Dimer Self-association Research Articles

Asymmetric horseshoe-like assembly of peroxisomal yeast oxalyl-CoA synthetase.

Oxalyl-CoA synthetase from Saccharomyces cerevisiae is one of the most abundant peroxisomal proteins in yeast and hence has become a model to study peroxisomal translocation. It contains a C-terminal Peroxisome Targeting Signal 1, which however is partly dispensable, suggesting additional receptor bindings sites. To unravel any additional features that may contribute to its capacity to be recognized as peroxisomal target, we determined its assembly and overall architecture by an integrated structural biology approach, including X-ray crystallography, single particle cryo-electron microscopy and small angle X-ray scattering. Surprisingly, it assembles into mixture of concentration-dependent dimers, tetramers and hexamers by dimer self-association. Hexameric particles form an unprecedented asymmetric horseshoe-like arrangement, which considerably differs from symmetric hexameric assembly found in many other protein structures. A single mutation within the self-association interface is sufficient to abolish any higher-level oligomerization, resulting in a homogenous dimeric assembly. The small C-terminal domain of yeast Oxalyl-CoA synthetase is connected by a partly flexible hinge with the large N-terminal domain, which provides the sole basis for oligomeric assembly. Our data provide a basis to mechanistically study peroxisomal translocation of this target.

To deform or not to deform: the evolutionary basis of mammalian red blood cell deformability

To deform or not to deform: the evolutionary basis of mammalian red blood cell deformability

Novel mechanism of hereditary pyropoikilocytosis phenotype due to co‐inheritance of β globin and α spectrin mutations

Novel mechanism of hereditary pyropoikilocytosis phenotype due to co‐inheritance of β globin and α spectrin mutations

New Disulphide Bond in Cystatin-Based Protein Scaffold Prevents Domain-Swap-Mediated Oligomerization and Stabilizes the Functionally Active Form.

Peptide aptamers built using engineered scaffolds are a valuable alternative to monoclonal antibodies in many research applications because of their smaller size, versatility, specificity for chosen targets, and ease of production. However, inserting peptides needed for target binding may affect the aptamer structure, in turn compromising its activity. We have shown previously that a stefin A-based protein scaffold with AU1 and Myc peptide insertions (SQT-1C) spontaneously forms dimers and tetramers and that inserted loops mediate this process. In the present study, we show that SQT-1C forms tetramers by self-association of dimers and determine the kinetics of monomer–dimer and dimer–tetramer transitions. Using site-directed mutagenesis, we show that while slow domain swapping defines the rate of dimerization, conserved proline P80 is involved in the tetramerization process. We also demonstrate that the addition of a disulphide bond at the base of the engineered loop prevents domain swapping and dimer formation, also preventing subsequent tetramerization. Formation of SQT-1C oligomers compromises the presentation of inserted peptides for target molecule binding, diminishing aptamer activity; however, the introduction of the disulphide bond locking the monomeric state enables maximum specific aptamer activity, while also increasing its thermal and colloidal stability. We conclude that stabilizing scaffold proteins by adding disulphide bonds at peptide insertion sites might be a useful approach in preventing binding-epitope-driven oligomerization, enabling creation of very stable aptamers with maximum binding activity.

The Early Phase of β2m Aggregation: An Integrative Computational Study Framed on the D76N Mutant and the ΔN6 Variant.

Human β2-microglobulin (b2m) protein is classically associated with dialysis-related amyloidosis (DRA). Recently, the single point mutant D76N was identified as the causative agent of a hereditary systemic amyloidosis affecting visceral organs. To get insight into the early stage of the β2m aggregation mechanism, we used molecular simulations to perform an in depth comparative analysis of the dimerization phase of the D76N mutant and the ΔN6 variant, a cleaved form lacking the first six N-terminal residues, which is a major component of ex vivo amyloid plaques from DRA patients. We also provide first glimpses into the tetramerization phase of D76N at physiological pH. Results from extensive protein–protein docking simulations predict an essential role of the C- and N-terminal regions (both variants), as well as of the BC-loop (ΔN6 variant), DE-loop (both variants) and EF-loop (D76N mutant) in dimerization. The terminal regions are more relevant under acidic conditions while the BC-, DE- and EF-loops gain importance at physiological pH. Our results recapitulate experimental evidence according to which Tyr10 (A-strand), Phe30 and His31 (BC-loop), Trp60 and Phe62 (DE-loop) and Arg97 (C-terminus) act as dimerization hot-spots, and further predict the occurrence of novel residues with the ability to nucleate dimerization, namely Lys-75 (EF-loop) and Trp-95 (C-terminus). We propose that D76N tetramerization is mainly driven by the self-association of dimers via the N-terminus and DE-loop, and identify Arg3 (N-terminus), Tyr10, Phe56 (D-strand) and Trp60 as potential tetramerization hot-spots.

Molecular basis for multimerization in the activation of the epidermal growth factor receptor.

The epidermal growth factor receptor (EGFR) is activated by dimerization, but activation also generates higher-order multimers, whose nature and function are poorly understood. We have characterized ligand-induced dimerization and multimerization of EGFR using single-molecule analysis, and show that multimerization can be blocked by mutations in a specific region of Domain IV of the extracellular module. These mutations reduce autophosphorylation of the C-terminal tail of EGFR and attenuate phosphorylation of phosphatidyl inositol 3-kinase, which is recruited by EGFR. The catalytic activity of EGFR is switched on through allosteric activation of one kinase domain by another, and we show that if this is restricted to dimers, then sites in the tail that are proximal to the kinase domain are phosphorylated in only one subunit. We propose a structural model for EGFR multimerization through self-association of ligand-bound dimers, in which the majority of kinase domains are activated cooperatively, thereby boosting tail phosphorylation.

Readily prepared inclusion forming chiral calixsalens.

Calixsalens, chiral triangular hexaimines are readily synthesized by [3 + 3] cyclocondensation of trans-(R,R)-1,2-diaminocyclohexane with 2-hydroxyisophthalaldehyde derivatives. The usually rigid calixsalen ring is able to invert its conformation as a consequence of steric repulsion between bulky substituents at the C5 positions of the aromatic rings. The steric and electronic nature of the substituents does not affect only the conformation of the macrocycle. Small polar substituents enforce dimeric self-association to form an apohost where each of the monomers simultaneously serves as the host and the guest of its partner. Non-associating calixsalens form assemblies in which two symmetry-related molecules are arranged in a head-to-head fashion to form a capsule, or unimolecular cages that are able to entrap solvent molecules in their intrinsic voids.

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.

IR Study of Monomer–Dimer Self-association of 2,2-Dimethyl-3-ethyl-3-pentanol in n-Octane: Determination of the Molar Absorptivities of Monomer and Dimer Bands, and Dimerization Constants Using Novel Equations

The monomer–dimer self-association equilibrium of 2,2-dimethyl-3-ethyl-3-pentanol in n-octane has been studied by IR spectroscopy at four temperatures (288, 298, 308, and 318 K). The solute was chosen to restrict the self-association between solute molecules to dimerization only, owing to steric hindrance of the bulky chains in the vicinity of the OH group. Two linear equations have been derived for the treatment of the experimental data. One of these equations was used to treat the data of the concentration dependent integrated absorbance of the monomer bands for each temperature to obtain the monomer molar absorptivity, ε m, and dimerization constant, K. The other equation was used to treat the data of concentration-dependent dimer bands to obtain the dimer molar absorptivity, ε d, and K. Thus, the dimerization constant was determined by two methods. Since the same thermodynamic quantity K is obtained from either the monomer bands or the dimer bands, the difference between them at a given temperature can serve as an assessment of the quality of the experiment. The standard enthalpy and entropy of dimerization were also obtained from a van’t Hoff plot.

Novel exon 2 spectrin mutation and intragenic crossover: three morphological phenotypes associated with four distinct spectrin defects

Hereditary pyropoikilocytosis is a severe hemolytic anemia caused by spectrin deficiency and defective spectrin dimer self-association, typically found in African populations. We describe two Utah families of northern European ancestry including 2 propositi with atypical non-microcytic hereditary pyropoikilocytosis, 7 hereditary elliptocytosis members and one asymptomatic carrier. The underlying molecular defect is a novel mutation in the alpha(α) spectrin gene, SPTA(R34P) that impairs spectrin tetramer formation. It is inherited in trans to the hypomorphic SPTA(αLELY) in the 2 propositi and 5 of 7 hereditary elliptocytosis individuals indicating that SPTA(αLELY) is not the sole determinant of the variable clinical expression. α Spectrin mRNA was mildly decreased in all hereditary elliptocytosis subjects, whereas both hereditary pyropoikilocytosis propositi had a severe decrease to ~10% of normal. Genotyping identified a unique SPTA intragenic crossover and uniparental disomy in one hereditary elliptocytosis individual. Two additional crossover events demonstrated the susceptibility of SPTA gene to rearrangement and revealed a novel segregation of the two SPTA(αLELY) mutations. We conclude that the profound phenotypic heterogeneity in these families can be attributed to the SPTA(R34P) mutation in combination with: 1) inheritance in trans of either SPTA(αLELY); or 2) the wild-type SPTA; 3) a decrease of α spectrin mRNA; and 4) SPTA intragenic crossover.

The molecular self-association of carboxylic acids in solution: testing the validity of the link hypothesis using a quantum mechanical continuum solvation approach

According to the “link hypothesis” there is a connection between the solvent-dependent most stable self-associated groups of molecules and the building unit present in the polymorph that crystallizes from solution. We have tested this hypothesis by computing the Gibbs free energies for the molecular self-association in solution of three selected monocarboxylic acids that crystallize in different polymorphic forms and exhibit selective crystallization depending on the solvent: tetrolic acid (TTA), m-aminobenzoic acid (mABA) and m-hydroxybenzoic acid (mHBA). Calculations have been conducted at the density functional theory (M06-2X) level with the SMD polarizable continuum model to simulate aqueous and organic solutions. For all three systems we have found that the solvation environment significantly affects the stability of dimers. The most stable dimer in solution is the classic carboxylic acid dimer, and its stability decreases on going from nonpolar solvents, such as chloroform or acetonitrile, to aqueous and alcoholic solutions. However, whilst for TTA and mABA there is a link between the carboxylic dimer synthon in solution and the structural synthon packed in the metastable α-TTA polymorph, which crystallizes from chloroform, and in the crystal form-II of mABA, which crystallizes from acetonitrile, for mHBA the stabilization of the centrosymmetric carboxylic dimeric in acetonitrile and ethyl acetate does not correspond with recent experimental observations that the polymorph containing this structural unit (form I) is not the crystal form that nucleates preferentially from these solvents (Cryst. Growth Des., 2013, 13, 1140). Starting from the carboxylic dimer of TTA, the processes of trimerization and tetramerization of TTA from solution have been also modelled. The calculations suggest that the formation of tetramers in chloroform occurs from the self-association of the carboxylic acid dimers and not through the association of the TTA monomers and trimers. The free energy formation of the ionized forms of the mABA dimer (non-zwitterionic–zwitterionic and zwitterionic–zwitterionic) has been evaluated and, according to our results, zwitterionic–zwitterionic mABA dimers might be abundant in supersaturated aqueous and alcoholic solutions of m-aminobenzoic acid.

Dimer self-association via hydrogen bonding: Measurement and comparison of binding constants with 2-amidopyrimidine derivatives

A method based on 1H NMR was used to measure the self-assembly equilibrium constants for three acylated derivatives of 2-aminopyrimidine: 2-acetamidopyrimidine (1), 2-isopropylamidopyrimidine (2), and 2-neopentylamidopyrimidine (3). The synthesis of the latter two compounds is described. The self-association constant decreases from 1 to 2 to 3, which is attributed to the syn/anti conformational preference of the amide bond. For 1, complexation with chloroform must be included in order to explain the observed chemical shift values. Complementary density functional theory calculations (MP2/cc-pVTZ//B3LYP-D/cc-pVDZ) suggest a direct relationship between conformational preference of the amide bond and the self-assembly constant.

Structure of the heterodimer of human NONO and paraspeckle protein component 1 and analysis of its role in subnuclear body formation

Proteins of the Drosophila behavior/human splicing (DBHS) family include mammalian SFPQ (PSF), NONO (p54nrb), PSPC1, and invertebrate NONA and Hrp65. DBHS proteins are predominately nuclear, and are involved in transcriptional and posttranscriptional gene regulatory functions as well as DNA repair. DBHS proteins influence a wide gamut of biological processes, including the regulation of circadian rhythm, carcinogenesis, and progression of cancer. Additionally, mammalian DBHS proteins associate with the architectural long noncoding RNA NEAT1 (Menε/β) to form paraspeckles, subnuclear bodies that alter gene expression via the nuclear retention of RNA. Here we describe the crystal structure of the heterodimer of the multidomain conserved region of the DBHS proteins, PSPC1 and NONO. These proteins form an extensively intertwined dimer, consistent with the observation that the different DBHS proteins are typically copurified from mammalian cells, and suggesting that they act as obligate heterodimers. The PSPC1/NONO heterodimer has a right-handed antiparallel coiled-coil that positions two of four RNA recognition motif domains in an unprecedented arrangement on either side of a 20-Å channel. This configuration is supported by a protein:protein interaction involving the NONA/paraspeckle domain, which is characteristic of the DBHS family. By examining various mutants and truncations in cell culture, we find that DBHS proteins require an additional antiparallel coiled-coil emanating from either end of the dimer for paraspeckle subnuclear body formation. These results suggest that paraspeckles may potentially form through self-association of DBHS dimers into higher-order structures.

Determinants of the Higher Order Association of the Restriction Factor TRIM5α and Other Tripartite Motif (TRIM) Proteins

Many tripartite motif (TRIM) proteins self-associate, forming dimers and higher order complexes. For example, dimers of TRIM5α, a host factor that restricts retrovirus infection, assemble into higher order arrays on the surface of the viral capsid, resulting in an increase in avidity. Here we show that the higher order association of different TRIM proteins exhibits a wide range of efficiencies. Homologous association (self-association) was more efficient than the heterologous association of different TRIM proteins, indicating that specificity determinants of higher order self-association exist. To investigate the structural determinants of higher order self-association, we studied TRIM mutants and chimeras. These studies revealed the following: 1) the RING domain contributes to the efficiency of higher order self-association, which enhances the binding of TRIM5α to the human immunodeficiency virus (HIV-1) capsid; 2) the RING and B-box 2 domains work together as a homologous unit to promote higher order association of dimers; 3) dimerization is probably required for efficient higher order self-association; 4) the Linker 2 region contributes to higher order self-association, independently of effects of Linker 2 changes on TRIM dimerization; and 5) for efficiently self-associating TRIM proteins, the B30.2(SPRY) domain is not required for higher order self-association. These results support a model in which both ends of the core TRIM dimer (RING-B-box 2 at one end and Linker 2 at the other) contribute to the formation of higher order arrays.

Dimeric self-association of an isophthalamide macrocycle in solution and the solid state

An isophthalamide macrocycle used in anion-templated interpenetrated structures is found to participate in dimeric self-assembly throughout eight different crystal forms. Analysis of these, in combination with solution-phase NMR studies, reveals the nature of the hydrogen-bonded self-association, yielding new perspectives and potential applications for the macrocycle system.

Rigid Dimers Formed through Strong Interdigitated H‐Bonds Yield Compact 1D Supramolecular Helical Polymers

Hierarchical self-assembly of small abiotic molecular modules interacting through noncovalent forces is increasingly being used to generate functional structures and materials for electronic, catalytic, and biomedical applications. The greatest control over the geometry in H-bond supramolecular architectures, especially in H-bonded supramolecular polymers, can be achieved by using conformationally rigid molecular modules undergoing self-assembly through strong H-bonds. Their binding strength depends on the multiplicity of the H-bonds, the nature of donor/acceptor pairs and their secondary attractive/repulsive interactions. Here a functionalized molecular module is described, which is capable of self-associating through self-complementary H-bonding patterns comprising four strong and two medium-strength H-bonds to form dimers. The self-association of these phenylpyrimidine-based dimers through directional H-bonding between two lateral pyridin-2(1H)-one units of neighboring molecules allows the formation of highly compact 1D supramolecular polymers by self-assembly on graphite. A concentration-dependent study by scanning tunneling microscopy at the solid-liquid interface, corroborated by dispersion-corrected density functional studies, reveals the controlled generation of either linear supramolecular 2D arrays, or long helical supramolecular polymers with a high shape persistence.

Phenylketonuria as a protein misfolding disease: The mutation pG46S in phenylalanine hydroxylase promotes self-association and fibril formation

The missense mutation pG46S in the regulatory (R) domain of human phenylalanine hydroxylase (hPAH), associated with a severe form of phenylketonuria, generates a misfolded protein which is rapidly degraded on expression in HEK293 cells. When overexpressed as a MBP-G46S fusion protein, soluble and fully active tetrameric/dimeric forms are assembled and recovered in a metastable conformational state. When MBP is cleaved off, G46S undergoes a conformational change and self-associates with a lag phase and an autocatalytic growth phase (tetramers ≫ dimers), as determined by light scattering. The self-association is controlled by pH, ionic strength, temperature, protein concentration and the phosphorylation state of Ser16; the net charge of the protein being a main modulator of the process. A superstoichiometric amount of WT dimers revealed a 2-fold enhancement of the rate of G46S dimer self-association. Electron microscopy demonstrates the formation of higher-order oligomers and linear polymers of variable length, partly as a branching network, and partly as individual long and twisted fibrils (diameter ~ 145–300 Å). The heat-shock proteins Hsp70/Hsp40, Hsp90 and a proposed pharmacological PAH chaperone (3-amino-2-benzyl-7-nitro-4-(2-quinolyl)-1,2-dihydroisoquinolin-1-one) partly inhibit the self-association process. Our data indicate that the G46S mutation results in a N-terminal extension of α-helix 1 which perturbs the wild-type α– β sandwich motif in the R-domain and promotes new intermolecular contacts, self-association and non-amyloid fibril formation. The metastable conformational state of G46S as a MBP fusion protein, and its self-association propensity when released from MBP, may represent a model system for the study of other hPAH missense mutations characterized by misfolded proteins.

The Impact of Viral RNA on the Association Rates of Capsid Protein Assembly: Bacteriophage MS2 as a Case Study

A large number of single-stranded RNA viruses, which form a major class of all viruses, co-assemble their protein container and their genomic material. The multiple roles of the viral genome in this process are presently only partly understood. Recent experimental results indicate that RNA, in addition to its function as a repository for genetic information, could play important functional roles during the assembly of the viral protein containers. An investigation of the impact of genomic RNA on the association of the protein subunits may therefore provide further insights into the mechanism of virus assembly. We study here the impact of viral RNA on the association rates of the capsid proteins during virus assembly. As a case study, we consider the viral capsid of bacteriophage MS2, which is formed from 60 asymmetric (AB) and 30 symmetric (CC) protein dimers. Using Brownian dynamics simulations, we investigate the effect of the binding of an RNA stem–loop (the translational repressor) on the association rates of the capsid protein dimers. Our analysis shows that translational repressor binding results in self-association of AB dimers being inhibited, whilst association of AB with CC dimers is greatly enhanced. This provides an explanation for experimental results in which an alternating assembly pattern of AB and CC dimer addition to the growing assembly intermediate has been observed to be the dominant mode of assembly. The presence of the RNA hence dramatically decreases the number of dominant assembly pathways and thereby reduces the complexity of the self-assembly process of these viruses.

αII-Spectrin Is Critical for Cell Adhesion and Cell Cycle

Spectrins are ubiquitous scaffolding components of the membrane skeleton that organize and stabilize microdomains on both the plasma membrane and the intracellular organelles. By way of their numerous interactions with diverse protein families, they are implicated in various cellular functions. Using small interfering RNA strategy in the WM-266 cell line derived from human melanoma, we found that alphaII-spectrin deficiency is associated with a defect in cell proliferation, which is related to a cell cycle arrest at the G1 phase (first gap phase), as evaluated by DNA analysis and Rb phosphorylation. These observations coincided with elevated expression of the cyclin-dependent kinase inhibitor, p21Cip. Concomitantly, spectrin loss impaired cell adhesion and spreading. These cell adhesion defects were associated with modifications of the actin cytoskeleton, such as loss of stress fibers, alterations of focal adhesions, and modified expression of some integrins. Our results provide novel insights into spectrin functions by demonstrating the involvement of alphaII-spectrin in cell cycle regulation and actin organization.

NMR and molecular modeling of the dimeric self-association of the enantiomers of 1,1′-bi-2-naphthol and 1-phenyl-2,2,2-trifluoroethanol in the solution state and their relevance to enantiomer self-disproportionation on achiral-phase chromatography (ESDAC)

Molecular modeling of the homo- and heterochiral dimeric self-associates of the enantiomers of 1,1'-bi-2-naphthol and 1-phenyl-2,2,2-trifluoroethanol in solution has been performed in order to understand their NMR behavior and in light of the phenomenon of "enantiomer self-disproportionation on achiral-phase chromatography" (ESDAC). For 1,1'-bi-2-naphthol in C(6)D(6), distinct NMR signals for each enantiomer arise for some spins in non-racemic mixtures-the phenomenon of self-induced diastereomeric anisochronism (SIDA). The linear divergence of these split signals across an enantiomeric titration (a series of samples in which the percentage of one enantiomer is varied from 50-100% whilst maintaining a constant total concentration), as well as the near linear migration of certain signals in CDCl(3) across a similar enantiomeric titration, where signals were not observed to be split, is consistent with the calculated small energy differences between the homo- and heterochiral associates. For an enantiomeric titration of 1-phenyl-2,2,2-trifluoroethanol in n-hexane, NMR signals also remained unsplit but the noticeable migration of some revealed a skew indicative of a preference for the heterochiral associate. This was duly reflected in the calculations which provided a DeltaG value favoring the heterochiral associate by 2.4 kJ mol(-1). The relevance of these results to evaluating the likely occurrence of ESDAC is considered.