Apolipoprotein E4 has extensive conformational heterogeneity in lipid-free and lipid-bound forms.

The ε4-allele variant of apolipoprotein E (ApoE4) is the strongest genetic risk factor for Alzheimer's disease, although it only differs from its neutral counterpart ApoE3 by a single amino acid substitution. While ApoE4 influences the formation of plaques and neurofibrillary tangles, the structural determinants of pathogenicity remain undetermined due to limited structural information. Previous studies have led to conflicting models of the C-terminal region positioning with respect to the N-terminal domain across isoforms largely because the data are potentially confounded by the presence of heterogeneous oligomers. Here, we apply a combination of single-molecule spectroscopy and molecular dynamics simulations to construct an atomically detailed model of monomeric ApoE4 and probe the effect of lipid association. Importantly, our approach overcomes previous limitations by allowing us to work at picomolar concentrations where only the monomer is present. Our data reveal that ApoE4 is far more disordered and extended than previously thought and retains significant conformational heterogeneity after binding lipids. Comparing the proximity of the N- and C-terminal domains across the three major isoforms (ApoE4, ApoE3, and ApoE2) suggests that all maintain heterogeneous conformations in their monomeric form, with ApoE2 adopting a slightly more compact ensemble. Overall, these data provide a foundation for understanding how ApoE4 differs from nonpathogenic and protective variants of the protein.

APOE3-Jacksonville (V236E) variant reduces self-aggregation and risk of dementia.

Apolipoprotein E (APOE) genetic variants have been shown to modify Alzheimer's disease (AD) risk. We previously identified an APOE3 variant (APOE3-V236E), named APOE3-Jacksonville (APOE3-Jac), associated with healthy brain aging and reduced risk for AD and dementia with Lewy bodies (DLB). Herein, we resolved the functional mechanism by which APOE3-Jac reduces APOE aggregation and enhances its lipidation in human brains, as well as in cellular and biochemical assays. Compared to APOE3, expression of APOE3-Jac in astrocytes increases several classes of lipids in the brain including phosphatidylserine, phosphatidylethanolamine, phosphatidic acid, and sulfatide, critical for synaptic functions. Mice expressing APOE3-Jac have reduced amyloid pathology, plaque-associated immune responses, and neuritic dystrophy. The V236E substitution is also sufficient to reduce the aggregation of APOE4, whose gene allele is a major genetic risk factor for AD and DLB. These findings suggest that targeting APOE aggregation might be an effective strategy for treating a subgroup of individuals with AD and DLB.

Bioorthogonal Chemistry Enables Single-Molecule FRET Measurements of Catalytically Active Protein Disulfide Isomerase.

Folding of newly synthesized proteins in the endoplasmic reticulum is assisted by several families of enzymes. One such family is the protein disulfide isomerases (PDIs). PDIs are oxidoreductases, capable of forming new disulfide bonds or breaking existing ones. Structural information on PDIs unbound and bound to substrates is highly desirable for developing targeted therapeutics, yet it has been difficult to obtain by using traditional approaches because of their relatively large size and remarkable flexibility. Single-molecule FRET (smFRET) could be a powerful tool to study PDIs' structure and dynamics under conditions relevant to physiology, but its implementation has been hindered by technical challenges of position-specific fluorophore labeling. We have overcome this limitation by site-specifically engineering fluorescent dyes into human PDI, the founding member of the family. Proof-of-concept smFRET measurements of catalytically active PDI demonstrate, for the first time, the feasibility of this approach, expanding the toolkit for structural studies of PDIs.

Functional insights from biophysical study of TREM2 interactions with apoE and Aß1-42.

INTRODUCTION: Triggering receptor expressed on myeloid cells-2 (TREM2) is an immune receptor expressed on microglia that also can become soluble (sTREM2). How TREM2 engages different ligands remains poorly understood. METHODS: We used comprehensive biolayer interferometry (BLI) analysis to investigate TREM2 and sTREM2 interactions with apolipoprotein E (apoE) and monomeric amyloid beta (Aß) (mAß42). RESULTS: TREM2 engagement of apoE was protein mediated with little effect of lipidation, showing slight affinity differences between isoforms (E4 > E3 > E2). Another family member, TREML2, did not bind apoE. Disease-linked TREM2 variants within a "basic patch" minimally impact apoE binding. Instead, TREM2 uses a unique hydrophobic surface to bind apoE, which requires the apoE hinge region. TREM2 and sTREM2 directly bind mAß42 and potently inhibit Aß42 polymerization, suggesting a potential role for soluble sTREM2 in preventing AD pathogenesis. DISCUSSION: These findings demonstrate that TREM2 has at least two ligand-binding surfaces that might be therapeutic targets and uncovers a potential function for sTREM2 in directly inhibiting Aß polymerization.

19F NMR reveals the conformational properties of free thrombin and its zymogen precursor prethrombin-2.

The conformational properties of trypsin-like proteases and their zymogen forms remain controversial because of a lack of sufficient information on their free forms. Specifically, it is unclear whether the free protease is zymogen-like and shifts to its mature form upon a ligand-induced fit or exists in multiple conformations in equilibrium from which the ligand selects the optimal fit via conformational selection. Here we report the results of 19F NMR measurements that reveal the conformational properties of a protease and its zymogen precursor in the free form. Using the trypsin-like, clotting protease thrombin as a relevant model system, we show that its conformation is quite different from that of its direct zymogen precursor prethrombin-2 and more similar to that of its fully active Na+-bound form. The results cast doubts on recent hypotheses that free thrombin is zymogen-like and transitions to protease-like forms upon ligand binding. Rather, they validate the scenario emerged from previous findings of X-ray crystallography and rapid kinetics supporting a pre-existing equilibrium between open (E) and closed (E*) forms of the active site. In this scenario, prethrombin-2 is more dynamic and exists predominantly in the E* form, whereas thrombin is more rigid and exists predominantly in the E form. Ligand binding to thrombin takes place exclusively in the E form without significant changes in the overall conformation. In summary, these results disclose the structural architecture of the free forms of thrombin and prethrombin-2, consistent with an E*-E equilibrium and providing no evidence that free thrombin is zymogen-like.

Charge Reduction of Membrane Proteins in Native Mass Spectrometry Using Alkali Metal Acetate Salts.

Native mass spectrometry (MS) provides the capacity to monitor membrane protein complexes and noncovalent binding of ligands and lipids to membrane proteins. The charge states produced by native MS of membrane proteins often result in gas-phase protein unfolding or loss of noncovalent interactions. In an effort to reduce the charge of membrane proteins, we examined the utility of alkali metal salts as a charge-reducing agent. Low concentrations of alkali metal salts caused marked charge reduction in the membrane protein, Erwinia ligand-gated ion channel (ELIC). The charge-reducing effect only occurred for membrane proteins and was detergent-dependent, being most pronounced in long polyethylene glycol (PEG)-based detergents such as C10E5 and C12E8. On the basis of these results, we propose a mechanism for alkali metal charge reduction of membrane proteins. Addition of low concentrations of alkali metals may provide an advantageous approach for charge reduction of detergent-solubilized membrane proteins by native MS.

Native Mass Spectrometry, Ion Mobility, Electron-Capture Dissociation, and Modeling Provide Structural Information for Gas-Phase Apolipoprotein E Oligomers.

Apolipoprotein E (apoE) is an essential protein in lipid and cholesterol metabolism. Although the three common isoforms in humans differ only at two sites, their consequences in Alzheimer's disease (AD) are dramatically different: only the ε4 allele is a major genetic risk factor for late-onset Alzheimer's disease. The isoforms exist as a mixture of oligomers, primarily tetramer, at low µM concentrations in a lipid-free environment. This self-association is involved in equilibrium with the lipid-free state, and the oligomerization interface overlaps with the lipid-binding region. Elucidation of apoE wild-type (WT) structures at an oligomeric state, however, has not yet been achieved. To address this need, we used native electrospray ionization and mass spectrometry (native MS) coupled with ion mobility (IM) to examine the monomer and tetramer of the three WT isoforms. Although collision-induced unfolding (CIU) cannot distinguish the WT isoforms, the monomeric mutant (MM) of apoE3 shows higher stability when submitted to CIU than the WT monomer. From ion-mobility measurements, we obtained the collision cross section and built a coarse-grained model for the tetramer. Application of electron-capture dissociation (ECD) to the tetramer causes unfolding starting from the C-terminal domain, in good agreement with solution denaturation data, and provides additional support for the C4 symmetry structure of the tetramer.

Protein oligomerization as a metabolic control mechanism: Application to apoE.

It has been estimated that 30%-50% of proteins self-assemble to form complexes consisting of multiple copies of themselves. If there is a functional difference between different molecular weight forms and if these forms interconvert on a reasonable time scale then oligomerization could be an important metabolic control mechanism. The example given here is of apoE for which the oligomerization process is measured in minutes to hours and the monomer binds lipids while the tetramer does not. Examination of the literature reveals few reports on the rate constants that control the interconversion of different molecular weight forms. Perhaps it is time to collect such data.

Structure-Function Analysis of the Curli Accessory Protein CsgE Defines Surfaces Essential for Coordinating Amyloid Fiber Formation.

Curli amyloid fibers are produced as part of the extracellular biofilm matrix and are composed primarily of the major structural subunit CsgA. The CsgE chaperone facilitates the secretion of CsgA through CsgG by forming a cap at the base of the nonameric CsgG outer membrane pore. We elucidated a series of finely tuned nonpolar and charge-charge interactions that facilitate the oligomerization of CsgE and its ability to transport unfolded CsgA to CsgG for translocation. CsgE oligomerization in vitro is temperature dependent and is disrupted by mutations in the W48 and F79 residues. Using nuclear magnetic resonance (NMR), we identified two regions of CsgE involved in the CsgE-CsgA interaction: a head comprising a positively charged patch centered around R47 and a stem comprising a negatively charged patch containing E31 and E85. Negatively charged residues in the intrinsically disordered N- and C-terminal "tails" were not implicated in this interaction. Head and stem residues were mutated and interrogated using in vivo measurements of curli production and in vitro amyloid polymerization assays. The R47 head residue of CsgE is required for stabilization of CsgA- and CsgE-mediated curli fiber formation. Mutation of the E31 and E85 stem residues to positively charged side chains decreased CsgE-mediated curli fiber formation but increased CsgE-mediated stabilization of CsgA. No single-amino-acid substitutions in the head, stem, or tail regions affected the ability of CsgE to cap the CsgG pore as determined by a bile salt sensitivity assay. These mechanistic insights into the directed assembly of functional amyloids in extracellular biofilms elucidate possible targets for biofilm-associated bacterial infections.IMPORTANCE Curli represent a class of functional amyloid fibers produced by Escherichia coli and other Gram-negative bacteria that serve as protein scaffolds in the extracellular biofilm matrix. Despite the lack of sequence conservation among different amyloidogenic proteins, the structural and biophysical properties of functional amyloids such as curli closely resemble those of amyloids associated with several common neurodegenerative diseases. These parallels are underscored by the observation that certain proteins and chemicals can prevent amyloid formation by the major curli subunit CsgA and by alpha-synuclein, the amyloid-forming protein found in Lewy bodies during Parkinson's disease. CsgA subunits are targeted to the CsgG outer membrane pore by CsgE prior to secretion and assembly into fibers. Here, we use biophysical, biochemical, and genetic approaches to elucidate a mechanistic understanding of CsgE function in curli biogenesis.

Understanding Curli Amyloid-Protein Aggregation by Hydrogen-Deuterium Exchange and Mass Spectrometry.

Bacteria within Curli biofilms are protected from environmental pressures (e.g., disinfectants, antibiotics), and this is responsible for intractable infections. Understanding aggregation of the major protein component of Curli, CsgA, may uncover disease-associated amyloidogenesis mechanisms. Here, we report the application of pulsed hydrogen-deuterium exchange and mass spectrometry (HDX-MS) to study CsgA aggregation, thereby obtaining region-specific information. By following time-dependent peptide signal depletion, presumably a result of insoluble fibril formation, we acquired sigmoidal profiles that are specific for regions (region-specific) of the protein. These signal-depletion profiles not only provide an alternative aggregation measurement, but also give insight on soluble species in the aggregation. The HDX data present as bimodal isotopic distributions, one representing a highly disordered species whereas the other a well-structured one. Although the extents of deuterium uptake of the two species remain the same with time, the relative abundance of the lower mass, less-exchanged species increases in a region-specific manner. The same region-specific aggregation properties also pertain to different aggregation conditions. Although CsgA is an intrinsically disordered protein, within the fibril it is thought to consist of five imperfect ß-strand repeating units (labeled R1-R5). We found that the exterior repeating units R1 and R5 have higher aggregation propensities than do the interior units R2, R3, and R4. We also employed TEM to obtain complementary information of the well-structured species. The results provide insight on aggregation and a new approach for further application of HDX-MS to unravel aggregation mechanisms of amyloid proteins.

Peptide-Level Interactions between Proteins and Small-Molecule Drug Candidates by Two Hydrogen-Deuterium Exchange MS-Based Methods: The Example of Apolipoprotein E3.

We describe a platform utilizing two methods based on hydrogen-deuterium exchange (HDX) coupled with mass spectrometry (MS) to characterize interactions between a protein and a small-molecule ligand. The model system is apolipoprotein E3 (apoE3) and a small-molecule drug candidate. We extended PLIMSTEX (protein-ligand interactions by mass spectrometry, titration, and H/D exchange) to the regional level by incorporating enzymatic digestion to acquire binding information for peptides. In a single experiment, we not only identified putative binding sites, but also obtained affinities of 6.0, 6.8, and 10.6 µM for the three different regions, giving an overall binding affinity of 7.4 µM. These values agree well with literature values determined by accepted methods. Unlike those methods, PLIMSTEX provides site-specific binding information. The second approach, modified SUPREX (stability of unpurified proteins from rates of H/D exchange) coupled with electrospray ionization (ESI), allowed us to obtain detailed understanding about apoE unfolding and its changes upon ligand binding. Three binding regions, along with an additional site, which may be important for lipid binding, show increased stability (less unfolding) upon ligand binding. By employing a single parameter, ΔC1/2%, we compared relative changes of denaturation between peptides. This integrated platform provides information orthogonal to commonly used HDX kinetics experiments, providing a general and novel approach for studying protein-ligand interactions.

A mechanism for lipid binding to apoE and the role of intrinsically disordered regions coupled to domain-domain interactions.

Relative to the apolipoprotein E (apoE) E3 allele of the APOE gene, apoE4 strongly increases the risk for the development of late-onset Alzheimer's disease. However, apoE4 differs from apoE3 by only a single amino acid at position 112, which is arginine in apoE4 and cysteine in apoE3. It remains unclear why apoE3 and apoE4 are functionally different. Described here is a proposal for understanding the functional differences between these two isoforms with respect to lipid binding. A mechanism is proposed that is based on the full-length monomeric structure of the protein, on hydrogen-deuterium exchange mass spectrometry data, and on the role of intrinsically disordered regions to control protein motions. It is proposed that lipid binds between the N-terminal and C-terminal domains and that separation of the two domains, along with the presence of intrinsically disordered regions, controls this process. The mechanism explains why apoE3 differs from apoE4 with respect to different lipid-binding specificities, why lipid increases the binding of apoE to its receptor, and why specific residues are conserved.

Deamidation Slows Curli Amyloid-Protein Aggregation.

Nonenzymatic deamidation of asparagine and glutamine in peptides and proteins is a frequent modification both in vivo and in vitro. The biological effect is not completely understood, but it is often associated with protein degradation and loss of biological function. Here we describe the deamidation of CsgA, the major protein subunit of curli, which are important proteinaceous components of biofilms. CsgA has a high content of Asn and Gln, a feature seen in a few proteins that self-aggregate. We have implemented an approach to monitor deamidation rapidly by following the globally centroid mass shift, providing guidance for studies at the residue level. From the global mass measurement, we identified, using LC-MS/MS, extensive deamidation of several Asn residues and discovered three "Asn-Gly" sites to be the hottest spots for deamidation. The fibrillization of deamidated CsgA was measured using thioflavin T (ThT) fluorescence, circular dichroism (CD), and a previously reported hydrogen-deuterium exchange (HDX) platform. Deamidated proteins exhibit a longer lag phase and lower final ThT fluorescence, strongly suggesting slower and less amyloid fibril formation. CD spectra show that extensively deamidated CsgA remains unstructured and loses its ability to form amyloids. Mass-spectrometry-based HDX also shows that deamidated CsgA aggregates more slowly than wild-type CsgA. Taken together, the results show that deamidation of CsgA slows its fibrillization and disrupts its function, suggesting an opportunity to modulate CsgA fibrillization and affect curli and biofilm formation.

Solution NMR structure of CsgE: Structural insights into a chaperone and regulator protein important for functional amyloid formation.

Curli, consisting primarily of major structural subunit CsgA, are functional amyloids produced on the surface of Escherichia coli, as well as many other enteric bacteria, and are involved in cell colonization and biofilm formation. CsgE is a periplasmic accessory protein that plays a crucial role in curli biogenesis. CsgE binds to both CsgA and the nonameric pore protein CsgG. The CsgG-CsgE complex is the curli secretion channel and is essential for the formation of the curli fibril in vivo. To better understand the role of CsgE in curli formation, we have determined the solution NMR structure of a double mutant of CsgE (W48A/F79A) that appears to be similar to the wild-type (WT) protein in overall structure and function but does not form mixed oligomers at NMR concentrations similar to the WT. The well-converged structure of this mutant has a core scaffold composed of a layer of two α-helices and a layer of three-stranded antiparallel ß-sheet with flexible N and C termini. The structure of CsgE fits well into the cryoelectron microscopy density map of the CsgG-CsgE complex. We highlight a striking feature of the electrostatic potential surface in CsgE structure and present an assembly model of the CsgG-CsgE complex. We suggest a structural mechanism of the interaction between CsgE and CsgA. Understanding curli formation can provide the information necessary to develop treatments and therapeutic agents for biofilm-related infections and may benefit the prevention and treatment of amyloid diseases. CsgE could establish a paradigm for the regulation of amyloidogenesis because of its unique role in curli formation.

ApoE: In Vitro Studies of a Small Molecule Effector.

Apolipoprotein E4 (apoE4), one of three isoforms of apoE, is the major risk factor for developing late onset Alzheimer's disease. The only differences among these isoforms (apoE2, apoE3, and apoE4) are single amino acid changes. Yet these proteins are functionally very different. One approach to ameliorating the effect of apoE4 with respect to Alzheimer's disease would be to find small molecular weight compounds that affect the behavior of apoE4. Few studies of this approach have been carried out in part because there was no complete structure of any full-length apoE isoform until 2011. Here, we focus on one small molecular weight compound, EZ-482, and explore the effects of its binding to apoE. Using hydrogen-deuterium exchange, we determined that EZ-482 binds to the C-terminal domains of both apoE3 and apoE4. The binding to apoE4, however, is accompanied by a unique N-terminal allosteric effect. Using fluorescence methods, we determined an apparent dissociation constant of approximately 8 µM. Although EZ-482 binds to the C-terminal domain, it blocks heparin binding to the N-terminal domain. The residues of apoE that bind heparin are the same as those involved in apoE binding to LDL and LRP-1 receptors. The methods and the data presented here may serve as a template for future studies using small molecular weight compounds to modulate the behavior of apoE.

Continuous and pulsed hydrogen-deuterium exchange and mass spectrometry characterize CsgE oligomerization.

We report the use of hydrogen-deuterium amide exchange coupled to mass spectrometry (HDX-MS) to study the interfaces of and conformational changes accompanying CsgE oligomerization. This protein plays an important role in enteric bacteria biofilm formation. Biofilms provide protection for enteric bacteria from environmental extremes and raise concerns about controlling bacteria and infectious disease. Their proteinaceous components, called curli, are extracellular functional amyloids that initiate surface contact and biofilm formation. The highly regulated curli biogenesis involves a major subunit, CsgA, a minor subunit CsgB, and a series of other accessory proteins. CsgE, possibly functioning as oligomer, is a chaperonin-like protein that delivers CsgA to an outer-membrane bound oligomeric CsgG complex. No higher-order structure, or interfaces and dynamics of its oligomerization, however, are known. In this work, we determined regions involved in CsgE self-association by continuous HDX, and, on the basis of that, prepared a double mutant W48A/F79A, derived from interface alanine scan, and verified that it exists as monomer. Using pulsed HDX and MS, we suggest there is a structural rearrangement occurring during the oligomerization of CsgE.

ApoE: the role of conserved residues in defining function.

The amino acid sequences of apolipoprotein E (apoE) from 63 different mammalian species have been downloaded from the protein database. The sequences were compared to human apoE4 to determine conserved and non-conserved sequences of amino acids. ApoE4 is the major risk factor for the development of late onset Alzheimer's disease while apoE3, which differs from apoE4 by a single amino acid change at position 112, poses little or no risk for the development of this disease. Thus, the two proteins appear to be structurally and functionally different. Seven highly conserved regions, representing approximately 47 amino acids (of 299) have been found. These regions are distributed throughout the protein and reflect ligand binding sites as well as regions proposed to be involved in the propagation of the cysteine-arginine change at position 112 to distant regions of the protein in the N- and C-terminal domains. Highly non-conserved regions are at the N- and C-terminal ends of the apoE protein.

The binding of apolipoprotein E to oligomers and fibrils of amyloid-ß alters the kinetics of amyloid aggregation.

Deposition of amyloid-ß (Aß) in Alzheimer's disease (AD) is strongly correlated with the APOE genotype. However, the role of apolipoprotein E (apoE) in Aß aggregation has remained unclear. Here we have used different apoE preparations, such as recombinant protein or protein isolated from cultured astrocytes, to examine the effect of apoE on the aggregation of both Aß1-40 and Aß1-42. The kinetics of aggregation, measured by the loss of fluorescence of tetramethylrhodamine-labeled Aß, is shown to be dramatically slowed by the presence of substoichiometric concentrations of apoE. Using these concentrations, we conclude that apoE binds primarily to and affects the growth of oligomers that lead to the nuclei required for fibril growth. At higher apoE concentrations, the protein also binds to Aß fibrils, resulting in fibril stabilization and a slower rate of fibril growth. The aggregation of Aß1-40 is dependent on the apoE isoform, being the most dramatic for apoE4 and less so for apoE3 and apoE2. Our results indicate that the detrimental role of apoE4 in AD could be related to apoE-induced stabilization of the soluble but cytotoxic oligomeric forms and intermediates of Aß, as well as fibril stabilization.

Unmasking the roles of N- and C-terminal flanking sequences from exon 1 of huntingtin as modulators of polyglutamine aggregation.

Huntington disease is caused by mutational expansion of the CAG trinucleotide within exon 1 of the huntingtin (Htt) gene. Exon 1 spanning N-terminal fragments (NTFs) of the Htt protein result from aberrant splicing of transcripts of mutant Htt. NTFs typically encompass a polyglutamine tract flanked by an N-terminal 17-residue amphipathic stretch (N17) and a C-terminal 38-residue proline-rich stretch (C38). We present results from in vitro biophysical studies that quantify the driving forces for and mechanisms of polyglutamine aggregation as modulated by N17 and C38. Although N17 is highly soluble by itself, it lowers the saturation concentration of soluble NTFs and increases the driving force, vis-à-vis homopolymeric polyglutamine, for forming insoluble aggregates. Kinetically, N17 accelerates fibril formation and destabilizes nonfibrillar intermediates. C38 is also highly soluble by itself, and it lends its high intrinsic solubility to lower the driving force for forming insoluble aggregates by increasing the saturation concentration of soluble NTFs. In NTFs with both modules, N17 and C38 act synergistically to destabilize nonfibrillar intermediates (N17 effect) and lower the driving force for forming insoluble aggregates (C38 effect). Morphological studies show that N17 and C38 promote the formation of ordered fibrils by NTFs. Homopolymeric polyglutamine forms a mixture of amorphous aggregates and fibrils, and its aggregation mechanisms involve early formation of heterogeneous distributions of nonfibrillar species. We propose that N17 and C38 act as gatekeepers that control the intrinsic heterogeneities of polyglutamine aggregation. This provides a biophysical explanation for the modulation of in vivo NTF toxicities by N17 and C38.

Concerning the structure of apoE.

Apolipoprotein E (apoE), first described in 1973, is a truly fascinating protein. While studies initially focused on its role in cholesterol and lipid metabolism, one apoE isoform (apoE4) is a major risk factor for development of late onset Alzheimer's disease. Yet the difference between apoE3, the common form, and apoE4 is a single amino acid of the 299 in this 34 kDa protein. Structure determination of the two domain full length apoE3 protein was only accomplished in 2011 and supports the notion that mutations in the N-terminal domain can be propagated through the structure to the C-terminal domain. Understanding the structural differences between apoE3 and apoE4 is critical for finding ways to modulate the deleterious effect of apoE4.