Trimer Of Dimers Research Articles

Oligomerization of Protein Arginine Methyltransferase 1 and Its Functional Impact on Substrate Arginine Methylation

Protein arginine methyltransferases (PRMTs) are important post-translational modifying enzymes in eukaryotic proteins and regulate diverse pathways from gene transcription, RNA splicing, and signal transduction to metabolism. Increasing evidence supports that PRMTs exhibit the capacity to form higher-order oligomeric structures, but the structural basis of PRMT oligomerization and its functional consequence are elusive. Herein, we revealed for the first time different oligomeric structural forms of the predominant arginine methyltransferase PRMT1 using cryogenic electron microscopy, which included tetramer (dimer of dimers), hexamer (trimer of dimers), octamer (tetramer of dimers), decamer (pentamer of dimers), and also helical filaments. Through a host of biochemical assays, we showed that PRMT1 methyltransferase activity was substantially enhanced as a result of the high-ordered oligomerization. High-ordered oligomerization increased the catalytic turnover and the multi-methylation processivity of PRMT1. Presence of a catalytically-dead PRMT1 mutant also abled enhanced activity of wild-type PRMT1, pointing out a non-catalytic role of oligomerization. Structural modeling demonstrates that oligomerization enhances substrate retention at the PRMT1 surface through electrostatic force. Our studies offered key insights into PRMT1 oligomerization and established that oligomerization constitutes a novel molecular mechanism that positively regulates the enzymatic activity of PRMTs in biology.

The CRISPR-associated adenosine deaminase Cad1 converts ATP to ITP to provide antiviral immunity.

Type III CRISPR systems provide immunity against genetic invaders through the production of cyclic oligo-adenylate (cAn) molecules that activate effector proteins that contain CRISPR-associated Rossman fold (CARF) domains. Here, we characterized the function and structure of an effector in which the CARF domain is fused to an adenosine deaminase domain, CRISPR-associated adenosine deaminase 1 (Cad1). We show that upon binding of cA4 or cA6 to its CARF domain, Cad1 converts ATP to ITP, both invivo and invitro. Cryoelectron microscopy (cryo-EM) structural studies on full-length Cad1 reveal an hexameric assembly composed of a trimer of dimers, with bound ATP at inter-domain sites required for activity and ATP/ITP within deaminase active sites. Upon synthesis of cAn during phage infection, Cad1 activation leads to a growth arrest of the host that prevents viral propagation. Our findings reveal that CRISPR-Cas systems employ a wide range of molecular mechanisms beyond nucleic acid degradation to provide adaptive immunity in prokaryotes.

De Novo Designed Cell-Penetrating Peptide Self-Assembly Featuring Distinctive Tertiary Structure.

Recent attention has focused on the de novo design of proteins, paralleling advancements in biopharmaceuticals. Achieving protein designs with both structure and function poses a significant challenge, particularly considering the importance of quaternary structures, such as oligomers, in protein function. The cell penetration properties of peptides are of particular interest as they involve the penetration of large molecules into cells. We previously suggested a link between the oligomerization propensity of amphipathic peptides and their cell penetration abilities, yet concrete evidence at cellular-relevant concentrations was lacking due to oligomers' instability. In this study, we sought to characterize oligomerization states using various techniques, including X-ray crystallography, acceptor photobleaching Förster resonance energy transfer (FRET), native mass spectrometry (MS), and differential scanning calorimetry (DSC), while exploring the function related to oligomer status. X-ray crystallography revealed the atomic structures of oligomers formed by LK-3, a bis-disulfide bridged dimer with amino acid sequence LKKLCLKLKKLCKLAG, and its derivatives, highlighting the formation of hexamers, specifically the trimer of dimers, which exhibited a stable hydrophobic core. FRET experiments showed that LK-3 oligomer formation was associated with cell penetration. Native MS confirmed higher-order oligomers of LK-3, while an intriguing finding was the enhanced cell-penetrating capability of a 1:1 mixture of l/d-peptide dimers compared to pure enantiomers. DSC analysis supported the notion that this enantiomeric mixture promotes the formation of functional oligomers, crucial for cell penetration. In conclusion, our study provides direct evidence that amphipathic peptide LK-3 forms oligomers at low nanomolar concentrations, underscoring their significance in cell penetration behavior.

Cyanobacterial α-carboxysome carbonic anhydrase is allosterically regulated by the Rubisco substrate RuBP.

Cyanobacterial CO2 concentrating mechanisms (CCMs) sequester a globally consequential proportion of carbon into the biosphere. Proteinaceous microcompartments, called carboxysomes, play a critical role in CCM function, housing two enzymes to enhance CO2 fixation: carbonic anhydrase (CA) and Rubisco. Despite its importance, our current understanding of the carboxysomal CAs found in α-cyanobacteria, CsoSCA, remains limited, particularly regarding the regulation of its activity. Here, we present a structural and biochemical study of CsoSCA from the cyanobacterium Cyanobium sp. PCC7001. Our results show that the Cyanobium CsoSCA is allosterically activated by the Rubisco substrate ribulose-1,5-bisphosphate and forms a hexameric trimer of dimers. Comprehensive phylogenetic and mutational analyses are consistent with this regulation appearing exclusively in cyanobacterial α-carboxysome CAs. These findings clarify the biologically relevant oligomeric state of α-carboxysomal CAs and advance our understanding of the regulation of photosynthesis in this globally dominant lineage.

Structure of the prenyltransferase in bifunctional copalyl diphosphate synthase from Penicillium fellutanum reveals an open hexamer conformation

Copalyl diphosphate synthase from Penicillium fellutanum (PfCPS) is an assembly-line terpene synthase that contains both prenyltransferase and class II cyclase activities. The prenyltransferase catalyzes processive chain elongation reactions using dimethylallyl diphosphate and three equivalents of isopentenyl diphosphate to yield geranylgeranyl diphosphate, which is then utilized as a substrate by the class II cyclase domain to generate copalyl diphosphate. Here, we report the 2.81 Å-resolution cryo-EM structure of the hexameric prenyltransferase of full-length PfCPS, which is surrounded by randomly splayed-out class II cyclase domains connected by disordered polypeptide linkers. The hexamer can be described as a trimer of dimers; surprisingly, one of the three dimer-dimer interfaces is separated to yield an open hexamer conformation, thus breaking the D3 symmetry typically observed in crystal structures of other prenyltransferase hexamers such as wild-type human GGPP synthase (hGGPPS). Interestingly, however, an open hexamer conformation was previously observed in the crystal structure of D188Y hGGPPS, apparently facilitated by hexamer-hexamer packing in the crystal lattice. The cryo-EM structure of the PfCPS prenyltransferase hexamer is the first to reveal that an open conformation can be achieved even in the absence of a point mutation or interaction with another hexamer. Even though PfCPS octamers are not detected, we suggest that the open hexamer conformation represents an intermediate in the hexamer-octamer equilibrium for those prenyltransferases that do exhibit oligomeric heterogeneity.

PtuA and PtuB assemble into an inflammasome-like oligomer for anti-phage defense.

Escherichia coli Septu system, an anti-phage defense system, comprises two components: PtuA and PtuB. PtuA contains an ATPase domain, while PtuB is predicted to function as a nuclease. Here we show that PtuA and PtuB form a stable complex with a 6:2 stoichiometry. Cryo-electron microscopy structure of PtuAB reveals a distinctive horseshoe-like configuration. PtuA adopts a hexameric arrangement, organized as an asymmetric trimer of dimers, contrasting the ring-like structure by other ATPases. Notably, the three pairs of PtuA dimers assume distinct conformations and fulfill unique roles in recruiting PtuB. Our functional assays have further illuminated the importance of the oligomeric assembly of PtuAB in anti-phage defense. Moreover, we have uncovered that ATP molecules can directly bind to PtuA and inhibit the activities of PtuAB. Together, the assembly and function of the Septu system shed light on understanding other ATPase-containing systems in bacterial immunity.

The cytoplasmic N-terminal tail of Zika virus NS4A protein forms oligomers in the absence of detergent or lipids

The non-structural (NS) NS4A protein in flaviviruses has three predicted transmembrane domains, is critical for virulence and participates in membrane morphogenesis. In Dengue virus (DENV), both hydrophylic N-terminal tail and its first transmembrane domain participate in the formation of oligomers which are important for pathogenicity. However, the relative importance of the N-terminal domain in oligomerization has been under debate. In particular, since in the absence of detergent or lipids, this domain (residues 1–48) in both DENV and Zika virus (ZIKV) NS4A, was found to be disordered. Recently, however, we reported preliminary data that showed that peptide ZIKV NS4A 4–58 adopts a defined secondary structure in aqueous solution and forms oligomers, signaling its importance for full length NS4A oligomerization. Herein we have performed detailed analytical ultracentrifugation experiments to further characterize the oligomerization of this peptide and also a shorter variant (residues 4–44). In both cases, sedimentation velocity produced a single species with concentration-dependent sedimentation coefficient, consistent with a fast equilibrium between at least two species. Combining sedimentation velocity and equilibrium experiments, data is best fitted to a monomer–dimer–trimer equilibrium. Possible models of NS4A oligomers obtained with AlphaFold-2 predict the stabilizing role for residues in this N-terminal domain, such as Arg20, Asn27, Ala44 and Glu50, all at highly conserved positions in flavivirus NS4A proteins. Our results are thus consistent with N-terminal domain interactions acting as one of the driving forces for NS4A homo-oligomerization.

Solubilization, purification, and characterization of the hexameric form of phosphatidylserine synthase from Candida albicans

Phosphatidylserine (PS) synthase from Candida albicans, encoded by the CHO1 gene, has been identified as a potential drug target for new antifungals against systemic candidiasis. Rational drug design or small molecule screening are effective ways to identify specific inhibitors of Cho1, but both will be facilitated by protein purification. Due to the transmembrane nature of Cho1, methods were needed to solubilize and purify the native form of Cho1. Here, we used six non-ionic detergents and three styrene maleic acids (SMAs) to solubilize an HA-tagged Cho1 protein from the total microsomal fractions. Blue native PAGE and immunoblot analysis revealed a single band corresponding to Cho1 in all detergent-solubilized fractions, while two bands were present in the SMA2000-solubilized fraction. Our enzymatic assay suggests that digitonin-or DDM-solubilized enzyme has the most PS synthase activity. Pull-downs of HA-tagged Cho1 from the digitonin-solubilized fraction reveal an apparent MW of Cho1 consistent with a hexamer. Furthermore, negative-staining electron microscopy analysis and AlphaFold2 structure prediction modeling suggest the hexamer is composed of a trimer of dimers. We purified Cho1 protein to near-homogeneity as a hexamer using affinity chromatography and TEV protease treatment, and optimized Cho1 enzyme activity for manganese and detergent concentrations, temperature (24 °C), and pH (8.0). The purified Cho1 has a Km for its substrate CDP-diacylglycerol of 72.20μM with a Vmax of 0.079nmol/(μg∗min) while exhibiting a sigmoidal kinetic curve for its other substrate serine, indicating cooperative binding. Purified hexameric Cho1 can potentially be used in downstream structure determination and small drug screening.

A dimer between monomers and hexamers-Oligomeric variations in glucosamine-6-phosphate deaminase family.

In bacteria that live in hosts whose terminal sugar is a sialic acid, Glucosamine-6-phosphate deaminase (NagB) catalyzes the last step in converting sialic acid into Fructose-6-phosphate. These bacteria then use the Fructose-6-phosphate as an energy source. The enzyme NagB exists as a hexamer in Gram-negative bacteria and is allosterically regulated. In Gram-positive bacteria, it exists as a monomer and lacks allosteric regulation. Our identification of a dimeric Gram-negative bacterial NagB motivated us to characterize the structural basis of two closely related oligomeric forms. We report here the crystal structures of NagB from two Gram-negative pathogens, Haemophilus influenzae (Hi) and Pasturella multocida (Pm). The Hi-NagB is active as a hexamer, while Pm-NagB is active as a dimer. Both Hi-NagB and Pm-NagB contain the C-terminal helix implicated as essential for hexamer formation. The hexamer is described as a dimer of trimers. In the Pm-NagB dimer, the dimeric interface is conserved. The conservation of the dimer interface suggests that the three possible oligomeric forms of NagB are a monomer, a dimer, and a trimer of dimers. Computational modeling and MD simulations indicate that the residues at the trimeric interface have less stabilizing energy of oligomer formation than those in the dimer interface. We propose that Pm-NagB is the evolutionary link between the monomer and the hexamer forms.

The apo‐form of the Vibrio cholerae replicative helicase DnaB is a labile and inactive planar trimer of dimers

To enable chromosomal replication, DNA is unwound by the ATPase molecular motor replicative helicase. The bacterial helicase DnaB is a ring-shaped homo-hexamer whose conformational dynamics are being studied through its different 3D structural states adopted along its functional cycle. Our findings describe a new crystal structure for the apo-DnaB from Vibrio cholerae, forming a planar hexamer with pseudo-symmetry, constituted by a trimer of dimers in which the C-terminal domains delimit a triskelion-shaped hole. This hexamer is labile and inactive. We suggest that it represents an intermediate state allowing the formation of the active NTP-bound hexamer from dimers.

Solubilization and purification of phosphatidylserine synthase from Candida albicans

Systemic infections by Candida spp. are associated with high mortality rates, and this is related, in part, to limitations in current antifungals. In this regard, the phosphatidylserine (PS) synthase from Candida albicans, encoded by the CHO1 gene, has been identified as a potential drug target for new antifungals. The reasons include: 1) Cho1 is required for oral and systemic Candida infection in the mouse model and is essential for viability in the fungal pathogen Cryptococcus neoformans; 2) this gene and gene product is absent in humans, and 3) it is a highly conserved protein among many human fungal pathogens. Inhibitors of Cho1 could be used as lead compounds for drug development. Small molecule screening is one way to develop specific inhibitors. This requires protein purification of the drug target. Due to the transmembrane nature of Cho1, therefore, we aim to solubilize and purify the native form of the Cho1 protein. Initially, four nonionic detergents (Digitonin, n‐Dodecyl‐b‐D‐ Maltopyranoside [DDM], n‐Tetradecyl‐b‐D‐Maltopyranoside [TDM], and Triton X‐100) were used to solubilize an HA‐tagged Cho1 protein from the total microsomal fraction. Analysis by Blue Native PAGE (BN‐PAGE) and immunoblotting revealed a single band corresponding to Cho1 protein in these four solubilized fractions. A radiochemical enzymatic assay suggests that the digitonin‐ and DDM‐solubilized fractions have more PS synthase activity than the TDM‐ and Triton X‐100‐ solubilized fractions. Using a pulldown assay for the HA‐tagged Cho1 in the digitonin‐solubilized fraction reveals a single band in BN‐PAGE, and a subsequent 2D gel electrophoresis confirms the purity of this protein following silver staining. The comparative mobility by BN‐PAGE gives an apparent MW of Cho1 of ~178 kDa. Based on the amino acid sequence, this would suggest a hexameric form of the CandidaCho1. This is quite different from the structure of other membrane phospholipid synthases, which all appear to be dimers. Preliminary negative staining and TEM single particle analysis suggests the presence of a hexamer population of Cho1 complex in the pulldown sample. The hexametric Cho1 is predicted to be assembled as a trimer of dimers. As expected, a subsequent 2D particle classification on the TEM images has shown different assemblies of Cho1 dimers, mostly trimers of dimers, strengthening the claim that Cho1 forms native trimers of dimers.In order to achieve large‐scale purification for small molecule screening, three protein affinity tags (His8‐, GST‐, & MBP‐tag) were added to the Cho1 protein, and a TEV protease recognition sequence was also added between Cho1 protein and the tags. All three different versions of Cho1 protein showed expression, but His8‐tagged Cho1 stands out among the others because it retains protein activity and is compatible with digitonin. Though nonspecific bands exist in the initial His‐tag affinity purification, a subsequent TEV protease treatment and a second His‐tag affinity purification were able to clear the nonspecific bands and result in a very homogeneous band of Cho1 protein. In sum, we are able to achieve an over‐500‐fold and microgram‐scale purification of Cho1. We plan to further characterize the pure Cho1 protein and use it in future small molecule screening.

Structure-function relationship of a novel fucoside-binding fruiting body lectin from Coprinopsis cinerea exhibiting nematotoxic activity.

Lectins are non-immunoglobulin-type proteins that bind to specific carbohydrate epitopes and play important roles in intra- and inter-organismic interactions. Here, we describe a novel fucose-specific lectin, termed CML1, which we identified from fruiting body extracts of Coprinopsis cinerea. For further characterization, the coding sequence for CML1 was cloned and heterologously expressed in Escherichia coli. Feeding of CML1-producing bacteria inhibited larval development of the bacterivorous nematode Caenorhabditis tropicalis, but not of C. elegans. The crystal structure of the recombinant protein in its apo-form and in complex with H type I or Lewis A blood group antigens was determined by X-ray crystallography. The protein folds as a sandwich of 2 antiparallel β-sheets and forms hexamers resulting from a trimer of dimers. The hexameric arrangement was confirmed by small-angle X-ray scattering (SAXS). One carbohydrate-binding site per protomer was found at the dimer interface with both protomers contributing to ligand binding, resulting in a hexavalent lectin. In terms of lectin activity of recombinant CML1, substitution of the carbohydrate-interacting residues His54, Asn55, Trp94, and Arg114 by Ala abolished carbohydrate-binding and nematotoxicity. Although no similarities to any characterized lectin were found, sequence alignments identified many non-characterized agaricomycete proteins. These results suggest that CML1 is the founding member of a novel family of fucoside-binding lectins involved in the defense of agaricomycete fruiting bodies against predation by fungivorous nematodes.

Hexameric rings of the scaffolding protein CheW enhance response sensitivity and cooperativity in Escherichia coli chemoreceptor arrays.

The Escherichia coli chemoreceptor array is a supramolecular assembly that enables cells to respond to extracellular cues dynamically and with great precision and sensitivity. In the array, transmembrane receptors organized as trimers of dimers are connected at their cytoplasmic tips by hexameric rings of alternating subunits of the kinase CheA and the scaffolding protein CheW (CheA-CheW rings). Interactions of CheW molecules with the members of receptor trimers not directly bound to CheA-CheW rings may lead to the formation of hexameric CheW rings in the chemoreceptor array. Here, we detected such CheW rings with a cellular cysteine-directed cross-linking assay and explored the requirements for their formation and their participation in array assembly. We found that CheW ring formation varied with cellular CheW abundance, depended on the presence of receptors capable of a trimer-of-dimers arrangement, and did not require CheA. Cross-linking studies of a CheA~CheW fusion protein incapable of forming homomeric CheW oligomers demonstrated that CheW rings were not essential for the assembly of CheA-containing arrays. Förster resonance energy transfer (FRET)-based kinase assays of arrays containing variable amounts of CheW rings revealed that CheW rings enhanced the cooperativity and the sensitivity of the responses to attractants. We propose that six-membered CheW rings provide the additional interconnectivity required for optimal signaling and gradient tracking performance by chemosensory arrays.

Pseudomonas aeruginosa C-Terminal Processing Protease CtpA Assembles into a Hexameric Structure That Requires Activation by a Spiral-Shaped Lipoprotein-Binding Partner.

ABSTRACTPseudomonas aeruginosa CtpA is a carboxyl-terminal processing protease that partners with the outer membrane lipoprotein LbcA to degrade at least five cell wall-associated proteins, four of which are cell wall hydrolases. This activity plays an important role in supporting P. aeruginosa virulence in a mouse model of acute pneumonia. However, almost nothing is known about the molecular mechanisms underlying CtpA and LbcA function. Here, we used structural analysis to show that CtpA alone assembles into an inactive hexamer comprising a trimer of dimers, which limits its substrate access and prevents nonspecific degradation. The adaptor protein LbcA is a right-handed open spiral with 11 tetratricopeptide repeats, which might wrap around a substrate to deliver it to CtpA for degradation. By structure-guided mutagenesis and functional assays, we also showed that the interfaces of the CtpA trimer of dimers and an N-terminal helix of LbcA are important for LbcA-mediated substrate degradation by CtpA both in vitro and in vivo. This work improves our understanding of the molecular mechanism of the LbcA-CtpA proteolytic system and reveals some striking differences from the arrangements found in some other bacterial CTPs.

X-ray diffraction and in vivo studies reveal the quinary structure of Trypanosoma cruzi nucleoside diphosphate kinase 1: a novel helical oligomer structure.

Trypanosoma cruzi is a flagellated protozoan parasite that causes Chagas disease, which represents a serious health problem in the Americas. Nucleoside diphosphate kinases (NDPKs) are key enzymes that are implicated in cellular energy management. TcNDPK1 is the canonical isoform in the T. cruzi parasite. TcNDPK1 has a cytosolic, perinuclear and nuclear distribution. It is also found in non-membrane-bound filaments adjacent to the nucleus. In the present work, X-ray diffraction and in vivo studies of TcNDPK1 are described. The structure reveals a novel, multi-hexameric, left-handed helical oligomer structure. The results of directed mutagenesis studies led to the conclusion that the microscopic TcNDPK1 granules observed in vivo in T. cruzi parasites are made up by the association of TcNDPK1 oligomers. In the absence of experimental data, analysis of the interactions in the X-ray structure of the TcNDPK1 oligomer suggests the probable assembly and disassembly steps: dimerization, assembly of the hexamer as a trimer of dimers, hexamer association to generate the left-handed helical oligomer structure and finally oligomer association in a parallel manner to form the microscopic TcNDPK1 filaments that are observed in vivo in T. cruzi parasites. Oligomer disassembly takes place on the binding of substrate in the active site of TcNDPK1, leading to dissociation of the hexamers. This study constitutes the first report of such a protein arrangement, which has never previously been seen for any protein or NDPK. Further studies are needed to determine its physiological role. However, it may suggest a paradigm for protein storage reflecting the complex mechanism of action of TcNDPK1.

Disassembly of Single Virus Capsids Monitored in Real Time with Multicycle Resistive-Pulse Sensing.

Virus assembly and disassembly are critical steps in the virus lifecycle; however, virus disassembly is much less well understood than assembly. For hepatitis B virus (HBV) capsids, disassembly of the virus capsid in the presence of guanidine hydrochloride (GuHCl) exhibits strong hysteresis that requires additional chemical energy to initiate disassembly and disrupt the capsid structure. To study disassembly of HBV capsids, we mixed T = 4 HBV capsids with 1.0-3.0 M GuHCl, monitored the reaction over time by randomly selecting particles, and measured their size with resistive-pulse sensing. Particles were cycled forward and backward multiple times to increase the observation time and likelihood of observing a disassembly event. The four-pore device used for resistive-pulse sensing produces four current pulses for each particle during translocation that improves tracking and identification of single particles and increases the precision of particle-size measurements when pulses are averaged. We studied disassembly at GuHCl concentrations below and above denaturing conditions of the dimer, the fundamental unit of HBV capsid assembly. As expected, capsids showed little disassembly at low GuHCl concentrations (e.g., 1.0 M GuHCl), whereas at higher GuHCl concentrations (≥1.5 M), capsids exhibited disassembly, sometimes as a complex series of events. In all cases, disassembly was an accelerating process, where capsids catastrophically disassembled within a few 100 ms of reaching critical stability; disassembly rates reached tens of dimers per second just before capsids fell apart. Some disassembly events exhibited metastable intermediates that appeared to lose one or more trimers of dimers in a stepwise fashion.

Structural analysis of the dNTP triphosphohydrolase PA1124 from Pseudomonas aeruginosa

dNTP triphosphohydrolase (TPH) belongs to the histidine/aspartate (HD) superfamily and catalyzes the hydrolysis of dNTPs into 2′-deoxyribonucleoside and inorganic triphosphate. TPHs are required for cellular dNTP homeostasis and DNA replication fidelity and are employed as a host defense mechanism. PA1124 from the pathogenic Pseudomonas aeruginosa bacterium functions as a dGTP and dTTP triphosphohydrolase. To reveal how PA1124 drives dNTP hydrolysis and is regulated, we performed a structural study of PA1124. PA1124 assembles into a hexameric architecture as a trimer of dimers. Each monomer has an interdomain dent where a metal ion is coordinated by conserved histidine and aspartate residues. A structure-based comparative analysis suggests that PA1124 accommodates the dNTP substrate into the interdomain dent near the metal ion. Interestingly, PA1124 interacts with ssDNA, presumably as an allosteric regulator, using a positively charged intersubunit cleft that is generated via dimerization. Furthermore, our phylogenetic analysis highlights similar or distinct oligomerization profiles across the TPH family.

Single tryptophan Y160W mutant of homooligomeric E. coli purine nucleoside phosphorylase implies that dimers forming the hexamer are functionally not equivalent

E. coli purine nucleoside phosphorylase is a homohexamer, which structure, in the apo form, can be described as a trimer of dimers. Earlier studies suggested that ligand binding and kinetic properties are well described by two binding constants and two sets of kinetic constants. However, most of the crystal structures of this enzyme complexes with ligands do not hold the three-fold symmetry, but only two-fold symmetry, as one of the three dimers is different (both active sites in the open conformation) from the other two (one active site in the open and one in the closed conformation). Our recent detailed studies conducted over broad ligand concentration range suggest that protein–ligand complex formation in solution actually deviates from the two-binding-site model. To reveal the details of interactions present in the hexameric molecule we have engineered a single tryptophan Y160W mutant, responding with substantial intrinsic fluorescence change upon ligand binding. By observing various physical properties of the protein and its various complexes with substrate and substrate analogues we have shown that indeed three-binding-site model is necessary to properly describe binding of ligands by both the wild type enzyme and the Y160W mutant. Thus we have pointed out that a symmetrical dimer with both active sites in the open conformation is not forced to adopt this conformation by interactions in the crystal, but most probably the dimers forming the hexamer in solution are not equivalent as well. This, in turn, implies that an allosteric cooperation occurs not only within a dimer, but also among all three dimers forming a hexameric molecule.

Molecular model of a sensor of two-component signaling system

Two-component systems (TCS) are widespread signaling systems present in all domains of life. TCS typically consist of a signal receptor/transducer and a response regulator. The receptors (histidine kinases, chemoreceptors and photoreceptors) are often embedded in the membrane and have a similar modular structure. Chemoreceptors were shown to function in highly ordered arrays, with trimers of dimers being the smallest functional unit. However, much less is known about photoreceptors. Here, we use small-angle scattering (SAS) to show that detergent-solubilized sensory rhodopsin II in complex with its cognate transducer forms dimers at low salt concentration, which associate into trimers of dimers at higher buffer molarities. We then fit an atomistic model of the whole complex into the SAS data. The obtained results suggest that the trimer of dimers is "tripod"-shaped and that the contacts between the dimers occur only through their cytoplasmic regions, whereas the transmembrane regions remain unconnected.

Structure and dynamics of a mycobacterial type\xa0VII secretion system

Mycobacterium tuberculosis is the cause of one of the most important infectious diseases in humans, which leads to 1.4 million deaths every year1. Specialized protein transport systems—known as type VII secretion systems (T7SSs)—are central to the virulence of this pathogen, and are also crucial for nutrient and metabolite transport across the mycobacterial cell envelope2,3. Here we present the structure of an intact T7SS inner-membrane complex of M. tuberculosis. We show how the 2.32-MDa ESX-5 assembly, which contains 165 transmembrane helices, is restructured and stabilized as a trimer of dimers by the MycP5 protease. A trimer of MycP5 caps a central periplasmic dome-like chamber that is formed by three EccB5 dimers, with the proteolytic sites of MycP5 facing towards the cavity. This chamber suggests a central secretion and processing conduit. Complexes without MycP5 show disruption of the EccB5 periplasmic assembly and increased flexibility, which highlights the importance of MycP5 for complex integrity. Beneath the EccB5–MycP5 chamber, dimers of the EccC5 ATPase assemble into three bundles of four transmembrane helices each, which together seal the potential central secretion channel. Individual cytoplasmic EccC5 domains adopt two distinctive conformations that probably reflect different secretion states. Our work suggests a previously undescribed mechanism of protein transport and provides a structural scaffold to aid in the development of drugs against this major human pathogen.