Disrupting FKF1 homodimerization increases FT transcript levels in the evening by enhancing CO stabilization.

KEY MESSAGE: FKF1 dimerization is crucial for proper FT levels to fine-tune flowering time. Attenuating FKF1 homodimerization increased CO abundance by enhancing its COP1 binding, thereby accelerating flowering under long days. In Arabidopsis (Arabidopsis thaliana), the blue-light photoreceptor FKF1 (FLAVIN-BINDING, KELCH REPEAT, F-BOX 1) plays a key role in inducing the expression of FLOWERING LOCUS T (FT), encoding the main florigenic signal in plants, in the late afternoon under long-day conditions (LDs) by forming dimers with FT regulators. Although structural studies have unveiled a variant of FKF1 (FKF1 I160R) that disrupts homodimer formation in vitro, the mechanism by which disrupted FKF1 homodimer formation regulates flowering time remains elusive. In this study, we determined that the attenuation of FKF1 homodimer formation enhances FT expression in the evening by promoting the increased stability of CONSTANS (CO), a primary activator of FT, in the afternoon, thereby contributing to early flowering. In contrast to wild-type FKF1, introducing the FKF1 I160R variant into the fkf1 mutant led to increased FT expression under LDs. In addition, the FKF1 I160R variant exhibited diminished dimerization with FKF1, while its interaction with GIGANTEA (GI), a modulator of FKF1 function, was enhanced under LDs. Furthermore, the FKF1 I160R variant increased the level of CO in the afternoon under LDs by enhancing its binding to COP1, an E3 ubiquitin ligase responsible for CO degradation. These findings suggest that the regulation of FKF1 homodimerization and heterodimerization allows plants to finely adjust FT expression levels around dusk by modulating its interactions with GI and COP1.

ß-sheets mediate the conformational change and allosteric signal transmission between the AsLOV2 termini.

Avena sativa phototropin 1 light-oxygen-voltage 2 domain (AsLOV2) is a model protein of Per-Arnt-Sim (PAS) superfamily, characterized by conformational changes in response to external environmental stimuli. This conformational change begins with the unfolding of the N-terminal A'α helix in the dark state followed by the unfolding of the C-terminal Jα helix. The light state is characterized by the unfolded termini and the subsequent modifications in hydrogen bond patterns. In this photoreceptor, ß-sheets are identified as crucial components for mediating allosteric signal transmission between the two termini. Through combined experimental and computational investigations, the Hß and Iß strands are recognized as the most critical and influential ß-sheets in AsLOV2's allosteric mechanism. To elucidate the role of these ß-sheets, we introduced 13 distinct mutations (F490L, N492A, L493A, F494L, H495L, L496F, Q497A, R500A, F509L, Q513A, L514A, D515V, and T517V) and conducted comprehensive molecular dynamics simulations. In-depth hydrogen bond analyses emphasized the role of two hydrogen bonds, Asn482-Leu453 and Gln479-Val520, in the observed distinct behaviors of L493A, L496F, Q497A, and D515V mutants. This illustrates the role of ß-sheets in the transmission of the allosteric signal upon the photoactivation of the light state.

Microsecond Molecular Dynamics Simulations and Markov State Models of Mutation-Induced Allosteric Mechanisms for the Light-Oxygen-Voltage 2 Protein : Revealing Structural Basis of Signal Transmission Induced by Photoactivation of the Light Protein State.

Avena Sativa phototropin 1 Light-oxygen-voltage 2 domain (AsLOV2) is the model protein of Per-Arnt-Sim (PAS) superfamily, characterized by conformational changes in response to external environmental stimuli. This conformational change is initiated by the unfolding of the N-terminal helix in the dark state followed by the unfolding of the C-terminal helix. The light state is defined by the unfolded termini and the subsequent modifications in hydrogen bond patterns. In this photoreceptor, ß-sheets have been identified as crucial components for mediating allosteric signal transmission between the two termini. In this study, we combined microsecond all-atm molecular dynamics simulations and Markov state modeling of conformational states to quantify molecular basis of mutation-induced allostery in the AsLOV2 protein. Through a combination of computational investigations, we determine that the Hß and Iß strands are the most critical structural elements involved in the allosteric mechanism. To elucidate the role of these ß-sheets, we introduced 13 distinct mutations (F490L, N492A, L493A, F494L, H495L, L496F, Q497A, R500A, F509L, Q513A, L514A, D515V, and T517V) and conducted comprehensive simulation analysis. The results highlighted the role of two hydrogen bond Asn482-Leu453 and Gln479-Val520 in the observed distinct behaviors of L493A, L496F, Q497A, and D515V mutants. The comprehensive atomistic-level analysis of the conformational landscapes revealed the critical functional role of ß-sheet segments in the transmission of the allosteric signal upon the photoactivation of the light state.

Allosteric regulation in STAT3 interdomains is mediated by a rigid core: SH2 domain regulation by CCD in D170A variant.

Signal Transducer and Activator of Transcription 3 (STAT3) plays a crucial role in cancer development and thus is a viable target for cancer treatment. STAT3 functions as a dimer mediated by phosphorylation of the SRC-homology 2 (SH2) domain, a key target for therapeutic drugs. While great efforts have been employed towards the development of compounds that directly target the SH2 domain, no compound has yet been approved by the FDA due to a lack of specificity and pharmacologic efficacy. Studies have shown that allosteric regulation of SH2 via the coiled-coil domain (CCD) is an alternative drug design strategy. Several CCD effectors have been shown to modulate SH2 binding and affinity, and at the time of writing at least one drug candidate has entered phase I clinical trials. However, the mechanism for SH2 regulation via CCD is poorly understood. Here, we investigate structural and dynamic features of STAT3 and compare the wild type to the reduced function variant D170A in order to delineate mechanistic differences and propose allosteric pathways. Molecular dynamics simulations were employed to explore conformational space of STAT3 and the variant, followed by structural, conformation, and dynamic analysis. The trajectories explored show distinctive conformational changes in the SH2 domain for the D170A variant, indicating long range allosteric effects. Multiple analyses provide evidence for long range communication pathways between the two STAT3 domains, which seem to be mediated by a rigid core which connects the CCD and SH2 domains via the linker domain (LD) and transmits conformational changes through a network of short-range interactions. The proposed allosteric mechanism provides new insight into the understanding of intramolecular signaling in STAT3 and potential pharmaceutical control of STAT3 specificity and activity.

Allosteric control of ACE2 peptidase domain dynamics.

The Angiotensin Converting Enzyme 2 (ACE2) assists the regulation of blood pressure and is the main target of the coronaviruses responsible for SARS and COVID19. The catalytic function of ACE2 relies on the opening and closing motion of its peptidase domain (PD). In this study, we investigated the possibility of allosterically controlling the ACE2 PD functional dynamics. After confirming that ACE2 PD binding site opening-closing motion is dominant in characterizing its conformational landscape, we observed that few mutations in the viral receptor binding domain fragments were able to impart different effects on the binding site opening of ACE2 PD. This showed that binding to the solvent exposed area of ACE2 PD can effectively alter the conformational profile of the protein, and thus likely its catalytic function. Using a targeted machine learning model and relative entropy-based statistical analysis, we proposed the mechanism for the allosteric perturbation that regulates the ACE2 PD binding site dynamics at atomistic level. The key residues and the source of the allosteric regulation of ACE PD dynamics are also presented.

Dimeric allostery mechanism of the plant circadian clock photoreceptor ZEITLUPE.

In Arabidopsis thaliana, the Light-Oxygen-Voltage (LOV) domain containing protein ZEITLUPE (ZTL) integrates light quality, intensity, and duration into regulation of the circadian clock. Recent structural and biochemical studies of ZTL indicate that the protein diverges from other members of the LOV superfamily in its allosteric mechanism, and that the divergent allosteric mechanism hinges upon conservation of two signaling residues G46 and V48 that alter dynamic motions of a Gln residue implicated in signal transduction in all LOV proteins. Here, we delineate the allosteric mechanism of ZTL via an integrated computational approach that employs atomistic simulations of wild type and allosteric variants of ZTL in the functional dark and light states, together with Markov state and supervised machine learning classification models. This approach has unveiled key factors of the ZTL allosteric mechanisms, and identified specific interactions and residues implicated in functional allosteric changes. The final results reveal atomic level insights into allosteric mechanisms of ZTL function that operate via a non-trivial combination of population-shift and dynamics-driven allosteric pathways.

Cryptochromes: Photochemical and structural insight into magnetoreception.

Cryptochromes (CRYs) function as blue light photoreceptors in diverse physiological processes in nearly all kingdoms of life. Over the past several decades, they have emerged as the most likely candidates for light-dependent magnetoreception in animals, however, a long history of conflicts between in vitro photochemistry and in vivo behavioral data complicate validation of CRYs as a magnetosensor. In this review, we highlight the origins of conflicts regarding CRY photochemistry and signal transduction, and identify recent data that provides clarity on potential mechanisms of signal transduction in magnetoreception. The review primarily focuses on examining differences in photochemistry and signal transduction in plant and animal CRYs, and identifies potential modes of convergent evolution within these independent lineages that may identify conserved signaling pathways.

Steric and Electronic Interactions at Gln154 in ZEITLUPE Induce Reorganization of the LOV Domain Dimer Interface.

Plants measure light quality, intensity, and duration to coordinate growth and development with daily and seasonal changes in environmental conditions; however, the molecular details linking photochemistry to signal transduction remain incomplete. Two closely related light, oxygen, or voltage (LOV) domain-containing photoreceptor proteins, ZEITLUPE (ZTL) and FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (FKF1), divergently regulate the protein stability of circadian clock and photoperiodic flowering components to mediate daily and seasonal development. Using structural approaches, we identified that mutations at the Gly46 position led to global rearrangements of the ZTL dimer interface in the isolated ZTL-LOV domain. Specifically, G46S and G46A variants induce a 180° rotation about the ZTL-LOV dimer interface that is coupled to ordering of N- and C-terminal signaling elements. These conformational changes hinge upon rotation of a C-terminal Gln residue (Gln154) analogous to that present in light-state structures of ZTL. In contrast to other LOV proteins, a Q154L variant retains light-state interactions with GIGANTEA (GI), thereby indicating N5 protonation is not required for ZTL signaling. The results presented herein confirm a divergent signaling mechanism within ZTL, whereby steric and electronic effects following adduct formation can be sufficient for signal propagation in LOV proteins containing a Gly residue at position 46. Examination of bacterial LOV structures with Gly residues at the equivalent position suggests that mechanisms of signal transduction in LOV proteins may be fluid across the LOV protein family.

Deciphering the Allosteric Process of the Phaeodactylum tricornutum Aureochrome 1a LOV Domain.

The conformational-driven allosteric protein diatom Phaeodactylum tricornutum aureochrome 1a (PtAu1a) differs from other light-oxygen-voltage (LOV) proteins for its uncommon structural topology. The mechanism of signaling transduction in the PtAu1a LOV domain (AuLOV) including flanking helices remains unclear because of this dissimilarity, which hinders the study of PtAu1a as an optogenetic tool. To clarify this mechanism, we employed a combination of tree-based machine learning models, Markov state models, machine-learning-based community analysis, and transition path theory to quantitatively analyze the allosteric process. Our results are in good agreement with the reported experimental findings and reveal a previously overlooked Cα helix and protein linkers as important in promoting the protein conformational changes. This integrated approach can be considered as a general workflow and applied on other allosteric proteins to provide detailed information about their allosteric mechanisms.

Chemical and structural analysis of a photoactive vertebrate cryptochrome from pigeon.

Computational and biochemical studies implicate the blue-light sensor cryptochrome (CRY) as an endogenous light-dependent magnetosensor enabling migratory birds to navigate using the Earth's magnetic field. Validation of such a mechanism has been hampered by the absence of structures of vertebrate CRYs that have functional photochemistry. Here we present crystal structures of Columba livia (pigeon) CRY4 that reveal evolutionarily conserved modifications to a sequence of Trp residues (Trp-triad) required for CRY photoreduction. In ClCRY4, the Trp-triad chain is extended to include a fourth Trp (W369) and a Tyr (Y319) residue at the protein surface that imparts an unusually high quantum yield of photoreduction. These results are consistent with observations of night migratory behavior in animals at low light levels and could have implications for photochemical pathways allowing magnetosensing.

Allosteric mechanism of the circadian protein Vivid resolved through Markov state model and machine learning analysis.

The fungal circadian clock photoreceptor Vivid (VVD) contains a photosensitive allosteric light, oxygen, voltage (LOV) domain that undergoes a large N-terminal conformational change. The mechanism by which a blue-light driven covalent bond formation leads to a global conformational change remains unclear, which hinders the further development of VVD as an optogenetic tool. We answered this question through a novel computational platform integrating Markov state models, machine learning methods, and newly developed community analysis algorithms. Applying this new integrative approach, we provided a quantitative evaluation of the contribution from the covalent bond to the protein global conformational change, and proposed an atomistic allosteric mechanism leading to the discovery of the unexpected importance of A'α/Aß and previously overlooked Eα/Fα loops in the conformational change. This approach could be applicable to other allosteric proteins in general to provide interpretable atomistic representations of their otherwise elusive allosteric mechanisms.

Characterization of a Vivid Homolog in Botrytis cinerea.

Blue light-signaling pathways regulated by members of the light-oxygen-voltage (LOV) domain family integrate stress responses, circadian rhythms and pathogenesis in fungi. The canonical signaling mechanism involves two LOV-containing proteins that maintain homology to Neurospora crassa Vivid (NcVVD) and White Collar 1 (NcWC1). These proteins engage in homo- and heterodimerization events that modulate gene transcription in response to light. Here, we clone and characterize the VVD homolog in Botrytis cinerea (BcVVD). BcVVD retains divergent photocycle kinetics and is incapable of LOV mediated homodimerization, indicating modification of the classical hetero/homodimerization mechanism of photoadaptation in fungi.

Revealing Hidden Conformational Space of LOV Protein VIVID Through Rigid Residue Scan Simulations.

VIVID(VVD) protein is a Light-Oxygen-Voltage(LOV) domain in circadian clock system. Upon blue light activation, a covalent bond is formed between VVD residue Cys108 and its cofactor flavin adenine dinucleotide(FAD), and prompts VVD switching from Dark state to Light state with significant conformational deviation. However, the mechanism of this local environment initiated global protein conformational change remains elusive. We employed a recently developed computational approach, rigid residue scan(RRS), to systematically probe the impact of the internal degrees of freedom in each amino acid residue of VVD on its overall dynamics by applying rigid body constraint on each residue in molecular dynamics simulations. Key residues were identified with distinctive impacts on Dark and Light states, respectively. All the simulations display wide range of distribution on a two-dimensional(2D) plot upon structural root-mean-square deviations(RMSD) from either Dark or Light state. Clustering analysis of the 2D RMSD distribution leads to 15 representative structures with drastically different conformation of N-terminus, which is also a key difference between Dark and Light states of VVD. Further principle component analyses(PCA) of RRS simulations agree with the observation of distinctive impact from individual residues on Dark and Light states.

Kinetics of the LOV domain of ZEITLUPE determine its circadian function in Arabidopsis.

A LOV (Light, Oxygen, or Voltage) domain containing blue-light photoreceptor ZEITLUPE (ZTL) directs circadian timing by degrading clock proteins in plants. Functions hinge upon allosteric differences coupled to the ZTL photocycle; however, structural and kinetic information was unavailable. Herein, we tune the ZTL photocycle over two orders of magnitude. These variants reveal that ZTL complexes with targets independent of light, but dictates enhanced protein degradation in the dark. In vivo experiments definitively show photocycle kinetics dictate the rate of clock component degradation, thereby impacting circadian period. Structural studies demonstrate that photocycle dependent activation of ZTL depends on an unusual dark-state conformation of ZTL. Crystal structures of ZTL LOV domain confirm delineation of structural and kinetic mechanisms and identify an evolutionarily selected allosteric hinge differentiating modes of PAS/LOV signal transduction. The combined biochemical, genetic and structural studies provide new mechanisms indicating how PAS/LOV proteins integrate environmental variables in complex networks.

A Native Threonine Coordinates Ordered Water to Tune Light-Oxygen-Voltage (LOV) Domain Photocycle Kinetics and Osmotic Stress Signaling in Trichoderma reesei ENVOY.

Light-oxygen-voltage (LOV) domain-containing proteins function as small light-activated modules capable of imparting blue light control of biological processes. Their small modular nature has made them model proteins for allosteric signal transduction and optogenetic devices. Despite intense research, key aspects of their signal transduction mechanisms and photochemistry remain poorly understood. In particular, ordered water has been identified as a possible key mediator of photocycle kinetics, despite the lack of ordered water in the LOV active site. Herein, we use recent crystal structures of a fungal LOV protein ENVOY to interrogate the role of Thr(101) in recruiting water to the flavin active site where it can function as an intrinsic base to accelerate photocycle kinetics. Kinetic and molecular dynamic simulations confirm a role in solvent recruitment to the active site and identify structural changes that correlate with solvent recruitment. In vivo analysis of T101I indicates a direct role of the Thr(101) position in mediating adaptation to osmotic stress, thereby verifying biological relevance of ordered water in LOV signaling. The combined studies identify position 101 as a mediator of both allostery and photocycle catalysis that can impact organism physiology.

Short LOV Proteins in Methylocystis Reveal Insight into LOV Domain Photocycle Mechanisms.

Light Oxygen Voltage (LOV) proteins are widely used in optogenetic devices, however universal signal transduction pathways and photocycle mechanisms remain elusive. In particular, short-LOV (sLOV) proteins have been discovered in bacteria and fungi, containing only the photoresponsive LOV element without any obvious signal transduction domains. These sLOV proteins may be ideal models for LOV domain function due to their ease of study as full-length proteins. Unfortunately, characterization of such proteins remains limited to select systems. Herein, we identify a family of bacterial sLOV proteins present in Methylocystis. Sequence analysis of Methylocystis LOV proteins (McLOV) demonstrates conservation with sLOV proteins from fungal systems that employ competitive dimerization as a signaling mechanism. Cloning and characterization of McLOV proteins confirms functional dimer formation and reveal unexpected photocycle mechanisms. Specifically, some McLOV photocycles are insensitive to external bases such as imidazole, in contrast to previously characterized LOV proteins. Mutational analysis identifies a key residue that imparts insensitivity to imidazole in two McLOV homologs and affects adduct decay by two orders of magnitude. The resultant data identifies a new family of LOV proteins that indicate a universal photocycle mechanism may not be present in LOV proteins.

LOV-based optogenetic devices: light-driven modules to impart photoregulated control of cellular signaling.

The Light-Oxygen-Voltage domain family of proteins is widespread in biology where they impart sensory responses to signal transduction domains. The small, light responsive LOV modules offer a novel platform for the construction of optogenetic tools. Currently, the design and implementation of these devices is partially hindered by a lack of understanding of how light drives allosteric changes in protein conformation to activate diverse signal transduction domains. Further, divergent photocycle properties amongst LOV family members complicate construction of highly sensitive devices with fast on/off kinetics. In the present review we discuss the history of LOV domain research with primary emphasis on tuning LOV domain chemistry and signal transduction to allow for improved optogenetic tools.

Structural biochemistry of a fungal LOV domain photoreceptor reveals an evolutionarily conserved pathway integrating light and oxidative stress.

Fungal LOV proteins facilitate photoadaptation via blue light regulation of dimer formation. Despite considerable homology of these proteins in closely related fungi, deviations in signaling exist. Here we report the crystal structure of ENVOY (ENV1), a homolog of N. crassa VVD in the fungus T. reesei, a model organism for plant cell wall degradation. Structural studies contradict a model of reversible competitive dimerization. Rather, evolutionary pressures have facilitated a two-residue shift in the position of a key Cys residue (Cys96) that enables the integration of environmental stress and light responses. A Cys96Thr variant abolishes adaptive responses to light and oxidative stress in a carbon source-dependent manner in vivo. Phylogenetic analysis verifies an evolutionary relevance of the Cys residue shift in different orders within Sordariomycetes. In this manner, we identified a widespread oxidative stress signaling mechanism that couples metabolic sensing and blue light responses not previously identified in LOV proteins.