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The development of near-infrared (NIR) fluorophores that have both excellent chemical stability and photostability, as well as efficient cell permeability, is highly demanded. In this study, we present phospha-rhodamine (POR) dyes which display significantly improved performance for protein labeling. This is achieved by incorporating a 2-carboxy-3-benzothiophenyl group at the 9-position of the xanthene scaffold. The resulting cis and trans isomers were successfully isolated and structurally characterized using X-ray diffraction. The HaloTag ligand conjugates of the two isomers exhibited different staining abilities in live cells. While the cis isomer showed non-specific accumulation on the organelle membranes, the trans isomer selectively labeled the HaloTag-fused proteins, enabling the long-term imaging of cell division and the 5-color imaging of cell organelles. Molecular dynamics simulations of the HaloTag ligand conjugates within the lipid membrane suggested that the cis isomer is more prone to forming oligomers in the membrane. In contrast, the oligomerization of the trans isomer is effectively suppressed by its interaction with the lipid molecules. By taking advantage of the superior labeling performance of the trans isomer and its NIR-emissive properties, multi-color time-lapse super-resolution 3D imaging, namely super-resolution 5D-imaging, of the interconnected network between the endoplasmic reticulum and microtubules was achieved in living cells.
Assuntos
Corantes Fluorescentes , Organelas , Rodaminas , Ligantes , Corantes Fluorescentes/química , Organelas/metabolismo , Proteínas , Microscopia de Fluorescência/métodos , LipídeosRESUMO
Single-particle imaging using X-ray free-electron lasers (XFELs) is a promising technique for observing nanoscale biological samples under near-physiological conditions. However, as the sample's orientation in each diffraction pattern is unknown, advanced algorithms are required to reconstruct the 3D diffraction intensity volume and subsequently the sample's density model. While most approaches perform 3D reconstruction via determining the orientation of each diffraction pattern, a correlation-based approach utilizes the averaged spatial correlations of diffraction intensities over all patterns, making it well suited for processing experimental data with a poor signal-to-noise ratio of individual patterns. Here, a method is proposed to determine the 3D structure of a sample by analyzing the double, triple and quadruple spatial correlations in diffraction patterns. This ab initio method can reconstruct the basic shape of an irregular unsymmetric 3D sample without requiring any prior knowledge of the sample. The impact of background and noise on correlations is investigated and corrected to ensure the success of reconstruction under simulated experimental conditions. Additionally, the feasibility of using the correlation-based approach to process incomplete partial diffraction patterns is demonstrated. The proposed method is a variable addition to existing algorithms for 3D reconstruction and will further promote the development and adoption of XFEL single-particle imaging techniques.
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Flexible fitting based on molecular dynamics simulation is a technique for structure modeling from cryo-EM data. It has been utilized for nearly two decades, and while cryo-EM resolution has improved significantly, it remains a powerful approach that can provide structural and dynamical insights that are not directly accessible from experimental data alone. Molecular dynamics simulations provide a means to extract atomistic details of conformational changes that are encoded in cryo-EM data and can also assist in improving the quality of structural models. Additionally, molecular dynamics simulations enable the characterization of conformational heterogeneity in cryo-EM data. We will summarize the advancements made in these techniques and highlight recent developments in this field.
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Simulação de Dinâmica Molecular , Microscopia Crioeletrônica/métodos , Conformação ProteicaRESUMO
Single-particle analysis using x-ray free-electron lasers (XFELs) is a novel method for obtaining structural information of samples in a state close to nature. In particular, it is suitable for observing the inner structure of large biomolecules by taking advantage of the high transmittance of x-rays. However, systematic studies on the resolution achievable for large molecules are lacking. In this study, the molecular size dependence of the resolution of a three-dimensional (3D) structure resulting from XFEL single-particle reconstruction is evaluated using synthetic data. Evidently, 3D structures of larger molecules can be restored with higher detail (defined relative to the molecular sizes) than smaller ones; however, reconstruction with high absolute resolution (defined in nm-1) is challenging. Our results provide useful information for the experimental design of 3D structure reconstruction using coherent x-ray diffraction patterns of single-particles.
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This article presents an original approach for extracting atomic-resolution landscapes of continuous conformational variability of biomolecular complexes from cryo electron microscopy (cryo-EM) single particle images. This approach is based on a new 3D-to-2D flexible fitting method, which uses molecular dynamics (MD) simulation and is embedded in an iterative conformational-landscape refinement scheme. This new approach is referred to as MDSPACE, which stands for Molecular Dynamics simulation for Single Particle Analysis of Continuous Conformational hEterogeneity. The article describes the MDSPACE approach and shows its performance using synthetic and experimental datasets.
Assuntos
Simulação de Dinâmica Molecular , Imagem Individual de Molécula , Microscopia Crioeletrônica/métodos , Conformação ProteicaRESUMO
SWEET sucrose transporters play important roles in the allocation of sucrose in plants. Some SWEETs were shown to also mediate transport of the plant growth regulator gibberellin (GA). The close physiological relationship between sucrose and GA raised the questions of whether there is a functional connection and whether one or both of the substrates are physiologically relevant. To dissect these two activities, molecular dynamics were used to map the binding sites of sucrose and GA in the pore of SWEET13 and predicted binding interactions that might be selective for sucrose or GA. Transport assays confirmed these predictions. In transport assays, the N76Q mutant had 7x higher relative GA3 activity, and the S142N mutant only transported sucrose. The impaired pollen viability and germination in sweet13;14 double mutants were complemented by the sucrose-selective SWEET13S142N, but not by the SWEET13N76Q mutant, indicating that sucrose is the physiologically relevant substrate and that GA transport capacity is dispensable in the context of male fertility. Therefore, GA supplementation to counter male sterility may act indirectly via stimulating sucrose supply in male sterile mutants. These findings are also relevant in the context of the role of SWEETs in pathogen susceptibility.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fertilidade/genética , Regulação da Expressão Gênica de Plantas , Giberelinas/metabolismo , Proteínas de Transporte de Monossacarídeos , Reguladores de Crescimento de Plantas/metabolismo , Sacarose/metabolismoRESUMO
The mammalian cryptochrome isoforms, CRY1 and CRY2, are core circadian clock regulators that work redundantly. Recent studies revealed distinct roles of these closely related homologs in clock output pathways. Isoform-selective control of CRY1 and CRY2 is critical for further understanding their redundant and distinct roles. KL001 was the first identified small-molecule CRY modulator that activates both CRY1 and CRY2. SHP656 is an orally available KL001 derivative and has shown efficacy in blood glucose control and inhibition of glioblastoma stem cell (GSC) growth in animal models. However, CRY isoform selectivity of SHP656 was uncharacterized, limiting understanding of the roles of CRY1 and CRY2. Here, we report the elucidation of CRY2 selectivity of SHP656. SHP656 lengthened cellular circadian period in a CRY2-dependent manner and selectively interacted with CRY2. By determining the X-ray crystal structure of CRY2 in complex with SHP656 and performing molecular dynamics simulations, we elucidated compound interaction mechanisms. SHP656 binding was compatible with the intrinsic CRY2 gatekeeper W417 "in" orientation and also a close "further in" conformation. Perturbation of W417 interaction with the lid loop resulted in a reduced effect of SHP656 on CRY2, supporting an important role of gatekeeper orientation in isoform selectivity. We also identified the R form of SHP656 (called SHP1703) as the active isomer. Treatment with SHP1703 effectively reduced GSC viability. Our results suggest a direct role of CRY2 in glioblastoma antitumorigenesis and provide a rationale for the selective modulation of CRY isoforms in the therapeutic treatment of glioblastoma and other circadian clock-related diseases.
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Relógios Circadianos , Glioblastoma , Animais , Carbazóis , Ritmo Circadiano/fisiologia , Criptocromos/metabolismo , Glioblastoma/tratamento farmacológico , Mamíferos/metabolismo , Isoformas de Proteínas/genética , SulfonamidasRESUMO
Protein kinases are responsible for healthy cellular processes and signalling pathways, and their dysfunction is the basis of many pathologies. There are numerous small molecule inhibitors of protein kinases that systemically regulate dysfunctional signalling processes. However, attaining selectivity in kinase inhibition within the complex human kinome is still a challenge that inspires unconventional approaches. One of those approaches is photopharmacology, which uses light-controlled bioactive molecules to selectively activate drugs only at the intended space and time, thereby avoiding side effects outside of the irradiated area. Still, in the context of kinase inhibition, photopharmacology has thus far been rather unsuccessful in providing light-controlled drugs. Here, we present the discovery and optimisation of a photoswitchable inhibitor of casein kinase 1δ (CK1δ), important for the control of cell differentiation, circadian rhythm, DNA repair, apoptosis, and numerous other signalling processes. Varying the position at which the light-responsive azobenzene moiety has been introduced into a known CK1δ inhibitor, LH846, revealed the preferred regioisomer for efficient photo-modulation of inhibitory activity, but the photoswitchable inhibitor suffered from sub-optimal (photo)chemical properties. Replacement of the bis-phenyl azobenzene group with the arylazopyrazole moiety yielded a superior photoswitch with very high photostationary state distributions, increased solubility and a 10-fold difference in activity between irradiated and thermally adapted samples. The reasons behind those findings are explored with molecular docking and molecular dynamics simulations. Results described here show how the evaluation of privileged molecular architecture, followed by the optimisation of the photoswitchable unit, is a valuable strategy for the challenging design of the photoswitchable kinase inhibitors.
Assuntos
Caseína Quinase Idelta , Inibidores de Proteínas Quinases , Pirazóis , Apoptose/efeitos dos fármacos , Caseína Quinase Idelta/antagonistas & inibidores , Caseína Quinase Idelta/metabolismo , Humanos , Simulação de Acoplamento Molecular , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologiaRESUMO
In bifidobacteria, phosphoketolase (PKT) plays a key role in the central hexose fermentation pathway called "bifid shunt." The three-dimensional structure of PKT from Bifidobacterium longum with co-enzyme thiamine diphosphate (ThDpp) was determined at 2.1 Å resolution by cryo-EM single-particle analysis using 196,147 particles to build up the structural model of a PKT octamer related by D4 symmetry. Although the cryo-EM structure of PKT was almost identical to the X-ray crystal structure previously determined at 2.2 Å resolution, several interesting structural features were observed in the cryo-EM structure. Because this structure was solved at relatively high resolution, it was observed that several amino acid residues adopt multiple conformations. Among them, Q546-D547-H548-N549 (the QN-loop) demonstrate the largest structural change, which seems to be related to the enzymatic function of PKT. The QN-loop is at the entrance to the substrate binding pocket. The minor conformer of the QN-loop is similar to the conformation of the QN-loop in the crystal structure. The major conformer is located further from ThDpp than the minor conformer. Interestingly, the major conformer in the cryo-EM structure of PKT resembles the corresponding loop structure of substrate-bound Escherichia coli transketolase. That is, the minor and major conformers may correspond to "closed" and "open" states for substrate access, respectively. Moreover, because of the high-resolution analysis, many water molecules were observed in the cryo-EM structure of PKT. Structural features of the water molecules in the cryo-EM structure are discussed and compared with water molecules observed in the crystal structure.
Assuntos
Aldeído Liases/química , Bifidobacterium longum/enzimologia , Microscopia Crioeletrônica/métodos , Escherichia coli , Modelos Moleculares , Tiamina Pirofosfato , ÁguaRESUMO
Atomic models of cryo electron microscopy (cryo-EM) maps of biomolecular conformations are often obtained by flexible fitting of the maps with available atomic structures of other conformations (e.g., obtained by X-ray crystallography). This article presents a new flexible fitting method, NMMD, which combines normal mode analysis (NMA) and molecular dynamics simulation (MD). Given an atomic structure and a cryo-EM map to fit, NMMD simultaneously estimates global atomic displacements based on NMA and local displacements based on MD. NMMD was implemented by modifying EMfit, a flexible fitting method using MD only, in GENESIS 1.4. As EMfit, NMMD can be run with replica exchange umbrella sampling procedure. The new method was tested using a variety of EM maps (synthetic and experimental, with different noise levels and resolutions). The results of the tests show that adding normal modes to MD-based fitting makes the fitting faster (40% in average) and, in the majority of cases, more accurate.
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Simulação de Dinâmica Molecular , Microscopia Crioeletrônica/métodos , Cristalografia por Raios X , Conformação Molecular , Conformação ProteicaRESUMO
Pannexin (PANX) family proteins form large-pore channels that mediate purinergic signaling. We analyzed the cryo-EM structures of human PANX1 in lipid nanodiscs to elucidate the gating mechanism and its regulation by the amino terminus in phospholipids. The wild-type channel has an amino-terminal funnel in the pore, but in the presence of the inhibitor probenecid, a cytoplasmically oriented amino terminus and phospholipids obstruct the pore. Functional analysis using whole-cell patch-clamp and oocyte voltage clamp showed that PANX1 lacking the amino terminus did not open and had a dominant negative effect on channel activity, thus confirming that the amino-terminal domain played an essential role in channel opening. These observations suggest that dynamic conformational changes in the amino terminus of human PANX1 are associated with lipid movement in and out of the pore. Moreover, the data provide insight into the gating mechanism of PANX1 and, more broadly, other large-pore channels.
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Conexinas , Fosfolipídeos , Conexinas/genética , Conexinas/metabolismo , Humanos , Proteínas do Tecido Nervoso/genética , Oócitos/metabolismo , Transdução de SinaisRESUMO
X-ray free-electron laser (XFEL) is the latest generation of the X-ray source that could become an invaluable technique in structural biology. XFEL has ultrashort pulse duration, extreme peak brilliance, and high spatial coherence, which could enable the observation of the biological molecules in near nature state at room temperature without crystallization. However, for biological systems, due to their low diffraction power and complexity of sample delivery, experiments and data analysis are not straightforward, making it extremely challenging to reconstruct three-dimensional (3D) structures from single particle XFEL data. Given the current limitations to the amount and resolution of the data from such XFEL experiments, we propose a new hybrid approach for characterizing biomolecular conformational transitions by using a single 2D low-resolution XFEL diffraction pattern in combination with another known conformation. In our method, we represent the molecular structure with a coarse-grained model, the Gaussian mixture model, to describe large conformational transitions from low-resolution XFEL data. We obtain plausible 3D structural models that are consistent with the XFEL diffraction pattern by deforming an initial structural model to maximize the similarity between the target pattern and the simulated diffraction patterns from the candidate models. We tested the proposed algorithm on two biomolecules of different sizes with different complexities of conformational transitions, adenylate kinase, and elongation factor 2, using synthetic XFEL data. The results show that, with the proposed algorithm, we can successfully describe the conformational transitions by flexibly fitting the coarse-grained model of one conformation to become consistent with an XFEL diffraction pattern simulated from another conformation. In addition, we showed that the incident beam orientation has some effect on the accuracy of the 3D structure modeling and discussed the reasons for the inaccuracies for certain orientations. The proposed method could serve as an alternative approach for retrieving information on 3D conformational transitions from the XFEL diffraction patterns to interpret experimental data. Since the molecules are represented by Gaussian kernels and no atomic structure is needed in principle, such a method could also be used as a tool to seek initial models for 3D reconstruction algorithms.
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ClpB belongs to the cellular disaggretase machinery involved in rescuing misfolded or aggregated proteins during heat or other cellular shocks. The function of this protein relies on the interconversion between different conformations in its native condition. A recent high-speed-atomic-force-microscopy (HS-AFM) experiment on ClpB from Thermus thermophilus shows four predominant conformational classes, namely, open, closed, spiral, and half-spiral. Analyses of AFM images provide only partial structural information regarding the molecular surface, and thus computational modeling of three-dimensional (3D) structures of these conformations should help interpret dynamical events related to ClpB functions. In this study, we reconstruct 3D models of ClpB from HS-AFM images in different conformational classes. We have applied our recently developed computational method based on a low-resolution representation of 3D structure using a Gaussian mixture model, combined with a Monte-Carlo sampling algorithm to optimize the agreement with target AFM images. After conformational sampling, we obtained models that reflect conformational variety embedded within the AFM images. From these reconstructed 3D models, we described, in terms of relative domain arrangement, the different types of ClpB oligomeric conformations observed by HS-AFM experiments. In particular, we highlighted the slippage of the monomeric components around the seam. This study demonstrates that such details of information, necessary for annotating the different conformational states involved in the ClpB function, can be obtained by combining HS-AFM images, even with limited resolution, and computational modeling.
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X-ray free-electron laser (XFEL) scattering promises to probe single biomolecular complexes without crystallization, enabling the study of biomolecular structures under near-physiological conditions at room temperature. However, such structural determination of biomolecules is extremely challenging thus far. In addition to the large numbers of diffraction patterns required, the orientation of each diffraction pattern needs to be accurately estimated and the missing phase information needs to be recovered for three-dimensional (3D) structure reconstruction. Given the current limitations to the amount and resolution of the data available from single-particle XFEL scattering experiments, we propose an alternative approach to find plausible 3D biological shapes from a limited number of diffraction patterns to serve as a starting point for further analyses. In our proposed strategy, small sets of input (e.g., five) XFEL diffraction patterns were matched against a library of diffraction patterns simulated from 1628 electron microscopy (EM) models to find potential matching 3D models that are consistent with the input diffraction patterns. This approach was tested for three example cases: EMD-3457 (Thermoplasma acidophilum 20S proteasome), EMD-5141 (Escherichia coli 70S ribosome complex), and EMD-5152 (budding yeast Nup84 complex). We observed that choosing the best strategy to define matching regions on the diffraction patterns is critical for identifying correctly matching diffraction patterns. While increasing the number of input diffraction patterns improved the matches in some cases, we found that the resulting matches are more dependent on the uniqueness or complexity of the shape as captured in the individual input diffraction patterns and the availability of a similar 3D biological shape in the search library. The protocol could be useful for finding candidate models for a limited amount of low-resolution data, even when insufficient for reconstruction, performing a quick exploration of new data upon collection, and the analysis of the conformational heterogeneity of the particle of interest as captured within the diffraction patterns.
Assuntos
Lasers , Cristalização , Conformação Molecular , Difração de Raios XRESUMO
The circadian clock is a biological timekeeper that operates through transcription-translation feedback loops in mammals. Cryptochrome 1 (CRY1) and Cryptochrome 2 (CRY2) are highly conserved core clock components having redundant and distinct functions. We recently identified the CRY1- and CRY2-selective compounds KL101 and TH301, respectively, which provide useful tools for the exploration of isoform-selective CRY regulation. However, intrinsic differences in the compound-binding FAD (flavin adenine dinucleotide) pockets between CRY1 and CRY2 are not well understood, partly because of nonoptimal properties of previously reported apo form structures in this particular region constituted by almost identical sequences. Here, we show unliganded CRY1 and CRY2 crystal structures with well-defined electron densities that are largely free of crystal contacts at the FAD pocket and nearby lid loop. We revealed conformational isomerism in key residues. In particular, CRY1 W399 and corresponding CRY2 W417 in the FAD pocket had distinct conformations ("out" for CRY1 and "in" for CRY2) by interacting with the lid loop residues CRY1 Q407 and CRY2 F424, respectively, resulting in different overall lid loop structures. Molecular dynamics simulations supported that these conformations were energetically favorable to each isoform. Isoform-selective compounds KL101 and TH301 preferred intrinsic "out" and "in" conformations of the tryptophan residue in CRY1 and CRY2, respectively, while the nonselective compound KL001 fit to both conformations. Mutations of lid loop residues designed to perturb their isoform-specific interaction with the tryptophan resulted in reversed responses of CRY1 and CRY2 to KL101 and TH301. We propose that these intrinsic structural differences of CRY1 and CRY2 can be targeted for isoform-selective regulation.
Assuntos
Criptocromos/química , Criptocromos/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Mamíferos/metabolismo , Animais , Sítios de Ligação , Criptocromos/genética , Cristalografia por Raios X , Simulação de Dinâmica Molecular , Mutação/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Estrutura Secundária de ProteínaRESUMO
The circadian clock controls daily rhythms of physiological processes. The presence of the clock mechanism throughout the body is hampering its local regulation by small molecules. A photoresponsive clock modulator would enable precise and reversible regulation of circadian rhythms using light as a bio-orthogonal external stimulus. Here we show, through judicious molecular design and state-of-the-art photopharmacological tools, the development of a visible light-responsive inhibitor of casein kinase I (CKI) that controls the period and phase of cellular and tissue circadian rhythms in a reversible manner. The dark isomer of photoswitchable inhibitor 9 exhibits almost identical affinity towards the CKIα and CKIδ isoforms, while upon irradiation it becomes more selective towards CKIδ, revealing the higher importance of CKIδ in the period regulation. Our studies enable long-term regulation of CKI activity in cells for multiple days and show the reversible modulation of circadian rhythms with a several hour period and phase change through chronophotopharmacology.
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Caseína Quinase Ialfa/antagonistas & inibidores , Caseína Quinase Idelta/antagonistas & inibidores , Ritmo Circadiano/efeitos dos fármacos , Cronofarmacoterapia , Inibidores de Proteínas Quinases/farmacologia , Animais , Caseína Quinase Ialfa/metabolismo , Caseína Quinase Ialfa/ultraestrutura , Caseína Quinase Idelta/metabolismo , Linhagem Celular Tumoral , Transtornos Cronobiológicos/tratamento farmacológico , Relógios Circadianos/efeitos da radiação , Avaliação Pré-Clínica de Medicamentos , Ensaios Enzimáticos , Humanos , Luz , Camundongos , Camundongos Transgênicos , Simulação de Acoplamento Molecular , Fotoperíodo , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/efeitos da radiação , Núcleo Supraquiasmático/efeitos dos fármacos , Núcleo Supraquiasmático/metabolismo , Técnicas de Cultura de TecidosRESUMO
CRY1 and CRY2 proteins are highly conserved components of the circadian clock that controls daily physiological rhythms. Disruption of CRY functions are related to many diseases, including circadian sleep phase disorder. Development of isoform-selective and spatiotemporally controllable tools will facilitate the understanding of shared and distinct functions of CRY1 and CRY2. Here, we developed CRY1-selective compounds that enable light-dependent manipulation of the circadian clock. From phenotypic chemical screening in human cells, we identified benzophenone derivatives that lengthened the circadian period. These compounds selectively interacted with the CRY1 photolyase homology region, resulting in activation of CRY1 but not CRY2. The benzophenone moiety rearranged a CRY1 region called the "lid loop" located outside of the compound-binding pocket and formed a unique interaction with Phe409 in the lid loop. Manipulation of this key interaction was achieved by rationally designed replacement of the benzophenone with a switchable azobenzene moiety whose cis-trans isomerization can be controlled by light. The metastable cis form exhibited sufficiently high half-life in aqueous solutions and structurally mimicked the benzophenone unit, enabling reversible period regulation over days by cellular irradiation with visible light. This study revealed an unprecedented role of the lid loop in CRY-compound interaction and paves the way for spatiotemporal regulation of CRY1 activity by photopharmacology for molecular understanding of CRY1-dependent functions in health and disease.
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Relógios Circadianos/efeitos dos fármacos , Criptocromos/efeitos dos fármacos , Animais , Relógios Circadianos/fisiologia , Humanos , LuzRESUMO
CRY1 and CRY2 are essential components of the circadian clock controlling daily physiological rhythms. Accumulating evidences indicate distinct roles of these highly homologous proteins, in addition to redundant functions. Therefore, the development of isoform-selective compounds represents an effective approach towards understanding the similarities and differences of CRY1 and CRY2 by controlling each isoform individually. We conducted phenotypic screenings of circadian clock modulators, and identified KL101 and TH301 that selectively stabilize CRY1 and CRY2, respectively. Crystal structures of CRY-compound complexes revealed conservation of compound-binding sites between CRY1 and CRY2. We further discovered a unique mechanism underlying compound selectivity in which the disordered C-terminal region outside the pocket was required for the differential effects of KL101 and TH301 against CRY isoforms. By using these compounds, we found a new role of CRY1 and CRY2 as enhancers of brown adipocyte differentiation, providing the basis of CRY-mediated regulation of energy expenditure.
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Criptocromos/química , Isoformas de Proteínas/química , Animais , Sítios de Ligação , Relógios Circadianos , Criptocromos/genética , Fibroblastos/metabolismo , Células HEK293 , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Masculino , Camundongos Knockout , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Isoformas de Proteínas/genética , TermodinâmicaRESUMO
Mammalian circadian rhythms are generated by a transcription-based feedback loop in which CLOCK:BMAL1 drives transcription of its repressors (PER1/2, CRY1/2), which ultimately interact with CLOCK:BMAL1 to close the feedback loop with ~24 hr periodicity. Here we pinpoint a key difference between CRY1 and CRY2 that underlies their differential strengths as transcriptional repressors. Both cryptochromes bind the BMAL1 transactivation domain similarly to sequester it from coactivators and repress CLOCK:BMAL1 activity. However, we find that CRY1 is recruited with much higher affinity to the PAS domain core of CLOCK:BMAL1, allowing it to serve as a stronger repressor that lengthens circadian period. We discovered a dynamic serine-rich loop adjacent to the secondary pocket in the photolyase homology region (PHR) domain that regulates differential binding of cryptochromes to the PAS domain core of CLOCK:BMAL1. Notably, binding of the co-repressor PER2 remodels the serine loop of CRY2, making it more CRY1-like and enhancing its affinity for CLOCK:BMAL1.
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Fatores de Transcrição ARNTL/fisiologia , Proteínas CLOCK/fisiologia , Ritmo Circadiano , Criptocromos/metabolismo , Fatores de Transcrição ARNTL/química , Fatores de Transcrição ARNTL/metabolismo , Animais , Proteínas CLOCK/química , Proteínas CLOCK/metabolismo , Ritmo Circadiano/fisiologia , Criptocromos/química , Criptocromos/fisiologia , Camundongos , Estrutura Terciária de Proteína , Serina/metabolismoRESUMO
The structural and dynamical characterization of biomolecules holds central importance in the endeavor to understand the molecular mechanisms regulating living systems. However, owing to the inherent heterogeneity of biomolecular interactions within cells, it is often difficult to understand the overall structure and dynamics of biomolecules using any experimental method in isolation. In this regard, hybrid methods that combine data from multiple experiments to generate a comprehensive model of biomolecular complexes have gained prominence in the last few years. In this article, we discuss the advancements in hybrid methods, with a particular focus on the role of computation in their development and application. We further outline the future directions that hybrid methods are likely to take, regarding the advancements in techniques such as X-ray free-electron laser single- particle imaging, and electron cryo-tomography. Finally, we conclude the review by highlighting the future goals of broader consensus and collaboration within the integrative/hybrid structural biology community and for disseminating the data generated by hybrid modeling efforts.