Anle138b interaction in α-synuclein aggregates by dynamic nuclear polarization NMR.

Small molecules that bind to oligomeric protein species such as membrane proteins and fibrils are of clinical interest for development of therapeutics and diagnostics. Definition of the binding site at atomic resolution via NMR is often challenging due to low binding stoichiometry of the small molecule. For fibrils and aggregation intermediates grown in the presence of lipids, we report atomic-resolution contacts to the small molecule at sub nm distance via solid-state NMR using dynamic nuclear polarization (DNP) and orthogonally labelled samples of the protein and the small molecule. We apply this approach to α-synuclein (αS) aggregates in complex with the small molecule anle138b, which is a clinical drug candidate for disease modifying therapy. The small central pyrazole moiety of anle138b is detected in close proximity to the protein backbone and differences in the contacts between fibrils and early intermediates are observed. For intermediate species, the 100 K condition for DNP helps to preserve the aggregation state, while for both fibrils and oligomers, the DNP enhancement is essential to obtain sufficient sensitivity.

The calcium-free form of atorvastatin inhibits amyloid-ß(1-42) aggregation in vitro.

Alzheimer's disease is characterized by the presence of extraneuronal amyloid plaques composed of amyloid-beta (Aß) fibrillar aggregates in the brains of patients. In mouse models, it has previously been shown that atorvastatin (Ator), a cholesterol-lowering drug, has some reducing effect on the production of cerebral Aß. A meta-analysis on humans showed moderate effects in the short term but no improvement in the Alzheimer's Disease Assessment Scale-Cognitive Subscale behavioral test. Here, we explore a potential direct effect of Ator on Aß42 aggregation. Using NMR-based monomer consumption assays and CD spectroscopy, we observed a promoting effect of Ator in its original form (Ator-calcium) on Aß42 aggregation, as expected because of the presence of calcium ions. The effect was reversed when applying a CaCO3-based calcium ion scavenging method, which was validated by the aforementioned methods as well as thioflavin-T fluorescence assays and transmission electron microscopy. We found that the aggregation was inhibited significantly when the concentration of calcium-free Ator exceeded that of Aß by at least a factor of 2. The 1H-15N heteronuclear single quantum correlation and saturation-transfer difference NMR data suggest that calcium-free Ator exerts its effect through interaction with the 16KLVF19 binding site on the Aß peptide via its aromatic rings as well as hydroxyl and methyl groups. On the other hand, molecular dynamics simulations confirmed that the increasing concentration of Ator is necessary for the inhibition of the conformational transition of Aß from an α-helix-dominant to a ß-sheet-dominant structure.

Maturation of amyloid ß fibrils alters their molecular stability.

Little is known about how maturation of Alzheimer's disease-related amyloid ß (Aß) fibrils alters their stability and potentially influences their spreading in the brain. Using high-pressure NMR, we show that progression from early to late Aß40 aggregates enhances the kinetic stability, while ageing during weeks to months enhances their thermodynamic stability.

Structure and Gating Behavior of the Human Integral Membrane Protein VDAC1 in a Lipid Bilayer.

The voltage-dependent anion channel (VDAC), the most abundant protein in the outer mitochondrial membrane, is responsible for the transport of all ions and metabolites into and out of mitochondria. Larger than any of the ß-barrel structures determined to date by magic-angle spinning (MAS) NMR, but smaller than the size limit of cryo-electron microscopy (cryo-EM), VDAC1's 31 kDa size has long been a bottleneck in determining its structure in a near-native lipid bilayer environment. Using a single two-dimensional (2D) crystalline sample of human VDAC1 in lipids, we applied proton-detected fast magic-angle spinning NMR spectroscopy to determine the arrangement of ß strands. Combining these data with long-range restraints from a spin-labeled sample, chemical shift-based secondary structure prediction, and previous MAS NMR and atomic force microscopy (AFM) data, we determined the channel's structure at a 2.2 Å root-mean-square deviation (RMSD). The structure, a 19-stranded ß-barrel, with an N-terminal α-helix in the pore is in agreement with previous data in detergent, which was questioned due to the potential for the detergent to perturb the protein's functional structure. Using a quintuple mutant implementing the channel's closed state, we found that dynamics are a key element in the protein's gating behavior, as channel closure leads to the destabilization of not only the C-terminal barrel residues but also the α2 helix. We showed that cholesterol, previously shown to reduce the frequency of channel closure, stabilizes the barrel relative to the N-terminal helix. Furthermore, we observed channel closure through steric blockage by a drug shown to selectively bind to the channel, the Bcl2-antisense oligonucleotide G3139.

Membrane-embedded TSPO: an NMR view.

Translocator Protein (18 kDa) (TSPO) is a mitochondrial transmembrane protein commonly used as a biomarker for neuroinflammation and is also a potential therapeutic target in neurodegenerative diseases. Despite intensive research efforts, the function of TSPO is still largely enigmatic. Deciphering TSPO structure in the native lipid environment is essential to gain insight into its cellular activities and to design improved diagnostic and therapeutic ligands. Here, we discuss the influence of lipid composition on the structure of mammalian TSPO embedded into lipid bilayers on the basis of solid-state NMR experiments. We further highlight that cholesterol can influence both the tertiary and quaternary TSPO structure and also influence TSPO localization in mitochondria-associated endoplasmic reticulum membranes.

Structure, gating and interactions of the voltage-dependent anion channel.

The voltage-dependent anion channel (VDAC) is one of the most highly abundant proteins found in the outer mitochondrial membrane, and was one of the earliest discovered. Here we review progress in understanding VDAC function with a focus on its structure, discussing various models proposed for voltage gating as well as potential drug targets to modulate the channel's function. In addition, we explore the sensitivity of VDAC structure to variations in the membrane environment, comparing DMPC-only, DMPC with cholesterol, and near-native lipid compositions, and use magic-angle spinning NMR spectroscopy to locate cholesterol on the outside of the ß-barrel. We find that the VDAC protein structure remains unchanged in different membrane compositions, including conditions with cholesterol.

Cryogenic optical localization provides 3D protein structure data with Angstrom resolution.

We introduce Cryogenic Optical Localization in 3D (COLD), a method to localize multiple fluorescent sites within a single small protein with Angstrom resolution. We demonstrate COLD by determining the conformational state of the cytosolic Per-ARNT-Sim domain from the histidine kinase CitA of Geobacillus thermodenitrificans and resolving the four biotin sites of streptavidin. COLD provides quantitative 3D information about small- to medium-sized biomolecules on the Angstrom scale and complements other techniques in structural biology.

Sensory domain contraction in histidine kinase CitA triggers transmembrane signaling in the membrane-bound sensor.

Bacteria use membrane-integral sensor histidine kinases (HK) to perceive stimuli and transduce signals from the environment to the cytosol. Information on how the signal is transmitted across the membrane by HKs is still scarce. Combining both liquid- and solid-state NMR, we demonstrate that structural rearrangements in the extracytoplasmic, citrate-sensing Per-Arnt-Sim (PAS) domain of HK CitA are identical for the isolated domain in solution and in a longer construct containing the membrane-embedded HK and lacking only the kinase core. We show that upon citrate binding, the PAS domain contracts, resulting in a shortening of the C-terminal ß-strand. We demonstrate that this contraction of the PAS domain, which is well characterized for the isolated domain, is the signal transmitted to the transmembrane (TM) helices in a CitA construct in liposomes. Putting the extracytoplasmic PAS domain into context of the membrane-embedded CitA construct slows down citrate-binding kinetics by at least a factor of 60, confirming that TM helix motions are linked to the citrate-binding event. Our results are confirmation of a hallmark of the HK signal transduction mechanism with atomic resolution on a full-length construct lacking only the kinase core domain.

Protein resonance assignment by BSH-CP-based 3D solid-state NMR experiments: A practical guide.

Solid-state NMR (ssNMR) spectroscopy has evolved into a powerful method to obtain structural information and to study the dynamics of proteins at atomic resolution and under physiological conditions. The method is especially well suited to investigate insoluble and noncrystalline proteins that cannot be investigated easily by X-ray crystallography or solution NMR. To allow for detailed analysis of ssNMR data, the assignment of resonances to the protein atoms is essential. For this purpose, a set of three-dimensional (3D) spectra needs to be acquired. Band-selective homo-nuclear cross-polarization (BSH-CP) is an effective method for magnetization transfer between carbonyl carbon (CO) and alpha carbon (CA) atoms, which is an important transfer step in multidimensional ssNMR experiments. This tutorial describes the detailed procedure for the chemical shift assignment of the backbone atoms of 13 C-15 N-labeled proteins by BSH-CP-based 13 C-detected ssNMR experiments. A set of six 3D experiments is used for unambiguous assignment of the protein backbone as well as certain side-chain resonances. The tutorial especially addresses scientists with little experience in the field of ssNMR and provides all the necessary information for protein assignment in an efficient, time-saving approach.

Comparison of the 3D structures of mouse and human α-synuclein fibrils by solid-state NMR and STEM.

Intra-neuronal aggregation of α-synuclein into fibrils is the molecular basis for α-synucleinopathies, such as Parkinson's disease. The atomic structure of human α-synuclein (hAS) fibrils was recently determined by Tuttle et al. using solid-state NMR (ssNMR). The previous study found that hAS fibrils are composed of a single protofilament. Here, we have investigated the structure of mouse α-synuclein (mAS) fibrils by STEM and isotope-dilution ssNMR experiments. We found that in contrast to hAS, mAS fibrils consist of two or even three protofilaments which are connected by rather weak interactions in between them. Although the number of protofilaments appears to be different between hAS and mAS, we found that they have a remarkably similar secondary structure and protofilament 3D structure as judged by secondary chemical shifts and intra-molecular distance restraints. We conclude that the two mutant sites between hAS and mAS (positions 53 and 87) in the fibril core region are crucial for determining the quaternary structure of α-synuclein fibrils.

Characterization of H/D exchange in type 1 pili by proton-detected solid-state NMR and molecular dynamics simulations.

Uropathogenic Escherichia coli invades and colonizes hosts by attaching to cells using adhesive pili on the bacterial surface. Although many biophysical techniques have been used to study the structure and mechanical properties of pili, many important details are still unknown. Here we use proton-detected solid-state NMR experiments to investigate solvent accessibility and structural dynamics. Deuterium back-exchange at labile sites of the perdeuterated, fully proton back-exchanged pili was conducted to investigate hydrogen/deuterium (H/D) exchange patterns of backbone amide protons in pre-assembled pili. We found distinct H/D exchange patterns in lateral and axial intermolecular interfaces in pili. Amide protons protected from H/D exchange in pili are mainly located in the core region of the monomeric subunit and in the lateral intermolecular interface, whereas the axial intermolecular interface and the exterior region of pili are highly exposed to H/D exchange. Additionally, we performed molecular dynamics simulations of the type 1 pilus rod and estimated the probability of H/D exchange based on hydrogen bond dynamics. The comparison of the experimental observables and simulation data provides insights into stability and mechanical properties of pili.

Alpha protons as NMR probes in deuterated proteins.

We describe a new labeling method that allows for full protonation at the backbone Hα position, maintaining protein side chains with a high level of deuteration. We refer to the method as alpha proton exchange by transamination (α-PET) since it relies on transaminase activity demonstrated here using Escherichia coli expression. We show that α-PET labeling is particularly useful in improving structural characterization of solid proteins by introduction of an additional proton reporter, while eliminating many strong dipolar couplings. The approach benefits from the high sensitivity associated with 1.3 mm samples, more abundant information including Hα resonances, and the narrow proton linewidths encountered for highly deuterated proteins. The labeling strategy solves amide proton exchange problems commonly encountered for membrane proteins when using perdeuteration and backexchange protocols, allowing access to alpha and all amide protons including those in exchange-protected regions. The incorporation of Hα protons provides new insights, as the close Hα-Hα and Hα-HN contacts present in ß-sheets become accessible, improving the chance to determine the protein structure as compared with HN-HN contacts alone. Protonation of the Hα position higher than 90% is achieved for Ile, Leu, Phe, Tyr, Met, Val, Ala, Gln, Asn, Thr, Ser, Glu, Asp even though LAAO is only active at this degree for Ile, Leu, Phe, Tyr, Trp, Met. Additionally, the glycine methylene carbon is labeled preferentially with a single deuteron, allowing stereospecific assignment of glycine alpha protons. In solution, we show that the high deuteration level dramatically reduces R2 relaxation rates, which is beneficial for the study of large proteins and protein dynamics. We demonstrate the method using two model systems, as well as a 32 kDa membrane protein, hVDAC1, showing the applicability of the method to study membrane proteins.

Correction to: Endogenous oligodendroglial alpha-synuclein and TPPP/p25α orchestrate alpha-synuclein pathology in experimental multiple system atrophy models.

he original version of this article.

Endogenous oligodendroglial alpha-synuclein and TPPP/p25α orchestrate alpha-synuclein pathology in experimental multiple system atrophy models.

Multiple system atrophy (MSA) is characterized by the presence of distinctive glial cytoplasmic inclusions (GCIs) within oligodendrocytes that contain the neuronal protein alpha-synuclein (aSyn) and the oligodendroglia-specific phosphoprotein TPPP/p25α. However, the role of oligodendroglial aSyn and p25α in the formation of aSyn-rich GCIs remains unclear. To address this conundrum, we have applied human aSyn (haSyn) pre-formed fibrils (PFFs) to rat wild-type (WT)-, haSyn-, or p25α-overexpressing oligodendroglial cells and to primary differentiated oligodendrocytes derived from WT, knockout (KO)-aSyn, and PLP-haSyn-transgenic mice. HaSyn PFFs are readily taken up by oligodendroglial cells and can recruit minute amounts of endogenous aSyn into the formation of insoluble, highly aggregated, pathological assemblies. The overexpression of haSyn or p25α accelerates the recruitment of endogenous protein and the generation of such aberrant species. In haSyn PFF-treated primary oligodendrocytes, the microtubule and myelin networks are disrupted, thus recapitulating a pathological hallmark of MSA, in a manner totally dependent upon the seeding of endogenous aSyn. Furthermore, using oligodendroglial and primary cortical cultures, we demonstrated that pathology-related S129 aSyn phosphorylation depends on aSyn and p25α protein load and may involve different aSyn "strains" present in oligodendroglial and neuronal synucleinopathies. Importantly, this hypothesis was further supported by data obtained from human post-mortem brain material derived from patients with MSA and dementia with Lewy bodies. Finally, delivery of haSyn PFFs into the mouse brain led to the formation of aberrant aSyn forms, including the endogenous protein, within oligodendroglia and evoked myelin decompaction in WT mice, but not in KO-aSyn mice. This line of research highlights the role of endogenous aSyn and p25α in the formation of pathological aSyn assemblies in oligodendrocytes and provides in vivo evidence of the contribution of oligodendroglial aSyn in the establishment of aSyn pathology in MSA.

Probing Membrane Protein Insertion into Lipid Bilayers by Solid-State NMR.

Determination of the environment surrounding a protein is often key to understanding its function and can also be used to infer the structural properties of the protein. By using proton-detected solid-state NMR, we show that reduced spin diffusion within the protein under conditions of fast magic-angle spinning, high magnetic field, and sample deuteration allows the efficient measurement of site-specific exposure to mobile water and lipids. We demonstrate this site specificity on two membrane proteins, the human voltage dependent anion channel, and the alkane transporter AlkL from Pseudomonas putida. Transfer from lipids is observed selectively in the membrane spanning region, and an average lipid-protein transfer rate of 6 s-1 was determined for residues protected from exchange. Transfer within the protein, as tracked in the 15 N-1 H 2D plane, was estimated from initial rates and found to be in a similar range of about 8 to 15 s-1 for several resolved residues, explaining the site specificity.

Allosteric switch regulates protein-protein binding through collective motion.

Many biological processes depend on allosteric communication between different parts of a protein, but the role of internal protein motion in propagating signals through the structure remains largely unknown. Through an experimental and computational analysis of the ground state dynamics in ubiquitin, we identify a collective global motion that is specifically linked to a conformational switch distant from the binding interface. This allosteric coupling is also present in crystal structures and is found to facilitate multispecificity, particularly binding to the ubiquitin-specific protease (USP) family of deubiquitinases. The collective motion that enables this allosteric communication does not affect binding through localized changes but, instead, depends on expansion and contraction of the entire protein domain. The characterization of these collective motions represents a promising avenue for finding and manipulating allosteric networks.

Insights into Cholesterol/Membrane Protein Interactions Using Paramagnetic Solid-State NMR.

Cholesterol is an essential component of animal cell membranes and impacts the structure and function of membrane proteins. But how cholesterol exerts its functions remains often enigmatic. Here, high-resolution solid-state NMR in combination with paramagnetic cholesterol analogues was shown to be a powerful approach to study the interaction of membrane proteins with cholesterol. Application of the method to the 169-residue translocator protein TSPO provides residue-specific information about its interaction with cholesterol. Comparison with NMR signal perturbations induced by diamagnetic cholesterol furthermore supports changes in the structure of mammalian TSPO caused by cholesterol binding.

Atomic model of the type III secretion system needle.

Pathogenic bacteria using a type III secretion system (T3SS) to manipulate host cells cause many different infections including Shigella dysentery, typhoid fever, enterohaemorrhagic colitis and bubonic plague. An essential part of the T3SS is a hollow needle-like protein filament through which effector proteins are injected into eukaryotic host cells. Currently, the three-dimensional structure of the needle is unknown because it is not amenable to X-ray crystallography and solution NMR, as a result of its inherent non-crystallinity and insolubility. Cryo-electron microscopy combined with crystal or solution NMR subunit structures has recently provided a powerful hybrid approach for studying supramolecular assemblies, resulting in low-resolution and medium-resolution models. However, such approaches cannot deliver atomic details, especially of the crucial subunit-subunit interfaces, because of the limited cryo-electron microscopic resolution obtained in these studies. Here we report an alternative approach combining recombinant wild-type needle production, solid-state NMR, electron microscopy and Rosetta modelling to reveal the supramolecular interfaces and ultimately the complete atomic structure of the Salmonella typhimurium T3SS needle. We show that the 80-residue subunits form a right-handed helical assembly with roughly 11 subunits per two turns, similar to that of the flagellar filament of S. typhimurium. In contrast to established models of the needle in which the amino terminus of the protein subunit was assumed to be α-helical and positioned inside the needle, our model reveals an extended amino-terminal domain that is positioned on the surface of the needle, while the highly conserved carboxy terminus points towards the lumen.

Non-equilibrium hydrogen exchange for determination of H-bond strength and water accessibility in solid proteins.

We demonstrate measurement of non-equilibrium backbone amide hydrogen-deuterium exchange rates (HDX) for solid proteins. The target of this study are the slowly exchanging residues in solid samples, which are associated with stable secondary-structural elements of proteins. These hydrogen exchange processes escape methods measuring equilibrium exchange rates of faster processes. The method was applied to a micro-crystalline preparation of the SH3 domain of chicken α-spectrin. Therefore, from a 100% back-exchanged micro-crystalline protein preparation, the supernatant buffer was exchanged by a partially deuterated buffer to reach a final protonation level of approximately 20% before packing the sample in a 1.3 mm rotor. Tracking of the HN peak intensities for 2 weeks reports on site-specific hydrogen bond strength and also likely reflects water accessibility in a qualitative manner. H/D exchange can be directly determined for hydrogen-bonded amides using 1H detection under fast magic angle spinning. This approach complements existing methods and provides the means to elucidate interesting site-specific characteristics for protein functionality in the solid state.

Voltage Dependence of Conformational Dynamics and Subconducting States of VDAC-1.

The voltage-dependent anion channel 1 (VDAC-1) is an important protein of the outer mitochondrial membrane that transports energy metabolites and is involved in apoptosis. The available structures of VDAC proteins show a wide ß-stranded barrel pore, with its N-terminal α-helix (N-α) bound to its interior. Electrophysiology experiments revealed that voltage, its polarity, and membrane composition modulate VDAC currents. Experiments with VDAC-1 mutants identified amino acids that regulate the gating process. However, the mechanisms for how these factors regulate VDAC-1, and which changes they trigger in the channel, are still unknown. In this study, molecular dynamics simulations and single-channel experiments of VDAC-1 show agreement for the current-voltage relationships of an "open" channel and they also show several subconducting transient states that are more cation selective in the simulations. We observed voltage-dependent asymmetric distortions of the VDAC-1 barrel and the displacement of particular charged amino acids. We constructed conformational models of the protein voltage response and the pore changes that consistently explain the protein conformations observed at opposite voltage polarities, either in phosphatidylethanolamine or phosphatidylcholine membranes. The submicrosecond VDAC-1 voltage response shows intrinsic structural changes that explain the role of key gating amino acids and support some of the current gating hypotheses. These voltage-dependent protein changes include asymmetric barrel distortion, its interaction with the membrane, and significant displacement of N-α amino acids.