Molecular structure of ß-amyloid fibrils in Alzheimer's disease brain tissue.

In vitro, ß-amyloid (Aß) peptides form polymorphic fibrils, with molecular structures that depend on growth conditions, plus various oligomeric and protofibrillar aggregates. Here, we investigate structures of human brain-derived Aß fibrils, using seeded fibril growth from brain extract and data from solid-state nuclear magnetic resonance and electron microscopy. Experiments on tissue from two Alzheimer's disease (AD) patients with distinct clinical histories showed a single predominant 40 residue Aß (Aß40) fibril structure in each patient; however, the structures were different from one another. A molecular structural model developed for Aß40 fibrils from one patient reveals features that distinguish in-vivo- from in-vitro-produced fibrils. The data suggest that fibrils in the brain may spread from a single nucleation site, that structural variations may correlate with variations in AD, and that structure-specific amyloid imaging agents may be an important future goal.

Structural variation in amyloid-ß fibrils from Alzheimer's disease clinical subtypes.

Aggregation of amyloid-ß peptides into fibrils or other self-assembled states is central to the pathogenesis of Alzheimer's disease. Fibrils formed in vitro by 40- and 42-residue amyloid-ß peptides (Aß40 and Aß42) are polymorphic, with variations in molecular structure that depend on fibril growth conditions. Recent experiments suggest that variations in amyloid-ß fibril structure in vivo may correlate with variations in Alzheimer's disease phenotype, in analogy to distinct prion strains that are associated with different clinical and pathological phenotypes. Here we investigate correlations between structural variation and Alzheimer's disease phenotype using solid-state nuclear magnetic resonance (ssNMR) measurements on Aß40 and Aß42 fibrils prepared by seeded growth from extracts of Alzheimer's disease brain cortex. We compared two atypical Alzheimer's disease clinical subtypes-the rapidly progressive form (r-AD) and the posterior cortical atrophy variant (PCA-AD)-with a typical prolonged-duration form (t-AD). On the basis of ssNMR data from 37 cortical tissue samples from 18 individuals, we find that a single Aß40 fibril structure is most abundant in samples from patients with t-AD and PCA-AD, whereas Aß40 fibrils from r-AD samples exhibit a significantly greater proportion of additional structures. Data for Aß42 fibrils indicate structural heterogeneity in most samples from all patient categories, with at least two prevalent structures. These results demonstrate the existence of a specific predominant Aß40 fibril structure in t-AD and PCA-AD, suggest that r-AD may relate to additional fibril structures and indicate that there is a qualitative difference between Aß40 and Aß42 aggregates in the brain tissue of patients with Alzheimer's disease.

Different Intermolecular Interactions Drive Nonpathogenic Liquid-Liquid Phase Separation and Potentially Pathogenic Fibril Formation by TDP-43.

The liquid-liquid phase separation (LLPS) of proteins has been found ubiquitously in eukaryotic cells, and is critical in the control of many biological processes by forming a temporary condensed phase with different bimolecular components. TDP-43 is recruited to stress granules in cells and is the main component of TDP-43 granules and proteinaceous amyloid inclusions in patients with amyotrophic lateral sclerosis (ALS). TDP-43 low complexity domain (LCD) is able to de-mix in solution, forming the protein condensed droplets, and amyloid aggregates would form from the droplets after incubation. The molecular interactions regulating TDP-43 LCD LLPS were investigated at the protein fusion equilibrium stage, when the droplets stopped growing after incubation. We found the molecules in the droplet were still liquid-like, but with enhanced intermolecular helix-helix interactions. The protein would only start to aggregate after a lag time and aggregate slower than at the condition when the protein does not phase separately into the droplets, or the molecules have a reduced intermolecular helix-helix interaction. In the protein condensed droplets, a structural transition intermediate toward protein aggregation was discovered involving a decrease in the intermolecular helix-helix interaction and a reduction in the helicity. Our results therefore indicate that different intermolecular interactions drive LLPS and fibril formation. The discovery that TDP-43 LCD aggregation was faster through the pathway without the first protein phase separation supports that LLPS and the intermolecular helical interaction could help maintain the stability of TDP-43 LCD.

Solid-State NMR Reveals the Structural Transformation of the TDP-43 Amyloidogenic Region upon Fibrillation.

TDP-43 is a primary pathological hallmark protein of amyotrophic lateral sclerosis and frontotemporal lobar degeneration, which may exist in the form of amyloid inclusions in the cells of patients. In addition to serving as a biomarker for these diseases, TDP-43 can also directly trigger neurodegeneration. We previously determined the amyloidogenic core region of TDP-43 (residues 311-360) and showed by solution NMR that this region includes two α-helices [(321-330) and (335-343)] in solution. We suggested that the helix-to-sheet structural transformation initiates TDP-43 aggregation. In the present study, X-ray diffraction shows that TDP-43 (311-360) aggregates adopt a cross-ß structure. Thioredoxin (Trx)-fused TDP-43 (311-360) can undergo liquid-liquid phase separation (LLPS) before fibrillation, suggesting that phase separation is an intermediate step before amyloid formation. Solid-state NMR (SSNMR), carried out to elucidate the structural changes of TDP-43 (311-360) at the atomic level, indicates five ß-strands of the amyloids formed, with the major two ß-strands contributed by the first helical region in the solution structure. The NMR evidence is also in support of the fibril having a parallel in-register conformation, implying a mechanism in which the helix-helix interactions in LLPS are converted into ß-strand parallel lateral association upon fibrillation. Our studies have assigned many key interresidue interactions that contribute to the stability of the fibril, including F316 with I318 and Q327 and W334 with A325, A326, A329, and S332. SSNMR with 1H detection reveals a unique close interaction between the indole Nε1-Hε1 of W334 and the side-chain carbonyl of Q343. This interaction could be a very important factor in initiating TDP-43 (311-360) folding/misfolding in LLPS.

Amyloid structure of high-order assembly of Leucine-rich amelogenin revealed by solid-state NMR.

High-order assemblies of amelogenin, the major protein in enamel protein matrix, are believed to act as the template for enamel mineral formation. The Leucine-rich amelogenin (LRAP) is a natural splice-variant of amelogenin, a functional protein in vivo, containing conserved domains of amelogenin. In this work, we showed LRAP aggregates hierarchically into assemblies with various sizes including scattered beads, beads-on-a-string and gel-like precipitations in the presence of both calcium and phosphate ions. Solid-state NMR combined with X-ray diffraction and microscopic techniques, was applied to give a picture of LRAP self-assemblies at the atomic level. Our results, for the first time, confirmed LRAP assemblies with different sizes all contained a consistent rigid segment with ß-sheet secondary structure (residues 12-27) and the ß-sheet segment would further assemble into amyloid-like structures.

Solid-State NMR Identification of Intermolecular Interactions in Amelogenin Bound to Hydroxyapatite.

Biomineralization processes govern the formation of hierarchical hard tissues such as bone and teeth in living organisms, and mimicking these processes could lead to the design of new materials with specialized properties. However, such advances require structural characterization of the proteins guiding biomineral formation to understand and mimic their impact. In their "active" form, biomineralization proteins are bound to a solid surface, severely limiting our ability to use many conventional structure characterization techniques. Here, solid-state NMR spectroscopy was applied to study the intermolecular interactions of amelogenin, the most abundant protein present during the early stages of enamel formation, in self-assembled oligomers bound to hydroxyapatite. Intermolecular dipolar couplings were identified that support amelogenin dimer formation stabilized by residues toward the C-termini. These dipolar interactions were corroborated by molecular dynamics simulations. A ß-sheet structure was identified in multiple regions of the protein, which is otherwise intrinsically disordered in the absence of hydroxyapatite. To our knowledge, this is the first intermolecular protein-protein interaction reported for a biomineralization protein, representing an advancement in understanding enamel development and a new general strategy toward investigating biomineralization proteins.

Major Variations in HIV-1 Capsid Assembly Morphologies Involve Minor Variations in Molecular Structures of Structurally Ordered Protein Segments.

We present the results of solid state nuclear magnetic resonance (NMR) experiments on HIV-1 capsid protein (CA) assemblies with three different morphologies, namely wild-type CA (WT-CA) tubes with 35-60 nm diameters, planar sheets formed by the Arg(18)-Leu mutant (R18L-CA), and R18L-CA spheres with 20-100 nm diameters. The experiments are intended to elucidate molecular structural variations that underlie these variations in CA assembly morphology. We find that multidimensional solid state NMR spectra of (15)N,(13)C-labeled CA assemblies are remarkably similar for the three morphologies, with only small differences in (15)N and (13)C chemical shifts, no significant differences in NMR line widths, and few differences in the number of detectable NMR cross-peaks. Thus, the pronounced differences in morphology do not involve major differences in the conformations and identities of structurally ordered protein segments. Instead, morphological variations are attributable to variations in conformational distributions within disordered segments, which do not contribute to the solid state NMR spectra. Variations in solid state NMR signals from certain amino acid side chains are also observed, suggesting differences in the intermolecular dimerization interface between curved and planar CA lattices, as well as possible differences in intramolecular helix-helix packing.

Highly selective and efficient biotransformation of linarin to produce tilianin by naringinase.

OBJECTIVES: To develop a practical method to prepare tilianin by highly selective and efficient hydrolysis of the C-7 rhamnosyl group from linarin. RESULTS: Naringinase was utilized to selectively catalyze the formation of tilianin using linarin as the starting material. The reaction conditions, including temperature, pH, metal ions, substrate concentration and enzyme concentration, were optimized. At 60 °C, naringinase showed enhanced α-L-rhamnosidase activity while the ß-D-glucosidase activity was abrogated. The addition of Mg(2+), Fe(2+) and Co(2+) was also beneficial for selective biotransformation of linarin to tilianin. Under the optimized conditions (pH 7.0 at 60 °C), linarin could be nearly completely transformed to tilianin with excellent selectivity (>98.9 %), while that of the by-product acacetin was less than 1.1 %. In addition, the structure of target product tilianin was fully characterized by HR-MS and (1)H-NMR. CONCLUSION: A highly selective and efficient biotransformation of linarin to tilianin was developed by the proper control of incubation temperature, which enhanced the α-L-rhamnosidase activity of naringinase and blocked its ß-D-glucosidase activity.

Phosphorylation and ionic strength alter the LRAP-HAP interface in the N-terminus.

The conditions present during enamel crystallite development change dramatically as a function of time, including the pH, protein concentration, surface type, and ionic strength. In this work, we investigate the role that two of these changing conditions, pH and ionic strength, have in modulating the interaction of the amelogenin, LRAP, with hydroxyapatite (HAP). Using solid-state NMR dipolar recoupling and chemical shift data, we investigate the structure, orientation, and dynamics of three regions in the N-terminus of the protein: L(15) to V(19), V(19) to L(23), and K(24) to S(28). These regions are also near the only phosphorylated residue in the protein pS(16); therefore, changes in the LRAP-HAP interaction as a function of phosphorylation (LRAP(-P) vs LRAP(+P)) were also investigated. All of the regions and conditions studied for the surface immobilized proteins showed restricted motion, with indications of slightly more mobility under all conditions for L(15)(+P) and K(24)(-P). The structure and orientation of the LRAP-HAP interaction in the N-terminus of the phosphorylated protein is very stable to changing solution conditions. From REDOR dipolar recoupling data, the structure and orientation in the region L(15)V(19)(+P) did not change significantly as a function of pH or ionic strength. The structure and orientation of the region V(19)L(23)(+P) were also stable to changes in pH, with the only significant change observed at high ionic strength, where the region becomes extended, suggesting this may be an important region in regulating mineral development. Chemical shift studies also suggest minimal changes in all three regions studied for both LRAP(-P) and LRAP(+P) as a function of pH or ionic strength, and also reveal that K(24) has multiple resolvable resonances, suggestive of two coexisting structures. Phosphorylation also alters the LRAP-HAP interface. All of the three residues investigated (L(15), V(19), and K(24)) are closer to the surface in LRAP(+P), but only K(24)S(28) changes structure as a result of phosphorylation, from a random coil to a largely helical structure, and V(19)L(23) becomes more extended at high ionic strength when phosphorylated. These observations suggest that ionic strength and dephosphorylation may provide switching mechanisms to trigger a change in the function of the N-terminus during enamel development.

Solid-state NMR studies of amyloids.

Amyloids have special structural properties and are involved in many aspects of biological function. In particular, amyloids are the cause or hallmarks of a group of notorious and incurable neurodegenerative diseases. The extraordinary high molecular weight and aggregation states of amyloids have posed a challenge for researchers studying them. Solid-state NMR (SSNMR) has been extensively applied to study the structures and dynamics of amyloids for the past 20 or more years and brought us tremendous progress in understanding their structure and related diseases. These studies, at the same time, helped to push SSNMR technical developments in sensitivity and resolution. In this review, some interesting research studies and important technical developments are highlighted to give the reader an overview of the current state of this field.

Corrigendum: Calcium interactions in amelogenin-derived peptide assembly.

[This corrects the article DOI: 10.3389/fphys.2022.1063970.].

Calcium interactions in amelogenin-derived peptide assembly.

Phosphorylation of serine residues has been recognized as a pivotal event in the evolution of mineralized tissues in many biological systems. During enamel development, the extracellular matrix protein amelogenin is most abundant and appears to be critical to the extreme high aspect ratios (length:width) of apatite mineral fibers reaching several millimeters in larger mammalian teeth. A 14-residue peptide (14P2, residues Gly8 to Thr21) was previously identified as a key sequence mediating amelogenin assembly formation, the domain also contains the native single phosphoserine residue (Ser16) of the full-length amelogenin. In this research, 14P2 and its phosphorylated form (p14P2) were investigated at pH 6.0 with various calcium and phosphate ion concentrations, indicating that both peptides could self-assemble into amyloid-like conformation but with differences in structural details. With calcium, the distance between 31P within the p14P2 self-assemblies is averaged to be 4.4 ± 0.2Å, determined by solid-state NMR 31P PITHIRDS-CT experiments. Combining with other experimental results, solid-state Nuclear Magnetic Resonance (SSNMR) suggests that the p14P2 self-assemblies are in parallel in-register ß-sheet conformation and divalent calcium ions most likely connect two adjacent peptide chains by binding to the phosphate group of Ser16 and the carboxylate of Glu18 side-chain. This study on the interactions between calcium ions and amelogenin-derived peptides provides insights on how amelogenin may self-assemble in the presence of calcium ions in early enamel development.

Solid-State Nuclear Magnetic Resonance (SSNMR) Characterization of Osteoblasts From Mesenchymal Stromal Cell Differentiation to Osteoblast Mineralization.

Solid-state nuclear magnetic resonance (SSNMR), a technique capable of studying solid or semisolid biological samples, was first applied to study the cell differentiation and mineralization using the whole-cell sample. Mesenchymal stromal cells (MSCs) with multipotent differentiation capacity were induced to differentiate into osteoblasts. The whole differentiation process, osteoblast mineralization and the mineral maturation, was investigated using SSNMR, providing intact, atomic level information on the cellular mineral structural transformation. Our research indicated the extent of osteoblast mineralization could vary significantly for different cell populations whereas the difference was not easily shown by other means of characterization. The SSNMR spectra revealed hydroxylapatite (or hydroxyapatite [HAP]) formation around 2 to 4 weeks after osteogenic induction for MSCs with a high differentiation potency. The early mineral phase deposit before HAP formation contained a high amount of HPO4 2-. The structures of minerals in the extracellular matrix (ECM) of osteoblasts could evolve for a period of time, even after the incubation of cells has been stopped. This observation was only possible by studying the sample in an intact state, where ECM was not disturbed. These findings improved our understanding of MSCs, which had wide applications in bone regeneration and tissue engineering. Meanwhile, this work demonstrated the advantage of studying these cellular systems as a whole without any mineral extraction, which had been largely overlooked. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Evidence from solid-state NMR for nonhelical conformations in the transmembrane domain of the amyloid precursor protein.

The amyloid precursor protein (APP) is subject to proteolytic processing by γ-secretase within neuronal membranes, leading to Alzheimer's disease-associated ß-amyloid peptide production by cleavage near the midpoint of the single transmembrane (TM) segment of APP. Conformational properties of the TM segment may affect its susceptibility to γ-secretase cleavage, but these properties have not been established definitively, especially in bilayer membranes with physiologically relevant lipid compositions. In this article, we report an investigation of the APP-TM conformation, using (13)C chemical shifts obtained with two-dimensional solid-state NMR spectroscopy as site-specific conformational probes. We find that the APP-TM conformation is not a simple α-helix, particularly at 37°C in multilamellar vesicles with compositions that mimic the composition of neuronal cell membranes. Instead, we observe a mixture of helical and nonhelical conformations at the N- and C-termini and in the vicinity of the γ-cleavage site. Conformational plasticity of the TM segment of APP may be an important factor in the γ-secretase cleavage mechanism.

Solid-State NMR Studies of the Succinate-Acetate Permease from Citrobacter Koseri in Liposomes and Native Nanodiscs.

The succinate-acetate permease (SatP) is an anion channel with six transmembrane domains. It forms different oligomers, especially hexamers in the detergent as well as in the membrane. Solid-state NMR studies of SatP were carried out successfully on SatP complexes by reconstructing the protein into liposomes or retaining the protein in the native membrane of E. coli., where it was expressed. The comparison of 13C-13C 2D correlation spectra between the two samples showed great similarity, opening the possibility to further study the acetate transport mechanism of SatP in its native membrane environment. Solid-state NMR studies also revealed small chemical shift differences of SatP in the two different membrane systems, indicating the importance of the lipid environment in determining the membrane protein structures and dynamics. Combining different 2D SSNMR spectra, chemical shift assignments were made on some sites, consistent with the helical structures in the transmembrane domains. In the end, we pointed out the limitation in the sensitivity for membrane proteins with such a size, and also indicated possible ways to overcome it.

The amyloid structure of mouse RIPK3 (receptor interacting protein kinase 3) in cell necroptosis.

RIPK3 amyloid complex plays crucial roles during TNF-induced necroptosis and in response to immune defense in both human and mouse. Here, we have structurally characterized mouse RIPK3 homogeneous self-assembly using solid-state NMR, revealing a well-ordered N-shaped amyloid core structure featured with 3 parallel in-register ß-sheets. This structure differs from previously published human RIPK1/RIPK3 hetero-amyloid complex structure, which adopted a serpentine fold. Functional studies indicate both RIPK1-RIPK3 binding and RIPK3 amyloid formation are essential but not sufficient for TNF-induced necroptosis. The structural integrity of RIPK3 fibril with three ß-strands is necessary for signaling. Molecular dynamics simulations with a mouse RIPK1/RIPK3 model indicate that the hetero-amyloid is less stable when adopting the RIPK3 fibril conformation, suggesting a structural transformation of RIPK3 from RIPK1-RIPK3 binding to RIPK3 amyloid formation. This structural transformation would provide the missing link connecting RIPK1-RIPK3 binding to RIPK3 homo-oligomer formation in the signal transduction.

The structural topology of wild-type phospholamban in oriented lipid bilayers using 15N solid-state NMR spectroscopy.

For the first time, 15N solid-state NMR experiments were conducted on wild-type phospholamban (WT-PLB) embedded inside mechanically oriented phospholipid bilayers to investigate the topology of its cytoplasmic and transmembrane domains. 15N solid-state NMR spectra of site-specific 15N-labeled WT-PLB indicate that the transmembrane domain has a tilt angle of 13 degrees+/-6 degrees with respect to the POPC (1-palmitoyl-2-oleoyl-sn-glycero-phosphocholine) bilayer normal and that the cytoplasmic domain of WT-PLB lies on the surface of the phospholipid bilayers. Comparable results were obtained from site-specific 15N-labeled WT-PLB embedded inside DOPC/DOPE (1,2-dioleoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) mechanically oriented phospholipids' bilayers. The new NMR data support a pinwheel geometry of WT-PLB, but disagree with a bellflower structure in micelles, and indicate that the orientation of the cytoplasmic domain of the WT-PLB is similar to that reported for the monomeric AFA-PLB mutant.

Exploring membrane selectivity of the antimicrobial peptide KIGAKI using solid-state NMR spectroscopy.

The designed antimicrobial peptide KIGAKIKIGAKIKIGAKI possesses enhanced membrane selectivity for bacterial lipids, such as phosphatidylethanolamine and phosphatidylglycerol. The perturbation of the bilayer by the peptide was first monitored using oriented bilayer samples on glass plates. The alignment of POPE/POPG model membranes with respect to the bilayer normal was severely altered at 4 mol% KIGAKI while the alignment of POPC bilayers was retained. The interaction mechanism between the peptide and POPE/POPG bilayers was investigated by carefully comparing three bilayer MLV samples (POPE bilayers, POPG bilayers, and POPE/POPG 4/1 bilayers). KIGAKI induces the formation of an isotropic phase for POPE/POPG bilayers, but only a slight change in the (31)P NMR CSA line shape for both POPE and POPG bilayers, indicating the synergistic roles of POPE and POPG lipids in the disruption of the membrane structure by KIGAKI. (2)H NMR powder spectra show no reduction of the lipid chain order for both POPG and POPE/POPG bilayers upon peptide incorporation, supporting the evidence that the peptide acts as a surface peptide. (31)P longitudinal relaxation studies confirmed that different dynamic changes occurred upon interaction of the peptide with the three different lipid bilayers, indicating that the strong electrostatic interaction between the cationic peptide KIGAKI and anionic POPG lipids is not the only factor in determining the antimicrobial activity. Furthermore, (31)P and (2)H NMR powder spectra demonstrated a change in membrane characteristics upon mixing of POPE and POPG lipids. The interaction between different lipids, such as POPE and POPG, in the mixed bilayers may provide the molecular basis for the KIGAKI carpet mechanism in the permeation of the membrane.

Solid-State Structure of Abeta (Aß) in Alzheimer's Disease.

Alzheimer's disease (AD) has become the most common neurodegenerative disease. The deposition of amyloid fibrils in the brain is one of the characteristics of AD. The fibrils are composed of amyloid-ß peptide (Aß). Aß is produced through a series event of protease cleavage of a transmembrane protein called ß-amyloid precursor protein (APP) which is commonly expressed in the brain. The production of Aß and its propensity to aggregation to form oligomers and fibrils are believed to initiate a sequence of events that lead to AD dementia. The production of Aß is influenced by the transmembrane domain (TM) structure of APP. The structure variety of different Aß assemblies including oligomers and fibrils may result in different neurotoxicity to the brain. Therefore, enormous work has been carried out to study the structure of APP TM and various Aß assemblies. Solid-state NMR has advantages in studying immobile protein structures with large molecular weight. In this review, solid-state NMR structure of APP TM and different Aß assemblies will be discussed, especially various Aß amyloid fibril structures. This structural information greatly enhanced our understanding in AD, providing fundamental knowledge that will help in finding a treatment for AD.

Probing membrane topology by high-resolution 1H-13C heteronuclear dipolar solid-state NMR spectroscopy.

Membrane topology changes introduced by the association of biologically pertinent molecules with membranes were analyzed utilizing the (1)H-(13)C heteronuclear dipolar solid-state NMR spectroscopy technique (SAMMY) on magnetically aligned phospholipid bilayers (bicelles). The phospholipids (1)H-(13)C dipolar coupling profiles lipid motions at the headgroup, glycerol backbone, and the acyl chain region. The transmembrane segment of phospholamban, the antimicrobial peptide (KIGAKI)(3) and cholesterol were incorporated into the bicelles, respectively. The lipids (1)H-(13)C dipolar coupling profiles exhibit different shifts in the dipolar coupling contour positions upon the addition of these molecules, demonstrating a variety of interaction mechanisms exist between the biological molecules and the membranes. The membrane topology changes revealed by the SAMMY pulse sequence provide a complete screening method for analyzing how these biologically active molecules interact with the membrane.