Monomeric Assembly Research Articles

A poly-pseudorotaxane constructed by threading pillar[5]arene onto an ion-pair recognition-based calix[4]pyrrole supramolecular polymer.

The ion-pair recognition ability of calix[4]pyrrole was utilized to form a multicomponent monomeric assembly and a linear supramolecular polymer via concurrent anion and bis-cation complexation. The inherent dynamic interactions of these assemblies were further used to construct pseudorotaxanes in monomeric and supramolecular polymer forms with pillar[5]arene.

BthTX-II from Bothrops jararacussu venom has variants with different oligomeric assemblies: An example of snake venom phospholipases A2 versatility

Phospholipases A2 (PLA2s) are found in almost every venomous snake family. In snakebites, some PLA2s can quickly cause local myonecrosis, which may lead to permanent sequelae if antivenom is administered belatedly. They hydrolyse phospholipids in membranes through a catalytic calcium ions-dependent mechanism. BthTX-II is a basic PLA2 and the second major component in the venom of Bothrops jararacussu. Herein, using the software SEQUENCE SLIDER, which integrates crystallographic, mass spectrometry and genetic data, we characterized the primary, tertiary and quaternary structure of two BthTX-II variants (called a and b), which diverge in 7 residues. Crystallographic structure BthTX-IIa is in a Tense-state with its distorted calcium binding loop buried in the dimer interface, contrarily, the novel BthTX-IIb structure is a monomer in a Relax-state with a fatty acid in the hydrophobic channel. Structural data in solution reveals that both variants are monomeric in neutral physiological conditions and mostly dimeric in an acidic environment, being catalytic active in both situations. Therefore, we propose two myotoxic mechanisms for BthTX-II, a catalytic one associated with the monomeric assembly, whereas the other has a calcium independent activity related to its C-terminal region, adopting a dimeric conformation similar to PLA2-like proteins.

The large bat Helitron DNA transposase forms a compact monomeric assembly that buries and protects its covalently bound 5′-transposon end

Helitrons are widespread eukaryotic DNA transposons that have significantly contributed to genome variability and evolution, in part because of their distinctive, replicative rolling-circle mechanism, which often mobilizes adjacent genes. Although most eukaryotic transposases form oligomers and use RNase H-like domains to break and rejoin double-stranded DNA (dsDNA), Helitron transposases contain a single-stranded DNA (ssDNA)-specific HUH endonuclease domain. Here, we report the cryo-electron microscopy structure of a Helitron transposase bound to the 5'-transposon end, providing insight into its multidomain architecture and function. The monomeric transposase forms a tightly packed assembly that buries the covalently attached cleaved end, protecting it until the second end becomes available. The structure reveals unexpected architectural similarity to TraI, a bacterial relaxase that also catalyzes ssDNA movement. The HUH active site suggests how two juxtaposed tyrosines, a feature of many replication initiators that use HUH nucleases, couple the conformational shift of an α-helix to control strand cleavage and ligation reactions.

Biochemical, pharmacological and structural characterization of BmooMP-I, a new P–I metalloproteinase from Bothrops moojeni venom

Snakebite envenoming is still a worrying health problem in countries under development, being recognized as a neglected disease by the World Health Organization. In Latin America, snakes from the genus Bothrops are widely spread and in Brazil, the Bothrops moojeni is a medically important species. The pharmacological effects of bothropic snake venoms include pain, blisters, bleeding, necrosis and even amputation of the affected limb. Snake venom metalloproteinases are enzymes abundantly present in venom from Bothrops snakes. These enzymes can cause hemorrhagic effects and lead to myonecrosis due to ischemia. Here, we present BmooMP-I, a new P–I class of metalloproteinase (this class only has the catalytic domain in the mature form) isolated from B. moojeni venom. This protein is able to express fibrinogenolytic and gelatinase activities, which play important roles in the prey’s immobilization and digestion, and also induces weak hemorrhagic effect. The primary sequence assignment was done by a novel method, SEQUENCE SLIDER, which combines crystallographic, bioinformatics and mass spectrometry data. The high-resolution crystal structure reveals the monomeric assembly and the conserved metal binding site H141ExxH145xxG148xxH151 with the natural substitution Gly148Asp that does not interfere in the zinc coordination. The presence of a structural calcium ion on the surface of the protein, which can play an important role in the stabilization of hemorrhagic toxins, was observed in the BmooMP-I structure. Due to the relevant local and systemic effects of snake venom metalloproteinases, studies involving these proteins help to better understand the pathological effects of snakebite envenoming.

The active form of quinol-dependent nitric oxide reductase from Neisseria meningitidis is a dimer.

Neisseria meningitidis is carried by nearly a billion humans, causing developmental impairment and over 100 000 deaths a year. A quinol-dependent nitric oxide reductase (qNOR) plays a critical role in the survival of the bacterium in the human host. X-ray crystallographic analyses of qNOR, including that from N. meningitidis (NmqNOR) reported here at 3.15 Å resolution, show monomeric assemblies, despite the more active dimeric sample being used for crystallization. Cryo-electron microscopic analysis of the same chromatographic fraction of NmqNOR, however, revealed a dimeric assembly at 3.06 Å resolution. It is shown that zinc (which is used in crystallization) binding near the dimer-stabilizing TMII region contributes to the disruption of the dimer. A similar destabilization is observed in the monomeric (∼85 kDa) cryo-EM structure of a mutant (Glu494Ala) qNOR from the opportunistic pathogen Alcaligenes (Achromobacter) xylosoxidans, which primarily migrates as a monomer. The monomer-dimer transition of qNORs seen in the cryo-EM and crystallographic structures has wider implications for structural studies of multimeric membrane proteins. X-ray crystallographic and cryo-EM structural analyses have been performed on the same chromatographic fraction of NmqNOR to high resolution. This represents one of the first examples in which the two approaches have been used to reveal a monomeric assembly in crystallo and a dimeric assembly in vitrified cryo-EM grids. A number of factors have been identified that may trigger the destabilization of helices that are necessary to preserve the integrity of the dimer. These include zinc binding near the entry of the putative proton-transfer channel and the preservation of the conformational integrity of the active site. The mutation near the active site results in disruption of the active site, causing an additional destabilization of helices (TMIX and TMX) that flank the proton-transfer channel helices, creating an inert monomeric enzyme.

A mechanistic model of tau amyloid aggregation based on direct observation of oligomers.

Protein aggregation plays a key role in neurodegenerative disease, giving rise to small oligomers that may become cytotoxic to cells. The fundamental microscopic reactions taking place during aggregation, and their rate constants, have been difficult to determine due to lack of suitable methods to identify and follow the low concentration of oligomers over time. Here we use single-molecule fluorescence to study the aggregation of the repeat domain of tau (K18), and two mutant forms linked with familial frontotemporal dementia, the deletion mutant ΔK280 and the point mutant P301L. Our kinetic analysis reveals that aggregation proceeds via monomeric assembly into small oligomers, and a subsequent slow structural conversion step before fibril formation. Using this approach, we have been able to quantitatively determine how these mutations alter the aggregation energy landscape.

Mutation of Y407 in the CH3 domain dramatically alters glycosylation and structure of human IgG

Antibody engineering is increasingly being used to influence the properties of monoclonal antibodies to improve their biotherapeutic potential. One important aspect of this is the modulation of glycosylation as a strategy to improve efficacy. Here, we describe mutations of Y407 in the CH3 domain of IgG1 and IgG4 that significantly increase sialylation, galactosylation, and branching of the N-linked glycans in the CH2 domain. These mutations also promote the formation of monomeric assemblies (one heavy-light chain pair). Hydrogen-deuterium exchange mass spectrometry was used to probe conformational changes in IgG1-Y407E, revealing, as expected, a more exposed CH3–CH3 dimerization interface. Additionally, allosteric structural effects in the CH2 domain and in the CH2–CH3 interface were identified, providing a possible explanation for the dramatic change in glycosylation. Thus, the mutation of Y407 in the CH3 domain remarkably affects both antibody conformation and glycosylation, which not only alters our understanding of antibody structure, but also reveals possibilities for obtaining recombinant IgG with glycosylation tailored for clinical applications.

Quaternary Structure Heterogeneity of Oligomeric Proteins: A SAXS and SANS Study of the Dissociation Products of Octopus vulgaris Hemocyanin

Octopus vulgaris hemocyanin shows a particular self-assembling pattern, characterized by a hierarchical organization of monomers. The highest molecular weight aggregate is a decamer, the stability of which in solution depends on several parameters. Different pH values, buffer compositions, H2O/D2O ratios and Hofmeister’s salts result in modifications of the aggregation state of Octopus vulgaris hemocyanin. The new QUAFIT method, recently applied to derive the structure of the decameric and the monomeric assembly from small-angle scattering data, is used here to model the polydisperse system that results from changing the solution conditions. A dataset of small-angle X-rays and neutron scattering curves is analysed by QUAFIT to derive structure, composition and concentration of different assemblies present in solution. According to the hierarchy of the association/dissociation processes and the possible number of different aggregation products in solution, each sample has been considered as a heterogeneous mixture composed of the entire decamer, the dissociated “loose” monomer and all the intermediate dissociation products. Scattering curves corresponding to given experimental conditions are well fitted by using a linear combination of single particle form factors. QUAFIT has proved to be a method of general validity to describe solutions of proteins that, even after purification processes, result to be intrinsically heterogeneous.

Crystal structure and biochemical studies of the trans-acting polyketide enoyl reductase LovC from lovastatin biosynthesis

Lovastatin is an important statin prescribed for the treatment and prevention of cardiovascular diseases. Biosynthesis of lovastatin uses an iterative type I polyketide synthase (PKS). LovC is a trans-acting enoyl reductase (ER) that specifically reduces three out of eight possible polyketide intermediates during lovastatin biosynthesis. Such trans-acting ERs have been reported across a variety of other fungal PKS enzymes as a strategy in nature to diversify polyketides. How LovC achieves such specificity is unknown. The 1.9-Å structure of LovC reveals that LovC possesses a medium-chain dehydrogenase/reductase (MDR) fold with a unique monomeric assembly. Two LovC cocrystal structures and enzymological studies help elucidate the molecular basis of LovC specificity, define stereochemistry, and identify active-site residues. Sequence alignment indicates a general applicability to trans-acting ERs of fungal PKSs, as well as their potential application to directing biosynthesis.

Efficient incorporation of monomeric anthracene into nanoporous silica/surfactant nanocomposite spheres using a mechanochemical solid state reaction

The incorporation of anthracene (Ant) into nanoporous silica/surfactant nanocomposite spheres was successfully achieved using a mechanochemical solid state reaction. The Ant molecules were incorporated into the nanopores while preserving the silica nanostructures, indicating the effective hydrophobic interactions with the alkyl chains of the surfactant. The blue photoluminescence intensity due to the monomer state was enhanced by increasing the mechanochemical milling force, suggesting this is an efficient preparation method for a luminescent medium by monomeric assembly of the Ant molecules.

Alternate States of Proteins Revealed by Detailed Energy Landscape Mapping

What conformations do protein molecules populate in solution? Crystallography provides a high-resolution description of protein structure in the crystal environment, while NMR describes structure in solution but using less data. NMR structures display more variability, but is this because crystal contacts are absent or because of fewer data constraints? Here we report unexpected insight into this issue obtained through analysis of detailed protein energy landscapes generated by large-scale, native-enhanced sampling of conformational space with Rosetta@home for 111 protein domains. In the absence of tightly associating binding partners or ligands, the lowest-energy Rosetta models were nearly all <2.5 Å CαRMSD from the experimental structure; this result demonstrates that structure prediction accuracy for globular proteins is limited mainly by the ability to sample close to the native structure. While the lowest-energy models are similar to deposited structures, they are not identical; the largest deviations are most often in regions involved in ligand, quaternary, or crystal contacts. For ligand binding proteins, the low energy models may resemble the apo structures, and for oligomeric proteins, the monomeric assembly intermediates. The deviations between the low energy models and crystal structures largely disappear when landscapes are computed in the context of the crystal lattice or multimer. The computed low-energy ensembles, with tight crystal-structure-like packing in the core, but more NMR-structure-like variability in loops, may in some cases resemble the native state ensembles of proteins better than individual crystal or NMR structures, and can suggest experimentally testable hypotheses relating alternative states and structural heterogeneity to function.

Assembly and oligomerization of human ATP synthase lacking mitochondrial subunits a and A6L

Here we study ATP synthase from human ρ0 (rho zero) cells by clear native electrophoresis (CNE or CN-PAGE) and show that ATP synthase is almost fully assembled in spite of the absence of subunits a and A6L. This identifies subunits a and A6L as two of the last subunits to complete the ATP synthase assembly. Minor amounts of dimeric and even tetrameric forms of the large assembly intermediate were preserved under the conditions of CNE, suggesting that it associated further into higher order structures in the mitochondrial membrane. This result was reminiscent to the reduced amounts of dimeric and tetrameric ATP synthase from yeast null mutants of subunits e and g detected by CNE. The dimer/oligomer-stabilizing effects of subunits e/g and a/A6L seem additive in human and yeast cells. The mature IF1 inhibitor was specifically bound to the dimeric/oligomeric forms of ATP synthase and not to the monomer. Conversely, nonprocessed pre-IF1 still containing the mitochondrial targeting sequence was selectively bound to the monomeric assembly intermediate in ρ0 cells and not to the dimeric form. This supports previous suggestions that IF1 plays an important role in the dimerization/oligomerization of mammalian ATP synthase and in the regulation of mitochondrial structure and function.

The syntheses, structures and fluorescent properties of two monomeric Zn(II) and Mn(II) assemblies containing bitriazole ligand

Two monomeric complexes, [Zn(trtr)2(H2O)4](NO3)2·2H2O (1) and [Mn(trtr)2(H2O)4](ClO4)2 (2), were obtained from the reactions of bitriazole ligand 4-[3-(1,2,4-triazolyl)-1,2,4-triazole] (trtr) with Zn(NO3)2 and Mn(ClO4)2, respectively. A 3-D supramolecular framework of 1 is constructed by the hydrogen bonds with lattice water molecule as linkers. In 2, a 2-D supramolecular layer is formed by hydrogen bonding interactions between the monomeric assembly and ClO4-, and further connected into a 3-D framework by d⋯π and π⋯π stacking interactions. The coordination of the metal centers to the new born triazole ring play an important role in their fluorescent properties, which present the emission peaks at 349nm and 358nm, respectively, originated from LMCT.

The syntheses, structures and fluorescent properties of two 3-D hydrogen-bonded frameworks constructed from monomeric Zn(II) and Cd(II) assemblies containing bitriazole

Two monomeric Cd(II) and Zn(II) complexes [M(trtr)2(H2O)4](SiF6) (M=Cd for 1; Zn for 2) were obtained from the reactions of bitriazole ligand 4-[3-(1,2,4-triazolyl)-1,2,4-triazole] with metal fluorosilicate. 3-D supramolecular frameworks are constructed by the hydrogen bondings between the monomeric assemblies and SiF62-. The fluorescence of 1 and 2 feature the emission peaks at 454 and 348nm, respectively, originating from the π–π∗ intraligand transition and LMCT, which are related to the triazole rings the metal centers coordinate with.

Thiol-reducing agents and calcium perturbants alter intracellular sorting of immunoglobulin M.

The secretory form of IgM (sIgM) undergoes developmentally regulated intracellular sorting and transport, as demonstrated by the distinct assembly patterns and intracellular fate exhibited by its mu heavy chain (microseconds). In the 38C B lymphocytes, microseconds-containing monomers are retained and degraded intracellularly, whereas in the 38C-derived D2 hybridoma, microseconds-containing polymers are secreted. Here we show that sorting of sIgM is impaired in the presence of the thiol-reducing agent beta-mercaptoethanol or when cellular calcium sequestration is perturbed either with the Ca2+ ionophore A23187 or with thapsigargin, an inhibitor of endoplasmic reticulum Ca(2+)-ATPase. Under these conditions, sIgM evades retention/degradation in the 38C cells, resulting in its enhanced secretion. Conversely, in the D2 cells, secretion is strongly attenuated, and sIgM is partly degraded. Both cell types secrete unusually processed sIgM, which is completely resistant to endoglycosidase H, unlike the partially sensitive sIgM secreted in the usual manner, indicating terminal glycosylation of all carbohydrate moieties of microseconds. Moreover, secretion is no longer restricted to fully assembled polymeric sIgM, and in both cell types under Ca2+ perturbation preferentially hemimeric and monomeric assembly intermediates are detected in the medium. Our results suggest that the intracellular fate of sIgM is collectively determined by several sorting events that confer sIgM retention/degradation, ensure sIgM secretion from D2 cells, and prevent the arrival of assembly intermediates to the cell surface. All of these sorting events appear to require high Ca2+ concentration and oxidizing thiol redox state, the conditions that prevail in the lumen of the endoplasmic reticulum. Thus, thiol and calcium are implicated as modulators of intracellular traffic.

Assembly of Torpedo acetylcholine receptors in Xenopus oocytes.

To study pathways by which acetylcholine receptor (AChR) subunits might assemble, Torpedo alpha subunits were expressed in Xenopus oocytes alone or in combination with beta, gamma, or delta subunits. The maturation of the conformation of the main immunogenic region (MIR) on alpha subunits was measured by binding of mAbs and the maturation of the conformation of the AChR binding site on alpha subunits was measured by binding of alpha-bungarotoxin (alpha Bgt) and cholinergic ligands. The size of subunits and subunit complexes was assayed by sedimentation on sucrose gradients. It is generally accepted that native AChRs have the subunit composition alpha 2 beta gamma delta. Torpedo alpha subunits expressed alone resulted in an amorphous range of complexes with little affinity for alpha Bgt or mAbs to the MIR, rather than in a unique 5S monomeric assembly intermediate species. A previously recognized temperature-dependent failure in alpha subunit maturation may cause instability of the monomeric assembly intermediate and accumulation of aggregated denatured alpha subunits. Coexpression of alpha with beta subunits also resulted in an amorphous range of complexes. However, coexpression of alpha subunits with gamma or delta subunits resulted in the efficient formation of 6.5S alpha gamma or alpha delta complexes with high affinity for mAbs to the MIR, alpha Bgt, and small cholinergic ligands. These alpha gamma and alpha delta subunit pairs may represent normal assembly intermediates in which Torpedo alpha is stabilized and matured in conformation. Coexpression of alpha, gamma, and delta efficiently formed 8.8S complexes, whereas complexes containing alpha beta and gamma or alpha beta and delta subunits are formed less efficiently. Assembly of beta subunits with complexes containing alpha gamma and delta subunits may normally be a rate-limiting step in assembly of AChRs.

Molecular composition of type VI collagen. Evidence for chain heterogeneity in mammalian tissues and cultured cells

The chain composition and relative abundance of type VI collagen synthesized by cells cultured from foetal bovine nuchal ligament and skin were compared with those of the type VI collagen present in these foetal tissues. Immunoprecipitation of intact collagen VI from medium and cell layers of nuchal ligament fibroblasts and skin fibroblasts at confluence revealed collagen type VI molecules with a chain composition consistent with an [alpha 1(VI)alpha 2(VI)alpha 3(VI)] monomeric assembly. Maintenance of cells in a post-confluent quiescent state promoted a marked phenotypic change in these ratios, with increased concentrations of assemblies composed of equimolar ratios of alpha 1(VI) and alpha 2(VI) chains detected in the medium of these cultures. Analysis of steady-state concentrations of mRNA for alpha 1(VI) and alpha 2(VI) chains revealed these species to be present in increased abundance at post-confluence in all the cultures, but no corresponding increase was observed in the alpha 3(VI) mRNA. In order to assess the physiological significance of these observations, the chain composition of the collagen VI content of the corresponding foetal tissues was assessed by Western blotting after extraction in guanidinium isothiocyanate under reducing conditions. Extracts of nuchal ligament revealed a collagen VI chain composition consistent with a heterotrimeric chain assembly. In contrast, the skin extracts revealed an abundance of alpha 1(VI) and alpha 2(VI) chains with only traces of the alpha 3(VI) chain detected. Increased equimolar concentrations of the alpha 1(VI)-chain and alpha 2(VI)-chain mRNAs in skin again reflected the increased concentrations of these polypeptide chains. Type VI collagen was present in greater abundance both in the nuchal ligament and in the corresponding nuchal-ligament fibroblast cultures. The results indicate that the chain composition of type VI collagen is subject to modulation at the level of transcription as a result of variations in the proliferative state of the cells, and demonstrate that different isoforms of collagen VI occur in foetal development.