Modeling glycosaminoglycan–protein complexes

Glycosaminoglycans are long linear and complex polysaccharides that are fundamental components of the mammalian extracellular matrix. Therefore, it is crucial to appropriately characterize molecular structure, dynamics, and interactions of protein-glycosaminoglycans complexes for improving understanding of molecular mechanisms underlying GAG biological function. Nevertheless, this proved challenging experimentally, and theoretical techniques are beneficial to construct new hypotheses and aid the interpretation of experimental data. The scope of this mini-review is to summarize four specific aspects of the current theoretical approaches for investigating noncovalent protein-glycosaminoglycan complexes such as molecular docking, free binding energy calculations, modeling ion impact, and addressing the phenomena of multipose binding of glycosaminoglycans to proteins.     

Molecular properties and antioxidant activities of polysaccharide–protein complexes from selected mushrooms by ultrasound-assisted extraction

Ultrasound-assisted extraction (UAE) was evaluated for isolation of polysaccharide–protein (PSP) complexes from three important medicinal mushrooms (Grifola frondosa, Coriolus versicolor and Lentinus edodes). Compared with those of conventional hot-water extraction (HWE), the PSP yield of UAE was similar with G. frondosa, notably higher with L. edodes but lower with C. versicolor, and the extraction rate of UAE was notably higher with G. frondosa and L. edodes but much lower with C. versicolor. The PSPs from all three mushrooms by UAE had higher protein but lower carbohydrate contents than those by HWE, and their molecular weight (MW) profiles exhibited an overall shift to lower MW and a major low-MW peak near 1.0 kDa. All PSPs from UAE but none from HWE exhibited 3–4 distinct protein bands between 10 and 130 kDa. The antioxidant activities of PSPs extracted by UAE were generally higher than those by HWE.     

Biochemical and biological properties of cortexillin III,a component of Dictyostelium DGAP1–cortexillin complexes

Cortexillins I–III are members of the α-actinin/spectrin subfamily of Dictyostelium calponin homology proteins. Unlike recombinant cortexillins I and II, which form homodimers as well as heterodimers in vitro, we find that recombinant cortexillin III is an unstable monomer but forms more stable heterodimers when coexpressed in Escherichia coli with cortexillin I or II. Expressed cortexillin III also forms heterodimers with both cortexillin I and II in vivo, and the heterodimers complex in vivo with DGAP1, a Dictyostelium GAP protein. Binding of cortexillin III to DGAP1 requires the presence of either cortexillin I or II; that is, cortexillin III binds to DGAP1 only as a heterodimer, and the heterodimers form in vivo in the absence of DGAP1. Expressed cortexillin III colocalizes with cortexillins I and II in the cortex of vegetative amoebae, the leading edge of motile cells, and the cleavage furrow of dividing cells. Colocalization of cortexillin III and F-actin may require the heterodimer/DGAP1 complex. Functionally, cortexillin III may be a negative regulator of cell growth, cytokinesis, pinocytosis, and phagocytosis, as all are enhanced in cortexillin III–null cells.     

Structures,magnetic properties,and XPS of cyanide-bridged NdIII/SmIII/GdIII–CrIII complexes

Synthesis, structural characterization, and spectroscopic and magnetic properties of three new cyano-bridged 3d–4f bimetallic complexes, Ln^III(DMF)₄(H₂O)₃Cr^III (CN)₆ · nH₂O (Ln = Nd, Sm, Gd), have been described. The Nd–Cr complex crystallizes in the monoclinic P2₁/n space group with the following unit cell parameters: a = 20.063(7) Å, b = 8.967(4) Å, c = 18.023(6) Å, b = 96.12(3)°, V = 3224(2) Å³, and Z = 4. The neodymium (III) ion, which adopts anti-prism eight-coordination environment, is linked to the [Cr^III(CN)₆]³⁻ moiety through a bridging cyanide ligand with Nd–N = 2.550(4) Å and Nd–N–C = 164.4(4)°. The variable-temperature (0.5 T at 2–300 K) and variable-field (0–5 T at 2 and 5 K) magnetic measurements reveal that the weak interaction of Gd–Cr complexes differs from that of Nd–Cr and Sm–Cr ones mainly because of the lack of orbital angular momentum. The XPS and diffuse reflectance electronic spectra were also measured to discuss charge transfer transitions concerning π-backdonation from the viewpoint of magneto-optical functions.     

Molecular and practical aspects of the enzymatic properties of human serum albumin and of albumin–ligand complexes

Background

Human serum albumin and some of its ligand complexes possess enzymatic properties which are useful both in vivo and in vitro.

Scope of review

This review summarizes present knowledge about molecular aspects, practical applications and potentials of these properties.

Major conclusions

The most pronounced activities of the protein are different types of hydrolysis. Key examples are esterase-like activities involving Tyr411 or Lys199 and the thioesterase activity of Cys34. In the first case, hydrolysis involves water and both products are released, whereas in the latter cases one of the products is set free, and the other stays covalently bound to the protein. However, the modified Cys34 can be converted back to its reduced form by another compound/enzymatic system. Among the other activities are glucuronidase, phosphatase and amidase as well as isomerase and dehydration properties. The protein has great impact on the metabolism of, for example, eicosanoids and xenobiotics. Albumin with a metal ion-containing complex is capable of facilitating reactions involving reactive oxygen and nitrogen species.

General significance

Albumin is useful in detoxification reactions, for activating prodrugs, and for binding and activating drug conjugates. The protein can be used to construct smart nanotubes with enzymatic properties useful for biomedical applications. Binding of organic compounds with a metal ion often results in metalloenzymes or can be used for nanoparticle formation. Because any compound acting as cofactor and/or the protein can be modified, enzymes can be constructed which are not naturally found and therefore can increase, often stereospecifically, the number of catalytic reactions. This article is part of a Special Issue entitled Serum Albumin.     

A Highlights from MBoC Selection: Neuromuscular synapse integrity requires linkage of acetylcholine receptors to postsynaptic intermediate filament networks via rapsyn–plectin 1f complexes

Mutations in the cytolinker protein plectin lead to grossly distorted morphology of neuromuscular junctions (NMJs) in patients suffering from epidermolysis bullosa simplex (EBS)-muscular dystrophy (MS) with myasthenic syndrome (MyS). Here we investigated whether plectin contributes to the structural integrity of NMJs by linking them to the postsynaptic intermediate filament (IF) network. Live imaging of acetylcholine receptors (AChRs) in cultured myotubes differentiated ex vivo from immortalized plectin-deficient myoblasts revealed them to be highly mobile and unable to coalesce into stable clusters, in contrast to wild-type cells. We found plectin isoform 1f (P1f) to bridge AChRs and IFs via direct interaction with the AChR-scaffolding protein rapsyn in an isoform-specific manner; forced expression of P1f in plectin-deficient cells rescued both compromised AChR clustering and IF network anchoring. In conditional plectin knockout mice with gene disruption in muscle precursor/satellite cells (Pax7-Cre/cKO), uncoupling of AChRs from IFs was shown to lead to loss of postsynaptic membrane infoldings and disorganization of the NMJ microenvironment, including its invasion by microtubules. In their phenotypic behavior, mutant mice closely mimicked EBS-MD-MyS patients, including impaired body balance, severe muscle weakness, and reduced life span. Our study demonstrates that linkage to desmin IF networks via plectin is crucial for formation and maintenance of AChR clusters, postsynaptic NMJ organization, and body locomotion.     

The DNA–DNA spacing in gemini surfactants–DOPE–DNA complexes

Gemini surfactants from the homologous series of alkane-α,ω-diyl-bis(dodecyldimethylammonium bromide) (CnCS12, number of spacer carbons n = 2 – 12) and dioleoylphosphatidylethanolamine (DOPE) were used for cationic liposome (CL) preparation. CLs condense highly polymerized DNA creating complexes. Small-angle X-ray diffraction identified them as condensed lamellar phase L_α^C in the studied range of molar ratios CnGS12/DOPE in the temperature range 20 – 60 °C. The DNA–DNA distance (d_DNA) is studied in dependence to CnGS12 spacer length and membrane surface charge density. The high membrane surface charge densities (CnGS12/DOPE = 0.35 and 0.4 mol/mol) lead to the linear dependence of d_DNA vs. n correlating with the interfacial area of the CnGS12 molecule.     

Hydrogen-bonding interactions in adrenaline–water complexes: DFT and QTAIM studies of structures, properties, and topologies

ωB97XD/6-311++G(d,p) calculations were carried out to investigate the hydrogen-bonding interactions between adrenaline (Ad) and water. Six Ad-H(2)O complexes possessing various types of hydrogen bonds (H-bonds) were characterized in terms of their geometries, energies, vibrational frequencies, and electron-density topology. Natural bond orbital (NBO) and quantum theory of atoms in molecules (QTAIM) analyses were performed to elucidate the nature of the hydrogen-bonding interactions in these complexes. The intramolecular H-bond between the amino and carboxyl oxygen atom of Ad was retained in most of the complexes, and cooperativity between the intra- and intermolecular H-bonds was present in some of the complexes. H-bonds in which hydroxyls of Ad/water acted as proton donors were stronger than other H-bonds. Both hydrogen-bonding interactions and structural deformation play important roles in the relative stabilities of the complexes. The intramolecular H-bond was broken during the formation of the most stable complex, which indicates that Ad tends to break the intramolecular H-bond and form two new intermolecular H-bonds with the first water molecule.     

Structural characterization of cationic lipid–tRNA complexes

Despite considerable interest and investigations on cationic lipid–DNA complexes, reports on lipid–RNA interaction are very limited. In contrast to lipid–DNA complexes where lipid binding induces partial B to A and B to C conformational changes, lipid–tRNA complexation preserves tRNA folded state. This study is the first attempt to investigate the binding of cationic lipid with transfer RNA and the effect of lipid complexation on tRNA aggregation and condensation. We examine the interaction of tRNA with cholesterol (Chol), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dioctadecyldimethylammoniumbromide (DDAB) and dioleoylphosphatidylethanolamine (DOPE), at physiological condition, using constant tRNA concentration and various lipid contents. FTIR, UV-visible, CD spectroscopic methods and atomic force microscopy (AFM) were used to analyze lipid binding site, the binding constant and the effects of lipid interaction on tRNA stability, conformation and condensation. Structural analysis showed lipid–tRNA interactions with G–C and A–U base pairs as well as the backbone phosphate group with overall binding constants of K_Chol = 5.94 (± 0.8) × 10⁴ M^–1, K_DDAB = 8.33 (± 0.90) × 10⁵ M^–1, K_DOTAP = 1.05 (± 0.30) × 10⁵ M^–1 and K_DOPE = 2.75 (± 0.50) × 10⁴ M^–1. The order of stability of lipid–tRNA complexation is DDAB > DOTAP > Chol > DOPE. Hydrophobic interactions between lipid aliphatic tails and tRNA were observed. RNA remains in A-family structure, while biopolymer aggregation and condensation occurred at high lipid concentrations.     

Footprinting analysis of BWYV pseudoknot–ribosome complexes

Many viruses regulate translation of polycistronic mRNA using a −1 ribosomal frameshift induced by an RNA pseudoknot. When the ribosome encounters the pseudoknot barrier that resists unraveling, transient mRNA–tRNA dissociation at the decoding site, results in a shift of the reading frame. The eukaryotic frameshifting pseudoknot from the beet western yellow virus (BWYV) has been well characterized, both structurally and functionally. Here, we show that in order to obtain eukaryotic levels of frameshifting efficiencies using prokaryotic Escherichia coli ribosomes, which depend upon the structural integrity of the BWYV pseudoknot, it is necessary to shorten the mRNA spacer between the slippery sequence and the pseudoknot by 1 or 2 nucleotides (nt). Shortening of the spacer is likely to re-establish tension and/or ribosomal contacts that were otherwise lost with the smaller E. coli ribosomes. Chemical probing experiments for frameshifting and nonframeshifting BWYV constructs were performed to investigate the structural integrity of the pseudoknot confined locally at the mRNA entry site. These data, obtained in the pretranslocation state, show a compact overall pseudoknot structure, with changes in the conformation of nucleotides (i.e., increase in reactivity to chemical probes) that are first “hit” by the ribosomal helicase center. Interestingly, with the 1-nt shortened spacer, this increase of reactivity extends to a downstream nucleotide in the first base pair (bp) of stem 1, consistent with melting of this base pair. Thus, the 3 bp that will unfold upon translocation are different in both constructs with likely consequences on unfolding kinetics.     

PCS-based structure determination of protein–protein complexes

A simple and fast nuclear magnetic resonance method for docking proteins using pseudo-contact shift (PCS) and ¹H^N/¹⁵N chemical shift perturbation is presented. PCS is induced by a paramagnetic lanthanide ion that is attached to a target protein using a lanthanide binding peptide tag anchored at two points. PCS provides long-range (~40 Å) distance and angular restraints between the lanthanide ion and the observed nuclei, while the ¹H^N/¹⁵N chemical shift perturbation data provide loose contact-surface information. The usefulness of this method was demonstrated through the structure determination of the p62 PB1-PB1 complex, which forms a front-to-back 20 kDa homo-oligomer. As p62 PB1 does not intrinsically bind metal ions, the lanthanide binding peptide tag was attached to one subunit of the dimer at two anchoring points. Each monomer was treated as a rigid body and was docked based on the backbone PCS and backbone chemical shift perturbation data. Unlike NOE-based structural determination, this method only requires resonance assignments of the backbone ¹H^N/¹⁵N signals and the PCS data obtained from several sets of two-dimensional ¹⁵N-heteronuclear single quantum coherence spectra, thus facilitating rapid structure determination of the protein–protein complex.     

Ginkgolides protect against amyloid-β1–42-mediated synapse damage in vitro

Background

The early stages of Alzheimer's disease (AD) are closely associated with the production of the Aβ_1–42 peptide, loss of synapses and gradual cognitive decline. Since some epidemiological studies showed that EGb 761, an extract from the leaves of the Ginkgo biloba tree, had a beneficial effect on mild forms of AD, the effects of some of the major components of the EGb 761 extract (ginkgolides A and B, myricetin and quercetin) on synapse damage in response to Aβ_1–42 were examined.

Results

The addition of Aβ_1–42 to cortical or hippocampal neurons reduced the amounts of cell associated synaptophysin, a pre-synaptic membrane protein that is essential for neurotransmission, indicating synapse damage. The effects of Aβ_1–42 on synapses were apparent at concentrations approximately 100 fold less than that required to kill neurons; the synaptophysin content of neuronal cultures was reduced by 50% by 50 nM Aβ_1–42. Pre-treatment of cortical or hippocampal neuronal cultures with ginkgolides A or B, but not with myrecitin or quercetin, protected against Aβ_1–42-induced loss of synaptophysin. This protective effect was achieved with nanomolar concentrations of ginkgolides. Previous studies indicated that the ginkgolides are platelet-activating factor (PAF) receptor antagonists and here we show that Aβ_1–42-induced loss of synaptophysin from neuronal cultures was also reduced by pre-treatment with other PAF antagonists (Hexa-PAF and CV6209). PAF, but not lyso-PAF, mimicked the effects Aβ_1–42 and caused a dose-dependent reduction in the synaptophysin content of neurons. This effect of PAF was greatly reduced by pre-treatment with ginkgolide B. In contrast, ginkgolide B did not affect the loss of synaptophysin in neurons incubated with prostaglandin E₂.

Conclusion

Pre-treatment with ginkgolides A or B protects neurons against Aβ_1–42-induced synapse damage. These ginkgolides also reduced the effects of PAF, but not those of prostaglandin E₂, on the synaptophysin content of neuronal cultures, results consistent with prior reports that ginkgolides act as PAF receptor antagonists. Such observations suggest that the ginkgolides are active components of Ginkgo biloba preparations and may protect against the synapse damage and the cognitive loss seen during the early stages of AD.     

What is an immunological synapse?

Immunological synapses (IS) are emerging as highly organized 3D structures -formed by surface and cytoplasmic signalling and cytoskeletal molecules – that assemble at the zone of contact between a T cell and an antigen presenting cell (APC). The IS control functions that allow APC and T cells modulate the immune response.