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.

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.

Effect of obstructions on growing Turing patterns.

We study how Turing pattern formation on a growing domain is affected by discrete domain discontinuities. We use the Lengyel-Epstein reaction-diffusion model to numerically simulate Turing pattern formation on radially expanding circular domains containing a variety of obstruction geometries, including obstructions spanning the length of the domain, such as walls and slits, and local obstructions, such as small blocks. The pattern formation is significantly affected by the obstructions, leading to novel pattern morphologies. We show that obstructions can induce growth mode switching and disrupt local pattern formation and that these effects depend on the shape and placement of the objects as well as the domain growth rate. This work provides a customizable framework to perform numerical simulations on different types of obstructions and other heterogeneous domains, which may guide future numerical and experimental studies. These results may also provide new insights into biological pattern growth and formation, especially in non-idealized domains containing noise or discontinuities.

Comparative analysis of 13C chemical shifts of ß-sheet amyloid proteins and outer membrane proteins.

Cross-ß amyloid fibrils and membrane-bound ß-barrels are two important classes of ß-sheet proteins. To investigate whether there are systematic differences in the backbone and sidechain conformations of these two families of proteins, here we analyze the 13C chemical shifts of 17 amyloid proteins and 7 ß-barrel membrane proteins whose high-resolution structures have been determined by NMR. These 24 proteins contain 373 ß-sheet residues in amyloid fibrils and 521 ß-sheet residues in ß-barrel membrane proteins. The 13C chemical shifts are shown in 2D 13C-13C correlation maps, and the amino acid residues are categorized by two criteria: (1) whether they occur in ß-strand segments or in loops and turns; (2) whether they are water-exposed or dry, facing other residues or lipids. We also examine the abundance of each amino acid in amyloid proteins and ß-barrels and compare the sidechain rotameric populations. The 13C chemical shifts indicate that hydrophobic methyl-rich residues and aromatic residues exhibit larger static sidechain conformational disorder in amyloid fibrils than in ß-barrels. In comparison, hydroxyl- and amide-containing polar residues have more ordered sidechains and more ordered backbones in amyloid fibrils than in ß-barrels. These trends can be explained by steric zipper interactions between ß-sheet planes in cross-ß fibrils, and by the interactions of ß-barrel residues with lipid and water in the membrane. These conformational trends should be useful for structural analysis of amyloid fibrils and ß-barrels based principally on NMR chemical shifts.

Turing patterns on radially growing domains: experiments and simulations.

We study Turing pattern formation in a system undergoing radial growth in two dimensions. The photosensitive chlorine dioxide-iodine-malonic acid reaction is illuminated to inhibit patterning, with a growing non-illuminated circular domain in which the pattern develops. We examine the relationship between the linear radial growth rate and the resulting pattern morphology. Faster growth causes the pattern to form parallel to the growing boundary as concentric rings, while slower growth leads to pattern formation perpendicular to the growing boundary. We observe three distinct growth modes for the Turing patterns, which also depend on the radial growth rate. The experimental results are qualitatively reproduced in numerical simulations using the Lengyel-Epstein model with an additional term to account for the photosensitivity of the reaction. These results may provide new insight into how patterns form in growing biological systems.

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.

Hexamethylene amiloride binds the SARS-CoV-2 envelope protein at the protein-lipid interface.

The SARS-CoV-2 envelope (E) protein forms a five-helix bundle in lipid bilayers whose cation-conducting activity is associated with the inflammatory response and respiratory distress symptoms of COVID-19. E channel activity is inhibited by the drug 5-(N,N-hexamethylene) amiloride (HMA). However, the binding site of HMA in E has not been determined. Here we use solid-state NMR to measure distances between HMA and the E transmembrane domain (ETM) in lipid bilayers. 13 C, 15 N-labeled HMA is combined with fluorinated or 13 C-labeled ETM. Conversely, fluorinated HMA is combined with 13 C, 15 N-labeled ETM. These orthogonal isotopic labeling patterns allow us to conduct dipolar recoupling NMR experiments to determine the HMA binding stoichiometry to ETM as well as HMA-protein distances. We find that HMA binds ETM with a stoichiometry of one drug per pentamer. Unexpectedly, the bound HMA is not centrally located within the channel pore, but lies on the lipid-facing surface in the middle of the TM domain. This result suggests that HMA may inhibit the E channel activity by interfering with the gating function of an aromatic network. These distance data are obtained under much lower drug concentrations than in previous chemical shift perturbation data, which showed the largest perturbation for N-terminal residues. This difference suggests that HMA has higher affinity for the protein-lipid interface than the channel pore. These results give insight into the inhibition mechanism of HMA for SARS-CoV-2 E.

Transient water wires mediate selective proton transport in designed channel proteins.

Selective proton transport through proteins is essential for forming and using proton gradients in cells. Protons are conducted along hydrogen-bonded 'wires' of water molecules and polar side chains, which, somewhat surprisingly, are often interrupted by dry apolar stretches in the conduction pathways, inferred from static protein structures. Here we hypothesize that protons are conducted through such dry spots by forming transient water wires, often highly correlated with the presence of the excess protons in the water wire. To test this hypothesis, we performed molecular dynamics simulations to design transmembrane channels with stable water pockets interspersed by apolar segments capable of forming flickering water wires. The minimalist designed channels conduct protons at rates similar to viral proton channels, and they are at least 106-fold more selective for H+ over Na+. These studies inform the mechanisms of biological proton conduction and the principles for engineering proton-conductive materials.