bioRxiv Science⌕ Search

Biology subjects

Schertler, G. F. X.

Publications and source records attributed to Schertler, G. F. X..

12 recordsLinked to original sources
bioRxiv2026

Covalent bond formation caught in a LOV photoreceptor

Light-oxygen-voltage (LOV) domains are blue-light photoreceptors of plants, algae and fungi, and among the most widely used tools in optogenetics. They switch on by forming a covalent thioether bond between a conserved cysteine and their flavin chromophore, in a reaction that needs a proton to cross from the cysteine to the flavin through a pocket containing essentially no water. Its mechanism has been debated for two decades1, and because the chemistry is over within a microsecond its elementary steps have stayed hidden. Here we combine 10 time-resolved serial femtosecond crystallography snapshots and infrared spectroscopy with QM/MM calculations to resolve the entire sequence of events at 1.4 [A] resolution: from excited-state distortion of the flavin ring (10-100 ps), through hydration of a surface channel (10 ns) and a single ordered water reaching the active site as the reactive cysteine shifts between its conformations (100-500 ns), to the thioether bond itself, caught half-formed at 1 {micro}s (half the molecules reacted, half still poised) and complete at 10-100 {micro}s. That water bridges the cysteine and the flavin and shuttles the proton, lowering the barrier from [~]35 to [~]15 kcal/mol and accelerating the reaction by roughly fourteen orders of magnitude (without it, the half-life would be [~]237,000 years), then departs before the bond forms. Proteins can therefore hydrate a dehydrated active site transiently and on demand to overcome otherwise prohibitive reaction barriers, a catalytic strategy that reaches well beyond photoreceptors.

biophysics↗
bioRxiv2026

Discovery of a Cell-Permeable Gi-Signaling Inhibitor

Heterotrimeric G proteins regulate diverse physiological processes, yet selective small molecule modulators for the Gi subfamily remain scarce. Here, we employed native metabolomics to screen a fungal culture collection for G protein binders, which led to the discovery of N-hydroxyapiosporamide (NHAP), a fungal specialized metabolite that functionally inhibits Gi-signaling. NHAP selectively binds Gi1 and diminishes Gi1-mediated GTP turnover in biochemical assays. In primary ventricular cardiomyocytes, NHAP largely reversed acetylcholine-induced negative inotropy, demonstrating functional Gi-signaling blockade in a physiological context without acute cytotoxicity. This work establishes NHAP as a first-in-class Gi-selective inhibitor, providing a cell-permeable chemical scaffold for the optimization of next-generation agents to control Gi-signaling in cell-based systems and, ultimately, to target Gi1-driven pathologies.

biochemistry↗
bioRxiv2026

Photoswitchable optoGPCRs for reversible control of Gs and arrestin signalling

OptoGPCRs are light-activatable G protein-coupled receptors (GPCRs) used for optogenetic control of physiological processes. Most existing optoGPCRs are based on monostable opsins, which are limited by photobleaching and irreversibility. The bistable jumping spider rhodopsin 1 (JSR1) carrying the single point mutant S199F introduces a [~]150 nm spectral separation between the active and inactive states, enabling bidirectional control with distinct wavelengths of light. Here, we show that JSR1-S199F demonstrates robust, light-reversible arrestin recruitment and Gq/i protein activity. We then engineered JSR1-S199F-based optoGPCRs with Gs protein activity, expanding the limited repertoire of bistable Gs-coupled opsins. Specifically, we present optoDRD1, a chimeric optoGPCR that redirects the native Gq/i protein activity of JSR1 towards the Gs pathway of the dopamine D1 receptor (DRD1). Through systematic screening of intracellular domain combinations, we identified an optimal chimeric configuration comprising ICL2, ICL3, helix 8, and the C-terminus from DRD1. The resulting optoGPCR is activated by violet light ({lambda}max = 397 nm) and deactivated by green light ({lambda}max = 531 nm) at physiologically relevant light intensities. A single violet light pulse drives sustained Gs signalling for several hours, while green light deactivation enables precise signal termination at any timepoint. OptoDRD1 closely mimics wild-type DRD1 signalling kinetics and G protein selectivity. Compared to JellyOp, the only previously characterised natively Gs-coupled opsin, optoDRD1 shows higher signal amplitude and reversibility over multiple light cycles. We further demonstrate optoDRD1s utility for optogenetic control of Gs-regulated processes in vitro, including insulin secretion in human {beta}-cells and signalling modulation in a neuronal cell line, supporting its potential for in vivo applications. The biochemical stability and known structure of JSR1 make it a robust scaffold for this rational engineering and for future biophysical characterization. Together, optoDRD1 and JSR1-S199F expand the optoGPCR toolkit and open new opportunities for dissecting dopaminergic signalling, Gs-mediated physiology, and GPCR signalling pharmacology.

synthetic biology↗
bioRxiv2024

Human Cells for Human Proteins: Isotope Labeling in Mammalian Cells for Functional NMR Studies of Disease-Relevant Proteins

In biological and biomedical research the focus progressively moves towards difficult human proteins, which often can only be expressed in higher eukaryotic cells. Nuclear magnetic resonance (NMR) could contribute significantly to the understanding of important proteins as it is one of the most information-rich methods: it allows studying structure, function and dynamics of biomolecules and, importantly, their interactions with natural ligands or drugs. However, to exploit the full potential of NMR, proteins must be isotope labeled. Although expression protocols in e.g. HEK293 cells are often established, isotope labeling is difficult and very expensive. To resolve this disparity, we have developed a comprehensive suite of protocols for isotope labeling in HEK293 cells. We demonstrate uniform 15N and 13C labeling, as well as specific labeling, with special focus on methyl bearing amino acids, including the popular ILV 13C-methyl labeling pattern. Labeling is achieved with a simple laboratory setup and affordable labeling media. These are based on either labeled amino acids, their precursors or amino extracts from microorganisms, like yeast, algae or bacteria. This enables NMR studies of important, but difficult to produce proteins, like receptors. We therefore expect that these new methods make many highly important proteins accessible to NMR studies and allow exploiting the high information content of this method for accelerating biological and pharmaceutical research.

biophysics↗
bioRxiv2024

Active state structures of a bistable visual opsin bound to G proteins

Opsins are G protein-coupled receptors (GPCRs) that have evolved to detect light stimuli and initiate intracellular signaling cascades. Their role as signal transducers is critical to light perception across the animal kingdom. Opsins covalently bind to the chromophore 11-cis retinal, which isomerizes to the all-trans isomer upon photon absorption, causing conformational changes that result in receptor activation. Monostable opsins, responsible for vision in vertebrates, release the chromophore after activation and must bind another retinal molecule to remain functional. In contrast, bistable opsins, responsible for non-visual light perception in vertebrates and for vision in invertebrates, absorb a second photon in the active state to return the chromophore and protein to the inactive state. Structures of bistable opsins in the activated state have proven elusive, limiting our understanding of how they function as bidirectional photoswitches. Here we present active state structures of a bistable opsin, jumping spider rhodopsin isoform-1 (JSR1), in complex with its downstream signaling partners, the Gi and Gq heterotrimers. These structures elucidate key differences in the activation mechanisms between monostable and bistable opsins, offering essential insights for the rational engineering of bistable opsins into diverse optogenetic tools to control G protein signaling pathways.

biochemistry↗
bioRxiv2024

Activating an invertebrate bistable opsin with the all-trans 6.11 retinal analogue

Animal vision depends on opsins, a category of G protein-coupled receptor (GPCR) that achieves light sensitivity by covalent attachment to retinal. Typically binding as an inverse agonist in the 11-cis form, retinal photoisomerizes to the all-trans isomer and activates the receptor, initiating downstream signaling cascades. Retinal bound to bistable opsins isomerizes back to the 11-cis state after absorption of a second photon, inactivating the receptor. Bistable opsins are essential for invertebrate vision and non-visual light perception across the animal kingdom. While crystal structures are available for bistable opsins in the inactive state, it has proven difficult to form homogeneous populations of activated bistable opsins either via illumination or reconstitution with all-trans retinal. Here we show that a non-natural retinal analogue, all-trans retinal 6.11 (ATR6.11), can be reconstituted with the invertebrate bistable opsin, Jumping Spider Rhodopsin-1 (JSR1). Biochemical activity assays demonstrate that ATR6.11 functions as an agonist of JSR1. ATR6.11 binding also enables complex formation between JSR1 and downstream signaling partners. Our findings demonstrate the utility of retinal analogues for biophysical characterization of bistable opsins, which will deepen our understanding of light perception in animals.

biochemistry↗
bioRxiv2023

Capturing the blue-light activated state of the Phot-LOV1 domain from Chlamydomonas reinhardtii using time-resolved serial synchrotron crystallography

Light-Oxygen-Voltage (LOV) domains are small photosensory flavoprotein modules that allow converting external stimuli (sunlight) into intracellular signals responsible for various cell behavior (e.g., phototropism and chloroplast relocation). This ability relies on the light-induced formation of a covalent thioether adduct between a flavin chromophore and a reactive cysteine from the protein environment, which triggers a cascade of structural changes that results in the activation of a serine/threonine (Ser/Thr) kinase. Recent developments in time-resolved crystallography may allow the observation of the activation cascade of the LOV domain in real-time, which has been elusive. In this study, we report a robust protocol for the production and stable delivery of microcrystals of the LOV domain of phototropin Phot-1 from Chlamydomonas reinhardtii (CrPhotLOV1) with a high-viscosity injector for time-resolved serial synchrotron crystallography (TR-SSX). The detailed process covers all aspects, from sample optimization to the actual data collection process, which may serve as a guide for soluble protein preparation for TR-SSX. In addition, we show that the obtained crystals preserve the photoreactivity using infrared spectroscopy. Furthermore, the results of the TR-SSX experiment provide high-resolution insights into structural alterations of CrPhotLOV1 from {Delta}t = 2.5 ms up to {Delta}t = 95 ms post-photoactivation, including resolving the geometry of the thioether adduct and the C-terminal region implicated in the signal transduction process.

biochemistry↗
bioRxiv2020

Structural basis of the activation of the CC chemokine receptor 5 by a chemokine agonist

The human CC chemokine receptor 5 (CCR5) is a G protein-coupled receptor (GPCR) that plays a major role in inflammation and is involved in the pathology of cancer, HIV, and COVID-19. Despite its significance as a drug target, the activation mechanism of CCR5, i.e. how chemokine agonists transduce the activation signal through the receptor, is yet unknown. Here, we report the cryo-EM structure of wild-type CCR5 in an active conformation bound to the chemokine super-agonist [6P4]CCL5 and the heterotrimeric Gi protein. The structure provides the rationale for the sequence-activity relation of agonist and antagonist chemokines. The N-terminus of agonist chemokines pushes onto an aromatic connector that transmits activation to the canonical GPCR microswitch network. This activation mechanism differs significantly from other CC chemokine receptors that bind shorter chemokines in a shallow binding mode and have unique sequence signatures and a specialized activation mechanism. One-sentence summaryThe structure of CCR5 in complex with the chemokine agonist [6P4]CCL5 and the heterotrimeric Gi protein reveals its activation mechanism

biophysics↗
bioRxiv2020

A set of common movements within GPCR-G-protein complexes from variability analysis of cryo-EM datasets

G-protein coupled receptors (GPCRs) are among the most versatile signal transducers in the cell. Once activated, GPCRs sample a large conformational space and couple to G-proteins to initiate distinct signaling pathways. The dynamical behavior of GPCR-G-protein complexes is difficult characterize structurally. Here, we report on the use of variability analysis to characterize the inherent flexibility within the cryo-EM dataset of the rhodopsin-Gi-protein complex (Tsai et al., 2019), on which this article builds on. We compare the outcome of this analysis with recently published results obtained on the cannabinoid-Gi- and secretin-Gs-receptor complexes. Despite differences related to the biochemical compositions of the three samples, a set of consensus movements emerges. We anticipate that systematic variability analysis on GPCR-G-protein complexes may provide useful information not only at the biological level, but also for improving the preparation of more stable samples for cryo-EM single-particle analysis.

bioinformatics↗
bioRxiv2020

Dimeric prion protein ligand activates Adgrg6 but does not rescue myelinopathy of PrP-deficient mice

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 peripheral demyelinating neuropathy 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 myelinotrophic therapies based on PrP-derived Adgrg6 ligands. While our treatment approach was not successful, Adgrg6 remains an attractive therapeutic target to be addressed in other disease models or by using different biologically active Adgrg6 ligands. Summary blurbA dimeric prion protein ligand activates Adgrg6 but fails to induce pro-myelination signaling upon chronic treatment in a mouse model of peripheral demyelination.

neuroscience↗
bioRxiv2020

A graph-based approach identifies dynamic H-bond communication networks in spike protein S of SARS-CoV-2

Corona virus spike protein S is a large homo-trimeric protein embedded in the membrane of the virion particle. Protein S binds to angiotensin-converting-enzyme 2, ACE2, of the host cell, followed by proteolysis of the spike protein, drastic protein conformational change with exposure of the fusion peptide of the virus, and entry of the virion into the host cell. The structural elements that govern conformational plasticity of the spike protein are largely unknown. Here, we present a methodology that relies upon graph and centrality analyses, augmented by bioinformatics, to identify and characterize large H-bond clusters in protein structures. We apply this methodology to protein S ectodomain and find that, in the closed conformation, the three protomers of protein S bring the same contribution to an extensive central network of H-bonds, has a relatively large H-bond cluster at the receptor binding domain, and a cluster near a protease cleavage site. Markedly different H-bonding at these three clusters in open and pre-fusion conformations suggest dynamic H-bond clusters could facilitate structural plasticity and selection of a protein S protomer for binding to the host receptor, and proteolytic cleavage. From analyses of spike protein sequences we identify patches of histidine and carboxylate groups that could be involved in transient proton binding.

bioinformatics↗