AB Dimer Research Articles

Fluoride-Triggered Self-Degradation of Poly(2,4-disubstitued 4-hydroxybutyric acid) Derivatives.

Self-immolative polymers are a special kind of degradable polymers that depolymerize into small molecules through a cascade of reactions upon stimuli-triggered cleavage of the polymer chain ends. This work reports the design and synthesis of a fluoride-triggered self-immolative polyester. A 2,4-disubstitued 4-hydroxy butyrate is first confirmed to quickly cyclize in solution to form a γ-butyrolactone derivative. Then, the Passerini three component reaction (P-3CR) of an AB dimer (A: aldehyde, B: carboxylic acid) with tert-butyl isocyanide or oligo(ethylene glycol) isocyanide affords two poly(2,4-disubstitued 4-hydroxybutyrate) derivatives (P2 and P3). Two silyl ether end-capped polymers (P4 and P5) are abtained from P2 and P3, and their degradation in solution is examined by NMR spectrum and size exclusion chromatography. Polymers P4 and P5 are stable in the absence of tetrabutylammonium fluoride (TBAF), while in the presence of TBAF, the molar masses of P4 and P5 gradually decrease with time together with the increase of the amount of formed 2,4-disubstitued γ-butyrolactone. The depolymerization mechanism is proposed. The first step is the fast removal of the silyl ether by fluoride. Then, the released hydroxyl group initiates the quick head-to-tail depolymerization of the polyester via intramolecular cyclization.

A Multiscale Model for the Self-Assembly of Coat Proteins in Bacteriophage MS2.

The self-assembly of viral capsids is an essential step to the formation of infectious viruses. Elucidating the kinetic mechanisms of how a capsid or virus-like particle assembles could advance our knowledge about the viral lifecycle, as well as the general principles in self-assembly of biomaterials. However, current understanding of capsid assembly remains incomplete for many viruses due to the fact that the transient intermediates along the assembling pathways are experimentally difficult to be detected. In this paper, we constructed a new multiscale computational framework to simulate the self-assembly of virus-like particles. We applied our method to the coat proteins of bacteriophage MS2 as a specific model system. This virus-like particle of bacteriophage MS2 has a unique feature that its 90 sequence-identical dimers can be classified into two structurally various groups: one is the symmetric CC dimer, and the other is the asymmetric AB dimer. The homotypic interactions between AB dimers result in a 5-fold symmetric contact, while the heterotypic interactions between AB and CC dimers result in 6-fold symmetric contact. We found that the assembly can be described as a physical process of phase transition that is regulated by various factors such as concentration and specific stoichiometry between AB and CC dimers. Our simulations also demonstrate that heterotypic and homotypic interfaces play distinctive roles in modulating the assembling kinetics. The interaction between AB and CC dimers is much more dynamic than that between two AB dimers. We therefore suggest that the alternate growth of viral capsid through the heterotypic dimer interactions dominates the assembling pathways. This is, to the best of our knowledge, the first multiscale model to simulate the assembling process of coat proteins in bacteriophage MS2. The generality of this approach opens the door to its further applications in assembly of other viral capsids, virus-like particles, and novel drug delivery systems.

Switchable Triggered Interconversion and Reconfiguration of DNA Origami Dimers and Their Use for Programmed Catalysis.

The switchable reconfiguration of a mixture of two dimers of DNA origami tiles, AB and CD, into a mixture of two DNA origami dimers composed of AD and CB, using a collection of fuel and antifuel strands, is presented. The reversible reconfiguration of the mixture of dimers AB and CD into AD and CB, is followed by labeling each of the tiles with 0, 1, 2, and 3 4× hairpins labels and by imaging the dimer structures by atomic force microscopy. Subjecting the reconfigurable dimer mixtures to a collection of Mg2+-ion-dependent DNAzyme subunits and the substrates consisting of the ROX/BHQ2-modified substrate and the FAM/BHQ1-modified substrate leads to the triggered and programmed switchable operation, in the presence of appropriate fuel and antifuel strands. In the presence of the AB and CD mixture, the DNAzyme subunits cleaving the ROX/BHQ2-modified substrate is switched on, leading to the fluorescence of ROX. The reconfiguration of the AB and CD dimer mixture to the AD and CB dimer mixture leads to the assembly of the DNAzyme subunits that switches on the cleavage of the FAM/BHQ1-modified substrate and to the fluorescence of FAM. By the cyclic and reversible reconfiguration of the AB and CD dimer mixture to the AD and CB dimer mixture, in the presence of the appropriate fuel and antifuel strands, the switchable catalytic operation of two Mg2+-ion-dependent DNAzymes, leading to the fluorescence of ROX or FAM, is demonstrated.

Heterodimer Autorepression Loop: A Robust and Flexible Pulse-Generating Genetic Module.

We investigate the dynamics of the heterodimer autorepression loop (HAL), a small genetic module in which a protein A acts as an autorepressor and binds to a second protein B to form an AB dimer. For suitable values of the rate constants, the HAL produces pulses of A alternating with pulses of B. By means of analytical and numerical calculations, we show that the duration of A pulses is extremely robust against variation of the rate constants while the duration of the B pulses can be flexibly adjusted. The HAL is thus a minimal genetic module generating robust pulses with a tunable duration, an interesting property for cellular signaling.

Atomic Level Description of the Immune Complex That Causes Heparin-Induced Thrombocytopenia (HIT)

Heparin-induced thrombocytopenia (HIT) is thrombotic disorder caused by immune complexes containing antibodies to an antigen composed of platelet factor 4 (PF4) and heparin or cellular glycosaminoglycans (GAGs). The structure of these immune complexes and how their composition might contribute to the difference between pathogenic and non-pathogenic anti-PF4 antibodies are unknown. To address these questions, we solved the crystal structures of human recombinant PF4 in complex with Fabs derived from KKO (a murine monoclonal HIT-like antibody that competes with pathogenic human HIT antibodies) and RTO (an isotype-matched non-HIT anti-PF4 antibody) combined with the crystal structure of PF4 complexed with the heparin-mimic pentasaccharide fondaparinux as a model sugar. The PF4 tetramer is asymmetric and is capable of accommodating only two fondaparinux molecules. Fondaparinux binds between monomers A, B and C or between monomers A, C, and D, which stabilizes the AB/CD and AC/BD associations and the resultant tetramer. KKO-Fab binds to the PF4 tetramer by making contacts with now identified residues within each of three PF4 monomers, indicating that tetramerization of PF4 is a critical initiating step in antigen formation. Mutations in the putative KKO epitopes in PF4 abolished antibody binding.Unexpectedly, RTO-Fab binds to the PF4 monomer between the AB dimer interface. Importantly, the amino acid sequence recognized by RTO and KKO show considerable overlap. However, the epitope for RTO is obscured upon tetramer formation, in direct contrast to binding of KKO, which requires tetramer formation to bind. Binding of RTO to the PF4 monomer prevents formation of AB dimers and subsequent tetramerization. In support of these findings, preincubation of PF4 with RTO inhibits KKO induced platelet activation and platelet aggregation in vitro. Based on the analyses of crystal lattices, we propose a new model of the heparin/PF4 complex, in which PF4 tetramers cluster around a semi-rigid linear heparin subunit. Clustering of PF4 on heparin might be required for apposition of sufficient HIT antibodies to induce persistent activation of cellular FcgIIA receptors. Heparin and pathogenic HIT antibodies collaborate to stabilize the ternary immune complex, which leads to the disappearance of binding sites for at least some non-pathogenic HIT antibodies. The balance between anti-monomer and anti-tetramer PF4 antibodies may help determine the probability of clinical disease. This model also helps to explain why RTO-like anti-PF4 antibodies are found so commonly in asymptomatic patients exposed to heparin and why fondaparinux may be antigenic but rarely causes HIT, whereas longer heparin fragments and GAGs extend and render the holo-complex more stable and thereby foster the formation of pathogenic immune complexes. In summary, these crystallographic studies lead to a new model to explain the formation of pathogenic immune complexes that lead to HIT. The inhibitory effect of the anti-PF4 antibody RTO provides a structural basis for the development of new diagnostics and non-anticoagulant therapeutics. DisclosuresNo relevant conflicts of interest to declare.

The existence of two nematic phases caused by dimer recombination

A mixture of A and B monomers without shape anisotropy but that are capable of forming three types, viz., AA, BB, and AB mesogenic dimers is investigated. The possibility of a temperature transition from one nematic phase consisting mainly of AB dimer formed by monomers of different types into another nematic phase consisting mainly of AA and BB dimers formed by monomers of the same type is shown.

Structure of a Protozoan Virus from the Human Genitourinary Parasite Trichomonas vaginalis

ABSTRACTThe flagellated protozoan Trichomonas vaginalis is an obligate human genitourinary parasite and the most frequent cause of sexually transmitted disease worldwide. Most clinical isolates of T. vaginalis are persistently infected with one or more double-stranded RNA (dsRNA) viruses from the genus Trichomonasvirus, family Totiviridae, which appear to influence not only protozoan biology but also human disease. Here we describe the three-dimensional structure of Trichomonas vaginalis virus 1 (TVV1) virions, as determined by electron cryomicroscopy and icosahedral image reconstruction. The structure reveals a T = 1 capsid comprising 120 subunits, 60 in each of two nonequivalent positions, designated A and B, as previously observed for fungal Totiviridae family members. The putative protomer is identified as an asymmetric AB dimer consistent with either decamer or tetramer assembly intermediates. The capsid surface is notable for raised plateaus around the icosahedral 5-fold axes, with canyons connecting the 2- and 3-fold axes. Capsid-spanning channels at the 5-fold axes are unusually wide and may facilitate release of the viral genome, promoting dsRNA-dependent immunoinflammatory responses, as recently shown upon the exposure of human cervicovaginal epithelial cells to either TVV-infected T. vaginalis or purified TVV1 virions. Despite extensive sequence divergence, conservative features of the capsid reveal a helix-rich fold probably derived from an ancestor shared with fungal Totiviridae family members. Also notable are mass spectrometry results assessing the virion proteins as a complement to structure determination, which suggest that translation of the TVV1 RNA-dependent RNA polymerase in fusion with its capsid protein involves −2, and not +1, ribosomal frameshifting, an uncommonly found mechanism to date.

Structural Determinants of the β-Selectivity of a Bacterial Aminotransferase

Chiral β-amino acids occur as constituents of various natural and synthetic compounds with potentially useful bioactivities. The pyridoxal 5'-phosphate (PLP)-dependent S-selective transaminase from Mesorhizobium sp. strain LUK (MesAT) is a fold type I aminotransferase that can be used for the preparation of enantiopure β-Phe and derivatives thereof. Using x-ray crystallography, we solved structures of MesAT in complex with (S)-β-Phe, (R)-3-amino-5-methylhexanoic acid, 2-oxoglutarate, and the inhibitor 2-aminooxyacetic acid, which allowed us to unveil the molecular basis of the amino acid specificity and enantioselectivity of this enzyme. The binding pocket of the side chain of a β-amino acid is located on the 3'-oxygen side of the PLP cofactor. The same binding pocket is utilized by MesAT to bind the α-carboxylate group of an α-amino acid. A β-amino acid thus binds in a reverse orientation in the active site of MesAT compared with an α-amino acid. Such a binding mode has not been reported before for any PLP-dependent aminotransferase and shows that the active site of MesAT has specifically evolved to accommodate both β- and α-amino acids.

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.

Structure of dehaloperoxidase B at 1.58 A resolution and structural characterization of the AB dimer from Amphitrite ornata.

As members of the globin superfamily, dehaloperoxidase (DHP) isoenzymes A and B from the marine annelid Amphitrite ornata possess hemoglobin function, but they also exhibit a biologically relevant peroxidase activity that is capable of converting 2,4,6-trihalophenols to the corresponding 2,6-dihaloquinones in the presence of hydrogen peroxide. Here, a comprehensive structural study of recombinant DHP B, both by itself and cocrystallized with isoenzyme A, using X-ray diffraction is presented. The structure of DHP B refined to 1.58 A resolution exhibits the same distal histidine (His55) conformational flexibility as that observed in isoenzyme A, as well as additional changes to the distal and proximal hydrogen-bonding networks. Furthermore, preliminary characterization of the DHP AB heterodimer is presented, which exhibits differences in the AB interface that are not observed in the A-only or B-only homodimers. These structural investigations of DHP B provide insights that may relate to the mechanistic details of the H(2)O(2)-dependent oxidative dehalogenation reaction catalyzed by dehaloperoxidase, present a clearer description of the function of specific residues in DHP at the molecular level and lead to a better understanding of the paradigms of globin structure-function relationships.

Crystal structure and association behaviour of the GluR2 amino-terminal domain

Fast excitatory neurotransmission is mediated largely by ionotropic glutamate receptors (iGluRs), tetrameric, ligand-gated ion channel proteins comprised of three subfamilies, AMPA, kainate and NMDA receptors, with each subfamily sharing a common, modular-domain architecture. For all receptor subfamilies, active channels are exclusively formed by assemblages of subunits within the same subfamily, a molecular process principally encoded by the amino-terminal domain (ATD). However, the molecular basis by which the ATD guides subfamily-specific receptor assembly is not known. Here we show that AMPA receptor GluR1- and GluR2-ATDs form tightly associated dimers and, by the analysis of crystal structures of the GluR2-ATD, propose mechanisms by which the ATD guides subfamily-specific receptor assembly.

Probing potential binding modes of the p53 tetramer to DNA based on the symmetries encoded in p53 response elements

Symmetries in the p53 response-element (p53RE) encode binding modes for p53 tetramer to recognize DNA. We investigated the molecular mechanisms and biological implications of the possible binding modes. The probabilities evaluated with molecular dynamics simulations and DNA sequence analyses were found to be correlated, indicating that p53 tetramer models studied here are able to read DNA sequence information. The traditionally believed mode with four p53 monomers binding at all four DNA quarter-sites does not cause linear DNA to bend. Alternatively, p53 tetramer can use only two monomers to recognize DNA sequence and induce DNA bending. With an arrangement of dimer of AB dimer observed in p53 trimer–DNA complex crystal, p53 can recognize supercoiled DNA sequence-specifically by binding to quarter-sites one and four (H14 mode) and recognize Holliday junction geometry-specifically. Examining R273H mutation and p53–DNA interactions, we found that at least three R273H monomers are needed to disable the p53 tetramer, consistent with experiments. But just one R273H monomer may greatly shift the binding mode probabilities. Our work suggests that p53 needs balanced binding modes to maintain genome stability. Inverse repeat p53REs favor the H14 mode and direct repeat p53REs may have high possibilities of other modes.

Conformational Dynamics of a Lipid-Interacting Protein: MD Simulations of Saposin B

Saposin B is a water soluble alpha-helical protein which can bind to membranes and extract selected lipids, especially cerebroside sulfates. The X-ray structure of saposin B is homodimeric. There are two conformations of the dimer in the crystal-one with a closed central cavity (the AB dimer) and one (the CD dimer) with a more open cavity. We have conducted a series of short (5 ns) molecular dynamics simulations of saposin B, starting from both the AB and CD conformations and with/without bound lipid and/or water molecules within the central hydrophobic cavity. The more open (CD) dimer showed greater conformational drift than the AB dimer. The conformational drift was also somewhat higher in the absence of bound lipid. Two more extended (30 ns) simulations of AB and CD dimers were performed and analyzed in terms of changes in intersubunit packing within the dimers. The AB dimer remained largely unchanged in conformation over the duration of the extended simulation. In contrast, the CD dimer underwent a substantial conformational change corresponding to a 'scissor' motion of the two monomers so as to compress the central cavity to a more closed conformation than that seen in the AB dimer structure. A H-bond between the Q53 and Y54 side chains of the alpha3 helices of the two opposing monomers seems to hold the dimer in this 'scissor-closed' conformation. We suggest that a cycle of conformational changes, expanding and compressing the central cavity of the saposin B dimer, may play a key role in facilitating lipid extraction from bilayers.

Molecular Mechanism of Allosteric Substrate Activation in a Thiamine Diphosphate-dependent Decarboxylase

Thiamine diphosphate-dependent enzymes are involved in a wide variety of metabolic pathways. The molecular mechanism behind active site communication and substrate activation, observed in some of these enzymes, has since long been an area of debate. Here, we report the crystal structures of a phenylpyruvate decarboxylase in complex with its substrates and a covalent reaction intermediate analogue. These structures reveal the regulatory site and unveil the mechanism of allosteric substrate activation. This signal transduction relies on quaternary structure reorganizations, domain rotations, and a pathway of local conformational changes that are relayed from the regulatory site to the active site. The current findings thus uncover the molecular mechanism by which the binding of a substrate in the regulatory site is linked to the mounting of the catalytic machinery in the active site in this thiamine diphosphate-dependent enzyme.

Crystal Structure of Helicobacter pylori Formamidase AmiF Reveals a Cysteine-Glutamate-Lysine Catalytic Triad

Helicobacter pylori AmiF formamidase that hydrolyzes formamide to produce formic acid and ammonia belongs to a member of the nitrilase superfamily. The crystal structure of AmiF was solved to 1.75A resolution using single-wavelength anomalous dispersion methods. The structure consists of a homohexamer related by 3-fold symmetry in which each subunit has an alpha-beta-beta-alpha four-layer architecture characteristic of the nitrilase superfamily. One exterior alpha layer faces the solvent, whereas the other one associates with that of the neighbor subunit, forming a tight alpha-beta-beta-alpha-alpha-beta-beta-alpha dimer. The apo and liganded crystal structures of an inactive mutant C166S were also determined to 2.50 and 2.30 A, respectively. These structures reveal a small formamide-binding pocket that includes Cys(166), Glu(60), and Lys(133) catalytic residues, in which Cys(166) acts as a nucleophile. Analysis of the liganded AmiF and N-carbamoyl d-amino acid amidohydrolase binding pockets reveals a common Cys-Glu-Lys triad, another conserved glutamate, and different subsets of ligand-binding residues. Molecular dynamic simulations show that the conserved triad has minimal fluctuations, catalyzing the hydrolysis of a specific nitrile or amide in the nitrilase superfamily efficiently.

Crystal structure of decameric peroxiredoxin (AhpC) from Amphibacillus xylanus

Crystal structure of decameric peroxiredoxin (AhpC) from <i>Amphibacillus xylanus</i>

Pairing of the nucleobases guanine and cytosine in the gas phase studied by IR–UV double-resonance spectroscopy and ab initio calculations

We present R2PI, IR–UV and UV–UV double resonance measurements of the guanine–cytosine (G–C) dimer formed in a supersonic jet. We show that there is only one isomer of G–C in the investigated wavelength range from 33200 to 34100 cm−1. We assigned the observed G–C isomer to a specific structure, based on comparisons of the IR spectra of the G and C monomers with the G–C dimer in the range of the OH and NH stretching vibrations and ab initio-calculated vibrational frequencies and dimer stabilities. The cluster exhibits an HNH⋯O/NH⋯N/CO⋯HNH bonding similar to the Watson–Crick G–C base pair bonding but with C as the enol tautomer. We did not observe any keto–keto or enol–enol G–C dimers in the investigated wavelength region.

Preliminary crystallographic studies of Y25F mutant of periplasmic Escherichia coli L-asparaginase.

Periplasmic Escherichia coli L-asparaginase II with Y25F mutation in the active-site cavity has been obtained by recombinant techniques. The protein was crystallized in a new hexagonal form (P6(5)22). Single crystals of this polymorph, suitable for X-ray diffraction, were obtained by vapor diffusion using 2-methyl-2,4-pentanediol as precipitant (pH 4.8). The crystals are characterized by a = 81.0, c = 341.1 A and diffract to 2.45 A resolution. The asymmetric unit contains two protein molecules arranged into an AB dimer. The physiologically relevant ABA'B' homotetramer is generated by the action of the crystallographic 2-fold axis along [1, -1, 0]. Kinetic studies show that the loss of the phenolic hydroxyl group at position 25 brought about by the replacement of Y with F strongly impairs kcat without significantly affecting Km.

Analysis of the Quaternary Structure, Substrate Specificity, and Catalytic Mechanism of Valine Dehydrogenase

The solution of the three-dimensional structure of Bacillus sphaericus leucine dehydrogenase has enabled us to undertake a homology-based modeling exercise on the sequence differences between the families of leucine (LeuDH) and valine (ValDH) dehydrogenases. This analysis indicates that the secondary structure elements in the core of the two domains of a single subunit of these enzymes are conserved, as are residues directly implicated in the recognition of the nucleotide cofactor and in catalysis. Comparison of the sequences indicates that the residues in the pocket accommodating the side chain of the amino acid substrate are conserved between these two enzymes, suggesting that the small differences in specificity arise from minor changes in molecular structure, possibly associated with shifts of the main chain rather than mutation of residues in the pocket itself. While B. sphaericus LeuDH is an octamer, both Streptomyces cinnamonensis and Streptomyces coelicolor ValDHs are dimers. The differences in quaternary structure can be understood in terms of the deletion in the latter of a C-terminal loop, which forms important interactions around the four-fold axis in LeuDH.

NMR solution structure of the 32-kDa platelet factor 4 ELR-motif N-terminal chimera: a symmetric tetramer.

Native human platelet factor 4 (PF4) is a homotetrameric protein (70 residues/subunit) known for its anticoagulant heparin binding activity. 2D 15N--1H HSQC NMR experiments of native PF4 in solution show the presence of conformational heterogeneity consistent with the formation of asymmetric homo-tetramers as observed in the X-ray crystal structure of both human and bovine PF4. A chimeric mutant of PF4 (called PF4-M2) which substitutes the first 11 N-terminal residues for the first eight residues from homologous interleukin-8 forms symmetric homo-tetramers with essentially the same heparin binding activity as native PF4. The solution structure of PF4-M2 has been investigated by using two- and three-dimensional 1H- and 15N-NMR spectroscopy and NOE-restrained simulated annealing molecular dynamics. As with other members of the CXC chemokine family whose structures are known, the PF4-M2 subunit monomer consists of a mostly hydrophobic, triple-stranded antiparallel beta-sheet onto which is folded an amphipathic C-terminal helix and a less periodic N-terminal domain. Although N-terminal substitution with the less acidic interleukin-8 sequence most affects the quarternary structure relative to native PF4 at the AC and AD dimer interfaces, AB dimer stability is weakened as reflected in reduced equilibrium association binding constants.