Lipidated apolipoprotein E4 structure and its receptor binding mechanism determined by a combined cross-linking coupled to mass spectrometry and molecular dynamics approach.

Apolipoprotein E (apoE) is a forefront actor in the transport of lipids and the maintenance of cholesterol homeostasis, and is also strongly implicated in Alzheimer's disease. Upon lipid-binding apoE adopts a conformational state that mediates the receptor-induced internalization of lipoproteins. Due to its inherent structural dynamics and the presence of lipids, the structure of the biologically active apoE remains so far poorly described. To address this issue, we developed an innovative hybrid method combining experimental data with molecular modeling and dynamics to generate comprehensive models of the lipidated apoE4 isoform. Chemical cross-linking combined with mass spectrometry provided distance restraints, characterizing the three-dimensional organization of apoE4 molecules at the surface of lipidic nanoparticles. The ensemble of spatial restraints was then rationalized in an original molecular modeling approach to generate monomeric models of apoE4 that advocated the existence of two alternative conformations. These two models point towards an activation mechanism of apoE4 relying on a regulation of the accessibility of its receptor binding region. Further, molecular dynamics simulations of the dimerized and lipidated apoE4 monomeric conformations revealed an elongation of the apoE N-terminal domain, whereby helix 4 is rearranged, together with Arg172, into a proper orientation essential for lipoprotein receptor association. Overall, our results show how apoE4 adapts its conformation for the recognition of the low density lipoprotein receptor and we propose a novel mechanism of activation for apoE4 that is based on accessibility and remodeling of the receptor binding region.

Bifunctional crosslinking ligands for transthyretin.

Wild-type and variant forms of transthyretin (TTR), a normal plasma protein, are amyloidogenic and can be deposited in the tissues as amyloid fibrils causing acquired and hereditary systemic TTR amyloidosis, a debilitating and usually fatal disease. Reduction in the abundance of amyloid fibril precursor proteins arrests amyloid deposition and halts disease progression in all forms of amyloidosis including TTR type. Our previous demonstration that circulating serum amyloid P component (SAP) is efficiently depleted by administration of a specific small molecule ligand compound, that non-covalently crosslinks pairs of SAP molecules, suggested that TTR may be also amenable to this approach. We first confirmed that chemically crosslinked human TTR is rapidly cleared from the circulation in mice. In order to crosslink pairs of TTR molecules, promote their accelerated clearance and thus therapeutically deplete plasma TTR, we prepared a range of bivalent specific ligands for the thyroxine binding sites of TTR. Non-covalently bound human TTR-ligand complexes were formed that were stable in vitro and in vivo, but they were not cleared from the plasma of mice in vivo more rapidly than native uncomplexed TTR. Therapeutic depletion of circulating TTR will require additional mechanisms.

Two distinct ß-sheet structures in Italian-mutant amyloid-beta fibrils: a potential link to different clinical phenotypes.

Most Alzheimer's disease (AD) cases are late-onset and characterized by the aggregation and deposition of the amyloid-beta (Aß) peptide in extracellular plaques in the brain. However, a few rare and hereditary Aß mutations, such as the Italian Glu22-to-Lys (E22K) mutation, guarantee the development of early-onset familial AD. This type of AD is associated with a younger age at disease onset, increased ß-amyloid accumulation, and Aß deposition in cerebral blood vessel walls, giving rise to cerebral amyloid angiopathy (CAA). It remains largely unknown how the Italian mutation results in the clinical phenotype that is characteristic of CAA. We therefore investigated how this single point mutation may affect the aggregation of Aß1-42 in vitro and structurally characterized the resulting fibrils using a biophysical approach. This paper reports that wild-type and Italian-mutant Aß both form fibrils characterized by the cross-ß architecture, but with distinct ß-sheet organizations, resulting in differences in thioflavin T fluorescence and solvent accessibility. E22K Aß1-42 oligomers and fibrils both display an antiparallel ß-sheet structure, in comparison with the parallel ß-sheet structure of wild-type fibrils, characteristic of most amyloid fibrils described in the literature. Moreover, we demonstrate structural plasticity for Italian-mutant Aß fibrils in a pH-dependent manner, in terms of their underlying ß-sheet arrangement. These findings are of interest in the ongoing debate that (1) antiparallel ß-sheet structure might represent a signature for toxicity, which could explain the higher toxicity reported for the Italian mutant, and that (2) fibril polymorphism might underlie differences in disease pathology and clinical manifestation.

Chemical cross-linking/mass spectrometry maps the amyloid ß peptide binding region on both apolipoprotein E domains.

Apolipoprotein E (apoE) binds the amyloid ß peptide (Aß), one of the major culprits in Alzheimer's disease development. The formation of apoE:Aß complexes is implicated in both Aß clearance and fibrillization. However, the binding interface between apoE and Aß is poorly defined despite substantial previous research efforts, and the exact role of apoE in the pathology of Alzheimer's disease remains largely elusive. Here, we compared the three main isoforms of apoE (E2, E3, and E4) for their interaction with Aß1-42 in an early stage of aggregation and at near physiological conditions. Using electron microscopy and Western blots, we showed that all three isoforms are able to prevent Aß fibrillization and form a noncovalent complex, with one molecule of Aß bound per apoE. Using chemical cross-linking coupled to mass spectrometry, we further examined the interface of interaction between apoE2/3/4 and Aß. Multiple high-confidence intermolecular apoE2/3/4:Aß cross-links confirmed that Lys16 is located in the region of Aß binding to apoE2/3/4. Further, we demonstrated that both N- and C-terminal domains of apoE2/3/4 are interacting with Aß. The cross-linked sites were mapped onto and evaluated in light of a recent structure of apoE. Our results support binding of the hydrophobic Aß at the apoE domain-domain interaction interface, which would explain how apoE is able to stabilize Aß and thereby prevent its subsequent aggregation.

Host-guest interactions in acid-porphyrin complexes.

In this report we use the weak interactions of acid-porphyrin complexes to selectively bind competing acids to the faces of a rigid cyclic porphyrin dimer, and characterise the resulting interactions by NMR spectroscopy and nano-electrospray ionisation spectrometry.

Mechanism and rates of exchange of L7/L12 between ribosomes and the effects of binding EF-G.

The ribosomal stalk complex binds and recruits translation factors to the ribosome during protein biosynthesis. In Escherichia coli the stalk is composed of protein L10 and four copies of L7/L12. Despite the crucial role of the stalk, mechanistic details of L7/L12 subunit exchange are not established. By incubating isotopically labeled intact ribosomes with their unlabeled counterparts we monitored the exchange of the labile stalk proteins by recording mass spectra as a function of time. On the basis of kinetic analysis, we proposed a mechanism whereby exchange proceeds via L7/L12 monomers and dimers. We also compared exchange of L7/L12 from free ribosomes with exchange from ribosomes in complex with elongation factor G (EF-G), trapped in the posttranslocational state by fusidic acid. Results showed that binding of EF-G reduces the L7/L12 exchange reaction of monomers by ~27% and of dimers by ~47% compared with exchange from free ribosomes. This is consistent with a model in which binding of EF-G does not modify interactions between the L7/L12 monomers but rather one of the four monomers, and as a result one of the two dimers, become anchored to the ribosome-EF-G complex preventing their free exchange. Overall therefore our results not only provide mechanistic insight into the exchange of L7/L12 monomers and dimers and the effects of EF-G binding but also have implications for modulating stability in response to environmental and functional stimuli within the cell.

Trapping of palindromic ligands within native transthyretin prevents amyloid formation.

Transthyretin (TTR) amyloidosis is a fatal disease for which new therapeutic approaches are urgently needed. We have designed two palindromic ligands, 2,2'-(4,4'-(heptane-1,7-diylbis(oxy))bis(3,5-dichloro-4,1-phenylene)) bis(azanediyl)dibenzoic acid (mds84) and 2,2'-(4,4'-(undecane-1,11-diylbis(oxy))bis(3,5-dichloro-4,1-phenylene)) bis(azanediyl)dibenzoic acid (4ajm15), that are rapidly bound by native wild-type TTR in whole serum and even more avidly by amyloidogenic TTR variants. One to one stoichiometry, demonstrable in solution and by MS, was confirmed by X-ray crystallographic analysis showing simultaneous occupation of both T4 binding sites in each tetrameric TTR molecule by the pair of ligand head groups. Ligand binding by native TTR was irreversible under physiological conditions, and it stabilized the tetrameric assembly and inhibited amyloidogenic aggregation more potently than other known ligands. These superstabilizers are orally bioavailable and exhibit low inhibitory activity against cyclooxygenase (COX). They offer a promising platform for development of drugs to treat and prevent TTR amyloidosis.

Retinol and retinol-binding protein stabilize transthyretin via formation of retinol transport complex.

Transthyretin (TTR) is a plasma hormone carrier protein associated with hereditary and senile forms of systemic amyloid disease, wherein slow tetramer disassembly is thought to be an obligatory step. Plasma transport of retinol is carried out exclusively by the retinol-binding protein (RBP), through complexation with transthyretin. Using mass spectrometry to examine the subunit exchange dynamics, we find that retinol stabilizes the quaternary structure of transthyretin, through its interactions with RBP, reducing the rate of transthyretin disassembly ∼17-fold compared to apoTTR. In the absence of retinol but in the presence of RBP, transthyretin is only marginally stabilized with the rate of disassembly reduced ∼two-fold with respect to apoTTR. Surprisingly, we found two retinoids that stabilize transthyretin directly, in the absence of RBP, whereas retinol itself requires RBP in order to stabilize transthyretin. Our results demonstrate new roles for RBP and retinoids as stabilizers of transthyretin.

Correlating solution binding and ESI-MS stabilities by incorporating solvation effects in a confined cucurbit[8]uril system.

The high-throughput characterization of solution binding equilibria is essential in biomedical research such as drug design as well as in material applications of synthetic systems in which reversible binding interactions play critical roles. Although isothermal titration calorimetry (ITC) has been widely employed for describing such binding events, factors such as speed, concentration, and sample complexity would principally favor a mass spectrometry approach. Here, we show a link between ITC and electrospray ionization mass spectrometry (ESI-MS) by incorporating solvation free energies in the study of the ternary complexes of the macrocyclic host cucurbit[8]uril (CB[8]). The binding affinities of 32 aromatic reference complexes were studied by ITC and ESI-MS and combined with solvation data of the guests from an implicit solvation model (SM8) to obtain a correlation between aqueous and gas-phase measurements. The data illustrates the critical importance of solvation on the binding strength in CB[8]'s ternary complexes. Finally, this treatment enabled us to predict association constants that were in excellent agreement with measured values, including several highly insoluble guest compounds.

Discrete, multi-component complexes with cucurbit[8]uril in the gas-phase.

The formation of modular, multi-component, host-guest complexes stable both in water and in the gas-phase is described; by modulating the stoichiometry of the different subunits in aqueous solution, quantitative self-assembly of the predicted and desired architecture is achieved, even allowing for ABA triblock copolymer assemblies stable in the gas-phase.

Acetylation of L12 increases interactions in the Escherichia coli ribosomal stalk complex.

The ribosomal stalk complex in Escherichia coli consists of L10 and four copies of L7/L12, and is largely responsible for binding and recruiting translation factors. Structural characterisation of this stalk complex is difficult, primarily due to its dynamics. Here, we apply mass spectrometry to follow post-translational modifications and their effect on structural changes of the stalk proteins on intact ribosomes. Our results show that increased acetylation of L12 occurs during the stationary phase on ribosomes harvested from cells grown under optimal conditions. For cells grown in minimal medium, L12 acetylation and processing is altered, resulting in deficient removal of N-terminal methionine in approximately 50% of the L12 population, while processed L12 is almost 100% acetylated. Our results show also that N-acetylation of L12 correlates with an increased stability of the stalk complex in the gas phase. To investigate further the basis of this increased stability, we applied a solution phase hydrogen deuterium exchange protocol to compare the rate of deuterium incorporation in the proteins L9, L10, L11 and L12 as well as the acetylated form of L12 (L7), in situ on the ribosome. Results show that deuterium incorporation is consistently slower for L7 relative to L12 and for L10 when L7 is predominant. Our results imply a tightening of the interaction between L7 and L10 relative to that between L12 and L10. Since acetylation is predominant when cells are grown in minimal medium, we propose that these modifications form part of the cell's strategy to increase stability of the stalk complex under conditions of stress. More generally, our results demonstrate that it is possible to discern the influence of a 42 Da post-translational modification by mass spectrometry and to record subtle changes in hydrogen/deuterium exchange within the context of an intact 2.5 MDa particle.

Photo-cross-linking between polymers derivatized with photoreactive ruthenium-1,4,5,8-tetraazaphenanthrene complexes and guanine-containing oligonucleotides.

We have shown previously that complexes containing 1,4,5,8-tetraazaphenanthrene (TAP) ligands are able to form photoadducts with the guanine bases of DNA and oligonucleotides. In this work, we have exploited this specific photoreaction for carrying out photo-cross-linkings between guanine-containing oligonucleotides (G-ODNs) and biodegradable polymers derivatized with the photoreactive Ru(II) compounds. The aim in the future is to use these polymer conjugates as vectorizing agents of the metallic compounds inside the cells. Thus, photooxidizing Ru(II) complexes such as [Ru(TAP)3]2+ and [Ru(TAP)2phen]2+ (phen = 1,10-phenanthroline) have been derivatized by an oxyamine function to attach them, via an oxime ether linkage, to a soluble 6 or 80 kDa poly-[N-(2-hydroxyethyl)-l-glutamine] polymer that contains pendent aldehyde groups. It is demonstrated that the resulting Ru-labeled polymers exhibit photophysical properties and a photochemistry that are comparable with those of the free, nonattached complexes. The photo-cross-linkings with the G-ODNs are clearly detected by gel electrophoresis with the 6 kDa Ru conjugates upon illumination.