Structural Impact of N-terminal Pyroglutamate in an Amyloid-ß(3-42) Fibril Probed by Solid-State NMR Spectroscopy.

Extracellular amyloid-ß (Aß) plaques, primarily formed by Aß(1-40) and Aß(1-42) fibrils, are a hallmark of Alzheimer's disease. The Aß peptide can undergo a high variety of different post-translational modifications including formation of a pyroglutamate (pGlu, pE) at N-terminal Glu3 or Glu11 of truncated Aß(3-x) or Aß(11-x), respectively. Here we studied structural similarities and differences between pEAß(3-42) and LS-shaped Aß(1-42) fibrils grown under identical conditions (pH 2) using solid-state NMR spectroscopy. We show that the central region of pEAß(3-42) fibrils including the turn region around V24 is almost identical to Aß(1-42) showing similar ß-strands also at the N-terminus. The missing N-terminal residues D1-A2 along with pE3 formation in pEAß(3-42) preclude a salt bridge between K28-D1' as in Aß(1-42) fibrils. G37 and G38 act as highly sensitive internal sensors for the modified N-terminus, which remains rigid over ~five pH units.

Amyloid fibril formation kinetics of low-pH denatured bovine PI3K-SH3 monitored by three different NMR techniques.

Introduction: Misfolding of amyloidogenic proteins is a molecular hallmark of neurodegenerative diseases in humans. A detailed understanding of the underlying molecular mechanisms is mandatory for developing innovative therapeutic approaches. The bovine PI3K-SH3 domain has been a model system for aggregation and fibril formation. Methods: We monitored the fibril formation kinetics of low pH-denatured recombinantly expressed [U-13C, 15N] labeled bovine PI3K-SH3 by a combination of solution NMR, high-resolution magic angle spinning (HR-MAS) NMR and solid-state NMR spectra. Solution NMR offers the highest sensitivity and, therefore, allows for the recording of two-dimensional NMR spectra with residue-specific resolution for individual time points of the time series. However, it can only follow the decay of the aggregating monomeric species. In solution NMR, aggregation occurs under quiescent experimental conditions. Solid-state NMR has lower sensitivity and allows only for the recording of one-dimensional spectra during the time series. Conversely, solid-state NMR is the only technique to detect disappearing monomers and aggregated species in the same sample by alternatingly recoding scalar coupling and dipolar coupling (CP)-based spectra. HR-MAS NMR is used here as a hybrid method bridging solution and solid-state NMR. In solid-state NMR and HR-MAS NMR the sample is agitated due to magic angle spinning. Results: Good agreement of the decay rate constants of monomeric SH3, measured by the three different NMR methods, is observed. Moderate MAS up to 8 kHz seems to influence the aggregation kinetics of seeded fibril formation only slightly. Therefore, under sufficient seeding (1% seeds used here), quiescent conditions (solution NMR), and agitated conditions deliver similar results, arguing against primary nucleation induced by MAS as a major contributor. Using solid-state NMR, we find that the amount of disappeared monomer corresponds approximately to the amount of aggregated species under the applied experimental conditions (250 µM PI3K-SH3, pH 2.5, 298 K, 1% seeds) and within the experimental error range. Data can be fitted by simple mono-exponential conversion kinetics, with lifetimes τ in the 14-38 h range. Atomic force microscopy confirms that fibrils substantially grew in length during the aggregation experiment. This argues for fibril elongation as the dominant growth mechanism in fibril mass (followed by the CP-based solid-state NMR signal). Conclusion: We suggest a combined approach employing both solution NMR and solid-state NMR, back-to-back, on two aliquots of the same sample under seeding conditions as an additional approach to follow monomer depletion and growth of fibril mass simultaneously. Atomic force microscopy images confirm fibril elongation as a major contributor to the increase in fibril mass.

Atomic Resolution Insights into pH Shift Induced Deprotonation Events in LS-Shaped Aß(1-42) Amyloid Fibrils.

Alzheimer's disease is a neurodegenerative disorder associated with the deposition of misfolded aggregates of the amyloid-ß protein (Aß). Aß(1-42) is one of the most aggregation-prone components in senile plaques of AD patients. We demonstrated that relatively homogeneous Aß(1-42) fibrils with one predominant fold visible in solid-state NMR spectra can be obtained at acidic pH. The structure of these fibrils differs remarkably from some other polymorphs obtained at neutral pH. In particular, the entire N-terminal region is part of the rigid fibril core. Here, we investigate the effects of a pH shift on the stability and the fold of these fibrils at higher pH values. Fibril bundling at neutral pH values renders cryo-EM studies impractical, but solid-state NMR spectroscopy, molecular dynamics simulations, and biophysical methods provide residue-specific structural information under these conditions. The LS-fold of the Aß(1-42) fibrils does not change over the complete pH range from pH 2 to pH 7; in particular, the N-terminus remains part of the fibril core. We observe changes in the protonation state of charged residues starting from pH 5 on a residue-specific level. The deprotonation of the C-terminal carboxyl group of A42 in the intermolecular salt bridge with D1 and K28 is slow on the NMR time scale, with a local pKa of 5.4, and local conformations of the involved residues are affected by deprotonation of A42. Thus, we demonstrate that this fibril form is stable at physiological pH values.

Conformational heterogeneity coupled with ß-fibril formation of a scaffold protein involved in chronic mental illnesses.

Chronic mental illnesses (CMIs) pose a significant challenge to global health due to their complex and poorly understood etiologies and hence, absence of causal therapies. Research of the past two decades has revealed dysfunction of the disrupted in schizophrenia 1 (DISC1) protein as a predisposing factor involved in several psychiatric disorders. DISC1 is a multifaceted protein that serves myriads of functions in mammalian cells, for instance, influencing neuronal development and synapse maintenance. It serves as a scaffold hub forming complexes with a variety (~300) of partners that constitute its interactome. Herein, using combinations of structural and biophysical tools, we demonstrate that the C-region of the DISC1 protein is highly polymorphic, with important consequences for its physiological role. Results from solid-state NMR spectroscopy and electron microscopy indicate that the protein not only forms symmetric oligomers but also gives rise to fibrils closely resembling those found in certain established amyloid proteinopathies. Furthermore, its aggregation as studied by isothermal titration calorimetry (ITC) is an exergonic process, involving a negative enthalpy change that drives the formation of oligomeric (presumably tetrameric) species as well as ß-fibrils. We have been able to narrow down the ß-core region participating in fibrillization to residues 716-761 of full-length human DISC1. This region is absent in the DISC1Δ22aa splice variant, resulting in reduced association with proteins from the dynein motor complex, viz., NDE-like 1 (NDEL1) and lissencephaly 1 (LIS1), which are crucial during mitosis. By employing surface plasmon resonance, we show that the oligomeric DISC1 C-region has an increased affinity and shows cooperativity in binding to LIS1 and NDEL1, in contrast to the noncooperative binding mode exhibited by the monomeric version. Based on the derived structural models, we propose that the association between the binding partners involves two neighboring subunits of DISC1 C-region oligomers. Altogether, our findings highlight the significance of the DISC1 C-region as a crucial factor governing the balance between its physiological role as a multifunctional scaffold protein and aggregation-related aberrations with potential significance for disease.

A novel strategy for site selective spin-labeling to investigate bioactive entities by DNP and EPR spectroscopy.

A novel specific spin-labeling strategy for bioactive molecules is presented for eptifibatide (integrilin) an antiplatelet aggregation inhibitor, which derives from the venom of certain rattlesnakes. By specifically labeling the disulfide bridge this molecule becomes accessible for analytical techniques such as Electron Paramagnetic Resonance (EPR) and solid state Dynamic Nuclear Polarization (DNP). The necessary spin-label was synthesized and inserted into the disulfide bridge of eptifibatide via reductive followed by insertion by a double Michael addition under physiological conditions. This procedure is universally applicable for disulfide containing biomolecules and is expected to preserve their tertiary structure with minimal change due to the small size of the label and restoring of the previous disulfide connection. HPLC and MS analysis show the successful introduction of the spin label and EPR spectroscopy confirms its activity. DNP-enhanced solid state NMR experiments show signal enhancement factors of up to 19 in 13C CP MAS experiments which corresponds to time saving factors of up to 361. This clearly shows the high potential of our new spin labeling strategy for the introduction of site selective radical spin labels into biomolecules and biosolids without compromising its conformational integrity for structural investigations employing solid-state DNP or advanced EPR techniques.

Amyloid-type Protein Aggregation and Prion-like Properties of Amyloids.

This review will focus on the process of amyloid-type protein aggregation. Amyloid fibrils are an important hallmark of protein misfolding diseases and therefore have been investigated for decades. Only recently, however, atomic or near-atomic resolution structures have been elucidated from various in vitro and ex vivo obtained fibrils. In parallel, the process of fibril formation has been studied in vitro under highly artificial but comparatively reproducible conditions. The review starts with a summary of what is known and speculated from artificial in vitro amyloid-type protein aggregation experiments. A partially hypothetic fibril selection model will be described that may be suitable to explain why amyloid fibrils look the way they do, in particular, why at least all so far reported high resolution cryo-electron microscopy obtained fibril structures are in register, parallel, cross-ß-sheet fibrils that mostly consist of two protofilaments twisted around each other. An intrinsic feature of the model is the prion-like nature of all amyloid assemblies. Transferring the model from the in vitro point of view to the in vivo situation is not straightforward, highly hypothetic, and leaves many open questions that need to be addressed in the future.

Structural details of amyloid ß oligomers in complex with human prion protein as revealed by solid-state MAS NMR spectroscopy.

Human PrP (huPrP) is a high-affinity receptor for oligomeric amyloid ß (Aß) protein aggregates. Binding of Aß oligomers to membrane-anchored huPrP has been suggested to trigger neurotoxic cell signaling in Alzheimer's disease, while an N-terminal soluble fragment of huPrP can sequester Aß oligomers and reduce their toxicity. Synthetic oligomeric Aß species are known to be heterogeneous, dynamic, and transient, rendering their structural investigation particularly challenging. Here, using huPrP to preserve Aß oligomers by coprecipitating them into large heteroassemblies, we investigated the conformations of Aß(1-42) oligomers and huPrP in the complex by solid-state MAS NMR spectroscopy. The disordered N-terminal region of huPrP becomes immobilized in the complex and therefore visible in dipolar spectra without adopting chemical shifts characteristic of a regular secondary structure. Most of the well-defined C-terminal part of huPrP is part of the rigid complex, and solid-state NMR spectra suggest a loss in regular secondary structure in the two C-terminal α-helices. For Aß(1-42) oligomers in complex with huPrP, secondary chemical shifts reveal substantial ß-strand content. Importantly, not all Aß(1-42) molecules within the complex have identical conformations. Comparison with the chemical shifts of synthetic Aß fibrils suggests that the Aß oligomer preparation represents a heterogeneous mixture of ß-strand-rich assemblies, of which some have the potential to evolve and elongate into different fibril polymorphs, reflecting a general propensity of Aß to adopt variable ß-strand-rich conformers. Taken together, our results reveal structural changes in huPrP upon binding to Aß oligomers that suggest a role of the C terminus of huPrP in cell signaling. Trapping Aß(1-42) oligomers by binding to huPrP has proved to be a useful tool for studying the structure of these highly heterogeneous ß-strand-rich assemblies.

Binding modes of thioflavin T and Congo red to the fibril structure of amyloid-ß(1-42).

Binding modes for the amyloid-ß(1-42) fibril fluorescent dyes thioflavin T and Congo red were predicted by molecular dynamics simulations and binding free energy calculations. Both probes bind on the fibril surface to primarily hydrophobic grooves, with their long axis oriented almost parallel to the fibril axis. The computed binding affinities are in agreement with experimental values. The binding modes also explain observables from previous structural studies and, thus, provide a starting point for the systematic search and design of novel molecules, which may improve in vitro diagnostics for Alzheimer's disease.

Heteronuclear cross-relaxation effect modulated by the dynamics of N-functional groups in the solid state under 15N DP-MAS DNP.

In a typical magic-angle spinning (MAS) dynamic nuclear polarization (DNP) nuclear magnetic resonance (NMR) experiment, several mechanisms are simultaneously involved when transferring much larger polarization of electron spins to NMR active nuclei of interest. Recently, specific cross-relaxation enhancement by active motions under DNP (SCREAM-DNP) [Daube et al. JACS 2016] has been reported as one of these mechanisms. Thereby 13C enhancement with inverted sign was observed in a direct polarization (DP) MAS DNP experiment, caused by reorientation dynamics of methyl that was not frozen out at 100 K. Here, we report on the spontaneous polarization transfer from hyperpolarized 1H to both primary amine and ammonium nitrogens, resulting in an additional positive signal enhancement in the 15N NMR spectra during 15N DP-MAS DNP. The cross-relaxation induced signal enhancement (CRE) for 15N is of opposite sign compared to that observed for 13C due to the negative sign of the gyromagnetic ratio of 15N. The influence on CRE efficiency caused by variation of the radical solution composition and by temperature was also investigated.

Determining isoleucine side-chain rotamer-sampling in proteins from 13C chemical shift.

Chemical shifts are often the only nuclear magnetic resonance parameter that can be obtained for challenging macromolecular systems. Here we present a framework to derive the conformational sampling of isoleucine side chains from 13C chemical shifts and demonstrate that side-chain conformations in a low-populated folding intermediate can be determined.

Development of a new largely scalable in vitro prion propagation method for the production of infectious recombinant prions for high resolution structural studies.

The resolution of the three-dimensional structure of infectious prions at the atomic level is pivotal to understand the pathobiology of Transmissible Spongiform Encephalopathies (TSE), but has been long hindered due to certain particularities of these proteinaceous pathogens. Difficulties related to their purification from brain homogenates of disease-affected animals were resolved almost a decade ago by the development of in vitro recombinant prion propagation systems giving rise to highly infectious recombinant prions. However, lack of knowledge about the molecular mechanisms of the misfolding event and the complexity of systems such as the Protein Misfolding Cyclic Amplification (PMCA), have limited generating the large amounts of homogeneous recombinant prion preparations required for high-resolution techniques such as solid state Nuclear Magnetic Resonance (ssNMR) imaging. Herein, we present a novel recombinant prion propagation system based on PMCA that substitutes sonication with shaking thereby allowing the production of unprecedented amounts of multi-labeled, infectious recombinant prions. The use of specific cofactors, such as dextran sulfate, limit the structural heterogeneity of the in vitro propagated prions and makes possible, for the first time, the generation of infectious and likely homogeneous samples in sufficient quantities for studies with high-resolution structural techniques as demonstrated by the preliminary ssNMR spectrum presented here. Overall, we consider that this new method named Protein Misfolding Shaking Amplification (PMSA), opens new avenues to finally elucidate the three-dimensional structure of infectious prions.

Hyperpolarized MAS NMR of unfolded and misfolded proteins.

In this article we give an overview over the use of DNP-enhanced solid-state NMR spectroscopy for the investigation of unfolded, disordered and misfolded proteins. We first provide an overview over studies in which DNP spectroscopy has successfully been applied for the structural investigation of well-folded amyloid fibrils formed by short peptides as well as full-length proteins. Sample cooling to cryogenic temperatures often leads to severe line broadening of resonance signals and thus a loss in resolution. However, inhomogeneous line broadening at low temperatures provides valuable information about residual dynamics and flexibility in proteins, and, in combination with appropriate selective isotope labeling techniques, inhomogeneous linewidths in disordered proteins or protein regions may be exploited for evaluation of conformational ensembles. In the last paragraph we highlight some recent studies where DNP-enhanced MAS-NMR-spectroscopy was applied to the study of disordered proteins/protein regions and inhomogeneous sample preparations.

Structural insights from lipid-bilayer nanodiscs link α-Synuclein membrane-binding modes to amyloid fibril formation.

The protein α-Synuclein (αS) is linked to Parkinson's disease through its abnormal aggregation, which is thought to involve cytosolic and membrane-bound forms of αS. Following previous studies using micelles and vesicles, we present a comprehensive study of αS interaction with phospholipid bilayer nanodiscs. Using a combination of NMR-spectroscopic, biophysical, and computational methods, we structurally and kinetically characterize αS interaction with different membrane discs in a quantitative and site-resolved way. We obtain global and residue-specific αS membrane affinities, and determine modulations of αS membrane binding due to αS acetylation, membrane plasticity, lipid charge density, and accessible membrane surface area, as well as the consequences of the different binding modes for αS amyloid fibril formation. Our results establish a structural and kinetic link between the observed dissimilar binding modes and either aggregation-inhibiting properties, largely unperturbed aggregation, or accelerated aggregation due to membrane-assisted fibril nucleation.

A d-enantiomeric peptide interferes with heteroassociation of amyloid-ß oligomers and prion protein.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that affects millions of people worldwide. One AD hallmark is the aggregation of ß-amyloid (Aß) into soluble oligomers and insoluble fibrils. Several studies have reported that oligomers rather than fibrils are the most toxic species in AD progression. Aß oligomers bind with high affinity to membrane-associated prion protein (PrP), leading to toxic signaling across the cell membrane, which makes the Aß-PrP interaction an attractive therapeutic target. Here, probing this interaction in more detail, we found that both full-length, soluble human (hu) PrP(23-230) and huPrP(23-144), lacking the globular C-terminal domain, bind to Aß oligomers to form large complexes above the megadalton size range. Following purification by sucrose density-gradient ultracentrifugation, the Aß and huPrP contents in these heteroassemblies were quantified by reversed-phase HPLC. The Aß:PrP molar ratio in these assemblies exhibited some limited variation depending on the molar ratio of the initial mixture. Specifically, a molar ratio of about four Aß to one huPrP in the presence of an excess of huPrP(23-230) or huPrP(23-144) suggested that four Aß units are required to form one huPrP-binding site. Of note, an Aß-binding all-d-enantiomeric peptide, RD2D3, competed with huPrP for Aß oligomers and interfered with Aß-PrP heteroassembly in a concentration-dependent manner. Our results highlight the importance of multivalent epitopes on Aß oligomers for Aß-PrP interactions and have yielded an all-d-peptide-based, therapeutically promising agent that competes with PrP for these interactions.

Origin of metastable oligomers and their effects on amyloid fibril self-assembly.

Assembly of rigid amyloid fibrils with their characteristic cross-ß sheet structure is a molecular signature of numerous neurodegenerative and non-neuropathic disorders. Frequently large populations of small globular amyloid oligomers (gOs) and curvilinear fibrils (CFs) precede the formation of late-stage rigid fibrils (RFs), and have been implicated in amyloid toxicity. Yet our understanding of the origin of these metastable oligomers, their role as on-pathway precursors or off-pathway competitors, and their effects on the self-assembly of amyloid fibrils remains incomplete. Using two unrelated amyloid proteins, amyloid-ß and lysozyme, we find that gO/CF formation, analogous to micelle formation by surfactants, is delineated by a "critical oligomer concentration" (COC). Below this COC, fibril assembly replicates the sigmoidal kinetics of nucleated polymerization. Upon crossing the COC, assembly kinetics becomes biphasic with gO/CF formation responsible for the lag-free initial phase, followed by a second upswing dominated by RF nucleation and growth. RF lag periods below the COC, as expected, decrease as a power law in monomer concentration. Surprisingly, the build-up of gO/CFs above the COC causes a progressive increase in RF lag periods. Our results suggest that metastable gO/CFs are off-pathway from RF formation, confined by a condition-dependent COC that is distinct from RF solubility, underlie a transition from sigmoidal to biphasic assembly kinetics and, most importantly, not only compete with RFs for the shared monomeric growth substrate but actively inhibit their nucleation and growth.

DNP-Enhanced MAS NMR: A Tool to Snapshot Conformational Ensembles of α-Synuclein in Different States.

Intrinsically disordered proteins dynamically sample a wide conformational space and therefore do not adopt a stable and defined three-dimensional conformation. The structural heterogeneity is related to their proper functioning in physiological processes. Knowledge of the conformational ensemble is crucial for a complete comprehension of this kind of proteins. We here present an approach that utilizes dynamic nuclear polarization-enhanced solid-state NMR spectroscopy of sparsely isotope-labeled proteins in frozen solution to take snapshots of the complete structural ensembles by exploiting the inhomogeneously broadened line-shapes. We investigated the intrinsically disordered protein α-synuclein (α-syn), which plays a key role in the etiology of Parkinson's disease, in three different physiologically relevant states. For the free monomer in frozen solution we could see that the so-called "random coil conformation" consists of α-helical and ß-sheet-like conformations, and that secondary chemical shifts of neighboring amino acids tend to be correlated, indicative of frequent formation of secondary structure elements. Based on these results, we could estimate the number of disordered regions in fibrillar α-syn as well as in α-syn bound to membranes in different protein-to-lipid ratios. Our approach thus provides quantitative information on the propensity to sample transient secondary structures in different functional states. Molecular dynamics simulations rationalize the results.

Molecular basis for diversification of yeast prion strain conformation.

Self-propagating ß-sheet-rich fibrillar protein aggregates, amyloid fibers, are often associated with cellular dysfunction and disease. Distinct amyloid conformations dictate different physiological consequences, such as cellular toxicity. However, the origin of the diversity of amyloid conformation remains unknown. Here, we suggest that altered conformational equilibrium in natively disordered monomeric proteins leads to the adaptation of alternate amyloid conformations that have different phenotypic effects. We performed a comprehensive high-resolution structural analysis of Sup35NM, an N-terminal fragment of the Sup35 yeast prion protein, and found that monomeric Sup35NM harbored latent local compact structures despite its overall disordered conformation. When the hidden local microstructures were relaxed by genetic mutations or solvent conditions, Sup35NM adopted a strikingly different amyloid conformation, which redirected chaperone-mediated fiber fragmentation and modulated prion strain phenotypes. Thus, dynamic conformational fluctuations in natively disordered monomeric proteins represent a posttranslational mechanism for diversification of aggregate structures and cellular phenotypes.

Advancements of the sFIDA method for oligomer-based diagnostics of neurodegenerative diseases.

Early diagnosis of Alzheimer's disease (AD) is of great importance for the development of therapeutics and their application in the clinical environment. Amyloid ß (Aß) oligomers are crucial for the onset and progression of AD and represent a popular drug target, being presumably the most direct biomarker. Efforts to measure Aß oligomers in body fluids are hampered by the low analyte concentration and presence of Aß monomers. The surface-based fluorescence intensity distribution analysis (sFIDA) features both highly specific and sensitive oligomer quantitation as well as total insensitivity towards monomers. In this Review, we highlight structural features of oligomeric and fibrillar Aß. Recent advancements in sFIDA assay development have been the successful automation, adaption for additional biomarkers such as α-synuclein oligomers, and significant improvement of essential assay parameters.

Fibril structure of amyloid-ß(1-42) by cryo-electron microscopy.

Amyloids are implicated in neurodegenerative diseases. Fibrillar aggregates of the amyloid-ß protein (Aß) are the main component of the senile plaques found in brains of Alzheimer's disease patients. We present the structure of an Aß(1-42) fibril composed of two intertwined protofilaments determined by cryo-electron microscopy (cryo-EM) to 4.0-angstrom resolution, complemented by solid-state nuclear magnetic resonance experiments. The backbone of all 42 residues and nearly all side chains are well resolved in the EM density map, including the entire N terminus, which is part of the cross-ß structure resulting in an overall "LS"-shaped topology of individual subunits. The dimer interface protects the hydrophobic C termini from the solvent. The characteristic staggering of the nonplanar subunits results in markedly different fibril ends, termed "groove" and "ridge," leading to different binding pathways on both fibril ends, which has implications for fibril growth.

Structural Characterization of Fibrils from Recombinant Human Islet Amyloid Polypeptide by Solid-State NMR: The Central FGAILS Segment Is Part of the ß-Sheet Core.

Amyloid deposits formed from islet amyloid polypeptide (IAPP) are a hallmark of type 2 diabetes mellitus and are known to be cytotoxic to pancreatic ß-cells. The molecular structure of the fibrillar form of IAPP is subject of intense research, and to date, different models exist. We present results of solid-state NMR experiments on fibrils of recombinantly expressed and uniformly 13C, 15N-labeled human IAPP in the non-amidated, free acid form. Complete sequential resonance assignments and resulting constraints on secondary structure are shown. A single set of chemical shifts is found for most residues, which is indicative of a high degree of homogeneity. The core region comprises three to four ß-sheets. We find that the central 23-FGAILS-28 segment, which is of critical importance for amyloid formation, is part of the core region and forms a ß-strand in our sample preparation. The eight N-terminal amino acid residues of IAPP, forming a ring-like structure due to a disulfide bridge between residues C2 and C7, appear to be well defined but with an increased degree of flexibility. This study supports the elucidation of the structural basis of IAPP amyloid formation and highlights the extent of amyloid fibril polymorphism.