Stable Tetramer Research Articles

Molten globule nature of Plasmodium falciparum P2 homo-tetramer

The P2 protein in Plasmodium falciparum has a high tendency to oligomerize, which seems to drive many of its non-ribosomal functions. During nuclear division of the parasite inside RBC, P2 translocates to the RBC surface as a tetramer. From a systematic study using variety of biophysical techniques, NMR spectral characteristics and relaxation dispersion measurements under different conditions of pH and/or urea concentrations, we deduce that (i) PfP2, an almost entirely helical protein, forms a molten globule monomer at low pH, (ii) at physiological pH, and at micro-molar concentrations, PfP2 is a stable tetramer wherein two dimmers associate sideways with close packing of helices at the interface, and (iii) the molten globule characteristic of the monomer is preserved in the tetramer. This dynamism in the structure of PfP2 may have functional implications since it is known that different kinds of oligomers are transiently formed in the parasite.

An evolutionarily conserved DNA architecture determines target specificity of the TWIST family bHLH transcription factors.

Basic helix-loop-helix (bHLH) transcription factors recognize the canonical E-box (CANNTG) to regulate gene transcription; however, given the prevalence of E-boxes in a genome, it has been puzzling how individual bHLH proteins selectively recognize E-box sequences on their targets. TWIST is a bHLH transcription factor that promotes epithelial-mesenchymal transition (EMT) during development and tumor metastasis. High-resolution mapping of TWIST occupancy in human and Drosophila genomes reveals that TWIST, but not other bHLH proteins, recognizes a unique double E-box motif with two E-boxes spaced preferentially by 5 nucleotides. Using molecular modeling and binding kinetic analyses, we found that the strict spatial configuration in the double E-box motif aligns two TWIST-E47 dimers on the same face of DNA, thus providing a high-affinity site for a highly stable intramolecular tetramer. Biochemical analyses showed that the WR domain of TWIST dimerizes to mediate tetramer formation, which is functionally required for TWIST-induced EMT. These results uncover a novel mechanism for a bHLH transcription factor to recognize a unique spatial configuration of E-boxes to achieve target specificity. The WR-WR domain interaction uncovered here sets an example of target gene specificity of a bHLH protein being controlled allosterically by a domain outside of the bHLH region.

Disordered interdomain region of Gins is important for functional tetramer formation to stimulate MCM helicase in Thermoplasma acidophilum.

The eukaryotic MCM is activated by forming the CMG complex with Cdc45 and GINS to work as a replicative helicase. The eukaryotic GINS consists of four different proteins to form tetrameric complex. In contrast, the TaGins51 protein from the thermophilic archaeon, Thermoplasma acidophilum forms a homotetramer (TaGINS), and interacts with the cognate MCM (TaMCM) to stimulate the DNA-binding, ATPase, and helicase activities of TaMCM. All Gins proteins from Archaea and Eukarya contain α-helical A- and β-stranded B-domains. Here, we found that TaGins51 forms the tetramer without the B-domain. However, the A-domain without the linker region between the A- and B-domains could not form a stable tetramer, and furthermore, the A-domain by itself could not stimulate the TaMCM activity. These results suggest that the formation of the Gins51 tetramer is necessary for MCM activation, and the disordered linker region between the two domains is critical for the functional complex formation.

Al12Cu Superatom as Stable Building Block of Ionic Salts

The neutral copper aluminum cluster complex Al12CuM3 [M = K, K3O, and K(2,2,2-crypt)] has been investigated at the PBE96 level of theory. It is found that Al12Cu could be considered a superatom mimic of a phosphorus atom. It shows large electron affinity and is able to receive three electrons from elements or compounds with low ionization energies like K, K3O, or K(2,2,2-crypt) to become a stable electronic closed shell with a large gap between the highest occupied and lowest unoccupied molecular orbitals (HOMO–LUMO gap). Theoretical calculations confirm that similar to the phosphorus atom in PK3, the superatom Al12Cu cluster could form ionic salts, as shown from stable dimers, trimers, and tetramers of the Al12Cu{K(2,2,2-crypt)}3 complex. On the basis of the maintenance of its integrity in these assemblies it could be predicted that Al12CuM3 holds great potential as a building block for the development of future nanostructured materials. Furthermore, the choice of the K(2,2,2-crypt) molecules to stabiliz...

Formation of Induced-Fit Chiral Templates by Amino Acid-Functionalized Pd(111) Surfaces

Chiral probe molecules, propylene oxide, and glycidol are used to measure the enantioselectivity of a range of amino acid-functionalized Pd(111) surfaces. Only those surfaces that contain tetrameric amino acid assemblies are found to be enantioselective, indicating that they act as chiral templates in which several modifiers operate in concert to form a chiral reaction pocket. It has previously been shown that the tetramers assemble from antiparallel anionic–zwitterionic dimers where three of the amino acids then undergo a concerted translational motion to form a more stable tetramer. However, density functional theory calculations reveal that the most stable tetramer has a pocket that is too small to accommodate the chiral probes, while the more open antiparallel anionic–zwitterionic dimer structure provides sufficient space for the epoxide to adsorb enantioselectively on the most stable atop palladium adsorption site. Amino acid destabilization is confirmed by its lower desorption temperature measured in temperature-programmed desorption.

Dissociation of glucocerebrosidase dimer in solution by its co-factor, saposin C

Mutations in the gene for the lysosomal enzyme glucocerebrosidase (GCase) cause Gaucher disease and are the most common risk factor for Parkinson disease (PD). Analytical ultracentrifugation of 8 μM GCase shows equilibrium between monomer and dimer forms. However, in the presence of its co-factor saposin C (Sap C), only monomer GCase is seen. Isothermal calorimetry confirms that Sap C associates with GCase in solution in a 1:1 complex (Kd = 2.1 ± 1.1 μM). Saturation cross-transfer NMR determined that the region of Sap C contacting GCase includes residues 63–66 and 74–76, which is distinct from the region known to enhance GCase activity. Because α-synuclein (α-syn), a protein closely associated with PD etiology, competes with Sap C for GCase binding, its interaction with GCase was also measured by ultracentrifugation and saturation cross-transfer. Unlike Sap C, binding of α-syn to GCase does not affect multimerization. However, adding α-syn reduces saturation cross-transfer from Sap C to GCase, confirming displacement. To explore where Sap C might disrupt multimeric GCase, GCase x-ray structures were analyzed using the program PISA, which predicted stable dimer and tetramer forms. For the most frequently predicted multimer interface, the GCase active sites are partially buried, suggesting that Sap C might disrupt the multimer by binding near the active site.

SAMHD1 Gene Mutations Are Associated with Cerebral Large-Artery Atherosclerosis.

Background. To investigate whether one or more SAMHD1 gene mutations are associated with cerebrovascular disease in the general population using a Chinese stroke cohort. Methods. Patients with a Chinese Han background (N = 300) diagnosed with either cerebral large-artery atherosclerosis (LAA, n = 100), cerebral small vessel disease (SVD, n = 100), or other stroke-free neurological disorders (control, n = 100) were recruited. Genomic DNA from the whole blood of each patient was isolated, and direct sequencing of the SAMHD1 gene was performed. Both wild type and mutant SAMHD1 proteins identified from the patients were expressed in E. coli and purified; then their dNTPase activities and ability to form stable tetramers were analysed in vitro. Results. Three heterozygous mutations, including two missense mutations c.64C>T (P22S) and c.841G>A (p.E281K) and one splice site mutation c.696+2T>A, were identified in the LAA group with a prevalence of 3%. No mutations were found in the patients with SVD or the controls (p = 0.05). The mutant SAMHD1 proteins were functionally impaired in terms of their catalytic activity as a dNTPase and ability to assemble stable tetramers. Conclusions. Heterozygous SAMHD1 gene mutations might cause genetic predispositions that interact with other risk factors, resulting in increased vulnerability to stroke.

Molecular Basis of Tetramerization and pH-Gating in the KcsA Potassium Channel Cytoplasmic Domain

The intracellular C-terminal domain (CTD) of KcsA, a bacterial homotetrameric potassium channel, is a 40-residue long segment which natively adopts a helical bundle conformation with four-fold symmetry. A hallmark of KcsA behavior is a pH-induced conformational change which leads to opening of the channel at acidic pH. While crystal structures of full-length KcsA failed to observe a pH-effect upon the CTD, other biophysical methods have presented evidence to the contrary. We approached the question of CTD structure and its pH-dependence by studying the behavior of soluble peptides corresponding to residues 128-160 of the CTD (CTD34). A combination of NMR and sedimentation equilibrium experiments established CTD34 to be a tetramer with a KD of (2.0 ± 0.5) x 10−11 M3 at neutral pH, and this tetrameric species undergoes pH-dependent dissociation, rendering CTD34 fully monomeric below pH 5.0. The structural basis for this phenomenon is destabilization of the tetrameric CTD34 by protonation of residue H145 in the monomeric form of the peptide.Molecular factors contributing to CTD tetramerization were investigated by comparing the tetrameric stability of single alanine mutants as determined by NMR, SE and molecular dynamics. Single-residue contributions to tetramer stability were in the 0.5-3.5 kcal/mol range. Hydrophobic interactions between residues lining the tetramer core generally contributed more to formation of tetramer than the inter-subunit salt-bridges between R147 and D149/E152. A third class of residues outside the helical interface influenced tetramer stability via the tetramerization on-rate by changing the inherent helical propensity of CTD34 which promotes tetramer formation. We conclude that the CTD is an independent tetramerization domain modulated by pH, and tetramer formation is controlled by a combination of on- and off-rate effects which conform to current paradigms of protein folding.

Density Functional Theory Study of the Oligomerization of Carboxylic Acids

We present a density functional theory [M06-2X/6-31+G(d,p)] study of the structures and free energies of formation of oligomers of four carboxylic acids (formic acid, acetic acid, tetrolic acid, and benzoic acid) in water, chloroform, and carbon tetrachloride. Solvation effects were treated using the SMD continuum solvation model. The low-lying energy structures of molecular complexes were located by adopting an efficient search procedure to probe the potential energy surfaces of the oligomers of carboxylic acids (CA)n (n = 2-6). The free energies of the isomers of (CA)n in solution were determined as the sum of the electronic energy, vibrational-rotational-translational gas-phase contribution, and solvation free energy. The assessment of the computational protocol adopted in this study with respect to the dimerization of acetic acid, (AA)2, and formic acid, (FA)2, located new isomers of (AA)2 and (FA)2 and gave dimerization constants in good agreement with the experimental values. The calculation of the self-association of acetic acid, tetrolic acid, and benzoic acid shows the following: (i) Classic carboxylic dimers are the most stable isomer of (CA)2 in both the gas phase and solution. (ii) Trimers of carboxylic acid are stable in apolar aprotic solvents. (iii) Molecular clusters consisting of two interacting classic carboxylic dimers (CA)4,(D+D) are the most stable type of tetramers, but their formation from the self-association of classic carboxylic dimers is highly unfavorable. (iv) For acetic acid and tetrolic acid the reactions (CA)2 + 2CA → (CA)4,(D+D) and (CA)3 + CA → (CA)4,(D+D) are exoergonic, but these aggregation pathways go through unstable clusters that could hinder the formation of tetrameric species. (v) For tetrolic acid the prenucleation species that are more likely to form in solution are dimeric and trimeric structures that have encoded structural motifs resembling the α and β solid forms of tetrolic acid. (vi) Stable tetramers of benzoic acid could form in carbon tetrachloride from the aggregation of trimers and monomers. (vii) Higher order clusters such as acetic acid pentamers and tetrolic acid hexamers are highly unstable in all solvation environments.

α+Thalassemia Antagonizes the Malaria-protective Effects of Sickle-Cell Trait

α+Thalassemia Antagonizes the Malaria-protective Effects of Sickle-Cell Trait

Instability, adiabaticity and controlling effects of external fields for the dark state in a heteronuclear atom–tetramer conversion system

We study the formation of stable heteronuclear tetramers from ultracold atoms via two different paths by generalized Raman adiabatic passage. The dynamical instability and adiabaticity of the dark state are investigated. The regions for the appearance of dynamical instability are analytically obtained and the adiabatic evolution is studied by adiabatic fidelity. Moreover, the effects of the external field parameters on the conversion efficiency are investigated, and a comparison is also drawn between the two different paths.

Tracing the Evolution of the p53 Tetramerization Domain

SummaryThe tetrameric transcription factors p53, p63, and p73 evolved from a common ancestor and play key roles in tumor suppression and development. Surprisingly, p63 and p73 require a second helix in their tetramerization domain for the formation of stable tetramers that is absent in human p53, raising questions about the evolutionary processes leading to diversification. Here we determined the crystal structure of the zebrafish p53 tetramerization domain, which contains a second helix, reminiscent of p63 and p73, combined with p53-like features. Through comprehensive phylogenetic analyses, we systematically traced the evolution of vertebrate p53 family oligomerization domains back to the beginning of multicellular life. We provide evidence that their last common ancestor also had an extended p63/p73-like domain and pinpoint evolutionary events that shaped this domain during vertebrate radiation. Domain compaction and transformation of a structured into a flexible, intrinsically disordered region may have contributed to the expansion of the human p53 interactome.

α-Synuclein Misfolding Versus Aggregation Relevance to Parkinson's Disease: Critical Assessment and Modeling.

α-Synuclein, an abundant and conserved presynaptic brain protein, is implicated as a critical factor in Parkinson's disease (PD). The aggregation of α-synuclein is believed to be a critical event in the disease process. α-Synuclein is characterized by a remarkable conformational plasticity, adopting different conformations depending on the environment. Therefore, it is classified as an "intrinsically disordered protein." Recently, a debate has challenged the view on the intrinsically disordered behavior of α-synuclein in the cell. It has been proposed that α-synuclein is a stable tetramer with a low propensity for aggregation; however, its destabilization leads to protein misfolding and its aggregation kinetics. In our critical analysis, we discussed about major issues: (i) why α-synuclein conformational behavior does not fit into the normal secondary structural characteristics of proteins, (ii) potential amino acids involved in the complexity of misfolding in α-synuclein that leads to aggregation, and (iii) the role of metals in misfolding and aggregation. To evaluate the above critical issues, we developed bioinformatics models related to secondary and tertiary conformations, Ramachandran plot, free energy change, intrinsic disordered prediction, solvent accessibility, and FoldIndex pattern. To the best of our knowledge, this is a novel critical assessment to understand the misfolding biology of synuclein and its relevance to Parkinson's disease.

Crystal structure of L-ribose isomerase from Cellulomonas parahominis MB426

Monosaccharides and their derivatives which hardly exist in nature are so-called "rare sugars". Rare sugars have significance not only in food industries but also pharmaceutical industries. We discovered a novel L-ribose isomerase from Cellulomonas parahominis (CpL-RbI, 249 amino acids), which catalyzes the reversible isomerization between L-ribose and L-ribulose, L-allose and L-psicose, and D-talose and D-tagatose. Since CpL-RbI has a broad substrate specificity, it is useful for the production of various rare sugars. To elucidate the molecular basis of unique enzymatic properties of CpL-RbI, we determined its crystal structure. The N-terminal His-tagged CpL-RbI overexpressed in Escherichia coli was purified using a nickel affinity column. Crystals of CpL-RbI were obtained from a reservoir solution of 0.1 M sodium acetate trihydrate (pH 4.6) with 3.9 M ammonium acetate, by a hanging-drop vapor-diffusion method at 293 K (Space group C2221, a = 76.8, b = 88.6, c = 152.3 Å). X-ray diffraction data were collected up to 2.10 Å resolution using a Rigaku R-AXIS VII on a RA-Micro7HF rotating anode generator (40 kV, 30 mA) at 100 K. The structure was solved by a molecular replacement method with a structure of Acinetobacter sp L-ribose isomerase (4NS7) as a search model, and refined to R-factor of 0.227. CpL-RbI had a cupin-type beta-barrel structure, and the catalytic site was found between two large beta-sheets with a bound metal ion (Fig. 1). There were two protein molecules in an asymmetric unit, forming a homo-dimer with a non-crystallographic 2-fold symmetry (Fig.1). Furthermore, the PISA server showed that two dimers in crystal were associated to form a stable tetramer. Complex structures with substrates, L-ribose, L-allose and L-psicose, were also successfully determined. We will discuss a broad substrate specificity and catalytic reaction mechanism of CpL-RbI based on its three-dimensional structure.

Structural characteristics of the nonallosteric human cytosolic malic enzyme

Human cytosolic NADP+-dependent malic enzyme (c-NADP-ME) is neither a cooperative nor an allosteric enzyme, whereas mitochondrial NAD(P)+-dependent malic enzyme (m-NAD(P)-ME) is allosterically activated by fumarate. This study examines the molecular basis for the different allosteric properties and quaternary structural stability of m-NAD(P)-ME and c-NADP-ME. Multiple residues corresponding to the fumarate-binding site were mutated in human c-NADP-ME to correspond to those found in human m-NAD(P)-ME. Additionally, the crystal structure of the apo (ligand-free) human c-NADP-ME conformation was determined. Kinetic studies indicated no significant difference between the wild-type and mutant enzymes in Km,NADP, Km,malate, and kcat. A chimeric enzyme, [51-105]_c-NADP-ME, was designed to include the putative fumarate-binding site of m-NAD(P)-ME at the dimer interface of c-NADP-ME; however, this chimera remained nonallosteric. In addition to fumarate activation, the quaternary structural stability of c-NADP-ME and m-NAD(P)-ME is quite different; c-NADP-ME is a stable tetramer, whereas m-NAD(P)-ME exists in equilibrium between a dimer and a tetramer. The quaternary structures for the S57K/N59E/E73K/S102D and S57K/N59E/E73K/S102D/H74K/D78P/D80E/D87G mutants of c-NADP-ME are tetrameric, whereas the K57S/E59N/K73E/D102S m-NAD(P)-ME quadruple mutant is primarily monomeric with some dimer formation. These results strongly suggest that the structural features near the fumarate-binding site and the dimer interface are highly related to the quaternary structural stability of c-NADP-ME and m-NAD(P)-ME. In this study, we attempt to delineate the structural features governing the fumarate-induced allosteric activation of malic enzyme.

Biochemical and Structural Analysis of the Hyperpolarization-Activated K+ Channel MVP

In contrast to the majority of voltage-gated ion channels, hyperpolarization-activated channels remain closed at depolarizing potentials and are activated at hyperpolarizing potentials. The basis for this reverse polarity is thought to be a result of differences in the way the voltage-sensing domain (VSD) couples to the pore domain. In the absence of structural data, the molecular mechanism of this reverse polarity coupling remains poorly characterized. Here we report the characterization of the structure and local dynamics of the closed activation gate (lower S6 region) of MVP, a hyperpolarization-activated potassium channel from Methanococcus jannaschii, by electron paramagnetic resonance (EPR) spectroscopy. We show that a codon-optimized version of MVP has high expression levels in Escherichia coli, is purified as a stable tetramer, and exhibits expected voltage-dependent activity when reconstituted in liposomes. EPR analysis of the mid to lower S6 region revealed positions exhibiting strong spin–spin coupling, indicating that the activation gate of MVP is closed at 0 mV. A comparison of local environmental parameters along the activation gate for MVP and KcsA indicates that MVP adopts a different closed conformation. These structural details set the stage for future evaluations of reverse electromechanical coupling in MVP.

Transthyretin Stabilization by Iododiflunisal Promotes Amyloid-β Peptide Clearance, Decreases its Deposition, and Ameliorates Cognitive Deficits in an Alzheimer's Disease Mouse Model

Alzheimer's disease (AD) is the most common form of dementia and now represents 50-70% of total dementia cases. Over the last two decades, transthyretin (TTR) has been associated with AD and, very recently, a novel concept of TTR stability has been established in vitro as a key factor in TTR/amyloid-β (Aβ) interaction. Small compounds, TTR stabilizers (usually non-steroid anti-inflammatory drugs), bind to the thyroxine (T4) central binding channel, increasing TTR tetrameric stability and TTR/Aβ interaction. In this work, we evaluated in vivo the effects of one of the TTR stabilizers identified as improving TTR/Aβ interaction, iododiflunisal (IDIF), in Aβ deposition and other AD features, using AβPPswe/PS1A246E transgenic mice, either carrying two or just one copy of the TTR gene (AD/TTR+/+ or AD/TTR+/-, respectively), available and characterized in our laboratory. The results showed that IDIF administered orally bound TTR in plasma and stabilized the protein, as assessed by T4 displacement assays, and was able to enter the brain as revealed by mass spectrometry analysis of cerebrospinal fluid. TTR levels, both in plasma and cerebrospinal fluid, were not altered. In AD/TTR+/- mice, IDIF administration resulted not only in decreased brain Aβ levels and deposition but also in improved cognitive function associated with the AD-like neuropathology in this mouse model, although no improvements were detectable in the AD/TTR+/+ animals. Further, in AD/TTR+/- mice, Aβ levels were reduced in plasma suggesting TTR promoted Aβ clearance from the brain and from the periphery. Taken together, these results strengthen the importance of TTR stability in the design of therapeutic drugs, highlighting the capacity of IDIF to be used in AD treatment to prevent and to slow the progression of the disease.

Crystal structure of the essential transcription antiterminator M2-1 protein of human respiratory syncytial virus and implications of its phosphorylation

The M2-1 protein of the important pathogen human respiratory syncytial virus is a zinc-binding transcription antiterminator that is essential for viral gene expression. We present the crystal structure of full-length M2-1 protein in its native tetrameric form at a resolution of 2.5 Å. The structure reveals that M2-1 forms a disk-like assembly with tetramerization driven by a long helix forming a four-helix bundle at its center, further stabilized by contact between the zinc-binding domain and adjacent protomers. The tetramerization helix is linked to a core domain responsible for RNA binding activity by a flexible region on which lie two functionally critical serine residues that are phosphorylated during infection. The crystal structure of a phosphomimetic M2-1 variant revealed altered charge density surrounding this flexible region although its position was unaffected. Structure-guided mutagenesis identified residues that contributed to RNA binding and antitermination activity, revealing a strong correlation between these two activities, and further defining the role of phosphorylation in M2-1 antitermination activity. The data we present here identify surfaces critical for M2-1 function that may be targeted by antiviral compounds.

MJK2, a K+Channel from M. Jannaschii Mediates pH Dependent Potassium Transport Activity

MjK2 was expressed in E. coli cells as a fusion protein containing N-or C-terminal an antibody binding site and a histidinehexamer. The C-terminal tagged fusion protein allows the expression and purification of an extra soluble RCK domain at p34 kDa, whereas this additional RCK domain was lost when the N-terminal tagged construct was used. Upon removal of the fusion peptide from the purified N-terminal tagged channel monomer, MjK2 occurred as a stable tetramer when incubated with synthetic lipid. The channel activity was studied after reconstitution into liposomes by single channel recording or by an optical assay with the potassium sensing dye, PBFI. First the channel function was improved by single channel recording. Single channel recording confirmed the pH dependence of the channel activity with single channel conductances of 42, 70, 85 and 202 pS and indicated that a functional K+ channel was formed. To study the function of the reconstituted MjK2 activity in an optical assay the potassium release was initiated when the external BaCl2 block was compensated by addition of EDTA. The release of potassium was mediated by reconstituted MjK2 at low pH or by the presence of internal calcium at high pH. MgCl2 had no or weak effect, whereas cAMP at low pH caused a complete loss of potassium during the preparation. Alignments studies revealed that MjK2 has different structural features in the channel pore and the RCK composition and therefore a different function can be expected. Amino acid sequence and structural alignments showed that a Ca2+ binding site and a typical nucleotide-binding site is not present in the RCK domain of MjK2 and therefore a different behavior could be expected. In addition a lysine reach linker region as found in human sperm K+ channels hslo1 and hslo3can play similar role in the gating behavior.

Cryo-Em Studies of DRP1 Self-Assembly Provide Insights into the Mechanism of Mitochondrial Fission

Dynamin-related protein 1 (Drp1) belongs to a family of large GTPase proteins that regulate membrane dynamics and morphology. Self-assembly of cytosolic Drp1 into larger oligomers on the surface of mitochondria is essential for ensuing membrane fission. Preliminary studies show that in vitro reconstitutions of Drp1 emulate key features of the mitochondrial division machinery and provide a model system for evaluating conformational changes that drive membrane remodeling. To understand Drp1 self-assembly, cryo-electron microscopy (cryo-EM) will be used to determine the 3D structures of both the pre-assembly state and helical oligomers. More specifically, recombinant Drp1 forms stable tetramers in solution that are amenable to EM analysis. In the presence of non-hydrolyzable GTP analogs and/or synthetic liposomes, Drp1 further assembles into helical oligomers with varying diameters, suggesting ligand-induced conformational changes. Cryo-EM studies will determine the 3D structures of reconstituted Drp1 oligomers to reveal key intermolecular interactions and conformational changes that drive Drp1 self-assembly and mediate mitochondrial membrane fission.