Structures of AT8 and PHF1 phosphomimetic tau: Insights into the posttranslational modification code of tau aggregation.

The microtubule-associated protein tau aggregates into amyloid fibrils in Alzheimer's disease and other neurodegenerative diseases. In these tauopathies, tau is hyperphosphorylated, suggesting that this posttranslational modification (PTM) may induce tau aggregation. Tau is also phosphorylated in normal developing brains. To investigate how tau phosphorylation induces amyloid fibrils, here we report the atomic structures of two phosphomimetic full-length tau fibrils assembled without anionic cofactors. We mutated key Ser and Thr residues to Glu in two regions of the protein. One construct contains three Glu mutations at the epitope of the anti-phospho-tau antibody AT8 (AT8-3E tau), whereas the other construct contains four Glu mutations at the epitope of the antibody PHF1 (PHF1-4E tau). Solid-state NMR data show that both phosphomimetic tau mutants form homogeneous fibrils with a single set of chemical shifts. The AT8-3E tau rigid core extends from the R3 repeat to the C terminus, whereas the PHF1-4E tau rigid core spans R2, R3, and R4 repeats. Cryoelectron microscopy data show that AT8-3E tau forms a triangular multi-layered core, whereas PHF1-4E tau forms a triple-stranded core. Interestingly, a construct combining all seven Glu mutations exhibits the same conformation as PHF1-4E tau. Scalar-coupled NMR data additionally reveal the dynamics and shape of the fuzzy coat surrounding the rigid cores. These results demonstrate that specific PTMs induce structurally specific tau aggregates, and the phosphorylation code of tau contains redundancy.

Structure of the nonhelical filament of the Alzheimer's disease tau core.

The microtubule-associated protein tau aggregates into neurofibrillary tangles in Alzheimer's disease (AD). The main type of aggregates, the paired helical filaments (PHF), incorporate about 20% of the full-length protein into the rigid core. Recently, cryo-electron microscopy data showed that a protease-resistant fragment of tau (residues 297-391) self-assembles in vitro in the presence of divalent cations to form twisted filaments whose molecular structure resembles that of AD PHF tau [S. Lövestam et al., Elife 11, e76494 (2022)]. To investigate whether this tau construct is uniquely predisposed to this morphology and structure, we fibrillized tau (297-391) under the reported conditions and determined its structure using solid-state NMR spectroscopy. Unexpectedly, the protein assembled predominantly into nontwisting ribbons whose rigid core spans residues 305-357. This rigid core forms a ß-arch that turns at residues 322CGS324. Two protofilaments stack together via a long interface that stretches from G323 to I354. Together, these two protofilaments form a four-layered ß-sheet core whose sidechains are stabilized by numerous polar and hydrophobic interactions. This structure gives insight into the fibril morphologies and molecular conformations that can be adopted by this protease-resistant core of AD tau under different pH and ionic conditions.

Alzheimer's disease seeded tau forms paired helical filaments yet lacks seeding potential.

Alzheimer's disease (AD) and many other neurodegenerative diseases are characterized by pathological aggregation of the protein tau. These tau aggregates spread in a stereotypical spatiotemporal pattern in the brain of each disease, suggesting that the misfolded tau can recruit soluble monomers to adopt the same pathological structure. To investigate whether recruited tau indeed adopts the same structure and properties as the original seed, here we template recombinant full-length 0N3R tau, 0N4R tau, and an equimolar mixture of the two using sarkosyl-insoluble tau extracted from AD brain and determine the structures of the resulting fibrils using cryoelectron microscopy. We show that these cell-free amplified tau fibrils adopt the same molecular structure as the AD paired-helical filament (PHF) tau but are unable to template additional monomers. Therefore, the PHF structure alone is insufficient for defining the pathological properties of AD tau, and other biochemical components such as tau posttranslational modifications, other proteins, polyanionic cofactors, and salt are required for the prion-like serial propagation of tauopathies.

Distinguishing Different Hydrogen-Bonded Helices in Proteins by Efficient 1H-Detected Three-Dimensional Solid-State NMR.

Helical structures in proteins include not only α-helices but also 310 and π helices. These secondary structures differ in the registry of the C═O···H-N hydrogen bonds, which are i to i + 4 for α-helices, i to i + 3 for 310 helices, and i to i + 5 for π-helices. The standard NMR observable of protein secondary structures are chemical shifts, which are, however, insensitive to the precise type of helices. Here, we introduce a three-dimensional (3D) 1H-detected experiment that measures and assigns CO-HN cross-peaks to distinguish the different types of hydrogen-bonded helices. This hCOhNH experiment combines efficient cross-polarization from CO to HN with 13C, 15N, and 1H chemical shift correlation to detect the relative proximities of the COi-Hi+jN spin pairs. We demonstrate this experiment on the membrane-bound transmembrane domain of the SARS-CoV-2 envelope (E) protein (ETM). We show that the C-terminal five residues of ETM form a 310-helix, whereas the rest of the transmembrane domain have COi-Hi+4N hydrogen bonds that are characteristic of α-helices. This result confirms the recent high-resolution solid-state NMR structure of the open state of ETM, which was solved in the absence of explicit hydrogen-bonding restraints. This C-terminal 310 helix may facilitate proton and calcium conduction across the hydrophobic gate of the channel. This hCOhNH experiment is generally applicable and can be used to distinguish not only different types of helices but also different types of ß-strands and other hydrogen-bonded conformations in proteins.

The Temperature Dependence of Hydrogen Bonds Is More Uniform in Stable Proteins: An Analysis of NMR h3JNC' Couplings in Four Different Protein Structures.

Long-range HNCO NMR spectra for proteins show crosspeaks due to 1JNC', 2JNC', 3JNCγ, and h3JNC' couplings. The h3JNC' couplings are transmitted through hydrogen bonds and their sizes are correlated to hydrogen bond lengths. We collected long-range HNCO data at a series of temperatures for four protein structures. P22i and CUS-3i are six-stranded beta-barrel I-domains from phages P22 and CUS-3 that share less than 40% sequence identity. The cis and trans states of the C-terminal domain from pore-forming toxin hemolysin ΙΙ (HlyIIC) arise from the isomerization of a single G404-P405 peptide bond. For P22i and CUS-3i, hydrogen bonds detected by NMR agree with those observed in the corresponding domains from cryoEM structures of the two phages. Hydrogen bond lengths derived from the h3JNC' couplings, however, are poorly conserved between the distantly related CUS-3i and P22i domains and show differences even between the closely related cis and trans state structures of HlyIIC. This is consistent with hydrogen bond lengths being determined by local differences in structure rather than the overall folding topology. With increasing temperature, hydrogen bonds typically show an apparent increase in length that has been attributed to protein thermal expansion. Some hydrogen bonds are invariant with temperature, however, while others show apparent decreases in length, suggesting they become stabilized with increasing temperature. Considering the data for the three proteins in this study and previously published data for ubiquitin and GB3, lowered protein folding stability and cooperativity corresponds with a larger range of temperature responses for hydrogen bonds. This suggests a partial uncoupling of hydrogen bond energetics from global unfolding cooperativity as protein stability decreases.

NMR detection and conformational dependence of two, three, and four-bond isotope shifts due to deuteration of backbone amides.

NMR isotope shifts occur due to small differences in nuclear shielding when nearby atoms are different isotopes. For molecules dissolved in 1:1 H2O:D2O, the resulting mixture of N-H and N-D isotopes leads to a small splitting of resonances from adjacent nuclei. We used multidimensional NMR to measure isotope shifts for the proteins CUS-3iD and CspA. We observed four-bond 4∆N(ND) isotope shifts in high-resolution 2D 15N-TROSY experiments of the perdeuterated proteins that correlate with the torsional angle psi. Three-bond 3∆C'(ND) isotope shifts detected in H(N)CO spectra correlate with the intraresidue H-O distance, and to a lesser extent with the dihedral angle phi. The conformational dependence of the isotope shifts agree with those previously reported in the literature. Both the 4∆N(ND) and 3∆C'(ND) isotope shifts are sensitive to distances between the atoms giving rise to the isotope shifts and the atoms experiencing the splitting, however, these distances are strongly correlated with backbone dihedral angles making it difficult to resolve distance from stereochemical contributions to the isotope shift. H(NCA)CO spectra were used to measure two-bond 2∆C'(ND) isotope shifts and [D]/[H] fractionation factors. Neither parameter showed significant differences for hydrogen-bonded sites, or changes over a 25° temperature range, suggesting they are not sensitive to hydrogen bonding. Finally, the quartet that arises from the combination of 2∆C'(ND) and 3∆C'(ND) isotope shifts in H(CA)CO spectra was used to measure synchronized hydrogen exchange for the sequence neighbors A315-S316 in the protein CUS-3iD. In many of our experiments we observed minor resonances due to the 10% D2O used for the sample deuterium lock, indicating isotope shifts can be a source of spectral heterogeneity in standard NMR experiments. We suggest that applications of isotope shifts such as conformational analysis and correlated hydrogen exchange could benefit from the larger magnetic fields becoming available.

SARS-CoV-2 Envelope Protein Forms Clustered Pentamers in Lipid Bilayers.

The SARS-CoV-2 envelope (E) protein is a viroporin associated with the acute respiratory symptoms of COVID-19. E forms cation-selective ion channels that assemble in the lipid membrane of the endoplasmic reticulum Golgi intermediate compartment. The channel activity of E is linked to the inflammatory response of the host cell to the virus. Like many viroporins, E is thought to oligomerize with a well-defined stoichiometry. However, attempts to determine the E stoichiometry have led to inconclusive results and suggested mixtures of oligomers whose exact nature might vary with the detergent used. Here, we employ 19F solid-state nuclear magnetic resonance and the centerband-only detection of exchange (CODEX) technique to determine the oligomeric number of E's transmembrane domain (ETM) in lipid bilayers. The CODEX equilibrium value, which corresponds to the inverse of the oligomeric number, indicates that ETM assembles into pentamers in lipid bilayers, without any detectable fraction of low-molecular-weight oligomers. Unexpectedly, at high peptide concentrations and in the presence of the lipid phosphatidylinositol, the CODEX data indicate that more than five 19F spins are within a detectable distance of about 2 nm, suggesting that the ETM pentamers cluster in the lipid bilayer. Monte Carlo simulations that take into account peptide-peptide and peptide-lipid interactions yielded pentamer clusters that reproduced the CODEX data. This supramolecular organization is likely important for E-mediated virus assembly and budding and for the channel function of the protein.

Binding Sites of a Positron Emission Tomography Imaging Agent in Alzheimer's ß-Amyloid Fibrils Studied Using 19F Solid-State NMR.

Amyloid imaging by positron emission tomography (PET) is an important method for diagnosing neurodegenerative disorders such as Alzheimer's disease. Many 11C- and 18F-labeled PET tracers show varying binding capacities, specificities, and affinities for their target proteins. The structural basis of these variations is poorly understood. Here we employ 19F and 13C solid-state NMR to investigate the binding sites of a PET ligand, flutemetamol, to the 40-residue Alzheimer's ß-amyloid peptide (Aß40). Analytical high-performance liquid chromatography and 19F NMR spectra show that flutemetamol binds the current Aß40 fibril polymorph with a stoichiometry of one ligand per four to five peptides. Half of the ligands are tightly bound while the other half are loosely bound. 13C and 15N chemical shifts indicate that this Aß40 polymorph has an immobilized N-terminus, a non-ß-sheet His14, and a non-ß-sheet C-terminus. We measured the proximity of the ligand fluorine to peptide residues using 19F-13C and 19F-1H rotational-echo double-resonance (REDOR) experiments. The spectra show that three segments in the peptide, 12VHH14, 18VFF20, and 39VV40, lie the closest to the ligand. REDOR-constrained docking simulations indicate that these three segments form multiple binding sites, and the ligand orientations and positions at these sites are similar across different Aß polymorphs. Comparison of the flutemetamol-interacting residues in Aß40 with the small-molecule binding sites in other amyloid proteins suggest that conjugated aromatic compounds preferentially bind ß-sheet surface grooves lined by aromatic, polar, and charged residues. These motifs may explain the specificity of different PET tracers to different amyloid proteins.

pH- and Calcium-Dependent Aromatic Network in the SARS-CoV-2 Envelope Protein.

The envelope (E) protein of the SARS-CoV-2 virus is a membrane-bound viroporin that conducts cations across the endoplasmic reticulum Golgi intermediate compartment (ERGIC) membrane of the host cell to cause virus pathogenicity. The structure of the closed state of the E transmembrane (TM) domain, ETM, was recently determined using solid-state NMR spectroscopy. However, how the channel pore opens to mediate cation transport is unclear. Here, we use 13C and 19F solid-state NMR spectroscopy to investigate the conformation and dynamics of ETM at acidic pH and in the presence of calcium ions, which mimic the ERGIC and lysosomal environment experienced by the E protein in the cell. Acidic pH and calcium ions increased the conformational disorder of the N- and C-terminal residues and also increased the water accessibility of the protein, indicating that the pore lumen has become more spacious. ETM contains three regularly spaced phenylalanine (Phe) residues in the center of the peptide. 19F NMR spectra of para-fluorinated Phe20 and Phe26 indicate that both residues exhibit two sidechain conformations, which coexist within each channel. These two Phe conformations differ in their water accessibility, lipid contact, and dynamics. Channel opening by acidic pH and Ca2+ increases the population of the dynamic lipid-facing conformation. These results suggest an intricate aromatic network that regulates the opening of the ETM channel pore. This aromatic network may be a target for E inhibitors against SARS-CoV-2 and related coronaviruses.

Solid-State NMR 19F-1H-15N Correlation Experiments for Resonance Assignment and Distance Measurements of Multifluorinated Proteins.

Several solid-state NMR techniques have been introduced recently to measure nanometer distances involving 19F, whose high gyromagnetic ratio makes it a potent nuclear spin for structural investigation. These solid-state NMR techniques either use 19F correlation with 1H or 13C to obtain qualitative interatomic contacts or use the rotational-echo double-resonance (REDOR) pulse sequence to measure quantitative distances. However, no NMR technique is yet available for disambiguating 1H-19F distances in multiply fluorinated proteins and protein-ligand complexes. Here, we introduce a three-dimensional (3D) 19F-15N-1H correlation experiment that resolves the distances of multiple fluorines to their adjacent amide protons. We show that optimal polarization transfer between 1H and 19F spins is achieved using an out-and-back 1H-19F REDOR sequence. We demonstrate this 3D correlation experiment on the model protein GB1 and apply it to the multidrug-resistance transporter, EmrE, complexed to a tetrafluorinated substrate. This technique should be useful for resolving and assigning distance constraints in multiply fluorinated proteins, leading to significant savings of time and precious samples compared to producing several singly fluorinated samples. Moreover, the method enables structural determination of protein-ligand complexes for ligands that contain multiple fluorines.

In vitro 0N4R tau fibrils contain a monomorphic ß-sheet core enclosed by dynamically heterogeneous fuzzy coat segments.

Misfolding of the microtubule-binding protein tau into filamentous aggregates is characteristic of many neurodegenerative diseases such as Alzheimer's disease and progressive supranuclear palsy. Determining the structures and dynamics of these tau fibrils is important for designing inhibitors against tau aggregation. Tau fibrils obtained from patient brains have been found by cryo-electron microscopy to adopt disease-specific molecular conformations. However, in vitro heparin-fibrillized 2N4R tau, which contains all four microtubule-binding repeats (4R), was recently found to adopt polymorphic structures. Here we use solid-state NMR spectroscopy to investigate the global fold and dynamics of heparin-fibrillized 0N4R tau. A single set of 13C and 15N chemical shifts was observed for residues in the four repeats, indicating a single ß-sheet conformation for the fibril core. This rigid core spans the R2 and R3 repeats and adopts a hairpin-like fold that has similarities to but also clear differences from any of the polymorphic 2N4R folds. Obtaining a homogeneous fibril sample required careful purification of the protein and removal of any proteolytic fragments. A variety of experiments and polarization transfer from water and mobile side chains indicate that 0N4R tau fibrils exhibit heterogeneous dynamics: Outside the rigid R2-R3 core, the R1 and R4 repeats are semirigid even though they exhibit ß-strand character and the proline-rich domains undergo large-amplitude anisotropic motions, whereas the two termini are nearly isotropically flexible. These results have significant implications for the structure and dynamics of 4R tau fibrils in vivo.

PLG-007 and Its Active Component Galactomannan-α Competitively Inhibit Enzymes That Hydrolyze Glucose Polymers.

PLG-007 is a developmental therapeutic compound that has been clinically shown to reduce the magnitude of postprandial glucose excursions and has the potential to be an adjunct treatment for diabetes and inflammatory-related diseases. The present investigation is aimed at understanding the molecular mechanism of action of PLG-007 and its galactomannan (GM) components GMα and GMß (in a 1:4 mass ratio, respectively) on enzyme (i.e., α-amylase, maltase, and lactase) hydrolysis of glucose polymers using colorimetric assays and 13C HSQC NMR spectroscopy. The starch-iodine colorimetric assay indicated that GMα strongly inhibits α-amylase activity (~16-fold more potent than GMß) and thus is the primary active component in PLG-007. 13C HSQC experiments, used to follow the α-amylase-mediated hydrolysis of starch and amylopectin, further demonstrate the α-amylase inhibitory effect of GMα via α-amylase-mediated hydrolysis of starch and amylopectin. Maltohexaose (MT6) was used to circumvent the relative kinetic complexity of starch/amylopectin degradation in Michaelis-Menten analyses. The Vmax, KM, and Ki parameters were determined using peak volume integrals from 13C HSQC NMR spectra. In the presence of PLG-007 with α-amylase and MT6, the increase in KM from 7.5 ± 0.6 × 10-3 M (control) to 21 ± 1.4 × 10-3 M, with no significant change in Vmax, indicates that PLG-007 is a competitive inhibitor of α-amylase. Using KM values, Ki was estimated to be 2.1 ± 0.9 × 10-6 M; however, the microscopic Ki value of GMα is expected to be larger as the binding stoichiometry is likely to be greater than 1:1. Colorimetric assays also demonstrated that GMα is a competitive inhibitor of the enzymes maltase and lactase. Overall, this study provides insight as to how PLG-007 (GMα) is likely to function in vivo.

Structurally Based Design of Glucagon Mutants That Inhibit Fibril Formation.

The peptide hormone glucagon is prescribed as a pharmaceutical compound to treat diabetic hypoglycemia. However, at the acidic pH where it is highly soluble, glucagon rapidly aggregates into inactive and cytotoxic amyloid fibrils. The recently determined high-resolution structure of these fibrils revealed various stabilizing molecular interactions. On the basis of this structure, we have now designed four arginine mutants of glucagon that resist fibrillization at pharmaceutical concentrations for weeks. An S2R, T29R double mutant and a T29R single mutant remove a hydrogen-bonding interaction in the wild-type fibril, whereas a Y13R, A19R double mutant and a Y13R mutant remove a cation-π interaction. 1H solution nuclear magnetic resonance spectra and ultraviolet absorbance data indicate that these mutants remain soluble in pH 2 buffer under quiescent conditions at concentrations of ≤4 mg/mL for weeks. Under stressed conditions with high salt concentrations and agitation, these mutants fibrillize significantly more slowly than the wild type. The S2R, T29R mutant and the T29R mutant exhibit a mixture of random coil and α-helical conformations, while the Y13R mutant is completely random coil. The mutation sites are chosen to be uninvolved in strong interactions with the glucagon receptor in the active structure of the peptide. Therefore, these arginine mutants of glucagon are promising alternative compounds for treating hypoglycemia.

Inclusion of the C-Terminal Domain in the ß-Sheet Core of Heparin-Fibrillized Three-Repeat Tau Protein Revealed by Solid-State Nuclear Magnetic Resonance Spectroscopy.

Many neurodegenerative diseases such as Alzheimer's disease are characterized by pathological ß-sheet filaments of the tau protein, which spread in a prion-like manner in patient brains. To date, high-resolution structures of tau filaments obtained from patient brains show that the ß-sheet core only includes portions of the microtubule-binding repeat domains and excludes the C-terminal residues, indicating that the C-terminus is dynamically disordered. Here, we use solid-state NMR spectroscopy to identify the ß-sheet core of full-length 0N3R tau fibrillized using heparin. Assignment of 13C and 15N chemical shifts of the rigid core of the protein revealed a single predominant ß-sheet conformation, which spans not only the R3, R4, R' repeats but also the entire C-terminal domain (CT) of the protein. This massive ß-sheet core qualitatively differs from all other tau fibril structures known to date. Using long-range correlation NMR experiments, we found that the R3 and R4 repeats form a ß-arch, similar to that seen in some of the brain-derived tau fibrils, but the R1 and R3 domains additionally stack against the CT, reminiscent of previously reported transient interactions of the CT with the microtubule-binding repeats. This expanded ß-sheet core structure suggests that the CT may have a protective effect against the formation of pathological tau fibrils by shielding the amyloidogenic R3 and R4 domains, preventing side-on nucleation. Truncation and post-translational modification of the CT in vivo may thus play an important role in the progression of tauopathies.

Hydration and Dynamics of Full-Length Tau Amyloid Fibrils Investigated by Solid-State Nuclear Magnetic Resonance.

The microtubule-associated protein tau aggregates into distinct neurofibrillary tangles in brains afflicted with multiple neurodegenerative diseases such as Alzheimer's disease and corticobasal degeneration (CBD). The mechanism of tau misfolding and aggregation is poorly understood. Determining the structure, dynamics, and water accessibility of tau filaments may provide insight into the pathway of tau misfolding. Here, we investigate the hydration and dynamics of the ß-sheet core of heparin-fibrillized 0N4R tau using solid-state nuclear magnetic resonance spectroscopy. This ß-sheet core consists of the second and third microtubule-binding repeats, R2 and R3, respectively, which form a hairpin. Water-edited two-dimensional (2D) 13C-13C and 15N-13C correlation spectra show that most residues in R2 and R3 domains have low water accessibility, indicating that this hairpin is surrounded by other proteinaceous segments. However, a small number of residues, especially S285 and S316, are well hydrated compared to other Ser and Thr residues, suggesting that there is a small water channel in the middle of the hairpin. To probe whether water accessibility correlates with protein dynamics, we measured the backbone N-H dipolar couplings of the ß-sheet core. Interestingly, residues in the fourth microtubule-binding repeat, R4, show rigid-limit N-H dipolar couplings, even though this domain exhibits weaker intensities in the 2D 15N-13C correlation spectra. These results suggest that the R4 domain participates in cross-ß hydrogen bonding in some of the subunits but exhibits dynamic disorder in other subunits. Taken together, these hydration and dynamics data indicate that the R2-R3 hairpin of 0N4R tau is shielded from water by other proteinaceous segments on the exterior but contains a small water pore in the interior. This structural topology has various similarities with the CBD tau fibril structure but also shows specific differences. The disorder of the R4 domain and the presence of a small water channel in the heparin-fibrillized 4R tau have implications for the structure of tau fibrils in diseased brains.

Amyloid fibril structures of tau: Conformational plasticity of the second microtubule-binding repeat.

The intrinsically disordered protein tau associates with microtubules in neurons but aggregates into cross-ß amyloid fibrils that propagate in neurodegenerative brains. Different tauopathies have different structures for the rigid fibril core. To understand the molecular basis of tau assembly into different polymorphs, here we use solid-state nuclear magnetic resonance (NMR) spectroscopy to determine the structure of a tau protein that includes all microtubule-binding repeats and a proline-rich domain. This P2R tau assembles into well-ordered filaments when induced by heparin. Two- and three-dimensional NMR spectra indicate that R2 and R3 repeats constitute the rigid ß-sheet core of the fibril. Unexpectedly, the amino-terminal half of R2 forms a ß-arch at ambient temperature (24°C) but a continuous ß-strand at 12°C, which dimerizes with the R2 of another protofilament. This temperature-dependent structure indicates that R2 is conformationally more plastic than the R3 domain. The distinct conformational stabilities of different microtubule-binding repeats give insight into the energy landscape of tau fibril formation.

Rapid Determination of the Topology of Oligomeric α-Helical Membrane Proteins by Water- and Lipid-Edited Methyl NMR.

Single-span oligomeric α-helical transmembrane proteins are common in virus ion channels, which are targets of antiviral drugs. Knowledge about the high-resolution structures of these oligomeric α-helical bundles is so far scarce. Structure determination of these membrane proteins by solid-state NMR traditionally requires resolving and assigning protein chemical shifts and measuring many interhelical distances, which are time-consuming. To accelerate experimental structure determination, here we introduce a simple solid-state NMR approach that uses magnetization transfer from water and lipid protons to the protein. By detecting the water- and lipid-transferred intensities of the high-sensitivity methyl 13C signals of Leu, Val, and Ile residues, which are highly enriched in these membrane proteins, we can derive models of the topology of these homo-oligomeric helical bundles. The topology is specified by the positions of amino acid residues in heptad repeats and the orientations of residues relative to the channel pore, lipids, and the helical interface. We demonstrate this water- and lipid-edited methyl NMR approach on the envelope (E) protein of SARS-CoV-2, the causative agent of the COVID-19 pandemic. We show that water-edited and lipid-edited 2D 13C-13C correlation spectra can be measured with sufficient sensitivity. Even without resolving multiple residues of the same type in the NMR spectra, we can obtain the helical bundle topology. We apply these experiments to the structurally unknown E proteins of the MERS coronavirus and the human coronavirus NL63. The resulting structural topologies show interesting differences in the positions of the aromatic residues in these three E proteins, suggesting that these viroporins may have different mechanisms of activation and ion conduction.

Atomic structure of the open SARS-CoV-2 E viroporin.

The envelope (E) protein of the SARS-CoV-2 virus forms cation-conducting channels in the endoplasmic reticulum Golgi intermediate compartment (ERGIC) of infected cells. The calcium channel activity of E is associated with the inflammatory responses of COVID-19. Using solid-state NMR (ssNMR) spectroscopy, we have determined the open-state structure of E's transmembrane domain (ETM) in lipid bilayers. Compared to the closed state, open ETM has an expansive water-filled amino-terminal chamber capped by key glutamate and threonine residues, a loose phenylalanine aromatic belt in the middle, and a constricted polar carboxyl-terminal pore filled with an arginine and a threonine residue. This structure gives insights into how protons and calcium ions are selected by ETM and how they permeate across the hydrophobic gate of this viroporin.

The Cytoplasmic Domain of the SARS-CoV-2 Envelope Protein Assembles into a ß-Sheet Bundle in Lipid Bilayers.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) envelope (E) protein forms a pentameric ion channel in the lipid membrane of the endoplasmic reticulum Golgi intermediate compartment (ERGIC) of the infected cell. The cytoplasmic domain of E interacts with host proteins to cause virus pathogenicity and may also mediate virus assembly and budding. To understand the structural basis of these functions, here we investigate the conformation and dynamics of an E protein construct (residues 8-65) that encompasses the transmembrane domain and the majority of the cytoplasmic domain using solid-state NMR. 13C and 15N chemical shifts indicate that the cytoplasmic domain adopts a ß-sheet-rich conformation that contains three ß-strands separated by turns. The five subunits associate into an umbrella-shaped bundle that is attached to the transmembrane helices by a disordered loop. Water-edited NMR spectra indicate that the third ß-strand at the C terminus of the protein is well hydrated, indicating that it is at the surface of the ß-bundle. The structure of the cytoplasmic domain cannot be uniquely determined from the inter-residue correlations obtained here due to ambiguities in distinguishing intermolecular and intramolecular contacts for a compact pentameric assembly of this small domain. Instead, we present four structural topologies that are consistent with the measured inter-residue contacts. These data indicate that the cytoplasmic domain of the SARS-CoV-2 E protein has a strong propensity to adopt ß-sheet conformations when the protein is present at high concentrations in lipid bilayers. The equilibrium between the ß-strand conformation and the previously reported α-helical conformation may underlie the multiple functions of E in the host cell and in the virion.

Microtubule-binding core of the tau protein.

The protein tau associates with microtubules to maintain neuronal health. Posttranslational modifications of tau interfere with this binding, leading to tau aggregation in neurodegenerative disorders. Here, we use solid-state nuclear magnetic resonance (NMR) to investigate the structure of the microtubule-binding domain of tau. Wild-type tau that contains four microtubule-binding repeats and a pseudorepeat R' is studied. Complexed with taxol-stabilized microtubules, the immobilized residues exhibit well-resolved two-dimensional spectra that can be assigned to the amino-terminal region of R4 and the R' domain. When tau coassembles with tubulin to form unstable microtubules, the R' signals remain, whereas the R4 signals disappear, indicating that R' remains immobilized, whereas R4 becomes more mobile. Therefore, R' outcompetes the other four repeats to associate with microtubules. These NMR data, together with previous cryo-electron microscopy densities, indicate an extended conformation for microtubule-bound R'. R' contains the largest number of charged residues among all repeats, suggesting that charge-charge interaction drives tau-microtubule association.