Dimeric Assembly Research Articles

Assembly of nuclear dimers of PI3K regulatory subunits is regulated by the Cdc42-activated tyrosine kinase ACK

Activated Cdc42-associated kinase (ACK) is an oncogenic nonreceptor tyrosine kinase associated with poor prognosis in several human cancers. ACK promotes proliferation, in part by contributing to the activation of Akt, the major effector of class 1A phosphoinositide 3-kinases (PI3Ks), which transduce signals via membrane phosphoinositol lipids. We now show that ACK also interacts with other key components of class 1A PI3K signaling, the PI3K regulatory subunits. We demonstrate ACK binds to all five PI3K regulatory subunit isoforms and directly phosphorylates p85α, p85β, p50α, and p55α on Tyr607 (or analogous residues). We found that phosphorylation of p85β promotes cell proliferation in HEK293T cells. We demonstrate that ACK interacts with p85α exclusively in nuclear-enriched cell fractions, where p85α phosphorylated at Tyr607 (pTyr607) also resides, and identify an interaction between pTyr607 and the N-terminal SH2 domain that supports dimerization of the regulatory subunits. We infer from this that ACK targets p110-independent p85 and further postulate that these regulatory subunit dimers undertake novel nuclear functions underpinning ACK activity. We conclude that these dimers represent a previously undescribed mode of regulation for the class1A PI3K regulatory subunits and potentially reveal additional avenues for therapeutic intervention.

Structural characterization of a dimerization interface in the CD28 transmembrane domain

CD28 has a crucial role in regulating immune responses by enhancing Tcell activation and differentiation. Recent studies have shown that the transmembrane helix (TMH) of CD28 mediates receptor assembly and activity, but a structural characterization of TMH is still lacking. Here, we determined the dimeric helix-helix packing of CD28-TMH using nuclear magnetic resonance (NMR) technology. Unexpectedly, wild-type CD28-TMH alone forms stable tetramers in lipid bicelles instead of dimers. The NMR structure of the CD28-TMH C165F mutant reveals that a GxxxA motif, which is highly conserved in many dimeric assemblies, is located at the dimerization interface. Mutating G160 and A164 can disrupt the transmembrane helix assembly and reduces CD28 enhancement in cells. In contrast, a previously proposed YxxxxT motif does not affect the dimerization of full-length CD28, but it does affect CD28 activity. These results imply that the transmembrane domain of CD28 regulates the signaling transduction in a complicated manner.

Asymmetric structure of the native Rhodobacter sphaeroides dimeric LH1\u2013RC complex

Rhodobacter sphaeroides is a model organism in bacterial photosynthesis, and its light-harvesting-reaction center (LH1–RC) complex contains both dimeric and monomeric forms. Here we present cryo-EM structures of the native LH1–RC dimer and an LH1–RC monomer lacking protein-U (ΔU). The native dimer reveals several asymmetric features including the arrangement of its two monomeric components, the structural integrity of protein-U, the overall organization of LH1, and rigidities of the proteins and pigments. PufX plays a critical role in connecting the two monomers in a dimer, with one PufX interacting at its N-terminus with another PufX and an LH1 β-polypeptide in the other monomer. One protein-U was only partially resolved in the dimeric structure, signaling different degrees of disorder in the two monomers. The ΔU LH1–RC monomer was half-moon-shaped and contained 11 α- and 10 β-polypeptides, indicating a critical role for protein-U in controlling the number of αβ-subunits required for dimer assembly and stabilization. These features are discussed in relation to membrane topology and an assembly model proposed for the native dimeric complex.

Plant phytochrome B is an asymmetric dimer with unique signalling potential.

Many aspects of plant photoperception are mediated by the phytochrome (Phy) family of bilin-containing photoreceptors that reversibly interconvert between inactive Pr and active Pfr conformers1,2. Despite extensive biochemical studies, full understanding of plant Phy signalling has remained unclear due to the absence of relevant 3D models. Here we report a cryo-electron microscopy structure of Arabidopsis PhyB in the Pr state that reveals a topologically complex dimeric organization that is substantially distinct from its prokaryotic relatives. Instead of an anticipated parallel architecture, the C-terminal histidine-kinase-related domains (HKRDs) associate head-to-head, whereas the N-terminal photosensory regions associate head-to-tail to form a parallelogram-shaped platform with near two-fold symmetry. The platform is internally linked by the second of two internal Per/Arnt/Sim domains that binds to the photosensory module of the opposing protomer and a preceding 'modulator' loop that assembles tightly with the photosensory module of its own protomer. Both connections accelerate the thermal reversion of Pfr back to Pr, consistent with an inverse relationship between dimer assembly and Pfr stability. Lopsided contacts between the HKRDs and the platform create profound asymmetry to PhyB that might imbue distinct signalling potentials to the protomers. We propose that this unique structural dynamism creates an extensive photostate-sensitive surface for conformation-dependent interactions between plant Phy photoreceptors and their signalling partners.

Structure-based molecular characterization of the YetL transcription factor from Bacillus subtilis

Bacillus subtilis is a gram-positive bacterium that has developed to coordinate gene expression and to survive against changes of nutrients and toxic chemicals. Flavonoids are exuded by plant cells and are abundant in the soil. To counteract the antibacterial effects of flavonoids, B. subtilis expresses flavonoid-detoxifying enzymes, and their expression is negatively regulated by transcription factors, including YetL. YetL was shown to control B. subtilis growth through the promoter regions of yetL and yetM genes in response to some flavonoids. Despite the functional significance of the YetL transcription factor in bacterial survival, no structural information is available for YetL. Here, we report the crystal structure of YetL and propose a flavonoid-induced regulatory mechanism. The YetL structure contains the canonical winged helix-turn-helix motif of the MarR superfamily but distinctly presents an additional N-terminal helix. In the dimeric assembly of YetL, the H1 helix intersects the YetL dimer and contributes to extensive intersubunit interactions. As a transcription factor, YetL recognizes a 28-mer operator of double-stranded DNA that contains a palindromic sequence. Moreover, our comparative structural analysis of YetL and other MarR members allows us to propose a flavonoid-induced transcription regulatory mechanism that is used for bacterial adaptation to environmental changes and stresses.

Sensitive Oligonucleotide Detection Using Resonant Coupling between Fano Resonance and Image Dipoles of Gold Nanoparticles.

The surface plasmon resonance (SPR)-based sensor has been widely used for biodetection. One of the attractive roles is the gold nanostructure with Fano resonance. Its sharp resonant profile takes advantage of the high figure of merit (FoM) in high-sensitivity detection. However, it is still difficult to detect small molecules at low concentrations due to the extremely low refractive index changes on the metallic surface. We propose using the coupling of image dipoles of gold nanoparticles (AuNPs) and Fano resonance of periodic capped gold nanoslits (CGNs) for sensitive small-molecule detections. The coupling mechanism was verified by three-dimensional finite-difference time-domain calculations and experiments. AuNPs on CGN form image dimer assemblies and induce image dipole with resonance wavelengths ranging from 730 to 550 nm. The surface plasmon polaritons (SPPs) interact with the image dipole of the AuNP on the CGNs and then scatter out through the periodic gold caps. The experimental results show that the peak intensity of grating resonance is decreased by the effect of image dipole and exhibits the maximum intensity change when the Fano resonance matches the resonance of image dipole. The 50 nm AuNPs can be detected with a surface density of less than one particle/μm2 by using the intensity change as the signal. With the resonant coupling between Fano resonance and image dipole extinction, the oligonucleotide with a molecular weight of 5.5 kDa can be detected at a concentration of 100 fM. The resonant coupling dramatically pushes the sensitivity boundary, and we report the limit of detection (LOD) to be 3 orders of magnitude lower than that of the prism-based SPR. This study provides a promising and efficient method for detecting low concentrations of small molecules such as aptamers, miRNA, mRNA, and peptides.

Identification of defined structural elements within TOR2 kinase required for TOR complex 2 assembly and function in Saccharomyces cerevisiae.

The mammalian target of rapamycin (mTOR) is a large protein kinase that assembles into two multisubunit protein complexes, mTORC1 and mTORC2, to regulate cell growth in eukaryotic cells. While significant progress has been made in our understanding of the composition and structure of these complexes, important questions remain regarding the role of specific sequences within mTOR important for complex formation and activity. To address these issues, we have used a molecular genetic approach to explore TOR complex assembly in budding yeast, where two closely related TOR paralogues, TOR1 and TOR2, partition preferentially into TORC1 versus TORC2, respectively. We previously identified an ∼500-amino-acid segment within the N-terminal half of each protein, termed the major assembly specificity (MAS) domain, which can govern specificity in formation of each complex. In this study, we have extended the use of chimeric TOR1-TOR2 genes as a “sensitized” genetic system to identify specific subdomains rendered essential for TORC2 function, using synthetic lethal interaction analyses. Our findings reveal important design principles underlying the dimeric assembly of TORC2 as well as identifying specific segments within the MAS domain critical for TORC2 function, to a level approaching single-amino-acid resolution. Together these findings highlight the complex and cooperative nature of TOR complex assembly and function.

A new inactive conformation of SARS-CoV-2 main protease.

The SARS‐CoV‐2 main protease (Mpro) has a pivotal role in mediating viral genome replication and transcription of the coronavirus, making it a promising target for drugs against the COVID‐19 pandemic. Here, a crystal structure is presented in which Mpro adopts an inactive state that has never been observed before, called new‐inactive. It is shown that the oxyanion loop, which is involved in substrate recognition and enzymatic activity, adopts a new catalytically incompetent conformation and that many of the key interactions of the active conformation of the enzyme around the active site are lost. Solvation/desolvation energetic contributions play an important role in the transition from the inactive to the active state, with Phe140 moving from an exposed to a buried environment and Asn142 moving from a buried environment to an exposed environment. In new‐inactive Mpro a new cavity is present near the S2′ subsite, and the N‐terminal and C‐terminal tails, as well as the dimeric interface, are perturbed, with partial destabilization of the dimeric assembly. This novel conformation is relevant both for comprehension of the mechanism of action of Mpro within the catalytic cycle and for the successful structure‐based drug design of antiviral drugs.

Trapped Cyclic Dimeric Oxyanion Assembly by a Urea-Functionalized Receptor: Potent Application in Linear Discriminant Analysis and as Antibacterial Agents

Trapped Cyclic Dimeric Oxyanion Assembly by a Urea-Functionalized Receptor: Potent Application in Linear Discriminant Analysis and as Antibacterial Agents

Early aggregation mechanism of Aβ16−22 revealed by Markov state models

Aβ16–22 is believed to have critical role in early aggregation of full length amyloids that are associated with the Alzheimer's disease and can aggregate to form amyloid fibrils. However, the early aggregation mechanism is still unsolved. Here, multiple long-term molecular dynamics simulations combining with Markov state model were used to probe the early oligomerization mechanism of Aβ16−22 peptides. The identified dimeric form adopted either globular random-coil or extended β-strand like conformations. The observed dimers of these variants shared many overall conformational characteristics but differed in several aspects at detailed level. In all cases, the most common type of secondary structure was intermolecular antiparallel β-sheets. The inter-state transitions were very frequent ranges from few to hundred nanoseconds. More strikingly, those states which contain fraction of β secondary structure and significant amount of extended coiled structures, therefore exposed to the solvent, were majorly participated in aggregation. The assembly of low-energy dimers, in which the peptides form antiparallel β sheets, occurred by multiple pathways with the formation of an obligatory intermediates. We proposed that these states might facilitate the Aβ16−22 aggregation through a significant component of the conformational selection mechanism, because they might increase the aggregates population by promoting the inter-chain hydrophobic and the hydrogen bond contacts. The formation of early stage antiparallel β sheet structures is critical for oligomerization, and at the same time provided a flat geometry to seed the ordered β-strand packing of the fibrils. Our findings hint at reorganization of this part of the molecule as a potentially critical step in Aβ aggregation and will insight into early oligomerization for large β amyloids.

Structure directing roles of weak noncovalent interactions and charge-assisted hydrogen bonds in the self-assembly of solvated podands: an example of an anion-assisted dimeric water capsule

A 4-aminopyridinium-functionalized mesitylene-based podand could facilitate the formation of a dimeric capsular assembly due to the complementary hydrogen bonding mediated by bromide counterions and lattice water.

Understanding tyrosine self-assembly: from dimer assembly to magnetized fluorescent nanotubes embedded into PVA films

Fate of tyrosine self assembly from dimer formation to photoluminescent and magnetically active ternary nanocomposite containing tyrosine nanotubes.

Mechanisms and physiological implications of cooperative gating of clustered ion channels.

Ion channels play a central role in the regulation of nearly every cellular process. Dating back to the classic 1952 Hodgkin-Huxley model of the generation of the action potential, ion channels have always been thought of as independent agents. A myriad of recent experimental findings exploiting advances in electrophysiology, structural biology, and imaging techniques, however, have posed a serious challenge to this long-held axiom, as several classes of ion channels appear to open and close in a coordinated, cooperative manner. Ion channel cooperativity ranges from variable-sized oligomeric cooperative gating in voltage-gated, dihydropyridine-sensitive CaV1.2 and CaV1.3 channels to obligatory dimeric assembly and gating of voltage-gated NaV1.5 channels. Potassium channels, transient receptor potential channels, hyperpolarization cyclic nucleotide-activated channels, ryanodine receptors (RyRs), and inositol trisphosphate receptors (IP3Rs) have also been shown to gate cooperatively. The implications of cooperative gating of these ion channels range from fine-tuning excitation-contraction coupling in muscle cells to regulating cardiac function and vascular tone, to modulation of action potential and conduction velocity in neurons and cardiac cells, and to control of pacemaking activity in the heart. In this review, we discuss the mechanisms leading to cooperative gating of ion channels, their physiological consequences, and how alterations in cooperative gating of ion channels may induce a range of clinically significant pathologies.

Structure of Mycobacterium tuberculosis cytochrome bcc in complex with Q203 and TB47, two anti-TB drug candidates.

Pathogenic mycobacteria pose a sustained threat to global human health. Recently, cytochrome bcc complexes have gained interest as targets for antibiotic drug development. However, there is currently no structural information for the cytochrome bcc complex from these pathogenic mycobacteria. Here, we report the structures of Mycobacterium tuberculosis cytochrome bcc alone (2.68 Å resolution) and in complex with clinical drug candidates Q203 (2.67 Å resolution) and TB47 (2.93 Å resolution) determined by single-particle cryo-electron microscopy. M. tuberculosis cytochrome bcc forms a dimeric assembly with endogenous menaquinone/menaquinol bound at the quinone/quinol-binding pockets. We observe Q203 and TB47 bound at the quinol-binding site and stabilized by hydrogen bonds with the side chains of QcrBThr313 and QcrBGlu314, residues that are conserved across pathogenic mycobacteria. These high-resolution images provide a basis for the design of new mycobacterial cytochrome bcc inhibitors that could be developed into broad-spectrum drugs to treat mycobacterial infections.

A comparative study of noncovalent interactions in various Ni-compounds containing nitrogen heteroaromatic ligands and pseudohalides: A combined experimental and theoretical studies

Five mononuclear nickel(II) complexes, [Ni(NCS)2(imz)4] (1) and [Ni(NCS)2(pyz)4] (2), [Ni(NCS)2(3-OHpy)4] (3), [Ni(NCS)2(3-Clpy)4] (4) and [Ni(NCO)2(3-Clpy)2(H2O)2] (5), where imz, pyz, 3-OHpy and 3-Clpy are imidazole, pyrazole, 3-hydroxypyridine and 3-chloropyridine, respectively, have been synthesized and characterized by X-ray crystallographic studies to explore the role of different heteroaromatic ligands and pseudohalides in the crystal packing. The noncovalent interactions witnessed in the crystal packing of these complexes have been well-defined focusing on the recurrent π–stacking motifs, leading to the π–stacked dimeric assemblies. Besides, noncovalent interactions such as, NH(CH)···π, lone pair···π, along with both conventional and nonconventional hydrogen bonding interactions play important roles in the stabilization of these complexes in the solid state. The energetic features of the π–stacking and the importance of additional noncovalent interactions towards cooperatively formed π–stacking dimers were investigated using DFT calculations in combination with the quantum theory of atoms-in-molecules (QTAIM) and noncovalent interaction plot (NCI plot) index computational tools. Remarkably, the aromatic ligands (imidazole, pyridine) through coordination generate favourable antiparallel orientation of the dipoles (180°) in the dimeric form of complexes 1, 3, 4 and 5, and thus exhibit strong π–stacking interaction in comparison to complex 2 (pyrazole) in its dimeric form with the perpendicular orientation of dipoles. This study may provide further insight into elucidating the role of weak noncovalent interactions in the supramolecular assemblies of metal-containing compounds.

Amino acid residues at core protein dimer-dimer interface modulate multiple steps of hepatitis B virus replication and HBeAg biogenesis.

The core protein (Cp) of hepatitis B virus (HBV) assembles pregenomic RNA (pgRNA) and viral DNA polymerase to form nucleocapsids where the reverse transcriptional viral DNA replication takes place. Core protein allosteric modulators (CpAMs) inhibit HBV replication by binding to a hydrophobic “HAP” pocket at Cp dimer-dimer interfaces to misdirect the assembly of Cp dimers into aberrant or morphologically “normal” capsids devoid of pgRNA. We report herein that a panel of CpAM-resistant Cp with single amino acid substitution of residues at the dimer-dimer interface not only disrupted pgRNA packaging, but also compromised nucleocapsid envelopment, virion infectivity and covalently closed circular (ccc) DNA biosynthesis. Interestingly, these mutations also significantly reduced the secretion of HBeAg. Biochemical analysis revealed that the CpAM-resistant mutations in the context of precore protein (p25) did not affect the levels of p22 produced by signal peptidase removal of N-terminal 19 amino acid residues, but significantly reduced p17, which is produced by furin cleavage of C-terminal arginine-rich domain of p22 and secreted as HBeAg. Interestingly, p22 existed as both unphosphorylated and phosphorylated forms. While the unphosphorylated p22 is in the membranous secretary organelles and the precursor of HBeAg, p22 in the cytosol and nuclei is hyperphosphorylated at the C-terminal arginine-rich domain and interacts with Cp to disrupt capsid assembly and viral DNA replication. The results thus indicate that in addition to nucleocapsid assembly, interaction of Cp at dimer-dimer interface also plays important roles in the production and infectivity of progeny virions through modulation of nucleocapsid envelopment and uncoating. Similar interaction at reduced p17 dimer-dimer interface appears to be important for its metabolic stability and sensitivity to CpAM suppression of HBeAg secretion.

Structural basis of cytokine-mediated activation of ALK family receptors.

Anaplastic lymphoma kinase (ALK)1 and the related leukocyte tyrosine kinase (LTK)2 are recently deorphanized receptor tyrosine kinases3. Together with their activating cytokines, ALKAL1 and ALKAL24-6 (also called FAM150A and FAM150B or AUGβ and AUGα, respectively), they are involved in neural development7, cancer7-9 and autoimmune diseases10. Furthermore, mammalian ALK recently emerged as a key regulator of energy expenditure and weight gain11, consistent with a metabolic role for Drosophila ALK12. Despite such functional pleiotropy and growing therapeutic relevance13,14, structural insights into ALK and LTK and their complexes with cognate cytokines have remained scarce. Here we show that the cytokine-binding segments of human ALK and LTK comprise a novel architectural chimera of a permuted TNF-like module that braces a glycine-rich subdomain featuring a hexagonal lattice of long polyglycine type II helices. The cognate cytokines ALKAL1 and ALKAL2 are monomeric three-helix bundles, yet their binding to ALK and LTK elicits similar dimeric assemblies with two-fold symmetry, that tent a single cytokine molecule proximal to the cell membrane. We show that the membrane-proximal EGF-like domain dictates the apparent cytokine preference of ALK. Assisted by these diverse structure-function findings, we propose a structural and mechanistic blueprint for complexes of ALK family receptors, and thereby extend the repertoire of ligand-mediated dimerization mechanisms adopted by receptor tyrosine kinases.

Oligomeric Structural Transition of HspB1 from Chinese Hamster.

HspB1 is a mammalian sHsp that is ubiquitously expressed in almost all tissues and involved in regulating many vital functions. Although the recent crystal structure of human HspB1 showed that 24 monomers form the oligomeric complex of human HspB1 in a spherical configuration, the molecular architecture of HspB1 is still controversial. In this study, we examined the oligomeric structural change of CgHspB1 by sedimentation velocity analytical ultracentrifugation. At the low temperature of 4 °C, CgHspB1 exists as an 18-mer, probably a trimeric complex of hexamers. It is relatively unstable and partially dissociates into small oligomers, hexamers, and dodecamers. At elevated temperatures, the 24-mer was more stable than the 18-mer. The 24-mer is also in dynamic equilibrium with the dissociated oligomers in the hexameric unit. The hexamer further dissociates to dimers. The disulfide bond between conserved cysteine residues seems to be partly responsible for the stabilization of hexamers. The N-terminal domain is involved in the assembly of dimers and the interaction between hexamers. It is plausible that CgHspB1 expresses a chaperone function in the 24-mer structure.

Ti3C2Tx MXenes loaded with Au nanoparticle dimers as a surface-enhanced Raman scattering aptasensor for AFB1 detection

Mycotoxin B1 (AFB1) contamination in agricultural products pose a deadlydangertoanimal and human health and its rapid and reliable detection is thus very important. Herein, a ratiometric surface-enhanced Raman scattering (SERS) aptasensor for AFB1 detection was developed, in which 1,2-bis(4-pyridyl) ethylene (BPE) was used to trigger the assembly of Au nanoparticle dimers (AuNP dimers) and form intensive SERS “hot spots”, and MXenes nanosheets could load aptamer-modified AuNP dimers due to the hydrogen bonding and the chelation between the phosphate groups of aptamers and the Ti ion of MXenes. With the presence of AFB1 preferentially binding to AFB1 aptamer, AuNP dimers were separated from MXenes nanosheets, leading to a decrease in SERS intensity. Regression analysis in the range from 0.001 to 100 ng·mL−1 showed the limit of detection (LOD) being 0.6 pg·mL−1 in standard solution, indicating that the great prospects of the AuNP dimers/MXenes SERS substrate for detecting AFB1.

The conformational stability of pro-apoptotic BAX is dictated by discrete residues of the protein core

BAX is a pro-apoptotic member of the BCL-2 family, which regulates the balance between cellular life and death. During homeostasis, BAX predominantly resides in the cytosol as a latent monomer but, in response to stress, transforms into an oligomeric protein that permeabilizes the mitochondria, leading to apoptosis. Because renegade BAX activation poses a grave risk to the cell, the architecture of BAX must ensure monomeric stability yet enable conformational change upon stress signaling. The specific structural features that afford both stability and dynamic flexibility remain ill-defined and represent a critical control point of BAX regulation. We identify a nexus of interactions involving four residues of the BAX core α5 helix that are individually essential to maintaining the structure and latency of monomeric BAX and are collectively required for dimeric assembly. The dual yet distinct roles of these residues reveals the intricacy of BAX conformational regulation and opportunities for therapeutic modulation.