecond structural dynamics of retinal isomerization in the light-driven proton pump bacteriorhodopsin using an x-ray laser. A series of structural snapshots with near-atomic spatial and temporal resolution in the femtosecond regime show how the excited all- retinal samples conformational states within the protein binding pocket prior to passing through a twisted geometry and emerging in the 13 conformation. Our findings suggest ultrafast collective motions of aspartic acid residues and functional water molecules in the proximity of the retinal Schiff base as a key ingredient for this stereo-selective and efficient photochemical reaction.
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We present the main specifications of the newly constructed Swiss Free Electron Laser, SwissFEL, and explore its potential impact on ultrafast science. In light of recent achievements at current X-ray free electron lasers, we discuss the... more
We present the main specifications of the newly constructed Swiss Free Electron Laser, SwissFEL, and explore its potential impact on ultrafast science. In light of recent achievements at current X-ray free electron lasers, we discuss the potential territory for new scientific breakthroughs offered by SwissFEL in Chemistry, Biology, and Materials Science, as well as nonlinear X-ray science.
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Structural information of the different conformational states of the two prototypical light-sensitive membrane proteins, bacteriorhodopsin and rhodopsin, has been obtained in the past by X-ray cryo-crystallography and cryo-electron... more
Structural information of the different conformational states of the two prototypical light-sensitive membrane proteins, bacteriorhodopsin and rhodopsin, has been obtained in the past by X-ray cryo-crystallography and cryo-electron microscopy. However, these methods do not allow for the structure determination of most intermediate conformations. Recently, the potential of X-Ray Free Electron Lasers (X-FELs) for tracking the dynamics of light-triggered processes by pump-probe serial femtosecond crystallography has been demonstrated using 3D-micron-sized crystals. In addition, X-FELs provide new opportunities for protein 2D-crystal diffraction, which would allow to observe the course of conformational changes of membrane proteins in a close-to-physiological lipid bilayer environment. Here, we describe the strategies towards structural dynamic studies of retinal proteins at room temperature, using injector or fixed-target based serial femtosecond crystallography at X-FELs. Thanks to re...
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Arrestin-1 desensitizes the activated and phosphorylated photoreceptor rhodopsin by forming transient rhodopsin−arrestin-1 complexes that eventually decay to opsin, retinal and arrestin-1. Via a multi-dimensional screening setup, we... more
Arrestin-1 desensitizes the activated and phosphorylated photoreceptor rhodopsin by forming transient rhodopsin−arrestin-1 complexes that eventually decay to opsin, retinal and arrestin-1. Via a multi-dimensional screening setup, we identified and combined arrestin-1 mutants that form lasting complexes with light-activated and phosphorylated rhodopsin in harsh conditions, such as high ionic salt concentration. Two quadruple mutants, D303A + T304A + E341A + F375A and R171A + T304A + E341A + F375A share similar heterologous expression and thermo-stability levels with wild type (WT) arrestin-1, but are able to stabilize complexes with rhodopsin with more than seven times higher half-maximal inhibitory concentration (IC50) values for NaCl compared to the WT arrestin-1 protein. These quadruple mutants are also characterized by higher binding affinities to phosphorylated rhodopsin, light-activated rhodopsin and phosphorylated opsin, as compared with WT arrestin-1. Furthermore, the assesse...
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Vision is initiated by the rhodopsin family of light-sensitive G protein-coupled receptors (GPCRs). A photon is absorbed by the 11-cisretinal chromophore of rhodopsin which isomerises within 200 femtoseconds to the all-transconformation,... more
Vision is initiated by the rhodopsin family of light-sensitive G protein-coupled receptors (GPCRs). A photon is absorbed by the 11-cisretinal chromophore of rhodopsin which isomerises within 200 femtoseconds to the all-transconformation, 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 elusive. In this work, we use ultrafast time-resolved crystallography at room temperature to determine how an isomerised twistedall-transretinal stores the photon energy required to initiate protein conformational changes associated with the formation of the G protein-binding signalling state. The distorted retinal at 1 ps time-delay of 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 th...
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Dynamic hydrogen-bond networks provide proteins with structural plasticity required to translate signals such as ligand binding into a cellular response or to transport ions and larger solutes across membranes and, thus, are of central... more
Dynamic hydrogen-bond networks provide proteins with structural plasticity required to translate signals such as ligand binding into a cellular response or to transport ions and larger solutes across membranes and, thus, are of central interest to understand protein reaction mechanisms. Here, we present C-Graphs, an efficient tool with graphical user interface that analyzes data sets of static protein structures or of independent numerical simulations to identify conserved, vs unique, hydrogen bonds and hydrogen-bond networks. For static structures, which may belong to the same protein or to proteins with different sequences, C-Graphs uses a clustering algorithm to identify sites of the hydrogen-bond network where waters are conserved among the structures. Using C-Graphs, we identify an internal protein-water hydrogen-bond network common to static structures of visual rhodopsins and adenosine A2A G protein-coupled receptors (GPCRs). Molecular dynamics simulations of a visual rhodopsin indicate that the conserved hydrogen-bond network from static structure can recruit dynamic hydrogen bonds and extend throughout most of the receptor. We release with this work the code for C-Graphs and its graphical user interface.
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Effective public-health measures and vaccination campaigns against SARS-CoV-2 require granular knowledge of population-level immune responses. We developed a Tripartite Automated Blood Immunoassay (TRABI) to assess the IgG response... more
Effective public-health measures and vaccination campaigns against SARS-CoV-2 require granular knowledge of population-level immune responses. We developed a Tripartite Automated Blood Immunoassay (TRABI) to assess the IgG response against the ectodomain and the receptor-binding domain of the spike protein as well as the nucleocapsid protein of SARS-CoV-2. We used TRABI for continuous seromonitoring of hospital patients and healthy blood donors (n=72’222) in the canton of Zurich from December 2019 to December 2020 (pre-vaccine period). Seroprevalence peaked in May 2020 and rose again in November 2020 in both cohorts. Validations of results included antibody diffusional sizing and Western Blotting. Using an extended Susceptible-Exposed-Infectious-Removed model, we found that antibodies waned with a half-life of 75 days, whereas the cumulative incidence rose from 2.3% in June 2020 to 12.2% in mid-December 2020 in the population of the canton of Zurich. A follow-up health survey indica...
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# The Author(s) 2015. This article is published with open access at Springerlink.com Abstract G-protein coupled receptors (GPCRs) are the targets of over half of all prescribed drugs today. The UniProt database has records for about 800... more
# The Author(s) 2015. This article is published with open access at Springerlink.com Abstract G-protein coupled receptors (GPCRs) are the targets of over half of all prescribed drugs today. The UniProt database has records for about 800 proteins classified as GPCRs, but drugs have only been developed against 50 of these. Thus, there is huge potential in terms of the number of targets for new therapies to be designed. Several breakthroughs in GPCRs biased pharmacology, structural biology, modelling and scoring have resulted in a resurgence of interest in GPCRs as drug targets. Therefore, an international conference, sponsored by the Royal Society, with world-renowned researchers from in-dustry and academia was recently held to discuss recent prog-
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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.... more
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.
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The adhesion G-protein coupled receptor Adgrg6 (formerly Gpr126) is instrumental in the development, maintenance and repair of peripheral nervous system myelin. The prion protein (PrP) is a potent activator of Adgrg6 and could be used as... more
The adhesion G-protein coupled receptor Adgrg6 (formerly Gpr126) is instrumental in the development, maintenance and repair of peripheral nervous system myelin. The prion protein (PrP) is a potent activator of Adgrg6 and could be used as a potential therapeutic agent in treating peripheral demyelinating and dysmyelinating diseases. We designed a dimeric Fc-fusion protein comprising the myelinotrophic domain of PrP (FT2Fc), which activated Adgrg6 in vitro and exhibited favorable pharmacokinetic properties for in vivo treatment of peripheral neuropathies. While chronic FT2Fc treatment elicited specific transcriptomic changes in the sciatic nerves of PrP knockout mice, no amelioration of the early molecular signs demyelination was detected. Instead, RNA sequencing of sciatic nerves revealed downregulation of cytoskeletal and sarcomere genes, akin to the gene expression changes seen in myopathic skeletal muscle of PrP overexpressing mice. These results call for caution when devising mye...
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G protein-coupled receptors (GPCRs) are cellular master regulators that translate extracellular stimuli such as light, small molecules or peptides into a cellular response. Upon ligand binding, they bind intracellular proteins such as G... more
G protein-coupled receptors (GPCRs) are cellular master regulators that translate extracellular stimuli such as light, small molecules or peptides into a cellular response. Upon ligand binding, they bind intracellular proteins such as G proteins or arrestins, modulating intracellular signaling cascades. Here, we use a protein-fragment complementation approach based on nanoluciferase (split luciferase assay) to assess interaction of all four known human arrestins with four different GPCRs (two class A and two class B receptors) in live cells. Besides directly tagging the 11S split-luciferase subunit to the receptor, we also could demonstrate that membrane localization of the 11S subunit with a CAAX-tag allowed us to probe arrestin recruitment by endogenously expressed GPCRs. Varying the expression levels of our reporter constructs changed the dynamic behavior of our assay, which we addressed with an advanced baculovirus-based multigene expression system. Our detection assay allowed u...
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Chemistry, Crystallography, Image Processing, Molecular Biology, Biology, and 15 moreCrystallization, Electron Diffraction, Medicine, Field emission, High Voltage, Molecular, Humans, Animals, Infant, Cattle, Electrons, Liquid Nitrogen, Biochemistry and cell biology, Electron Microscope, and Electron Crystallography
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Biophysics, Chemistry, Crystallography, Immunology, Transmission Electron Microscopy, and 14 moreMedicine, Humans, Animals, Bacteriorhodopsin, Clinical Sciences, Eye, Optometry and Ophthalmology, Protein Secondary Structure Prediction, Extracellular, Gtp Binding Proteins, Rhodopsin, Transducin, Molecular Structure, and Electron Crystallography
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Biophysics, Chemistry, Crystallography, Molecular Biology, Medicine, and 13 moreCell line, Sequence alignment, Animals, Sodium channel, Gating, Voltage-Gated Sodium Channels, Protein Conformation, Amino Acid Sequence, Structure activity Relationship, Lipid Bilayer, Biochemistry and cell biology, Linker, and Molecular Sequence Data
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The molecular mechanism of arrestin–G protein-coupled receptor (GPCR ) complex formation has been analyzed using inter alia spectroscopic, mutagenesis and structure determination methods. The latest crystal structure elucidating one... more
The molecular mechanism of arrestin–G protein-coupled receptor (GPCR ) complex formation has been analyzed using inter alia spectroscopic, mutagenesis and structure determination methods. The latest crystal structure elucidating one conformation of the rhodopsin -bound arrestin-1 complex confirmed our structural model of an overall complex conformation and experimentally based assumptions about single residues located at the direct binding interface. Comprehensive analyses of single amino acid contributions in arrestin-1 and other methodologies expand our knowledge of structure–function relationships for receptor recognition and arrestin–GPCR complex formation. With a lot of progress made in the last few years, we review most recent literature covering the roles of essential arrestin-1 residues for pre-activation, receptor recognition and receptor binding. Mechanistic understanding of these processes at the atomic level is key for the identification of receptor conformations trigger...
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G protein–coupled receptors (GPCRs) are one of the most important drug–target classes in pharmaceutical industry. Their diversity in signaling, which can be modulated with drugs, permits the design of more effective and better-tolerated... more
G protein–coupled receptors (GPCRs) are one of the most important drug–target classes in pharmaceutical industry. Their diversity in signaling, which can be modulated with drugs, permits the design of more effective and better-tolerated therapeutics. In this work, we have used rigid oligoproline backbones to generate bivalent ligands for the gastrin-releasing peptide receptor (GRPR) with a fixed distance between their recognition motifs. This allows the stabilization of GPCR dimers irrespective of their physiological occurrence and relevance, thus expanding the space for medicinal chemistry. Specifically, we observed that compounds presenting agonists or antagonists at 20- and 30-Å distance induce GRPR dimerization. Furthermore, we found that 1) compounds with two agonists at 20- and 30-Å distance that induce dimer formation show bias toward Gq efficacy, 2) dimers with 20- and 30-Å distance have different potencies toward β-arrestin-1 and β-arrestin-2, and 3) the divalent agonistic ...
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Long-wavelength pulses from the Swiss X-ray free-electron laser (XFEL) have been used for de novo protein structure determination by native single-wavelength anomalous diffraction (native-SAD) phasing of serial femtosecond crystallography... more
Long-wavelength pulses from the Swiss X-ray free-electron laser (XFEL) have been used for de novo protein structure determination by native single-wavelength anomalous diffraction (native-SAD) phasing of serial femtosecond crystallography (SFX) data. In this work, sensitive anomalous data-quality indicators and model proteins were used to quantify improvements in native-SAD at XFELs such as utilization of longer wavelengths, careful experimental geometry optimization, and better post-refinement and partiality correction. Compared with studies using shorter wavelengths at other XFELs and older software versions, up to one order of magnitude reduction in the required number of indexed images for native-SAD was achieved, hence lowering sample consumption and beam-time requirements significantly. Improved data quality and higher anomalous signal facilitate so-far underutilized de novo structure determination of challenging proteins at XFELs. Improvements presented in this work can be us...
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Label free, biosensor-based screening approaches can be easily adapted for testing interactions of arrestins with different GPCRs.