Optogenetic control of phosphate-responsive genes using single component fusion proteins in Saccharomyces cerevisiae.

Blue light illumination can be detected by Light-Oxygen-Voltage (LOV) photosensing proteins and translated into a range of biochemical responses, facilitating the generation of novel optogenetic tools to control cellular function. Here we develop new variants of our previously described VP-EL222 light-dependent transcription factor and apply them to study the phosphate-responsive signaling ( PHO ) pathway in the budding yeast Saccharomyces cerevisiae , exemplifying the utilities of these new tools. Focusing first on the VP-EL222 protein itself, we quantified the tunability of gene expression as a function of light intensity and duration, and demonstrated that this system can tolerate the addition of substantially larger effector domains without impacting function. We further demonstrated the utility of several EL222-driven transcriptional controllers in both plasmid and genomic settings, using the PHO5 and PHO84 promoters in their native chromosomal contexts as examples. These studies highlight the utility of light-controlled gene activation using EL222 tethered to either artificial transcription domains or yeast activator proteins (Pho4). Similarly, we demonstrate the ability to optogenetically repress gene expression with EL222 fused to the yeast Ume6 protein. We finally investigated the effects of moving EL222 recruitment sites to different locations within the PHO5 and PHO84 promoters, as well as determining how this artificial light-controlled regulation could be integrated with the native controls dependent on inorganic phosphate (P i ) availability. Taken together, our work expands the applicability of these versatile optogenetic tools in the types of functionality they can deliver and biological questions that can be probed.

Identification of Small Molecule Ligand Binding Sites On and In the ARNT PAS-B Domain.

Transcription factors are challenging to target with small molecule inhibitors due to their structural plasticity and lack of catalytic sites. Notable exceptions include naturally ligand-regulated transcription factors, including our prior work with the HIF-2 transcription factor, showing that small molecule binding within an internal pocket of the HIF-2α PAS-B domain can disrupt its interactions with its dimerization partner, ARNT. Here, we explore the feasibility of targeting small molecules to the analogous ARNT PAS-B domain itself, potentially opening a promising route to modulate several ARNT-mediated signaling pathways. Using solution NMR fragment screening, we previously identified several compounds that bind ARNT PAS-B and, in certain cases, antagonize ARNT association with the TACC3 transcriptional coactivator. However, these ligands have only modest binding affinities, complicating characterization of their binding sites. We address this challenge by combining NMR, MD simulations, and ensemble docking to identify ligand-binding 'hotspots' on and within the ARNT PAS-B domain. Our data indicate that the two ARNT/TACC3 inhibitors, KG-548 and KG-655, bind to a ß-sheet surface implicated in both HIF-2 dimerization and coactivator recruitment. Furthermore, while KG-548 binds exclusively to the ß-sheet surface, KG-655 can additionally bind within a water-accessible internal cavity in ARNT PAS-B. Finally, KG-279, while not a coactivator inhibitor, exemplifies ligands that preferentially bind only to the internal cavity. All three ligands promoted ARNT PAS-B homodimerization, albeit to varying degrees. Taken together, our findings provide a comprehensive overview of ARNT PAS-B ligand-binding sites and may guide the development of more potent coactivator inhibitors for cellular and functional studies.

Signal-regulated Unmasking of Nuclear Localization Motif in the PAS Domain Regulates the Nuclear Translocation of PASK.

The ligand-regulated PAS domains are one of the most diverse signal-integrating domains found in proteins from prokaryotes to humans. By biochemically connecting cellular processes with their environment, PAS domains facilitate an appropriate cellular response. PAS domain-containing Kinase (PASK) is an evolutionarily conserved protein kinase that plays important signaling roles in mammalian stem cells to establish stem cell fate. We have shown that the nuclear translocation of PASK is stimulated by differentiation signaling cues in muscle stem cells. However, the mechanistic basis of the regulation of PASK nucleo-cytoplasmic translocation remains unknown. Here, we show that the PAS-A domain of PASK contains a putative monopartite nuclear localization sequence (NLS) motif. This NLS is inhibited in cells through intramolecular association with a short linear motif, termed the PAS Interacting Motif (PIM), found upstream of the kinase domain. This interaction serves to retain PASK in the cytosol in the absence of signaling cues. Consistent with that, we show that metabolic inputs induce PASK nuclear import, likely by disrupting this association. We suggest that a route for such linkage may occur through the PAS-A ligand binding cavity. We show that PIM recruitment and artificial ligand binding to the PAS-A domain occur at neighboring locations that could facilitate metabolic control of the PAS-PIM interaction. Thus, the intramolecular interaction in PASK integrates metabolic signaling cues for nuclear translocation and could be targeted to control the balance between self-renewal and differentiation in stem cells.

Identification of Small Molecule Ligand Binding Sites On and In the ARNT PAS-B Domain.

Transcription factors are generally challenging to target with small molecule inhibitors due to their structural plasticity and lack of catalytic sites. Notable exceptions to this include a number of transcription factors which are naturally ligand-regulated, a strategy we have successfully exploited with the heterodimeric HIF-2 transcription factor, showing that a ligand-binding internal pocket in the HIF-2α PAS-B domain could be utilized to disrupt its dimerization with its partner, ARNT. Here, we explore the feasibility of directly targeting small molecules to the structurally similar ARNT PAS-B domain, potentially opening a promising route to simultaneously modulate several ARNT-mediated signaling pathways. Using solution NMR screening of an in-house fragment library, we previously identified several compounds that bind ARNT PAS-B and, in certain cases, antagonize ARNT association with the TACC3 transcriptional coactivator. However, these ligands only have mid-micromolar binding affinities, complicating characterization of their binding sites. Here we combine NMR, MD simulations, and ensemble docking to identify ligand-binding 'hotspots' on and within the ARNT PAS-B domain. Our data indicate that the two ARNT/TACC3 inhibitors, KG-548 and KG-655, bind to a ß-sheet surface implicated in both HIF-2 dimerization and coactivator recruitment. Furthermore, KG-548 binds exclusively to the ß-sheet surface, while KG-655 binds to the same site but can also enter a water-accessible internal cavity in ARNT PAS-B. Finally, KG-279, while not a coactivator inhibitor, exemplifies ligands that preferentially bind only to the internal cavity. Taken together, our findings provide a comprehensive overview of ARNT PAS-B ligand-binding sites and may guide the development of more potent coactivator inhibitors for cellular and functional studies.

Signal-regulated unmasking of the nuclear localization motif in the PAS domain regulates the nuclear translocation of PASK.

The ligand-regulated PAS domains are one of the most diverse signal-integrating domains found in proteins from prokaryotes to humans. By biochemically connecting cellular processes with their environment, PAS domains facilitate an appropriate cellular response. PAS domain-containing Kinase (PASK) is an evolutionarily conserved protein kinase that plays important signaling roles in mammalian stem cells to establish stem cell fate. We have shown that the nuclear translocation of PASK is stimulated by differentiation signaling cues in muscle stem cells. However, the mechanistic basis of the regulation of PASK nucleo-cytoplasmic translocation remains unknown. Here, we show that the PAS-A domain of PASK contains a putative monopartite nuclear localization sequence (NLS) motif. This NLS is inhibited in cells via intramolecular association with a short linear motif, termed the PAS Interacting Motif (PIM), found upstream of the kinase domain. The interaction between the PAS-A domain and PIM is evolutionarily conserved and serves to retain PASK in the cytosol in the absence of signaling cues. Consistent with that, we show that metabolic inputs induce PASK nuclear import, likely by disrupting the PAS-A: PIM association. We suggest that a route for such linkage may occur through the PAS-A ligand binding cavity. We show that PIM recruitment and artificial ligand binding to the PAS-A domain occur at neighboring locations that could facilitate metabolic control of the PAS-PIM interaction. Thus, the PAS-A domain of PASK integrates metabolic signaling cues for nuclear translocation and could be targeted to control the balance between self-renewal and differentiation in stem cells.

Diversity of function and higher-order structure within HWE sensor histidine kinases.

Integral to the protein structure/function paradigm, oligomeric state is typically conserved along with function across evolution. However, notable exceptions such as the hemoglobins show how evolution can alter oligomerization to enable new regulatory mechanisms. Here, we examine this linkage in histidine kinases (HKs), a large class of widely distributed prokaryotic environmental sensors. While the majority of HKs are transmembrane homodimers, members of the HWE/HisKA2 family can deviate from this architecture as exemplified by our finding of a monomeric soluble HWE/HisKA2 HK (EL346, a photosensing light-oxygen-voltage [LOV]-HK). To further explore the diversity of oligomerization states and regulation within this family, we biophysically and biochemically characterized multiple EL346 homologs and found a range of HK oligomeric states and functions. Three LOV-HK homologs are primarily dimeric with differing structural and functional responses to light, while two Per-ARNT-Sim-HKs interconvert between differentially active monomers and dimers, suggesting dimerization might control enzymatic activity for these proteins. Finally, we examined putative interfaces in a dimeric LOV-HK, finding that multiple regions contribute to dimerization. Our findings suggest the potential for novel regulatory modes and oligomeric states beyond those traditionally defined for this important family of environmental sensors.

Designing Single-Component Optogenetic Membrane Recruitment Systems: The Rho-Family GTPase Signaling Toolbox.

We describe the efficient creation of single-component optogenetic tools for membrane recruitment-based signaling perturbation using BcLOV4 technology. The workflow requires two plasmids to create six different domain arrangements of the dynamic membrane binder BcLOV4, a fluorescent reporter, and the fused signaling protein of interest. Screening of this limited set of genetic constructs for expression characteristics and dynamic translocation in response to one pulse of light is sufficient to identify viable signaling control tools. The reliability of this streamlined approach is demonstrated by the creation of an optogenetic Cdc42 GTPase and Rac1-activating Tiam1 GEF protein, which together with our other recently reported technologies, completes a toolbox for spatiotemporally precise induction of Rho-family GTPase signaling at the GEF or GTPase level, for driving filopodial protrusions, lamellipodial protrusions, and cell contractility, respectively mediated by Cdc42, Rac1, and RhoA.

Two steps, one ligand: How PPARγ binds small-molecule agonists.

In this issue of Structure,Shang and Kojetin (2021) present insights into the binding mechanism of artificial agonists to the PPARγ nuclear receptor. These data support a two-step model with induced fit and conformational selection aspects. This mechanism may exist in related receptors, providing new opportunities for drug development.

Lighting the way: Recent insights into the structure and regulation of phototropin blue light receptors.

The phototropins (phots) are light-activated kinases that are critical for plant physiology and the many diverse optogenetic tools that they have inspired. Phototropins combine two blue-light-sensing Light-Oxygen-Voltage (LOV) domains (LOV1 and LOV2) and a C-terminal serine/threonine kinase domain, using the LOV domains to control the catalytic activity of the kinase. While much is known about the structure and photochemistry of the light-perceiving LOV domains, particularly in how activation of the LOV2 domain triggers the unfolding of alpha helices that communicate the light signal to the kinase domain, many questions about phot structure and mechanism remain. Recent studies have made progress addressing these questions by utilizing small-angle X-ray scattering (SAXS) and other biophysical approaches to study multidomain phots from Chlamydomonas and Arabidopsis, leading to models where the domains have an extended linear arrangement, with the regulatory LOV2 domain contacting the kinase domain N-lobe. We discuss this and other advances that have improved structural and mechanistic understanding of phot regulation in this review, along with the challenges that will have to be overcome to obtain high-resolution structural information on these exciting photoreceptors. Such information will be essential to advancing fundamental understanding of plant physiology while enabling engineering efforts at both the whole plant and molecular levels.

Volume and compressibility differences between protein conformations revealed by high-pressure NMR.

Proteins often interconvert between different conformations in ways critical to their function. Although manipulating such equilibria for biophysical study is often challenging, the application of pressure is a potential route to achieve such control by favoring the population of lower volume states. Here, we use this feature to study the interconversion of ARNT PAS-B Y456T, which undergoes a dramatic +3 slip in the ß-strand register as it switches between two stably folded conformations. Using high-pressure biomolecular NMR approaches, we obtained the first, to our knowledge, quantitative data testing two key hypotheses of this process: the slipped conformation is both smaller and less compressible than the wild-type equivalent, and the interconversion proceeds through a chiefly unfolded intermediate state. Data collected in steady-state pressure and time-resolved pressure-jump modes, including observed pressure-dependent changes in the populations of the two conformers and increased rate of interconversion between conformers, support both hypotheses. Our work exemplifies how these approaches, which can be generally applied to protein conformational switches, can provide unique information that is not easily accessible through other techniques.

TAEL 2.0: An Improved Optogenetic Expression System for Zebrafish.

Inducible gene expression systems are valuable tools for studying biological processes. We previously developed an optogenetic gene expression system called TAEL that is optimized for use in zebrafish. When illuminated with blue light, TAEL transcription factors dimerize and activate gene expression downstream of the TAEL-responsive C120 promoter. By using light as the inducing agent, the TAEL/C120 system overcomes limitations of traditional inducible expression systems by enabling fine spatial and temporal regulation of gene expression. In this study, we describe ongoing efforts to improve the TAEL/C120 system. We made modifications to both the TAEL transcriptional activator and the C120 regulatory element, collectively referred to as TAEL 2.0. We demonstrate that TAEL 2.0 consistently induces higher levels of reporter gene expression and at a faster rate, but with comparable background and toxicity as the original TAEL system. With these improvements, we were able to create functional stable transgenic lines to express the TAEL 2.0 transcription factor either ubiquitously or with a tissue-specific promoter. We demonstrate that the ubiquitous line in particular can be used to induce expression at late embryonic and larval stages, addressing a major deficiency of the original TAEL system. This improved optogenetic expression system will be a broadly useful resource for the zebrafish community.

Fragile protein folds: Sequence and environmental factors affecting the equilibrium of two interconverting, stably folded protein conformations.

Recent research on fold-switching metamorphic proteins has revealed some notable exceptions to Anfinsen's hypothesis of protein folding. We have previously described how a single point mutation can enable a well-folded protein domain, one of the two PAS (Per-ARNT-Sim) domains of the human ARNT (aryl hydrocarbon receptor nuclear translocator) protein, to interconvert between two conformers related by a slip of an internal ß-strand. Using this protein as a test case, we advance the concept of a "fragile fold," a protein fold that can reversibly rearrange into another fold that differs by a substantial number of hydrogen bonds, entailing reorganization of single secondary structure elements to more drastic changes seen in metamorphic proteins. Here we use a battery of biophysical tests to examine several factors affecting the equilibrium between the two conformations of the switching ARNT PAS-B Y456T protein. Of note, we find that factors which impact the HI loop preceding the shifted Iß-strand affect both the equilibrium levels of the two conformers and the denatured state which links them in the interconversion process. Finally, we describe small molecules that selectively bind to and stabilize the wildtype conformation of ARNT PAS-B. These studies form a toolkit for studying fragile protein folds and could enable ways to modulate the biological functions of such fragile folds, both in natural and engineered proteins.

Unraveling the Mechanism of a LOV Domain Optogenetic Sensor: A Glutamine Lever Induces Unfolding of the Jα Helix.

Light-activated protein domains provide a convenient, modular, and genetically encodable sensor for optogenetics and optobiology. Although these domains have now been deployed in numerous systems, the precise mechanism of photoactivation and the accompanying structural dynamics that modulate output domain activity remain to be fully elucidated. In the C-terminal light-oxygen-voltage (LOV) domain of plant phototropins (LOV2), blue light activation leads to formation of an adduct between a conserved Cys residue and the embedded FMN chromophore, rotation of a conserved Gln (Q513), and unfolding of a helix (Jα-helix) which is coupled to the output domain. In the present work, we focus on the allosteric pathways leading to Jα helix unfolding in Avena sativa LOV2 (AsLOV2) using an interdisciplinary approach involving molecular dynamics simulations extending to 7 µs, time-resolved infrared spectroscopy, solution NMR spectroscopy, and in-cell optogenetic experiments. In the dark state, the side chain of N414 is hydrogen bonded to the backbone N-H of Q513. The simulations predict a lever-like motion of Q513 after Cys adduct formation resulting in a loss of the interaction between the side chain of N414 and the backbone C═O of Q513, and formation of a transient hydrogen bond between the Q513 and N414 side chains. The central role of N414 in signal transduction was evaluated by site-directed mutagenesis supporting a direct link between Jα helix unfolding dynamics and the cellular function of the Zdk2-AsLOV2 optogenetic construct. Through this multifaceted approach, we show that Q513 and N414 are critical mediators of protein structural dynamics, linking the ultrafast (sub-ps) excitation of the FMN chromophore to the microsecond conformational changes that result in photoreceptor activation and biological function.

Shining light on the alphaproteobacterial general stress response: Comment on: Fiebig et al., Mol Microbiol, 2019.

The general stress response (GSR) allows many bacterial species to react to myriad different stressors. In Alphaproteobacteria, this signaling pathway proceeds through the partner-switching PhyR-EcfG sigma-factor mechanism and is involved in multiple life processes, including virulence in Brucella abortus. To date, details of the alphaproteobacterial GSR signaling pathway have been determined using genetic and biochemical work on a diverse set of species distributed throughout the clade. Fiebig and co-workers establish Erythrobacter litoralis DSM 8509 as a genetically tractable lab strain and use it to both directly and indirectly delineate photoresponsive GSR pathways mediated by multiple HWE/HisKA_2 histidine kinases. The existence of a new phototrophic lab strain allows researchers to compare the GSR across different Alphaproteobacteria, as well as study the interplay between the GSR and phototrophy. Additionally, the discovery of new HWE/HisKA_2 kinases regulating the GSR poses new questions about how different stimuli feed into this widespread stress pathway.

Insights into histidine kinase activation mechanisms from the monomeric blue light sensor EL346.

Translation of environmental cues into cellular behavior is a necessary process in all forms of life. In bacteria, this process frequently involves two-component systems in which a sensor histidine kinase (HK) autophosphorylates in response to a stimulus before subsequently transferring the phosphoryl group to a response regulator that controls downstream effectors. Many details of the molecular mechanisms of HK activation are still unclear due to complications associated with the multiple signaling states of these large, multidomain proteins. To address these challenges, we combined complementary solution biophysical approaches to examine the conformational changes upon activation of a minimal, blue-light-sensing histidine kinase from Erythrobacter litoralis HTCC2594, EL346. Our data show that multiple conformations coexist in the dark state of EL346 in solution, which may explain the enzyme's residual dark-state activity. We also observe that activation involves destabilization of the helices in the dimerization and histidine phosphotransfer-like domain, where the phosphoacceptor histidine resides, and their interactions with the catalytic domain. Similar light-induced changes occur to some extent even in constitutively active or inactive mutants, showing that light sensing can be decoupled from activation of kinase activity. These structural changes mirror those inferred by comparing X-ray crystal structures of inactive and active HK fragments, suggesting that they are at the core of conformational changes leading to HK activation. More broadly, our findings uncover surprising complexity in this simple system and allow us to outline a mechanism of the multiple steps of HK activation.

Isotopic Labeling of Eukaryotic Membrane Proteins for NMR Studies of Interactions and Dynamics.

Membrane proteins, and especially G-protein coupled receptors (GPCRs), are increasingly important targets of structural biology studies due to their involvement in many biomedically critical pathways in humans. These proteins are often highly dynamic and thus benefit from studies by NMR spectroscopy in parallel with complementary crystallographic and cryo-EM analyses. However, such studies are often complicated by a range of practical concerns, including challenges in preparing suitably isotopically labeled membrane protein samples, large sizes of protein/detergent or protein/lipid complexes, and limitations on sample concentrations and stabilities. Here we describe our approach to addressing these challenges via the use of simple eukaryotic expression systems and modified NMR experiments, using the human adenosine A2A receptor as an example. Protocols are provided for the preparation of U-2H (13C,1H-Ile δ1)-labeled membrane proteins from overexpression in the methylotrophic yeast Pichia pastoris, as well as techniques for studying the fast ns-ps sidechain dynamics of the methyl groups of such samples. We believe that, with the proper optimization, these protocols should be generalizable to other GPCRs and human membrane proteins.

On the use of Pichia pastoris for isotopic labeling of human GPCRs for NMR studies.

NMR studies of human integral membrane proteins provide unique opportunities to probe structure and dynamics at specific locations and on multiple timescales, often with significant implications for disease mechanism and drug development. Since membrane proteins such as G protein-coupled receptors (GPCRs) are highly dynamic and regulated by ligands or other perturbations, NMR methods are potentially well suited to answer basic functional questions (such as addressing the biophysical basis of ligand efficacy) as well as guiding applications (such as novel ligand design). However, such studies on eukaryotic membrane proteins have often been limited by the inability to incorporate optimal isotopic labels for NMR methods developed for large protein/lipid complexes, including methyl TROSY. We review the different expression systems for production of isotopically labeled membrane proteins and highlight the use of the yeast Pichia pastoris to achieve perdeuteration and 13C methyl probe incorporation within isoleucine sidechains. We further illustrate the use of this method for labeling of several biomedically significant GPCRs.

Directly light-regulated binding of RGS-LOV photoreceptors to anionic membrane phospholipids.

We report natural light-oxygen-voltage (LOV) photoreceptors with a blue light-switched, high-affinity (KD ∼ 10-7 M), and direct electrostatic interaction with anionic phospholipids. Membrane localization of one such photoreceptor, BcLOV4 from Botrytis cinerea, is directly coupled to its flavin photocycle, and is mediated by a polybasic amphipathic helix in the linker region between the LOV sensor and its C-terminal domain of unknown function (DUF), as revealed through a combination of bioinformatics, computational protein modeling, structure-function studies, and optogenetic assays in yeast and mammalian cell line expression systems. In model systems, BcLOV4 rapidly translocates from the cytosol to plasma membrane (∼1 second). The reversible electrostatic interaction is nonselective among anionic phospholipids, exhibiting binding strengths dependent on the total anionic content of the membrane without preference for a specific headgroup. The in vitro and cellular responses were also observed with a BcLOV4 homolog and thus are likely to be general across the dikarya LOV class, whose members are associated with regulator of G-protein signaling (RGS) domains. Natural photoreceptors are not previously known to directly associate with membrane phospholipids in a light-dependent manner, and thus this work establishes both a photosensory signal transmission mode and a single-component optogenetic tool with rapid membrane localization kinetics that approaches the diffusion limit.

Blue-Light Receptors for Optogenetics.

Sensory photoreceptors underpin light-dependent adaptations of organismal physiology, development, and behavior in nature. Adapted for optogenetics, sensory photoreceptors become genetically encoded actuators and reporters to enable the noninvasive, spatiotemporally accurate and reversible control by light of cellular processes. Rooted in a mechanistic understanding of natural photoreceptors, artificial photoreceptors with customized light-gated function have been engineered that greatly expand the scope of optogenetics beyond the original application of light-controlled ion flow. As we survey presently, UV/blue-light-sensitive photoreceptors have particularly allowed optogenetics to transcend its initial neuroscience applications by unlocking numerous additional cellular processes and parameters for optogenetic intervention, including gene expression, DNA recombination, subcellular localization, cytoskeleton dynamics, intracellular protein stability, signal transduction cascades, apoptosis, and enzyme activity. The engineering of novel photoreceptors benefits from powerful and reusable design strategies, most importantly light-dependent protein association and (un)folding reactions. Additionally, modified versions of these same sensory photoreceptors serve as fluorescent proteins and generators of singlet oxygen, thereby further enriching the optogenetic toolkit. The available and upcoming UV/blue-light-sensitive actuators and reporters enable the detailed and quantitative interrogation of cellular signal networks and processes in increasingly more precise and illuminating manners.