Anne K. Schütz

Atomic-Resolution Three-Dimensional Structure of Amyloid β Fibrils Bearing the Osaka Mutation†

Despite its central importance for understanding the molecular basis of Alzheimers disease (AD), high-resolution structural information on amyloid β-peptide (Aβ) fibrils, which are intimately linked with AD, is scarce. We report an atomic-resolution fibril structure of the Aβ1-40 peptide with the Osaka mutation (E22Δ), associated with early-onset AD. The structure, which differs substantially from all previously proposed models, is based on a large number of unambiguous intra- and intermolecular solid-state NMR distance restraints.

The Amyloid–Congo Red Interface at Atomic Resolution

Amyloids are b-sheet-rich proteinaceous aggregates that are a pathological hallmark of a number of human diseases. They can replicate by seeded nucleation and propagate as prions, and may even exert important physiological activities. Amyloids are universally defined by their stainability with Congo red and the resulting green birefringence. Yet, remarkably, the binding mechanism, geometry, and fine structure of the Congo red/amyloid complex are not known. By using solid-state NMR spectroscopy we have characterized, at atomic resolution, the binding interface between Congo red and amyloid fibrils formed by the prion domain of the fungal HET-s protein. The dye binds highly sitespecifically by interacting with residues flanking a groove in the vicinity of a b-arc. The three-dimensional (3D) structure of the fibril is strongly conserved upon the binding of Congo red. Remarkably, a single point mutation, designed according to the binding information, provides an artificial amyloid structurally indistinguishable from HET-s but not stainable by Congo red. The methods used require no isotope labeling of the small molecule and can be used to characterize the interaction of a broad range of dyes, drugs, and tracers with amyloids or other insoluble proteins. Congo red (Figure S1 in the Supporting Information) is a small molecule that reacts specifically with amyloids and has been used since the 1920s as the analytical “gold standard” for amyloid characterization and diagnostics. Despite its binding specificity and its antiamyloidogenic properties, the use of Congo red as a molecular amyloid tracer in vivo and as an antiamyloid drug has been hindered by its high toxicity and poor pharmacokinetics. Contradictory reports exist on the nature of the interaction between Congo red and amyloid fibrils, but the following facts are generally agreed upon: Congo red binds roughly stochiometrically, 9] and the bound form displays a characteristic red-shift in its absorbance maximum, and shows dichroism and birefringence. 10] The negatively charged sulfate groups of Congo red are believed to be relevant for binding to two sites separated by a distance of approximately 20 . In the absence of atomic-resolution structure information, the binding mode is still debated. The long axis of the dye has been postulated to orient parallel or perpendicular to the fibril axis, and the dye binds as a monomer, oligomer, or micelle, or intercalated between the b-sheets. Hydrogen bonds and hydrophobic, aromatic, and ionic interactions have been assumed to contribute to the binding of Congo red to amyloid fibrils. Also, different side chains from residues such as histidine, arginine, and lysine have been suspected to promote congophilia. Atomic-resolution structures of amyloids, which have become available recently, enable studies of the interplay between these proteins and their binding partners in detail. HET-s(218–289), the prion-forming domain of the HET-s prion from the filamentous fungus Podospora anserina, is presently one of the structurally most precisely defined amyloids. To identify the protein surface that interacts with binding partners by NMR spectroscopy, we set out to detect characteristic chemical-shift perturbations (CSP) upon binding by comparing two-dimensional (2D) C–C correlation spectra (proton-driven spin diffusion, PDSD) of stained and unstained [C,N]-labeled fibrils. Since CSPs can be allosteric, we also performed polarization-transfer (PT) experiments which directly probe the spatial proximity of ligand and protein by exploiting the pronounced distance dependence (r ) of the dipolar interaction. Previously published PT approches have proven difficult for our system as they require isotope-labeled Congo red. We have therefore devised a method relying on polarization transfer from ligand H atoms to protein C atoms, which uses standard [H,C,N]-isotope-labeled protein but does not require isotopically labeled ligand. In such samples, the protons from Congo red are the unique polarization source in H–C polarization-transfer experiments, and the C signals detected identify residues in spatial proximity to Congo red (< 4 ). For enhanced spectral resolution, the C atoms are detected in a two-dimensional correlation experiment (dipolar recoupling enhanced by amplitude modulation, DREAM). The main results of the NMR experiments are summarized in Figure 1; details are given in Figures S2–S4 in the [*] A. K. Sch tz, A. Soragni, Dr. M. Ernst, Prof. B. H. Meier Physikalische Chemie, ETH Z rich, 8093 Z rich (Switzerland) E-mail: beme@ethz.ch

A Sedimented Sample of a 59 kDa Dodecameric Helicase Yields High‐Resolution Solid‐State NMR Spectra

Crystal clear: Preparing solid-state NMR samples that yield high-resolution spectra displaying high sensitivity is time-consuming and complicated. A sample of the 59 kDa protein DnaB, prepared simply by preparative centrifugation, provides spectra that are as good as the ones from carefully grown microcrystals.

The Molecular Organization of the Fungal Prion HET-s in Its Amyloid Form

The prion hypothesis states that it is solely the three-dimensional structure of the polypeptide chain that distinguishes the prion and nonprion forms of the protein. For HET-s, the atomic-resolution structure of the isolated prion domain HET-s(218-289), consisting of a highly ordered triangular cross-beta arrangement, is known. Here we present a solid-state NMR study of fibrils of the full-length HET-s prion in which we compare their spectra with spectra from isolated C-terminal prion domain fibrils and the crystalline N-terminal globular domain HET-s(1-227). The spectra reveal unequivocally that the highly ordered structure of the isolated prion domain HET-s(218-289) is conserved in the context of the full-length fibrils investigated here. However, the globular domain loses much of its tertiary structure while partly retaining its secondary structure, thus exhibiting behavior reminiscent of a molten globule. Flexible residues that may constitute the linker connecting the two domains are detected using INEPT (insensitive nuclei enhanced by polarization transfer) spectroscopy. Based on our data, we propose a structural model that is in line with a general model developed for amyloid fibrils built from a cross-beta core decorated with globular domains. The loss of structure in the HET-s globular domain sharply contrasts with the behavior observed for fibrils of Ure2p and suggests that there is considerable structural diversity in the fibrils of globular-domain-containing prions despite their similar appearances at the microscopic level.

Prion Fibrils of Ure2p Assembled under Physiological Conditions Contain Highly Ordered, Natively Folded Modules

The difference between the prion and the non-prion form of a protein is given solely by its three-dimensional structure, according to the prion hypothesis. It has been shown that solid-state NMR can unravel the atomic-resolution three-dimensional structure of prion fragments but, in the case of Ure2p, no highly resolved spectra are obtained from the isolated prion domain. Here, we demonstrate that the spectra of full-length fibrils of Ure2p interestingly lead to highly resolved solid-state NMR spectra. Prion fibrils formed under physiological conditions are therefore well-ordered objects on the molecular level. Comparing the full-length NMR spectra with the corresponding spectra of the prion and globular domains in isolation reveals that the globular part in particular shows almost perfect structural order. The NMR linewidths in these spectra are as narrow as the ones observed in crystals of the isolated globular domain. For the prion domain, the spectra reflect partial disorder, suggesting structural heterogeneity, both in isolation and in full-length Ure2p fibrils, although to different extents. The spectral quality is surprising in the light of existing structural models for Ure2p and in comparison to the corresponding spectra of the only other full-length prion fibrils (HET-s) investigated so far. This opens the exciting perspective of an atomic-resolution structure determination of the fibrillar form of a prion whose assembly is not accompanied by significant conformational changes and documents the structural diversity underlying prion propagation.

Structure-based drug design identifies polythiophenes as antiprion compounds

The targeted chemical design of luminescent conjugated polythiophenes may yield new therapeutic compounds for treating prion diseases. Putting prions in their place In a mouse model of prion disease, Herrmann et al. evaluated the therapeutic efficacy of luminescent conjugated polythiophenes (LCPs), which are molecules with a high affinity for ordered protein aggregates. Intracerebral administration of LCPs into prion-infected mice using osmotic pumps increased survival. Solid-state nuclear magnetic resonance and in silico binding studies of LCPs to simplified model fibrils allowed the authors to define structural rules, which they then used for the design of LCPs with superior prophylactic and therapeutic potency. The new work demonstrates the feasibility of rational drug design for developing therapeutics to treat prion diseases. Prions cause transmissible spongiform encephalopathies for which no treatment exists. Prions consist of PrPSc, a misfolded and aggregated form of the cellular prion protein (PrPC). We explore the antiprion properties of luminescent conjugated polythiophenes (LCPs) that bind and stabilize ordered protein aggregates. By administering a library of structurally diverse LCPs to the brains of prion-infected mice via osmotic minipumps, we found that antiprion activity required a minimum of five thiophene rings bearing regularly spaced carboxyl side groups. Solid-state nuclear magnetic resonance analyses and molecular dynamics simulations revealed that anionic side chains interacted with complementary, regularly spaced cationic amyloid residues of model prions. These findings allowed us to extract structural rules governing the interaction between LCPs and protein aggregates, which we then used to design a new set of LCPs with optimized binding. The new set of LCPs showed robust prophylactic and therapeutic potency in prion-infected mice, with the lead compound extending survival by >80% and showing activity against both mouse and hamster prions as well as efficacy upon intraperitoneal administration into mice. These results demonstrate the feasibility of targeted chemical design of compounds that may be useful for treating diseases of aberrant protein aggregation such as prion disease.

The conformation of the prion domain of Sup35p in isolation and in the full-length protein.

The yeast protein Sup35p has prion properties, and it aggregates into fibrillar assemblies. It is at the origin of the [PSI] trait in baker s yeast, Saccharomyces cerevisiae. The Sup35p yeast prion is an important model system to investigate the structure–function relationship of prions. To reduce the complexity of the problem, the Sup35pNM fragment is often used as a convenient model to document the assembly and infectious properties of the full-length prion, as fibrillar Sup35pNM is biologically relevant in the sense that it induces [PSI] when introduced into [psi ] cells. The notion that fibrillar Sup35pNM perfectly mimics Sup35p fibrils suggests that the N and M domains of Sup35p adopt identical conformations in Sup35pNM and Sup35p fibrils. We question this assumption in the following: Using solid-state NMR measurements performed on Sup35pNM and full-length Sup35p fibrils assembled under identical physiological conditions, and both inducing [PSI] strains (Supporting Information, Figure S1), we demonstrate that fibrillar Sup35pNM and full-length Sup35p show significant structural differences, although both have a high b-sheet content. Sup35p is a three-domain polypeptide (Supporting Information, Table S1); the N-terminal domain is critical for prion propagation, while the C-terminal domain has GTPase activity and is involved in translation termination. The middle domain connects the two domains. The N domain alone or together with the M domain, and also the full-length Sup35p, assemble into amyloid fibrils. Fibrils of both Sup35p and Sup35pNM give rise to high-resolution solid-state NMR spectra, as can be seen in the 2D spectra in Figure 1 for Sup35pNM and Sup35p (overlaid in the Supporting Information, Figure S2), with the corresponding electron micro-

Contribution of Specific Residues of the β-Solenoid Fold to HET-s Prion Function, Amyloid Structure and Stability

The [Het-s] prion of the fungus Podospora anserina represents a good model system for studying the structure-function relationship in amyloid proteins because a high resolution solid-state NMR structure of the amyloid prion form of the HET-s prion forming domain (PFD) is available. The HET-s PFD adopts a specific β-solenoid fold with two rungs of β-strands delimiting a triangular hydrophobic core. A C-terminal loop folds back onto the rigid core region and forms a more dynamic semi-hydrophobic pocket extending the hydrophobic core. Herein, an alanine scanning mutagenesis of the HET-s PFD was conducted. Different structural elements identified in the prion fold such as the triangular hydrophobic core, the salt bridges, the asparagines ladders and the C-terminal loop were altered and the effect of these mutations on prion function, fibril structure and stability was assayed. Prion activity and structure were found to be very robust; only a few key mutations were able to corrupt structure and function. While some mutations strongly destabilize the fold, many substitutions in fact increase stability of the fold. This increase in structural stability did not influence prion formation propensity in vivo. However, if an Ala replacement did alter the structure of the core or did influence the shape of the denaturation curve, the corresponding variant showed a decreased prion efficacy. It is also the finding that in addition to the structural elements of the rigid core region, the aromatic residues in the C-terminal semi-hydrophobic pocket are critical for prion propagation. Mutations in the latter region either positively or negatively affected prion formation. We thus identify a region that modulates prion formation although it is not part of the rigid cross-β core, an observation that might be relevant to other amyloid models.

Solid-state NMR sequential assignment of Osaka-mutant amyloid-beta (Aβ1−40 E22Δ) fibrils

AbstractAlzheimer’s disease (AD) is the most common form of dementia. Aggregation of amyloid β (Aβ), a peptide of 39−43 residues length, into insoluble fibrils is considered to initiate the disease. Determination of the molecular structure of Aβ fibrils is technically challenging and is a significant goal in AD research that may lead to design of effective therapeutical inhibitors of Aβ aggregation. Here, we present chemical-shift assignments for fibrils formed by highly pure recombinant Aβ1−40 with the Osaka E22Δ mutation that is found in familial AD. We show that that all regions of the peptide are rigid, including the N-terminal part often believed to be flexible in Aβ wt.

Solid-state NMR sequential assignments of the amyloid core of full-length Sup35p

Abstract Sup35p is a yeast prion and is responsible for the [PSI+] trait in Saccharomyces cerevisiae. With 685 amino acids, full-length soluble and fibrillar Sup35p are challenging targets for structural biology as they cannot be investigated by X-ray crystallography or NMR in solution. We present solid-state NMR studies of fibrils formed by the full-length Sup35 protein. We detect an ordered and rigid core of the protein that gives rise to narrow and strong peaks, while large parts of the protein show either static disorder or dynamics on time scales which interfere with dipolar polarization transfer or shorten the coherence lifetime. Thus, only a small subset of resonances is observed in 3D spectra. Here we describe in detail the sequential assignments of the 22 residues for which resonances are observed in 3D spectra: their chemical shifts mostly corresponding to β-sheet secondary structure. We suspect that these residues form the amyloid core of the fibril.