THERMODESTRUCTION OF Lа(III) COORDINATION COMPOUNDS WITH ALIPHATIC β-KETO­ESTERS

Mono- and mixed-ligand complexes of La (III) with aliphatic β-ketoesters were synthesized in the solid state. The complexes have the general formulas LаL2OH·H2O (L=meacac, etacac, alacac) and La(meacac)2X·nCH3OH(X = NO3, CH3COO; n = 1, 2). Their composition, structure, and thermal properties were established by chemical and thermal analysis, IR spectroscopy. It is shown that β-ketoesters are coordinated to the La (III) ion bidentate-cyclically into monoligand hydroxocomp­lexes. Ligand complexes with methylacetoacetate have an oligomeric structure. They consist of cationic fragments [La(meacac)2]+ with bridged connection of the nitrate or acetate anions. The thermal destructions of LaL2OH·H2O (L = meacac, etacac, alacac), La(meacac)2NO3· 2CH3OH and La(meacac)2(CH3COO)·CH3OH were studied for the first time in the helium dynamic atmosphere by TGA-MS in the temperature range of 25–900 °C. Depending on the ligand, dehydratation of the hydroxo-complexes takes place in the 120–180 (meacac), 120–190 (etacac) or 110–160 °C (alacac) temperature range, and the mass loss corresponds with the detachment of one water molecule. Decomposition of mixed-ligand complexes starts with the detachment of methanol in the 60–100 °C range. For La(meacac)2NO3·2CH3OH the decomposition process is attended with oxidation of methanol to carbon dioxide due to reduction of the nitrate-ion to nitrogen dioxide. Further heating to 300–400 °C leads to destruction of organic parts of the complexes attended with the release of low-molecular oxygen-containing organic compounds (aldehydes, ketones, alcohols), carbon dioxide and water. At ~500 °C all the La(III) complexes under study totally decompose, yielding the oxycarbonate La2O2CO3, which was fixed by IR spectroscopy. Under further heating to 850 °С oxycarbonate gradually decomposes to La2O3 liberating CO2.

Nanobody-aided crystallization of the transcription regulator PaaR2 from Escherichia coli O157:H7

paaR2–paaA2–parE2 is a three-component toxin–antitoxin module found in prophage CP-993P of Escherichia coli O157:H7. Transcription regulation of this module occurs via the 123-amino-acid regulator PaaR2, which forms a large oligomeric structure. Despite appearing to be well folded, PaaR2 withstands crystallization, as does its N-terminal DNA-binding domain. Native mass spectrometry was used to screen for nanobodies that form a unique complex and stabilize the octameric structure of PaaR2. One such nanobody, Nb33, allowed crystallization of the protein. The resulting crystals belong to space group F432, with unit-cell parameter a = 317 Å, diffract to 4.0 Å resolution and are likely to contain four PaaR2 monomers and four nanobody monomers in the asymmetric unit. Crystals of two truncates containing the N-terminal helix–turn–helix domain also interact with Nb33, and the corresponding co-crystals diffracted to 1.6 and 1.75 Å resolution.

Specificity of Human Sulfiredoxin for Reductant and Peroxiredoxin Oligomeric State

Human peroxiredoxins (Prx) are a family of antioxidant enzymes involved in a myriad of cellular functions and diseases. During the reaction with peroxides (e.g., H2O2), the typical 2-Cys Prxs change oligomeric structure between higher order (do)decamers and disulfide-linked dimers, with the hyperoxidized inactive state (-SO2H) favoring the multimeric structure of the reduced enzyme. Here, we present a study on the structural requirements for the repair of hyperoxidized 2-Cys Prxs by human sulfiredoxin (Srx) and the relative efficacy of physiological reductants hydrogen sulfide (H2S) and glutathione (GSH) in this reaction. The crystal structure of the toroidal Prx1-Srx complex shows an extended active site interface. The loss of this interface within engineered Prx2 and Prx3 dimers yielded variants more resistant to hyperoxidation and repair by Srx. Finally, we reveal for the first time Prx isoform-dependent use of and potential cooperation between GSH and H2S in supporting Srx activity.

Seipin accumulates and traps diacylglycerols and triglycerides in its ring-like structure

Lipid droplets (LDs) are intracellular organelles responsible for lipid storage, and they emerge from the endoplasmic reticulum (ER) upon the accumulation of neutral lipids, mostly triglycerides (TG), between the two leaflets of the ER membrane. LD biogenesis takes place at ER sites that are marked by the protein seipin, which subsequently recruits additional proteins to catalyze LD formation. Deletion of seipin, however, does not abolish LD biogenesis, and its precise role in controlling LD assembly remains unclear. Here, we use molecular dynamics simulations to investigate the molecular mechanism through which seipin promotes LD formation. We find that seipin clusters TG, as well as its precursor diacylglycerol, inside its unconventional ring-like oligomeric structure and that both its luminal and transmembrane regions contribute to this process. This mechanism is abolished upon mutations of polar residues involved in protein–TG interactions into hydrophobic residues. Our results suggest that seipin remodels the membrane of specific ER sites to prime them for LD biogenesis.

Heme Binding to HupZ with a C-Terminal Tag from Group A Streptococcus

HupZ is an expected heme degrading enzyme in the heme acquisition and utilization pathway in Group A Streptococcus. The isolated HupZ protein containing a C-terminal V5-His6 tag exhibits a weak heme degradation activity. Here, we revisited and characterized the HupZ-V5-His6 protein via biochemical, mutagenesis, protein quaternary structure, UV–vis, EPR, and resonance Raman spectroscopies. The results show that the ferric heme-protein complex did not display an expected ferric EPR signal and that heme binding to HupZ triggered the formation of higher oligomeric states. We found that heme binding to HupZ was an O2-dependent process. The single histidine residue in the HupZ sequence, His111, did not bind to the ferric heme, nor was it involved with the weak heme-degradation activity. Our results do not favor the heme oxygenase assignment because of the slow binding of heme and the newly discovered association of the weak heme degradation activity with the His6-tag. Altogether, the data suggest that the protein binds heme by its His6-tag, resulting in a heme-induced higher-order oligomeric structure and heme stacking. This work emphasizes the importance of considering exogenous tags when interpreting experimental observations during the study of heme utilization proteins.

Few Percent Efficient Polarization-Sensitive Conversion in Nonlinear Plasmonic Interactions Inside Oligomeric Gold Structures

The backscattering spectra of a 500 nm thick gold film, which was excited near the 525 nm transverse localized plasmon resonance of its constituent, self-organized, vertically-aligned nanorods by normally incident 515 nm, 300 fs laser pulses with linear, radial, azimuthal and circular polarizations, revealed a few-percent conversion into Stokes and anti-Stokes side-band peaks. The investigation of these spectral features based on the nanoscale characterization of the oligomeric structure and numerical simulations of its backscattering response indicated nonlinear Fano-like plasmonic interactions, particularly the partially degenerate four-wave mixing comprised by the visible-range transverse plasmon resonance of the individual nanorods and an IR-range collective mode of the oligomeric structure. Such oligomeric structures in plasmonic films may greatly enhance inner nonlinear electromagnetic interactions and inner near-IR hotspots, paving the way for their engineered IR tunability for broad applications in chemosensing and biosensing.

Chemokine receptor CXCR4 oligomerization is disrupted selectively by the antagonist ligand IT1t

CXCR4, a member of the family of chemokine-activated G protein-coupled receptors, is widely expressed in immune response cells. It is involved in both cancer development and progression as well as viral infection, notably by HIV-1. A variety of methods, including structural information, have suggested the receptor may exist as a dimer or oligomer. However, the mechanistic details surrounding receptor oligomerization and its potential dynamic regulation remain unclear. Using both biochemical and biophysical means we confirm that CXCR4 can exist as a mixture of monomers, dimers and higher-order oligomers in cell membranes and show that oligomeric structure becomes more complex as receptor expression levels increase. Mutations of CXCR4 residues located at a putative dimerization interface result in monomerization of the receptor. Additionally, binding of the CXCR4 antagonist IT1t— a small, drug-like isothiourea derivative — rapidly destabilizes the oligomeric structure, while AMD3100, another well-characterized CXCR4 antagonist, does not. Although a mutation that regulates constitutive activity of CXCR4 also results in monomerization of the receptor, binding of IT1t to this variant promotes receptor dimerization. These results provide novel insights into the basal organization of CXCR4 and how antagonist ligands of different chemotypes differentially regulate its oligomerization state.

Mechanism of negative membrane curvature generation by I-BAR domains

The membrane sculpting ability of BAR domains has been attributed to the intrinsic curvature of their banana-shaped dimeric structure. However, there is often a mismatch between this intrinsic curvature and the diameter of the membrane tubules generated. I-BAR domains have been especially mysterious: they are almost flat but generate high negative membrane curvature. Here, we use atomistic implicit-solvent computer modeling to show that the membrane bending of the IRSP53 I-BAR domain is dictated by its higher oligomeric structure, whose curvature is completely unrelated to the intrinsic curvature of the dimer. Two other I-BARs gave similar results, whereas a flat F-BAR sheet developed a concave membrane binding interface, consistent with its observed positive membrane curvature generation. Laterally interacting helical spirals of I-BAR dimers on tube interiors are stable and have an enhanced binding energy that is sufficient for membrane bending to experimentally observed tubule diameters at a reasonable surface density.