The three-dimensional structure of human ß-endorphin amyloid fibrils.

In the pituitary gland, hormones are stored in a functional amyloid state within acidic secretory granules before they are released into the blood. To gain a detailed understanding of the structure-function relationship of amyloids in hormone secretion, the three-dimensional (3D) structure of the amyloid fibril of the human hormone ß-endorphin was determined by solid-state NMR. We find that ß-endorphin fibrils are in a ß-solenoid conformation with a protonated glutamate residue in their fibrillar core. During exocytosis of the hormone amyloid the pH increases from acidic in the secretory granule to neutral level in the blood, thus it is suggested-and supported with mutagenesis data-that the pH change in the cellular milieu acts through the deprotonation of glutamate 8 to release the hormone from the amyloid. For amyloid disassembly in the blood, it is proposed that the pH change acts together with a buffer composition change and hormone dilution. In the pituitary gland, peptide hormones can be stored as amyloid fibrils within acidic secretory granules before release into the blood stream. Here, we use solid-state NMR to determine the 3D structure of the amyloid fiber formed by the human hormone ß-endorphin. We find that ß-endorphin fibrils are in a ß-solenoid conformation that is generally reminiscent of other functional amyloids. In the ß-endorphin amyloid, every layer of the ß-solenoid is composed of a single peptide and protonated Glu8 is located in the fibrillar core. The secretory granule has an acidic pH but, on exocytosis, the ß-endorphin fibril would encounter neutral pH conditions (pH 7.4) in the blood; this pH change would result in deprotonation of Glu8 to release the hormone peptide from the amyloid. Analyses of ß-endorphin variants carrying mutations in Glu8 support the role of the protonation state of this residue in fibril disassembly, among other environmental changes.

Two new polymorphic structures of human full-length alpha-synuclein fibrils solved by cryo-electron microscopy.

Intracellular inclusions rich in alpha-synuclein are a hallmark of several neuropathological diseases including Parkinson's disease (PD). Previously, we reported the structure of alpha-synuclein fibrils (residues 1-121), composed of two protofibrils that are connected via a densely-packed interface formed by residues 50-57 (Guerrero-Ferreira, eLife 218;7:e36402). We here report two new polymorphic atomic structures of alpha-synuclein fibrils termed polymorphs 2a and 2b, at 3.0 Å and 3.4 Å resolution, respectively. These polymorphs show a radically different structure compared to previously reported polymorphs. The new structures have a 10 nm fibril diameter and are composed of two protofilaments which interact via intermolecular salt-bridges between amino acids K45, E57 (polymorph 2a) or E46 (polymorph 2b). The non-amyloid component (NAC) region of alpha-synuclein is fully buried by previously non-described interactions with the N-terminus. A hydrophobic cleft, the location of familial PD mutation sites, and the nature of the protofilament interface now invite to formulate hypotheses about fibril formation, growth and stability.

CONFINE-MAS: a magic-angle spinning NMR probe that confines the sample in case of a rotor explosion.

Magic-angle spinning (MAS) is mandatory in solid-state NMR experiments to achieve resolved spectra. In rare cases, instabilities in the rotation or damage of either the rotor or the rotor cap can lead to a so called "rotor crash" involving a disintegration of the sample container and possibly the release of an aerosol or of dust. We present a modified design of a 3.2 mm probe with a confining chamber which in case of a rotor crash prevents the release of aerosols and possibly hazardous materials. 1D and 2D NMR experiments show that such a hazardous material-confining MAS probe ("CONFINE-MAS" probe) has a similar sensitivity compared to a standard probe and performs equally well in terms of spinning stability. We illustrate the CONFINE-MAS probe properties and performance by application to a fungal amyloid.

Amyloid Fibril Polymorphism: Almost Identical on the Atomic Level, Mesoscopically Very Different.

Amyloid polymorphism of twisted and straight ß-endorphin fibrils was studied by negative-stain transmission electron microscopy, scanning transmission electron microscopy, and solid-state nuclear magnetic resonance spectroscopy. Whereas fibrils assembled in the presence of salt formed flat, striated ribbons, in the absence of salt they formed mainly twisted filaments. To get insights into their structural differences at the atomic level, 3D solid-state NMR spectra of both fibril types were acquired, allowing the detection of the differences in chemical shifts of 13C and 15N atoms in both preparations. The spectral fingerprints and therefore the chemical shifts are very similar for both fibril types. This indicates that the monomer structure and the molecular interfaces are almost the same but that these small differences do propagate to produce flat and twisted morphologies at the mesoscopic scale. This finding is in agreement with both experimental and theoretical considerations on the assembly of polymers (including amyloids) under different salt conditions, which attribute the mesoscopic difference of flat versus twisted fibrils to electrostatic intermolecular repulsions.

Solid-state NMR sequential assignment of the ß-endorphin peptide in its amyloid form.

Insights into the three-dimensional structure of hormone fibrils are crucial for a detailed understanding of how an amyloid structure allows the storage of hormones in secretory vesicles prior to hormone secretion into the blood stream. As an example for various hormone amyloids, we have studied the endogenous opioid neuropeptide ß-endorphin in one of its fibril forms. We have achieved the sequential assignment of the chemical shifts of the backbone and side-chain heavy atoms of the fibril. The secondary chemical shift analysis revealed that the ß-endorphin peptide adopts three ß-strands in its fibril state. This finding fosters the amyloid nature of a hormone at the atomic level.

Preparation and Characterization of Stable α-Synuclein Lipoprotein Particles.

Multiple neurodegenerative diseases are caused by the aggregation of the human α-Synuclein (α-Syn) protein. α-Syn possesses high structural plasticity and the capability of interacting with membranes. Both features are not only essential for its physiological function but also play a role in the aggregation process. Recently it has been proposed that α-Syn is able to form lipid-protein particles reminiscent of high-density lipoproteins. Here, we present a method to obtain a stable and homogeneous population of nanometer-sized particles composed of α-Syn and anionic phospholipids. These particles are called α-Syn lipoprotein (nano)particles to indicate their relationship to high-density lipoproteins formed by human apolipoproteins in vivo and of in vitro self-assembling phospholipid bilayer nanodiscs. Structural investigations of the α-Syn lipoprotein particles by circular dichroism (CD) and magic angle solid-state nuclear magnetic resonance (MAS SS-NMR) spectroscopy establish that α-Syn adopts a helical secondary structure within these particles. Based on cryo-electron microscopy (cryo-EM) and dynamic light scattering (DLS) α-Syn lipoprotein particles have a defined size with a diameter of ∼23 nm. Chemical cross-linking in combination with solution-state NMR and multiangle static light scattering (MALS) of α-Syn particles reveal a high-order protein-lipid entity composed of ∼8-10 α-Syn molecules. The close resemblance in size between cross-linked in vitro-derived α-Syn lipoprotein particles and a cross-linked species of endogenous α-Syn from SH-SY5Y human neuroblastoma cells indicates a potential functional relevance of α-Syn lipoprotein nanoparticles.

Further exploration of the conformational space of α-synuclein fibrils: solid-state NMR assignment of a high-pH polymorph.

Polymorphism is a common and important phenomenon for protein fibrils which has been linked to the appearance of strains in prion and other neurodegenerative diseases. Parkinson disease is a frequently occurring neurodegenerative pathology, tightly associated with the formation of Lewy bodies. These deposits mainly consist of α-synuclein in fibrillar, ß-sheet-rich form. α-synuclein is known to form numerous different polymorphs, which show distinct structural features. Here, we describe the chemical shift assignments, and derive the secondary structure, of a polymorph that was fibrillized at higher-than-physiological pH conditions. The fibrillar core contains residues 40-95, with both the C- and N-terminus not showing any ordered, rigid parts. The chemical shifts are similar to those recorded previously for an assigned polymorph that was fibrillized at neutral pH.

Reassessment of MxiH subunit orientation and fold within native Shigella T3SS needles using surface labelling and solid-state NMR.

T3SSs are essential virulence determinants of many Gram-negative bacteria, used to inject bacterial effectors of virulence into eukaryotic host cells. Their major extracellular portion, a ∼50 nm hollow, needle-like structure, is essential to host cell sensing and the conduit for effector secretion. It is formed of a small, conserved subunit arranged as a helical polymer. The structure of the subunit has been studied by electron cryomicroscopy within native polymers and by solid-state NMR in recombinant polymers, yielding two incompatible atomic models. To resolve this controversy, we re-examined the native polymer used for electron cryomicroscopy via surface labelling and solid-state NMR. Our data show the orientation and overall fold of the subunit within this polymer is as established by solid-state NMR for recombinant polymers.