An essential protein of the SARS-CoV-2 virus, the envelope protein E, forms a homopentameric cation channel that is important for virus pathogenicity. Here we report a 2.1 Å structure and the drug-binding site of E’s transmembrane domain (ETM), determined using solid-state NMR spectroscopy. In lipid bilayers that mimic the endoplasmic-reticulum Golgi intermediate compartment (ERGIC) membrane, ETM forms a five-helix bundle surrounding a narrow pore. The protein deviates from the ideal α-helical geometry due to three phenylalanine residues, which stack within each helix and between helices. Together with valine and leucine interdigitation, these cause a dehydrated pore compared to viroporins of influenza and HIV viruses. Hexamethylene amiloride binds the polar N-terminal lumen whereas acidic pH affects the C-terminal conformation. Thus, the N- and C-terminal halves of this bipartite channel may interact with other viral and host proteins semi-independently. The structure sets the stage for designing E inhibitors as antiviral drugs.

Internuclear distances measured using NMR provide crucial constraints of three-dimensional structures but are often restricted to about 5 Å due to the weakness of nuclear-spin dipolar couplings. For studying macromolecular assemblies in biology and materials science, distance constraints beyond 1 nm will be extremely valuable. Here we present an extensive and quantitative analysis of the feasibility of F spin exchange NMR for precise and robust measurements of interatomic distances up to 1.6 nm at a magnetic field of 14.1 T, under 20-40 kHz magic-angle spinning (MAS). The measured distances are comparable to those achievable from paramagnetic relaxation enhancement but have higher precision, which is better than ±1 Å for short distances and ±2 Å for long distances. ForF spins with the same isotropic chemical shift but different anisotropic chemical shifts, intermediate MAS frequencies of 15-25 kHz without H irradiation accelerate spin exchange. For spectrally resolvedF-F spin exchange, H-F dipolar recoupling significantly speeds up F-F spin exchange. On the basis of data from five fluorinated synthetic, pharmaceutical, and biological compounds, we obtained two general curves for spin exchange between CF groups and between CF and CF groups. These curves allow F-F distances to be extracted from the measured spin exchange rates after taking into account F chemical shifts. These results demonstrate the robustness ofF spin exchange NMR for distance measurements in a wide range of biological and chemical systems.

The influenza B M2 (BM2) proton channel is activated by acidic pH to mediate virus uncoating. Unlike influenza A M2 (AM2), which conducts protons with strong inward rectification, BM2 conducts protons both inward and outward. Here we report 1.4- and 1.5-angstrom solid-state NMR structures of the transmembrane domain of the closed and open BM2 channels in phospholipid environment. Upon activation, the transmembrane helices increase the tilt angle by 6˚ and the average pore diameter enlarges by 2.1 Å. BM2 thus undergoes a scissor motion for activation, which differs from the alternating-access motion of AM2. These results indicate that asymmetric proton conduction requires a backbone hinge motion whereas bidirectional conduction is achieved by a symmetric scissor motion. The proton-selective histidine and gating tryptophan in the open BM2 reorient on the microsecond timescale, similar to AM2, indicating that sidechain dynamics are the essential driver of proton shuttling.

The ability to simultaneously measure many long-range distances is critical to efficient and accurate determination of protein structures by solid-state NMR (SSNMR). So far, the most common distance constraints for proteins are C-N distances, which are usually measured using the rotational-echo double-resonance (REDOR) technique. However, these measurements are restricted to distances of up to ~ 5 Å due to the low gyromagnetic ratios of N andC. Here we present a robust 2D C-F REDOR experiment to measure multiple distances to ~ 10 Å. The technique targets proteins that contain a small number of recombinantly or synthetically incorporated fluorines. The C-F REDOR sequence is combined with 2D C-C correlation to resolve multiple distances in highly C-labeled proteins. We show that, at the high magnetic fields which are important for obtaining well resolvedC spectra, the deleterious effect of the large F chemical shift anisotropy for REDOR is ameliorated by fast magic-angle spinning and is further taken into account in numerical simulations. We demonstrate this 2DC-C resolved C-F REDOR technique on C,N-labeled GB1. A 5-F-Trp tagged GB1 sample shows the extraction of distances to a single fluorine atom, while a 3-F-Tyr labeled GB1 sample allows us to evaluate the effects of multi-spin coupling and statistical F labeling on distance measurement. Finally, we apply this 2D REDOR experiment to membrane-bound influenza B M2 transmembrane peptide, and show that the distance between the proton-selective histidine residue and the gating tryptophan residue differs from the distances in the solution NMR structure of detergent-bound BM2. This 2DC-F REDOR technique should facilitate SSNMR-based protein structure determination by increasing the measurable distances to the ~ 10 Å range.

The dimeric transporter, EmrE, effluxes polyaromatic cationic drugs in a proton-coupled manner to confer multidrug resistance in bacteria. Although the protein is known to adopt an antiparallel asymmetric topology, its high-resolution drug-bound structure is so far unknown, limiting our understanding of the molecular basis of promiscuous transport. Here we report an experimental structure of drug-bound EmrE in phospholipid bilayers, determined using 19F and 1H solid-state NMR and a fluorinated substrate, tetra(4-fluorophenyl) phosphonium (F4-TPP+). The drug-binding site, constrained by 214 protein-substrate distances, is dominated by aromatic residues such as W63 and Y60, but is sufficiently spacious for the tetrahedral drug to reorient at physiological temperature. F4-TPP+ lies closer to the proton-binding residue E14 in subunit A than in subunit B, explaining the asymmetric protonation of the protein. The structure gives insight into the molecular mechanism of multidrug recognition by EmrE and establishes the basis for future design of substrate inhibitors to combat antibiotic resistance.