Solution and Solvent-Free Stopper Exchange Reactions for the Preparation of Pillar[5]arene-containing [2] and [3]Rotaxanes.

Diamine reagents have been used to functionalize a [2]rotaxane building block bearing an activated pentafluorophenyl ester stopper. Upon a first acylation, an intermediate host-guest complex with a terminal amine function is obtained. Dissociation of the intermediate occurs in solution and acylation of the released axle generates a [2]rotaxane with an elongated axle subunit. In contrast, the corresponding [3]rotaxane can be obtained if the reaction conditions are appropriate to stabilize the inclusion complex of the mono-amine intermediate and the pillar[5]arene. This is the case when the stopper exchange is performed under mechanochemical solvent-free conditions. Alternatively, if the newly introduced terminal amide group is large enough to prevent the dissociation, the second acylation provides exclusively a [3]rotaxane. On the other hand, detailed conformational analysis has been also carried out by variable temperature NMR investigations. A complete understanding of the shuttling motions of the pillar[5]arene subunit along the axles of the rotaxanes reported therein has been achieved with the help of density functional theory calculations.

Stepwise Functionalization of a Pillar[5]arene-Containing [2]Rotaxane with Pentafluorophenyl Ester Stoppers.

Invited for the cover of this issue are the group of Iwona and Jean-François Nierengarten from the University of Strasbourg (LIMA, UMR 7042, CNRS) and collaborators from the University of Carthage. The image depicts a sky of umbrellas floating in the air to illustrate the protective effect allowing the efficient mono-acylation of a symmetrical rotaxane building block with two pentafluorophenyl ester stoppers. Read the full text of the article at 10.1002/chem.2023003501.

Solution and Solvent-Free Stopper Exchange Reactions for the Preparation of Pillar[5]arene-Containing [2] and [3]Rotaxanes.

Invited for the cover of this issue are the group of Iwona and Jean-François Nierengarten from the University of Strasbourg (LIMA, UMR 7042, CNRS) and collaborators from the University of Carthage and the IPHC (University of Strasbourg and CNRS, UMR 7178). The image illustrates the fast motions of a pillar[5]arene subunit along the axle of a rotaxane, reminiscent of those of a guitarist's hand along the neck allowing him to use random parts of a scale with certain sweet spots when improvising a solo. Read the full text of the article at 10.1002/chem.202304131.

Stepwise Functionalization of a Pillar[5]arene-Containing [2]Rotaxane with Pentafluorophenyl Ester Stoppers.

Detailed investigations into the stepwise bis-functionalization of a pillar[5]arene-containing rotaxane building block have been carried out. Upon a first stopper exchange, the pillar[5]arene moiety of the mono-acylated product is preferentially located close to its reactive pentafluorophenyl ester stopper, thus limiting the accessibility to the reactive carbonyl group by the nucleophilic reagents. Selective mono-functionalization is thus very efficient. Introduction of a second stopper is then possible to generate dissymmetrical rotaxanes with different amide stoppers. Moreover, when dethreading is possible upon the second acylation, the pillar[5]arene plays the role of a protecting group allowing the synthesis of dissymmetrical axles that are particularly difficult to prepare under statistical conditions. Finally, detailed conformation analysis of the rotaxanes revealed that the position of the pillar[5]arene moiety on its axle subunit is mainly governed by polar interactions in nonpolar organic solvents, whereas solvophobic effects play a major role in polar solvents.

Quantification of Cholesterol Solubilized in Dietary Micelles: Dependence on Human Bile Salt Variability and the Presence of Dietary Food Ingredients.

The solubility of cholesterol in bile salt (BS) micelles is important to understand the availability of cholesterol for absorption in the intestinal epithelium and to develop strategies to decrease cholesterol intake from the intestinal lumen. This has been the subject of intense investigation, due to the established relation between the development of diseases such as atherosclerosis and high levels of cholesterol in the blood. In this work we quantify the effect of BS variability on the amount of cholesterol solubilized. The effect of some known hypocholesterolemic agents usually found in the diet is also evaluated, as well as some insight regarding the mechanisms involved. The results show that, depending on the bile salt composition, the average value of sterol per micelle is equal to or lower than 1. The amount of cholesterol solubilized in the BS micelles is essentially equal to its total concentration until the solubility limit is reached. Altogether, this indicates that the maximum cholesterol solubility in the BS micellar solution is the result of saturation of the aqueous phase and depends on the partition coefficient of cholesterol between the aqueous phase and the micellar pseudophase. The effect on cholesterol maximum solubility for several food ingredients usually encountered in the diet was characterized using methodology developed recently by us. This method allows the simultaneous quantification of both cholesterol and food ingredient solubilized in the BS micelles even in the presence of larger aggregates, therefore avoiding their physical separation with possible impacts on the overall equilibrium. The phytosterols stigmasterol and stigmastanol significantly decreased cholesterol solubility with a concomitant reduction in the total amount of sterol solubilized, most pronounced for stigmasterol. Those results point toward coprecipitation being the major cause for the decrease in cholesterol solubilization by the BS micelles. The presence of tocopherol and oleic acid leads to a small decrease in the amount of cholesterol solubilized while palmitic acid slightly increases the solubility of cholesterol. Those dietary food ingredients are completely solubilized by the BS micelles, indicating that the effects on cholesterol solubility are due to changes in the properties of the mixed micelles.

Biophysical characterization of laforin-carbohydrate interaction.

Laforin is a human dual-specificity phosphatase (DSP) involved in glycogen metabolism regulation containing a carbohydrate-binding module (CBM). Mutations in the gene coding for laforin are responsible for the development of Lafora disease, a progressive fatal myoclonus epilepsy with early onset, characterized by the intracellular deposition of abnormally branched, hyperphosphorylated insoluble glycogen-like polymers, called Lafora bodies. Despite the known importance of the CBM domain of laforin in the regulation of glycogen metabolism, the molecular mechanism of laforin-glycogen interaction is still poorly understood. Recently, the structure of laforin with bound maltohexaose was determined and despite the importance of such breakthrough, some molecular interaction details remained missing. We herein report a thorough biophysical characterization of laforin-carbohydrate interaction using soluble glycans. We demonstrated an increased preference of laforin for the interaction with glycans with higher order of polymerization and confirmed the importance of tryptophan residues for glycan interaction. Moreover, and in line with what has been described for other CBMs and lectins, our results confirmed that laforin-glycan interactions occur with a favourable enthalpic contribution counter-balanced by an unfavourable entropic contribution. The analysis of laforin-glycan interaction through the glycan side by saturation transfer difference (STD)-NMR has shown that the CBM-binding site can accommodate between 5 and 6 sugar units, which is in line with the recently obtained crystal structure of laforin. Overall, the work in the present study complements the structural characterization of laforin and sheds light on the molecular mechanism of laforin-glycan interaction, which is a pivotal requisite to understand the physiological and pathological roles of laforin.

Quantification of cholesterol solubilized in bile salt micellar aqueous solutions using (13)C nuclear magnetic resonance.

In this work, we develop a methodology to quantitatively follow the solubilization of cholesterol on glycodeoxycholic acid (GDCA) micelles using (13)C nuclear magnetic resonance (NMR). The amount of solubilized cholesterol enriched in (13)C at position 4, [4-(13)C]cholesterol, was quantified from the area of its resonance, at 44.5 ppm, using the CH(2) groups from GDCA as an internal reference. The loading of the micelles with cholesterol leads to a quantitative upper field shift of most carbons in the nonpolar surface of GDCA, and this was used to follow the solubilization of unlabeled cholesterol. The solubilization followed a pseudo first-order kinetics with a characteristic time constant of 3.6 h, and the maximum solubility of cholesterol in 50 mM total lipid (GDCA + cholesterol) is 3.0 ± 0.1mM, corresponding to a mean occupation number per micelle ≥1. The solubilization profile indicates that the affinity of cholesterol for the GDCA micelles is unaffected by the presence of the solute, leading essentially to full solubilization up to the saturation limit. The relaxation times of GDCA carbons at 50mM give information regarding its aggregation and indicate that GDCA is associated in small micelles (hydrodynamic [Rh] = 1.1 nm) without any evidence for formation of larger secondary micelles. This was confirmed by dynamic light scattering results.

Molecular basis for the association of human E4B U box ubiquitin ligase with E2-conjugating enzymes UbcH5c and Ubc4.

Human E4B, also called UFD2a, is a U box-containing protein that functions as an E3 ubiquitin ligase and an E4 polyubiquitin chain elongation factor. E4B is thought to participate in the proteasomal degradation of misfolded or damaged proteins through association with chaperones. The U box domain is an anchor site for E2 ubiquitin-conjugating enzymes, but little is known of the binding mechanism. Using X-ray crystallography and NMR spectroscopy, we determined the structures of E4B U box free and bound to UbcH5c and Ubc4 E2s. Whereas previously characterized U box domains are homodimeric, we show that E4B U box is a monomer stabilized by a network of hydrogen bonds identified from scalar coupling measurements. These structural studies, complemented by calorimetry- and NMR-based binding assays, suggest an allosteric regulation of UbcH5c and Ubc4 by E4B U box and provide a molecular basis to understand how the ubiquitylation machinery involving E4B assembles.

Kinetic analysis of interaction of BRCA1 tandem breast cancer c-terminal domains with phosphorylated peptides reveals two binding conformations.

Tandem breast cancer C-terminal (BRCT) domains, present in many DNA repair and cell cycle checkpoint signaling proteins, are phosphoprotein binding modules. The best-characterized tandem BRCT domains to date are from the protein BRCA1 (BRCA1-BRCT), an E3 ubiquitin ligase that has been linked to breast and ovarian cancer. While X-ray crystallography and NMR spectroscopy studies have uncovered the structural determinants of specificity of BRCA1-BRCT for phosphorylated peptides, a detailed kinetic and thermodynamic characterization of the interaction is also required to understand how structure and dynamics are connected and therefore better probe the mechanism of phosphopeptide recognition by BRCT domains. Through a global analysis of binding kinetics data obtained from surface plasmon resonance (SPR) and stopped-flow fluorescence spectroscopy, we show that the recognition mechanism is complex and best modeled by two equilibrium conformations of BRCA1-BRCT in the free state that both interact with a phosphopeptide, with dissociation constants ( K d) in the micromolar range. We show that the apparent global dissociation constant derived from this kinetic analysis is similar to the K d values measured using steady-state SPR, isothermal titration calorimetry, and fluorescence anisotropy. The dynamic nature of BRCA1-BRCT may facilitate the binding of BRCA1 to different phosphorylated protein targets.

Multiple conformations of the metal-bound pyoverdine PvdI, a siderophore of Pseudomonas aeruginosa: a nuclear magnetic resonance study.

Under iron-deficient conditions, the Gram-negative bacterium Pseudomonas aeruginosa ATCC 15692 secretes a peptidic siderophore, pyoverdine PvdI, composed of an aromatic chromophore derived from 2,3-diamino-6,7-dihydroxyquinoline and a partially cyclized octapeptide, d-Ser- l-Arg- d-Ser- l-FoOHOrn-( l-Lys- l-FoOHOrn- l-Thr- l-Thr), in which the C-terminal carboxyl group forms a peptidic bond with the primary amine of the l-Lys side chain. In aqueous solution at room temperature, the (1)H NMR spectrum of pyoverdine PvdI-Ga(III) showed clear evidence of exchange broadening. At 253 K, two distinct conformations were observed and the measurement of structural constraints was possible. The three-dimensional structures of the two PvdI-Ga(III) conformers were determined, and analysis of the structures indicates that the observed conformational exchange involves a stereoisomerization of the metal binding coordination accompanied by a change in the global shape of the siderophore. This conformational transition was further characterized by heteronuclear relaxation experiments. The possible implications of this dynamic behavior for siderophore recognition by the receptor FpvAI are discussed.

A low-temperature heteronuclear NMR study of two exchanging conformations of metal-bound pyoverdin PaA from Pseudomonas aeruginosa.

Under iron-deficient conditions, the Gram-negative bacterium Pseudomonas aeruginosa ATCC 15692 secretes a peptidic siderophore, pyoverdin PaA, composed of an aromatic chromophore derived from 2,3-diamino-6,7-dihydroxyquinoline and a partially cyclized octapeptide, D-Ser-L-Arg-D-Ser-L-FoOHOrn-(L-Lys-L-FoOHOrn-L-Thr-L-Thr) (FoOHOrn: delta N-formyl-delta N-hydroxyornithine), in which the C-terminal carboxyl group forms a peptidic bond with the primary amine of the L-Lys side chain. Ferric iron is chelated by the catechol group on the chromophore and the two hydroxyornithine side chains. In aqueous solution, the (1)H-NMR spectrum of pyoverdin PaA-Ga(III), in which Ga(III) is used instead of Fe(III) for spectroscopic purposes, showed clear evidence of exchange broadening, preventing further structural characterization. The use of cryo-solvents allowed measurements to be made at temperatures as low as 253 K where two distinct conformations with roughly equivalent populations could be observed. (13)C and (15)N labeling of pyoverdin PaA enabled complete assignment of both forms of pyoverdin PaA-Ga(III) at 253 and 267 K, using triple-resonance multidimensional NMR experiments commonly applied to doubly labeled proteins.

Dynamics and metal exchange properties of C4C4 RING domains from CNOT4 and the p44 subunit of TFIIH.

Zinc fingers are small structured protein domains that require the coordination of zinc for a stable tertiary fold. Together with FYVE and PHD, the RING domain forms a distinct class of zinc-binding domains, where two zinc ions are ligated in a cross-braced manner, with the first and third pairs of ligands coordinating one zinc ion, while the second and fourth pairs ligate the other zinc ion. To investigate the relationship between the stability and dynamic behaviour of the domains and the stability of the metal-binding site, we studied metal exchange for the C4C4 RING domains of CNOT4 and the p44 subunit of TFIIH. We found that Zn(2+)-Cd(2+) exchange is different between the two metal-binding sites in the C4C4 RING domains of the two proteins. In order to understand the origins of these distinct exchange rates, we studied the backbone dynamics of both domains in the presence of zinc and of cadmium by NMR spectroscopy. The differential stability of the two metal-binding sites in the RING domains, as reflected by the different metal exchange rates, can be explained by a combination of accessibility and an electrostatic ion interaction model. A greater backbone flexibility for the p44 RING domain as compared to CNOT4 may be related to the distinct types of protein-protein interactions in which the two C4C4 RING domains are involved.

Solution structure of the C-terminal domain of TFIIH P44 subunit reveals a novel type of C4C4 ring domain involved in protein-protein interactions.

The human general transcription factor TFIIH is involved in both transcription and DNA nucleotide excision repair. Among the 10 subunits of the complex, p44 subunit plays a crucial role in both mechanisms. Its N-terminal domain interacts with the XPD helicase, whereas its C-terminal domain is involved specifically in the promoter escape activity. By mutating an exposed and non-conserved cysteine residue into a serine, we produced a soluble mutant of p44-(321-395) suitable for solution structure determination. The domain adopts a C4C4 RING domain structure with sequential organization of beta-strands that is related to canonical RING domains by a circular permutation of the beta-sheet elements. Analysis of the molecular surface and mutagenesis experiments suggests that the binding of p44-(321-395) to TFIIH p34 subunit is not mediated by electrostatic interactions and, thus, differs from previously reported interaction mechanisms involving RING domains.

TFIIH contains a PH domain involved in DNA nucleotide excision repair.

The human general transcription factor TFIIH is involved in both transcription and DNA repair. We have identified a structural domain in the core subunit of TFIIH, p62, which is absolutely required for DNA repair activity through the nucleotide excision repair pathway. Using coimmunoprecipitation experiments, we showed that this activity involves the interaction between the N-terminal domain of p62 and the 3' endonuclease XPG, a major component of the nucleotide excision repair machinery. Furthermore, we reconstituted a functional TFIIH particle with a mutant of p62 lacking the N-terminal domain, showing that this domain is not required for assembly of the TFIIH complex and basal transcription. We solved its three-dimensional structure and found an unpredicted pleckstrin homology and phosphotyrosine binding (PH/PTB) domain, uncovering a new class of activity for this fold.

The three-dimensional structure of the gallium complex of azoverdin, a siderophore of Azomonas macrocytogenes ATCC 12334, determined by NMR using residual dipolar coupling constants.

In iron-deficient conditions, Azomonas macrocytogenes ATCC 12334 excretes a fluorescent siderophore called azoverdin, which is composed of a six-amino-acid peptide chain linked to a chromophore. Azoverdin chelates iron(III) very strongly, solubilizing it and transporting it back into the cells using an outer-membrane receptor. This compound is related to the pyoverdins, the peptidic siderophores of Pseudomonas, but differs in the site on the chromophore at which the peptide is covalently linked. This feature identifies azoverdin as a member of a new class of pyoverdins: the isopyoverdins. We report the three-dimensional structure of azoverdin-Ga(III) in solution. The use of orientational constraints obtained from the measurement of residual dipolar couplings using samples dissolved in a liquid crystalline medium allowed us to define the absolute configuration of the metal complex, which is Delta. The structure is characterized by a U-shape adopted by the peptide chain, with the N(delta)-acetyl-N(delta)-hydroxyornithine side chains adopting extended conformations in order to chelate the gallium ion. This conformation leaves a large open space permitting access to the gallium ion. The structural consequences of the particular isopyoverdin chemical structure are discussed in the context of the three-dimensional structures of other pyoverdins.