Disulfide bridge-dependent dimerization triggers FGF2 membrane translocation into the extracellular space.

Fibroblast growth factor 2 (FGF2) exits cells by direct translocation across the plasma membrane, a type I pathway of unconventional protein secretion. This process is initiated by phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2)-dependent formation of highly dynamic FGF2 oligomers at the inner plasma membrane leaflet, inducing the formation of lipidic membrane pores. Cell surface heparan sulfate chains linked to glypican-1 (GPC1) capture FGF2 at the outer plasma membrane leaflet, completing FGF2 membrane translocation into the extracellular space. While the basic steps of this pathway are well understood, the molecular mechanism by which FGF2 oligomerizes on membrane surfaces remains unclear. In the current study, we demonstrate the initial step of this process to depend on C95-C95 disulfide-bridge-mediated FGF2 dimerization on membrane surfaces, producing the building blocks for higher FGF2 oligomers that drive the formation of membrane pores. We find FGF2 with a C95A substitution to be defective in oligomerization, pore formation, and membrane translocation. Consistently, we demonstrate a C95A variant of FGF2 to be characterized by a severe secretion phenotype. By contrast, while also important for efficient FGF2 secretion from cells, a second cysteine residue on the molecular surface of FGF2 (C77) is not involved in FGF2 oligomerization. Rather, we find C77 to be part of the interaction interface through which FGF2 binds to the α1 subunit of the Na,K-ATPase, the landing platform for FGF2 at the inner plasma membrane leaflet. Using cross-linking mass spectrometry, atomistic molecular dynamics simulations combined with a machine learning analysis and cryo-electron tomography, we propose a mechanism by which disulfide-bridged FGF2 dimers bind with high avidity to PI(4,5)P2 on membrane surfaces. We further propose a tight coupling between FGF2 secretion and the formation of ternary signaling complexes on cell surfaces, hypothesizing that C95-C95-bridged FGF2 dimers are functioning as the molecular units triggering autocrine and paracrine FGF2 signaling.

Nanostructure-Driven Indocyanine Green Dimerization Generates Ultra-Stable Phototheranostics Nanoparticles.

Indocyanine green (ICG) is the only near-infrared (NIR) dye approved for clinical use. Despite its versatility in photonic applications and potential for photothermal therapy, its photobleaching hinders its application. Here we discovered a nanostructure of dimeric ICG (Nano-dICG) generated by using ICG to stabilize nanoemulsions, after which ICG enabled complete dimerization on the nanoemulsion shell, followed by J-aggregation of ICG-dimer, resulting in a narrow, red-shifted (780 nm→894 nm) and intense (≈2-fold) absorbance. Compared to ICG, Nano-dICG demonstrated superior photothermal conversion (2-fold higher), significantly reduced photodegradation (-9.6 % vs. -46.3 %), and undiminished photothermal effect (7 vs. 2 cycles) under repeated irradiations, in addition to excellent colloidal and structural stabilities. Following intravenous injection, Nano-dICG enabled real-time tracking of its delivery to mouse tumors within 24 h by photoacoustic imaging at NIR wavelength (890 nm) distinct from the endogenous signal to guide effective photothermal therapy. The unprecedented finding of nanostructure-driven ICG dimerization leads to an ultra-stable phototheranostic platform.

Promising disruptors of p53-MDM2 dimerization from some medicinal plant phytochemicals: a molecular modeling study.

Cancer is a major global health issue that has a high mortality rate. p53, which functions as a tumor suppressor, is critical in preventing tumor development by regulating the cell cycle and inducing apoptosis in damaged cells. However, the tumor suppressor function of p53 is effectively inhibited by its direct interaction with the hydrophobic cleft of MDM2 protein via multiple mechanisms As a result, restoring p53 activity by blocking the p53-MDM2 protein-protein interaction has been proposed as a compelling therapeutic strategy for cancer treatment. The use of molecular docking and phytochemical screening procedures are appraised to inhibit MDM2's hydrophobic cleft and disrupt the p53-MDM2 interaction. For this purpose, a library of 51 bioactive compounds from 10 medicinal plants was compiled and subjected to structure-based virtual screening. Out of these, only 3 compounds (Atalantoflavone, Cudraxanthone 1, and Ursolic acid) emerged as promising inhibitors of MDM2-p53 based on their binding affinities (-9.1 kcal/mol, -8.8 kcal/mol, and -8.8 kcal/mol respectively) when compared to the standard (-8.8 kcal/mol). Moreover, these compounds showed better pharmacokinetic and drug-like profiling than the standard inhibitor (Chromonotriazolopyrimidine 1). Finally, the 100 ns MD simulation analysis confirmed no significant perturbation in the conformational dynamics of the simulated binary complexes when compared to the standard. In particular, Ursolic acid was found to satisfy the molecular enumeration the most compared to the other inhibitors. Our overall molecular modeling finding shows why these compounds may emerge as potent arsenals for cancer therapeutics. Nonetheless, extensive experimental and clinical research is needed to augment their use in clinics.Communicated by Ramaswamy H. Sarma.

Zinc controls operator affinity of human transcription factor YY1 by mediating dimerization via its N-terminal region.

Human protein Yin Yang 1 (YY1) controls the transcription of hundreds of genes both positively and negatively through interactions with a wide range of partner proteins. Results presented here from proteolytic sensitivity, calorimetry, circular dichroism, fluorescence, NMR, size-exclusion chromatography, SELEX, and EMSA show that purified YY1 forms dimers via its disordered N-terminal region with strong zinc-ion concentration dependence. The YY1 dimer is shown to bind tandem repeats of a canonical recognition DNA sequence with high affinity, and analysis of human YY1 regulatory sites shows that many contain repeats of its recognition elements. YY1 dimerization may compete with partner protein interactions, making control by zinc ion concentration a previously unrecognized factor affecting YY1 gene regulation. Indeed, YY1 is known to be important in many pathogenic processes, including neoplasia, in which zinc ion concentrations are altered. The present results incentivize studies in vivo or in vitro that explore the role of zinc ion concentration in YY1-mediated gene expression.

Conserved Residues Lys64 and Glu78 at the Subunit Surface of Tau Glutathione Transferase in Rice Affect Structure and Enzymatic Properties.

Glutathione transferases (GSTs) are a superfamily of dimeric proteins associated with the detoxification of various reactive electrophiles and responsive to a multitude of stressors. We individually substituted Lys64 and Glu78 with Ala using site-directed mutagenesis to understand the role of subunit interactions in the structure and enzymatic properties of a rice GST (OsGSTU17). The wild-type OsGSTU17 lost the conserved hydrogen bond between subunits in tau class GSTs due to conserved Tyr92 replaced with Phe92, but still exhibited high substrate activities, and thermal stability remained in its dimeric structure. The significant decrease in thermal stability and obvious changes in the structure of mutant K64A implied that conserved Lys64 might play an essential role in the structural stability of tau class GSTs. The mutant E78A, supposed to be deprived of hydrogen and salt bonds between subunits, appeared in the soluble form of dimers, even though its tertiary structure altered and stability declined dramatically. These results suggest that the hydrogen and ionic bonds provided by conserved residues are not as important for OsGSTU17 dimerization and enzymatic properties. These results further supplement our understanding of the relationship between the structure and function of GSTs and provide a theoretical basis for improving crop resistance through targeted modification of GSTs.

A novel nuclear receptor subfamily enlightens the origin of heterodimerization.

BACKGROUND: Nuclear receptors are transcription factors of central importance in human biology and associated diseases. Much of the knowledge related to their major functions, such as ligand and DNA binding or dimerization, derives from functional studies undertaken in classical model animals. It has become evident, however, that a deeper understanding of these molecular functions requires uncovering how these characteristics originated and diversified during evolution, by looking at more species. In particular, the comprehension of how dimerization evolved from ancestral homodimers to a more sophisticated state of heterodimers has been missing, due to a too narrow phylogenetic sampling. Here, we experimentally and phylogenetically define the evolutionary trajectory of nuclear receptor dimerization by analyzing a novel NR7 subgroup, present in various metazoan groups, including cnidarians, annelids, mollusks, sea urchins, and amphioxus, but lost in vertebrates, arthropods, and nematodes. RESULTS: We focused on NR7 of the cephalochordate amphioxus B. lanceolatum. We present a complementary set of functional, structural, and evolutionary analyses that establish that NR7 lies at a pivotal point in the evolutionary trajectory from homodimerizing to heterodimerizing nuclear receptors. The crystal structure of the NR7 ligand-binding domain suggests that the isolated domain is not capable of dimerizing with the ubiquitous dimerization partner RXR. In contrast, the full-length NR7 dimerizes with RXR in a DNA-dependent manner and acts as a constitutively active receptor. The phylogenetic and sequence analyses position NR7 at a pivotal point, just between the basal class I nuclear receptors that form monomers or homodimers on DNA and the derived class II nuclear receptors that exhibit the classical DNA-independent RXR heterodimers. CONCLUSIONS: Our data suggest that NR7 represents the "missing link" in the transition between class I and class II nuclear receptors and that the DNA independency of heterodimer formation is a feature that was acquired during evolution. Our studies define a novel paradigm of nuclear receptor dimerization that evolved from DNA-dependent to DNA-independent requirements. This new concept emphasizes the importance of DNA in the dimerization of nuclear receptors, such as the glucocorticoid receptor and other members of this pharmacologically important oxosteroid receptor subfamily. Our studies further underline the importance of studying emerging model organisms for supporting cutting-edge research.

Human Serum Albumin Dimerization Enhances the S2 Emission of Bound Cyanine IR806.

Cyanine molecules are important phototheranostic compounds given their high fluorescence yield in the near-infrared region of the spectrum. We report on the frequency and time-resolved spectroscopy of the S2 state of IR806, which demonstrates enhanced emission upon binding to the hydrophobic pocket of human serum albumin (HSA). From excitation-emission matrix spectra and electronic structure calculations, we identify the emission as one associated with a state having the polymethine chain twisted out of plane by 103°. In addition, we find that this configuration is significantly stabilized as the concentration of HSA increases. Spectroscopic changes associated with the S1 and S2 states of IR806 as a function of HSA concentration, as well as anisotropy measurements, confirm the formation of HSA dimers at concentrations greater than 10 µM. These findings imply that the longer-lived S2 state configuration can lead to more efficient phototherapy agents, and cyanine S2 spectroscopy may be a useful tool to determine the oligomerization state of HSA.

Novel Antifungal Dimers from the Roots of Taiwania cryptomerioides.

Five new dimer compounds, namely Taiwaniacryptodimers A-E (1-5), were isolated from the methanol extract of the roots of Taiwania cryptomerioides. Their structures were established by mean of spectroscopic analysis and comparison of NMR data with those of known analogues. Their antifungal activities were also evaluated. Our results indicated that metabolites 1, 2, 4, and 5 displayed moderate antifungal activities against Aspergillus niger, Penicillium italicum, Candida albicans, and Saccharomyces cerevisiae.

Discovery of an unprecedented benz[α]anthraquinone-type heterodimer from a rare actinomycete Amycolatopsis sp. HCa1.

The angucylines are a family of aromatic polyketides featuring a tetracyclic benz[a]anthraquinone skeleton. This class of polycyclic aromatic polyketides are exclusively associated with actinomycetes and can undergo many modifications such as oxidation, ring cleavage, glycosylation and dimerization. Here we report the discovery of a new ether-linked benz[a]anthraquinone heterodimer, named mycolatone (1), from a grasshopper-derived actinomycete, Amycolatopsis sp. HCa1. The structure of mycolatone (1) was determined by comprehensive two-dimensional NMR analysis, high-resolution electrospray ionization mass spectrometry and biogenetic consideration. This new heterodimeric molecule is structurally derived from the dimerization of two tetracyclic angucylines, 2-hydroxy-5-O-methyltetragomycin and PD116779, through an ether bond between C-8 and C-8'. This new structural feature enrich the structural diversity of angucylines. Additionally, the surface tension activity and cytotoxic activities of 1 against human cervical cancer cell line (Hela), human gastric adenocarcinoma cell line (SGC-7901) and human lung adenocarcinoma cell line (SPC-A-1) were evaluated.

8-, 9-, and 11-Aryloxy Dimeric Aporphines and Their Pharmacological Activities.

Aporphines, a major group of aporphinoid alkaloids, exhibit interesting and diverse pharmacological activities. A set of dimeric aporphines with an aryloxy group at C8, C9, and C11 have been isolated from six genera and shown to elicit various biological activities such as antitumor, antimalarial, antimicrobial, antiplatelet aggregation, antifibrotic, immunosuppressive, and vasorelaxant properties. In this review, the nomenclature, chemical structures, botanical sources, pharmacological activities, and synthetic approaches of this set of dimeric alkaloids are presented.

Alkyl-benzofuran dimers from Eupatorium chinense with insulin-sensitizing and anti-inflammatory activities.

Five new racemic alkyl-benzofuran dimers, (±)-dieupachinins I-M (1-5), were isolated from the root tubers of Eupatorium chinense, a well-known traditional Chinese medicine for the treatment of diphtheria in Guangdong province. The structures of these compounds, especially the first examples of 12,10'-epoxy dimer dieupachinin I (1), 12-nor-dimer dieupachinin J (2), and 12,12'-dinor-dimer dieupachinin K (3), were elucidated by spectroscopic data analysis. Chiral resolution were further carried out on a cellulose column by HPLC, and compounds 2-5 were successfully separated into two enantiomers, respectively. The absolute configurations of (+)-(2-5) and (-)-(2-5) were established by theoretical ECD calculation. All the compounds were evaluated for insulin-stimulated glucose uptake in C2C12 myotubes and (±)-dieupachinin I (1) exhibited the best activity. Compound 1 enhanced insulin-stimulated glucose uptake via activating the insulin receptor substrate 1/protein kinase B/glycogen synthase kinase-3ß signaling pathway. Moreover, all the isolates were tested for their nitric oxygen (NO) inhibitory effects in lipopolysaccharide-treated RAW264.7 macrophages, and compounds (±)-1, (±)-2, and (±)-4 showed promising inhibitory effects with IC50 values of 6.42 ± 1.85, 6.29 ± 1.94, and 16.03 ± 2.07 µM, respectively. (±)-Dieupachinin I (1) again dose-dependently suppressed LPS-induced expression of inducible NO synthase and nuclear translocation of p65.

Design and synthesis of novel tacrine-indole hybrids as potential multitarget-directed ligands for the treatment of Alzheimer's disease.

The authors report on the synthesis and biological evaluation of new compounds whose structure combines tacrine and indole moieties. Tacrine-indole heterodimers were designed to inhibit cholinesterases and ß-amyloid formation, and to cross the blood-brain barrier. The most potent new acetylcholinesterase inhibitors were compounds 3c and 4d (IC50 = 25 and 39 nM, respectively). Compound 3c displayed considerably higher selectivity for acetylcholinesterase relative to human plasma butyrylcholinesterase in comparison to compound 4d (selectivity index: IC50 [butyrylcholinesterase]/IC50 [acetylcholinesterase] = 3 and 0.6, respectively). Furthermore, compound 3c inhibited ß-amyloid-dependent amyloid nucleation in the yeast-based prion nucleation assay and displayed no dsDNA destabilizing interactions with DNA. Compounds 3c and 4d displayed a high probability of crossing the blood-brain barrier. The results support the potential of 3c for future development as a dual-acting therapeutic agent in the prevention and/or treatment of Alzheimer's disease.

Structure-Based Virtual Screening of Tumor Necrosis Factor-α Inhibitors by Cheminformatics Approaches and Bio-Molecular Simulation.

Tumor necrosis factor-α (TNF-α) is a drug target in rheumatoid arthritis and several other auto-immune disorders. TNF-α binds with TNF receptors (TNFR), located on the surface of several immunological cells to exert its effect. Hence, the use of inhibitors that can hinder the complex formation of TNF-α/TNFR can be of medicinal significance. In this study, multiple chem-informatics approaches, including descriptor-based screening, 2D-similarity searching, and pharmacophore modelling were applied to screen new TNF-α inhibitors. Subsequently, multiple-docking protocols were used, and four-fold post-docking results were analyzed by consensus approach. After structure-based virtual screening, seventeen compounds were mutually ranked in top-ranked position by all the docking programs. Those identified hits target TNF-α dimer and effectively block TNF-α/TNFR interface. The predicted pharmacokinetics and physiological properties of the selected hits revealed that, out of seventeen, seven compounds (4, 5, 10, 11, 13-15) possessed excellent ADMET profile. These seven compounds plus three more molecules (7, 8 and 9) were chosen for molecular dynamics simulation studies to probe into ligand-induced structural and dynamic behavior of TNF-α, followed by ligand-TNF-α binding free energy calculation using MM-PBSA. The MM-PBSA calculations revealed that compounds 4, 5, 7 and 9 possess highest affinity for TNF-α; 8, 11, 13-15 exhibited moderate affinities, while compound 10 showed weaker binding affinity with TNF-α. This study provides valuable insights to design more potent and selective inhibitors of TNF-α, that will help to treat inflammatory disorders.

Coumarin-based derivatives with potential anti-HIV activity.

Acquired immunodeficiency syndrome (AIDS), as a result of human immunodeficiency virus (HIV) infection which leads to severe suppression of immune functions, is an enormous world-wide health threat. The anti-HIV agents are critical for the HIV/AIDS therapy, but the generation of viral mutants and the severe side effects of the anti-HIV agents pose serious hurdles in the treatment of HIV infection, and creat an urgent need to develop novel anti-HIV agents. The plant-derived compounds possess structural and mechanistic diversity, and among them, coumarin-based derivatives have the potential to inhibit different stages in the HIV replication cycle, inclusive of virus-host cell attachment, cell membrane fusion, integration, assembly besides the conventional target like inhibition of the reverse transcriptase, protease, and integrase. Moreover, (+)-calanolide A, a coumarin-based natural product, is a potential anti-HIV agent. Thus, coumarin-based derivatives are useful scaffolds for the development of anti-HIV agents. This review article describes the recent progress in the discovery, structural modification, and structure-activity relationship studies of potent anti-HIV coumarin-based derivatives including natural coumarin compounds, synthetic hybrids, dimers, and other synthetic derivatives covering articles published between 2000 and 2020.

Discovery and development of novel pyrimidine and pyrazolo/thieno-fused pyrimidine derivatives as potent and orally active inducible nitric oxide synthase dimerization inhibitor with efficacy for arthritis.

In order to discover and develop drug-like anti-inflammatory agents against arthritis, based on "Hit" we found earlier and to overcome drawbacks of toxicity, twelve series of total 89 novel pyrimidine, pyrazolo[4,3-d]pyrimidine and thieno[3,2-d]pyrimidine derivatives were designed, synthesized and screened for their anti-inflammatory activity against NO and toxicity for normal liver cells (LO2). Relationships of balance toxicity and activity have been summarized through multi-steps, and title compounds 22o, 22l were found to show lower toxicity (against LO2: IC50 = 2934, 2301 µM, respectively) and potent effect against NO release (IR = 98.3, 97.67%, at 10 µM, respectively). Furthermore, compound 22o showed potent iNOS inhibitory activity with value of IC50 is 0.96 µM and could interfere stability and formation of the active dimeric iNOS. It's anti-inflammatory activity in vivo was assessed by AIA rat model. Furthermore, the results of metabolic stability, CYP, PK study in vivo, acute toxicity study and subacute toxicity assessment indicated this compound had good drug-like properties for treatment.

Development and validation of an UHPLC-qTOF-MS method for the quantification of cyclic polyesters oligomers in pasta by applying a modified QuEChERS clean-up.

An Ultra High-Performance Liquid chromatography method quadruple time-of-flight mass spectrometry has been developed for the analysis of 11 cyclic polyesters oligomers, following a modified QuEChERS clean-up with alumina/primary secondary amine, in pasta. Target analytes were polyethylene terephthalate (PET) 1st series cyclic dimer to heptamer, polybutylene terephthalate (PBT) dimer to pentamer and a polyurethane oligomer. Standard addition method was applied for the calibration, and the limits of quantification ranged from 3.2 to 17.2 ng g-1. Recoveries ranged from 86.4 to 109.8%, RSDs were lower than 12% for all analytes, and matrix effect never exceeded ± 2.5%. The method was successfully applied to real commercial pasta samples, where the PET 1st series cyclic trimer was the most abundant oligomer, being found in all tested samples. The 1st series PET cyclic dimer and tetramer, as well as 1,4,7-trioxacyclotridecane-8,13-dione, were found in considerable amounts. Traces of the 2nd and 3rd series PET cyclic dimers were also found.

Lyciyunin, a new dimer of feruloyltyramine and five bioactive tyramines from the root of Lycium yunnanense Kuang.

Lyciyunin, a new dimer of feruloyltyramine (1), together with five known tyramines (2-6), was isolated from the water-soluble fraction of an EtOH extract of the root of L. yunnanense. Based on HR-TOF-MS, NMR spectral data and quantum chemistry ECD calculations, the structure of this new compound was determined, including its absolute configuration. Compounds (1-6) were tested for their antioxidant activity using in vitro DPPH radical scavenging assay, and 1-6 showed the moderate antioxidant activities with IC50 values of 12.44 ± 0.39, 21.29 ± 0.75, 24.44 ± 1.63, 21.15 ± 0.66, 21.15 ± 0.66 and 45.15 ± 0.56 µM, respectively. Compounds (5-6) showed anti-inflammatory activity in LPS-induced RAW 264.7 macrophages with the IC50 values of 43.95 ± 6.11 and 33.50 ± 2.04 µM, respectively.

Lignocellulosic fibres surface interactions in enzymatic reaction using data-mining.

Lignocellulosic fibres modification focused so far essentially on the resulting material properties to create functional fibres instead of determining the reaction influencing parameters. Using a data-mining algorithm, surface chemical composition of the fibres after modifications was compared to multiple signals. A 24 h reaction at either 25 °C or 60 °C, pH5 was conducted in presence of trans-ferulic acid, laccase, and lignocellulosic fibres (flax, hemp, or cellulose) having different chemical surface composition. Dimers and trimers were detected in variable concentrations in the reaction filtrate and extractive. At 25 °C, crystalline cellulose, amorphous cellulose, xylans, mannans, and lignins were well correlated to specific reaction products while at 60 °C, only lignins and xylan were found correlated to reaction products. Fibres surface composition affected the extractive profile. Lignocellulosic surface composition influence on the product formed was unveiled using a data mining approach. This study presents a way to unveil non-evident chemical interface interaction in reactions.

Chalcones Display Anti-NLRP3 Inflammasome Activity in Macrophages through Inhibition of Both Priming and Activation Steps-Structure-Activity-Relationship and Mechanism Studies.

Chalcones are responsible for biological activity throughout fruits, vegetables, and medicinal plants in preventing and treating a variety of inflammation-related diseases. However, their structure-activity relationship (SAR) in inhibiting inflammasome activation has not been explored. We synthesized numerous chalcones and determined their SAR on lipopolysaccharide (LPS)-primed ATP-induced NLRP3 inflammasome activation. 11Cha1 displayed good inhibitory activity on release reaction of caspase-1, IL-1ß, and IL-18. It significantly inhibited LPS-induced phosphorylation and proteolytic degradation of IĸB-α and nuclear translocation of NF-ĸB, but had little effect on mitogen-activated protein kinases (MAPKs) activities. Furthermore, 11Cha1 blocked LPS-induced up-regulation of NLRP3, pro-caspase-1, ASC, IL-18, and IL-1ß, indicating the suppression on priming step of inflammasome activation. ASC dimerization and oligomerization are considered to be direct evidence for inflammasome activation. 11Cha1 profoundly inhibited ATP-induced formation of ASC dimers, trimers, and oligomers, and the assembly of ASC, pro-caspase-1, and NLRP3 in inflammasome formation. Decrease of intracellular K+ levels is the common cellular activity elicited by all NLRP3 inflammasome activators. 11Cha1 substantially diminished ATP-mediated K+ efflux, confirming the anti-NLRP3 inflammasome activity of 11Cha1. In summary, the SAR of chalcone derivatives in anti-inflammasome activities was examined. Besides, 11Cha1 inhibited both priming and activation steps of NLRP3 inflammasome activation. It inhibited NF-ĸB activation and subsequently suppressed the up-regulation of NLRP3 inflammasome components including NLRP3, ASC, pro-caspase-1, pro-IL-18, and pro-IL-1ß. Next, 11Cha1 blocked ATP-mediated K+ efflux and suppressed the assembly and activation of NLRP3 inflammasome, leading to the inhibition of caspase-1 activation and proteolytic cleavage, maturation, and secretion of IL-1ß and IL-18.

Optimized Vivid-derived Magnets photodimerizers for subcellular optogenetics in mammalian cells.

Light-inducible dimerization protein modules enable precise temporal and spatial control of biological processes in non-invasive fashion. Among them, Magnets are small modules engineered from the Neurospora crassa photoreceptor Vivid by orthogonalizing the homodimerization interface into complementary heterodimers. Both Magnets components, which are well-tolerated as protein fusion partners, are photoreceptors requiring simultaneous photoactivation to interact, enabling high spatiotemporal confinement of dimerization with a single excitation wavelength. However, Magnets require concatemerization for efficient responses and cell preincubation at 28°C to be functional. Here we overcome these limitations by engineering an optimized Magnets pair requiring neither concatemerization nor low temperature preincubation. We validated these 'enhanced' Magnets (eMags) by using them to rapidly and reversibly recruit proteins to subcellular organelles, to induce organelle contacts, and to reconstitute OSBP-VAP ER-Golgi tethering implicated in phosphatidylinositol-4-phosphate transport and metabolism. eMags represent a very effective tool to optogenetically manipulate physiological processes over whole cells or in small subcellular volumes.

The cell relies on direct interactions among proteins and compartments called organelles to stay alive. Manipulating these interactions allows researchers to control a wide variety of cell behaviors. A system called 'Magnets' uses light to trigger interactions between proteins. Magnets uses a segment of a protein called Vivid from a common bread mold that responds to light. When light shines on two of these segments, it causes them to bind together, in a process known as dimerization. In the Magnets system, Vivid segments are attached to specific proteins or organelles. By using light, researchers can force their target molecules to come together and trigger signals that can change cell behavior. However, the Magnets system has limitations: its stability and low efficiency mean that the cells need to be kept at low temperatures and that several copies of Vivid are needed. These conditions can interfere with the activity of the target proteins. To expand the technique, Benedetti et al. added mutations to make the Vivid protein more similar to proteins found in fungi that thrive at temperatures around 50°C. These changes meant that the enhanced system could work at body temperature in mammals. Further mutations at the interface between the two Vivid segments improved the efficiency of dimerization. This enhanced version was put to the test in different applications, including delivering proteins to different organelles and bringing organelles together. The enhanced Magnets system should enable researchers to control a greater variety of signaling events in the cell. In addition, the methodology established for improving the efficiency of the Magnets system could be useful to researchers working on other proteins.