Defining Requirements for Heme Binding in PGRMC1 and Identifying Key Elements that Influence Protein Dimerization.

Progesterone receptor membrane component 1 (PGRMC1) binds heme via a surface-exposed site and displays some structural resemblance to cytochrome b5 despite their different functions. In the case of PGRMC1, it is the protein interaction with drug-metabolizing cytochrome P450s and the epidermal growth factor receptor that has garnered the most attention. These interactions are thought to result in a compromised ability to metabolize common chemotherapy agents and to enhance cancer cell proliferation. X-ray crystallography and immunoprecipitation data have suggested that heme-mediated PGRMC1 dimers are important for facilitating these interactions. However, more recent studies have called into question the requirement of heme binding for PGRMC1 dimerization. Our study employs spectroscopic and computational methods to probe and define heme binding and its impact on PGRMC1 dimerization. Fluorescence, electron paramagnetic resonance and circular dichroism spectroscopies confirm heme binding to apo-PGRMC1 and were used to demonstrate the stabilizing effect of heme on the wild-type protein. We also utilized variants (C129S and Y113F) to precisely define the contributions of disulfide bonds and direct heme coordination to PGRMC1 dimerization. Understanding the key factors involved in these processes has important implications for downstream protein-protein interactions that may influence the metabolism of chemotherapeutic agents. This work opens avenues for deeper exploration into the physiological significance of the truncated-PGRMC1 model and developing design principles for potential therapeutics to target PGRMC1 dimerization and downstream interactions.

The ß2-adrenergic receptor associates with CXCR4 multimers in human cancer cells.

While the existence and functional role of class C G-protein-coupled receptors (GPCR) dimers is well established, there is still a lack of consensus regarding class A and B GPCR multimerization. This lack of consensus is largely due to the inherent challenges of demonstrating the presence of multimeric receptor complexes in a physiologically relevant cellular context. The C-X-C motif chemokine receptor 4 (CXCR4) is a class A GPCR that is a promising target of anticancer therapy. Here, we investigated the potential of CXCR4 to form multimeric complexes with other GPCRs and characterized the relative size of the complexes in a live-cell environment. Using a bimolecular fluorescence complementation (BiFC) assay, we identified the ß2 adrenergic receptor (ß2AR) as an interaction partner. To investigate the molecular scale details of CXCR4-ß2AR interactions, we used a time-resolved fluorescence spectroscopy method called pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). PIE-FCCS can resolve membrane protein density, diffusion, and multimerization state in live cells at physiological expression levels. We probed CXCR4 and ß2AR homo- and heteromultimerization in model cell lines and found that CXCR4 assembles into multimeric complexes larger than dimers in MDA-MB-231 human breast cancer cells and in HCC4006 human lung cancer cells. We also found that ß2AR associates with CXCR4 multimers in MDA-MB-231 and HCC4006 cells to a higher degree than in COS-7 and CHO cells and in a ligand-dependent manner. These results suggest that CXCR4-ß2AR heteromers are present in human cancer cells and that GPCR multimerization is significantly affected by the plasma membrane environment.

The disordered C-terminal tail of fungal LPMOs from phytopathogens mediates protein dimerization and impacts plant penetration.

Lytic polysaccharide monooxygenases (LPMOs) are monocopper enzymes that oxidatively degrade various polysaccharides, such as cellulose. Despite extensive research on this class of enzymes, the role played by their C-terminal regions predicted to be intrinsically disordered (dCTR) has been overlooked. Here, we investigated the function of the dCTR of an LPMO, called CoAA9A, up-regulated during plant infection by Colletotrichum orbiculare, the causative agent of anthracnose. After recombinant production of the full-length protein, we found that the dCTR mediates CoAA9A dimerization in vitro, via a disulfide bridge, a hitherto-never-reported property that positively affects both binding and activity on cellulose. Using SAXS experiments, we show that the homodimer is in an extended conformation. In vivo, we demonstrate that gene deletion impairs formation of the infection-specialized cell called appressorium and delays penetration of the plant. Using immunochemistry, we show that the protein is a dimer not only in vitro but also in vivo when secreted by the appressorium. As these peculiar LPMOs are also found in other plant pathogens, our findings open up broad avenues for crop protection.

Parental histone transfer caught at the replication fork.

In eukaryotes, DNA compacts into chromatin through nucleosomes1,2. Replication of the eukaryotic genome must be coupled to the transmission of the epigenome encoded in the chromatin3,4. Here we report cryo-electron microscopy structures of yeast (Saccharomyces cerevisiae) replisomes associated with the FACT (facilitates chromatin transactions) complex (comprising Spt16 and Pob3) and an evicted histone hexamer. In these structures, FACT is positioned at the front end of the replisome by engaging with the parental DNA duplex to capture the histones through the middle domain and the acidic carboxyl-terminal domain of Spt16. The H2A-H2B dimer chaperoned by the carboxyl-terminal domain of Spt16 is stably tethered to the H3-H4 tetramer, while the vacant H2A-H2B site is occupied by the histone-binding domain of Mcm2. The Mcm2 histone-binding domain wraps around the DNA-binding surface of one H3-H4 dimer and extends across the tetramerization interface of the H3-H4 tetramer to the binding site of Spt16 middle domain before becoming disordered. This arrangement leaves the remaining DNA-binding surface of the other H3-H4 dimer exposed to additional interactions for further processing. The Mcm2 histone-binding domain and its downstream linker region are nested on top of Tof1, relocating the parental histones to the replisome front for transfer to the newly synthesized lagging-strand DNA. Our findings offer crucial structural insights into the mechanism of replication-coupled histone recycling for maintaining epigenetic inheritance.

Paired plant immune CHS3-CSA1 receptor alleles form distinct hetero-oligomeric complexes.

Plant intracellular nucleotide-binding leucine-rich repeat receptors (NLRs) analyzed to date oligomerize and form resistosomes upon activation to initiate immune responses. Some NLRs are encoded in tightly linked co-regulated head-to-head genes whose products function together as pairs. We uncover the oligomerization requirements for different Arabidopsis paired CHS3-CSA1 alleles. These pairs form resting-state heterodimers that oligomerize into complexes distinct from NLRs analyzed previously. Oligomerization requires both conserved and allele-specific features of the respective CHS3 and CSA1 Toll-like interleukin-1 receptor (TIR) domains. The receptor kinases BAK1 and BIRs inhibit CHS3-CSA1 pair oligomerization to maintain the CHS3-CSA1 heterodimer in an inactive state. Our study reveals that paired NLRs hetero-oligomerize and likely form a distinctive "dimer of heterodimers" and that structural heterogeneity is expected even among alleles of closely related paired NLRs.

Optimizing the Multimerization Properties of Quinoline-Based Allosteric HIV-1 Integrase Inhibitors.

Allosteric HIV-1 Integrase (IN) Inhibitors or ALLINIs bind at the dimer interface of the IN, away from the enzymatic catalytic site, and disable viral replication by inducing over-multimerization of IN. Interestingly, these inhibitors are capable of impacting both the early and late stages of viral replication. To better understand the important binding features of multi-substituted quinoline-based ALLINIs, we have surveyed published studies on IN multimerization and antiviral properties of various substituted quinolines at the 4, 6, 7, and 8 positions. Here we show how the efficacy of these inhibitors can be modulated by the nature of the substitutions at those positions. These features not only improve the overall antiviral potencies of these compounds but also significantly shift the selectivity toward the viral maturation stage. Thus, to fully maximize the potency of ALLINIs, the interactions between the inhibitor and multiple IN subunits need to be simultaneously optimized.

A Comprehensive Analysis of the Structural Recognition between KCTD Proteins and Cullin 3.

KCTD ((K)potassium Channel Tetramerization Domain-containing) proteins constitute an emerging class of proteins involved in fundamental physio-pathological processes. In these proteins, the BTB domain, which represents the defining element of the family, may have the dual role of promoting oligomerization and favoring functionally important partnerships with different interactors. Here, by exploiting the potential of recently developed methodologies for protein structure prediction, we report a comprehensive analysis of the interactions of all KCTD proteins with their most common partner Cullin 3 (Cul3). The data here presented demonstrate the impressive ability of this approach to discriminate between KCTDs that interact with Cul3 and those that do not. Indeed, reliable and stable models of the complexes were only obtained for the 15 members of the family that are known to interact with Cul3. The generation of three-dimensional models for all KCTD-Cul3 complexes provides interesting clues on the determinants of the structural basis of this partnership as clear structural differences emerged between KCTDs that bind or do not bind Cul3. Finally, the availability of accurate three-dimensional models for KCTD-Cul3 interactions may be valuable for the ad hoc design and development of compounds targeting specific KCTDs that are involved in several common diseases.

Synonymous Mutations Can Alter Protein Dimerization Through Localized Interface Misfolding Involving Self-entanglements.

Synonymous mutations in messenger RNAs (mRNAs) can reduce protein-protein binding substantially without changing the protein's amino acid sequence. Here, we use coarse-grain simulations of protein synthesis, post-translational dynamics, and dimerization to understand how synonymous mutations can influence the dimerization of two E. coli homodimers, oligoribonuclease and ribonuclease T. We synthesize each protein from its wildtype, fastest- and slowest-translating synonymous mRNAs in silico and calculate the ensemble-averaged interaction energy between the resulting dimers. We find synonymous mutations alter oligoribonuclease's dimer properties. Relative to wildtype, the dimer interaction energy becomes 4% and 10% stronger, respectively, when translated from its fastest- and slowest-translating mRNAs. Ribonuclease T dimerization, however, is insensitive to synonymous mutations. The structural and kinetic origin of these changes are misfolded states containing non-covalent lasso-entanglements, many of which structurally perturb the dimer interface, and whose probability of occurrence depends on translation speed. These entangled states are kinetic traps that persist for long time scales. Entanglements cause altered dimerization energies for oligoribonuclease, as there is a large association (odds ratio: 52) between the co-occurrence of non-native self-entanglements and weak-binding dimer conformations. Simulated at all-atom resolution, these entangled structures persist for long timescales, indicating the conclusions are independent of model resolution. Finally, we show that regions of the protein we predict to have changes in entanglement are also structurally perturbed during refolding, as detected by limited-proteolysis mass spectrometry. Thus, non-native changes in entanglement at dimer interfaces is a mechanism through which oligomer structure and stability can be altered.

Activation of Thoeris antiviral system via SIR2 effector filament assembly.

To survive bacteriophage (phage) infections, bacteria developed numerous anti-phage defence systems1-7. Some of them (for example, type III CRISPR-Cas, CBASS, Pycsar and Thoeris) consist of two modules: a sensor responsible for infection recognition and an effector that stops viral replication by destroying key cellular components8-12. In the Thoeris system, a Toll/interleukin-1 receptor (TIR)-domain protein, ThsB, acts as a sensor that synthesizes an isomer of cyclic ADP ribose, 1''-3' glycocyclic ADP ribose (gcADPR), which is bound in the Smf/DprA-LOG (SLOG) domain of the ThsA effector and activates the silent information regulator 2 (SIR2)-domain-mediated hydrolysis of a key cell metabolite, NAD+ (refs. 12-14). Although the structure of ThsA has been solved15, the ThsA activation mechanism remained incompletely understood. Here we show that 1''-3' gcADPR, synthesized in vitro by the dimeric ThsB' protein, binds to the ThsA SLOG domain, thereby activating ThsA by triggering helical filament assembly of ThsA tetramers. The cryogenic electron microscopy (cryo-EM) structure of activated ThsA revealed that filament assembly stabilizes the active conformation of the ThsA SIR2 domain, enabling rapid NAD+ depletion. Furthermore, we demonstrate that filament formation enables a switch-like response of ThsA to the 1''-3' gcADPR signal.

Microsecond dynamics control the HIV-1 Envelope conformation.

The HIV-1 Envelope (Env) glycoprotein facilitates host cell fusion through a complex series of receptor-induced structural changes. Although remarkable progress has been made in understanding the structures of various Env conformations, microsecond timescale dynamics have not been studied experimentally. Here, we used time-resolved, temperature-jump small-angle x-ray scattering to monitor structural rearrangements in an HIV-1 Env SOSIP ectodomain construct with microsecond precision. In two distinct Env variants, we detected a transition that correlated with known Env structure rearrangements with a time constant in the hundreds of microseconds range. A previously unknown structural transition was also observed, which occurred with a time constant below 10 µs, and involved an order-to-disorder transition in the trimer apex. Using this information, we engineered an Env SOSIP construct that locks the trimer in the prefusion closed state by connecting adjacent protomers via disulfides. Our findings show that the microsecond timescale structural dynamics play an essential role in controlling the Env conformation with impacts on vaccine design.

Roles of RNA scaffolding in nanoscale Gag multimerization and selective protein sorting at HIV membranes.

HIV-1 Gag proteins can multimerize upon the viral genomic RNA or multiple random cellular messenger RNAs to form a virus particle or a virus-like particle, respectively. To date, whether the two types of particles form via the same Gag multimerization process has remained unclarified. Using photoactivated localization microscopy to illuminate Gag organizations and dynamics at the nanoscale, here, we showed that genomic RNA mediates Gag multimerization in a more cluster-centric, cooperative, and spatiotemporally coordinated fashion, with the ability to drive dense Gag clustering dependent on its ability to act as a long-stranded scaffold not easily attainable by cellular messenger RNAs. These differences in Gag multimerization were further shown to affect downstream selective protein sorting into HIV membranes, indicating that the choice of RNA for packaging can modulate viral membrane compositions. These findings should advance the understanding of HIV assembly and further benefit the development of virus-like particle-based therapeutics.

DNA Sequence Control of Enzyme Filamentation and Activation of the SgrAI Endonuclease.

Enzyme polymerization (also known as filamentation) has emerged as a new layer of enzyme regulation. SgrAI is a sequence-dependent DNA endonuclease that forms polymeric filaments with enhanced DNA cleavage activity as well as altered DNA sequence specificity. To better understand this unusual regulatory mechanism, full global kinetic modeling of the reaction pathway, including the enzyme filamentation steps, has been undertaken. Prior work with the primary DNA recognition sequence cleaved by SgrAI has shown how the kinetic rate constants of each reaction step are tuned to maximize activation and DNA cleavage while minimizing the extent of DNA cleavage to the host genome. In the current work, we expand on our prior study by now including DNA cleavage of a secondary recognition sequence, to understand how the sequence of the bound DNA modulates filamentation and activation of SgrAI. The work shows that an allosteric equilibrium between low and high activity states is modulated by the sequence of bound DNA, with primary sequences more prone to activation and filament formation, while SgrAI bound to secondary recognition sequences favor the low (and nonfilamenting) state by up to 40-fold. In addition, the degree of methylation of secondary sequences in the host organism, Streptomyces griseus, is now reported for the first time and shows that as predicted, these sequences are left unprotected from the SgrAI endonuclease making sequence specificity critical in this unusual filament-forming enzyme.

Amplified and Specific Staining of Protein Dimerization on Cell Membrane Catalyzed by Responsively Installed DNA Nanomachines for Cancer Diagnosis.

In situ staining of protein dimerization on cell membrane has an important significance in accurate diagnosis during perioperative period, yet facile integration of specific recognition function and local signal conversion/amplification abilities on membrane surface remains a great challenge. Herein, a two-stage catalytic strategy is developed by installing DNA nanomachines and employing. Specifically, dual-aptamer-assisted DNA scaffold perform a "bispecific recognition-then-computing" operation and the output signal initiate a membrane-anchored biocatalysis for self-assembly of DNA catalytic converters, that is, G-quadruplex nanowire/hemin DNAzyme. Then, localized-deposition of chromogenic polydopamine is chemically catalyzed by horseradish peroxidase-mimicking DNAzyme and guided by supramolecular interactions between conjugate rigid plane of G-tetrad and polydopamine oligomer. The catalytic products exhibit nanofiber morphology with a diameter of 80-120 nm and a length of 1-10 µm, and one-to-one localize on DNA scaffold for amplified and specific staining of protein dimers. The bispecific staining leads to a higher (≈3.4-fold) signal intensity than traditional immunohistochemistry, which is beneficial for direct visualization. Moreover, an efficient discrimination ability of the bispecific staining strategy is observed in co-culture model staining. This study provides a novel catalytic method for controlling deposition of chromogens and paves a new avenue to sensitively stain of protein-protein interactions in disease diagnosis.

Rapid Optogenetic Clustering in the Cytoplasm with BcLOVclust.

Protein clustering is a powerful form of optogenetic control, yet remarkably few proteins are known to oligomerize with light. Recently, the photoreceptor BcLOV4 was found to form protein clusters in mammalian cells in response to blue light, although clustering coincided with its translocation to the plasma membrane, potentially constraining its application as an optogenetic clustering module. Herein we identify key amino acids that couple BcLOV4 clustering to membrane binding, allowing us to engineer a variant that clusters in the cytoplasm and does not associate with the membrane in response to blue light. This variant-called BcLOVclust-clustered over many cycles with substantially faster clustering and de-clustering kinetics compared to the widely used optogenetic clustering protein Cry2. The magnitude of clustering could be strengthened by appending an intrinsically disordered region from the fused in sarcoma (FUS) protein, or by selecting the appropriate fluorescent protein to which it was fused. Like wt BcLOV4, BcLOVclust activity was sensitive to temperature: light-induced clusters spontaneously dissolved at a rate that increased with temperature despite constant illumination. At low temperatures, BcLOVclust and Cry2 could be multiplexed in the same cells, allowing light control of independent protein condensates. BcLOVclust could also be applied to control signaling proteins and stress granules in mammalian cells. While its usage is currently best suited in cells and organisms that can be cultured below ∼30 °C, a deeper understanding of BcLOVclust thermal response will further enable its use at physiological mammalian temperatures.

Comparative analysis of two Caenorhabditis elegans kinesins KLP-6 and UNC-104 reveals a common and distinct activation mechanism in kinesin-3.

Kinesin-3 is a family of microtubule-dependent motor proteins that transport various cargos within the cell. However, the mechanism underlying kinesin-3 activations remains largely elusive. In this study, we compared the biochemical properties of two Caenorhabditis elegans kinesin-3 family proteins, KLP-6 and UNC-104. Both KLP-6 and UNC-104 are predominantly monomeric in solution. As previously shown for UNC-104, non-processive KLP-6 monomer is converted to a processive motor when artificially dimerized. We present evidence that releasing the autoinhibition is sufficient to trigger dimerization of monomeric UNC-104 at nanomolar concentrations, which results in processive movement of UNC-104 on microtubules, although it has long been thought that enrichment in the phospholipid microdomain on cargo vesicles is required for the dimerization and processive movement of UNC-104. In contrast, KLP-6 remains to be a non-processive monomer even when its autoinhibition is unlocked, suggesting a requirement of other factors for full activation. By examining the differences between KLP-6 and UNC-104, we identified a coiled-coil domain called coiled-coil 2 (CC2) that is required for the efficient dimerization and processive movement of UNC-104. Our results suggest a common activation mechanism for kinesin-3 family members, while also highlighting their diversification.

PAS Dimerization at the Nexus of the Mammalian Circadian Clock.

Circadian rhythms are genetically encoded molecular clocks for internal biological timekeeping. Organisms from single-cell bacteria to humans use these clocks to adapt to the external environment and synchronize their physiology and behavior to solar light/dark cycles. Although the proteins that constitute the molecular 'cogs' and give rise to circadian rhythms are now known, we still lack a detailed understanding of how these proteins interact to generate and sustain the ∼24-hour circadian clock. Structural studies have helped to expand the architecture of clock proteins and have revealed the abundance of the only well-defined structured regions in the mammalian clock called Per-ARNT-Sim (PAS) domains. PAS domains are modular, evolutionarily conserved sensory and signaling domains that typically mediate protein-protein interactions. In the mammalian circadian clock, PAS domains modulate homo and heterodimerization of several core clock proteins that assemble into transcription factors or repressors. This review will focus on the functional importance of the PAS domains in the circadian clock from a biophysical and biochemical standpoint and describe their roles in clock protein interactions and circadian timekeeping.

Structural basis of Gabija anti-phage defence and viral immune evasion.

Bacteria encode hundreds of diverse defence systems that protect them from viral infection and inhibit phage propagation1-5. Gabija is one of the most prevalent anti-phage defence systems, occurring in more than 15% of all sequenced bacterial and archaeal genomes1,6,7, but the molecular basis of how Gabija defends cells from viral infection remains poorly understood. Here we use X-ray crystallography and cryo-electron microscopy (cryo-EM) to define how Gabija proteins assemble into a supramolecular complex of around 500 kDa that degrades phage DNA. Gabija protein A (GajA) is a DNA endonuclease that tetramerizes to form the core of the anti-phage defence complex. Two sets of Gabija protein B (GajB) dimers dock at opposite sides of the complex and create a 4:4 GajA-GajB assembly (hereafter, GajAB) that is essential for phage resistance in vivo. We show that a phage-encoded protein, Gabija anti-defence 1 (Gad1), directly binds to the Gabija GajAB complex and inactivates defence. A cryo-EM structure of the virally inhibited state shows that Gad1 forms an octameric web that encases the GajAB complex and inhibits DNA recognition and cleavage. Our results reveal the structural basis of assembly of the Gabija anti-phage defence complex and define a unique mechanism of viral immune evasion.

Characterization of erythroferrone oligomerization and its impact on BMP antagonism.

Hepcidin, a peptide hormone that negatively regulates iron metabolism, is expressed by bone morphogenetic protein (BMP) signaling. Erythroferrone (ERFE) is an extracellular protein that binds and inhibits BMP ligands, thus positively regulating iron import by indirectly suppressing hepcidin. This allows for rapid erythrocyte regeneration after blood loss. ERFE belongs to the C1Q/TNF-related protein family and is suggested to adopt multiple oligomeric forms: a trimer, a hexamer, and a high molecular weight species. The molecular basis for how ERFE binds BMP ligands and how the different oligomeric states impact BMP inhibition are poorly understood. In this study, we demonstrated that ERFE activity is dependent on the presence of stable dimeric or trimeric ERFE and that larger species are dispensable for BMP inhibition. Additionally, we used an in silico approach to identify a helix, termed the ligand-binding domain, that was predicted to bind BMPs and occlude the type I receptor pocket. We provide evidence that the ligand-binding domain is crucial for activity through luciferase assays and surface plasmon resonance analysis. Our findings provide new insight into how ERFE oligomerization impacts BMP inhibition, while identifying critical molecular features of ERFE essential for binding BMP ligands.

Decoding Allosteric Control in Hypoxia-Inducible Factors.

The mammalian family of basic helix-loop-helix-PER-ARNT-SIM (bHLH-PAS) transcription factors possess the ability to sense and respond to diverse environmental and physiological cues. These proteins all share a common structural framework, comprising a bHLH domain, two PAS domains, and transcriptional activation or repression domain. To function effectively as transcription factors, members of the family must form dimers, bringing together bHLH segments to create a functional unit that allows for DNA response element binding. The significance of bHLH-PAS family is underscored by their involvement in many major human diseases, offering potential avenues for therapeutic intervention. Notably, the clear identification of ligand-binding cavities within their PAS domains enables the development of targeted small molecules. Two examples are Belzutifan, targeting hypoxia-inducible factor (HIF)-2α, and Tapinarof, targeting the aryl hydrocarbon receptor (AHR), both of which have gained regulatory approval recently. Here, we focus on the HIF subfamily. The crystal structures of all three HIF-α proteins have been elucidated, revealing their bHLH and tandem PAS domains are used to engage their dimerization partner aryl hydrocarbon receptor nuclear translocator (ARNT, also called HIF-1ß). A broad range of recent findings point to a shared allosteric modulation mechanism among these proteins, whereby small-molecules at the PAS-B domains exert direct influence over the HIF-α transcriptional functions. As our understanding of the architectural and allosteric mechanisms of bHLH-PAS proteins continues to advance, the possibility of discovering new therapeutic drugs becomes increasingly promising.

The AhR Signaling Mechanism: A Structural Point of View.

The Aryl hydrocarbon Receptor (AhR) is a well-known sensor of xenobiotics; moreover, it is considered a promising drug target as it is involved in the regulation of many patho-physiological processes. For these reasons the study of its ligand-activated transcription mechanism has stimulated several studies for over twenty years. In this review we highlight the key role of molecular structural information in understanding the different steps of the signaling mechanism. The architecture of the AhR cytosolic complex, encompassing the hsp90 chaperone protein and the XAP2 and p23 co-chaperones, has become available in the last year thanks to Cryo-EM experiments. The structure of the AhR ligand-binding (PAS-B) domain has remained elusive for a long time; it has been predicted by homology modelling, based on known PAS systems, and its ligand-bound forms were modelled through ligand molecular docking. Although very recently some structural information on this domain has become available, considerable efforts are still needed to determine the binding geometries of the AhR key ligands by experimental high-resolution studies. On the other hand, the dimeric structure of AhR with the ARNT protein, bound to the specific DNA responsive element, was partially determined by X-ray crystallography and it was completed by homology modelling. On the whole the current structural knowledge of the main protein complexes that form over the AhR mechanism opens the way to confirm and further investigate the main steps of the proposed ligand-activated transcription mechanism of the AhR.