Structural Basis for Asymmetric Conductance of the Influenza M2 Proton Channel Investigated by Solid-State NMR Spectroscopy

The influenza M2 protein forms an acid-activated proton channel that is essential for virus replication. The transmembrane H37 selects for protons under low external pH while W41 ensures proton conduction only from the N terminus to the C terminus and prevents reverse current under low internal pH. Here, we address the molecular basis for this asymmetric conduction by investigating the structure and dynamics of a mutant channel, W41F, which permits reverse current under low internal pH. Solid-state NMR experiments show that W41F M2 retains the pH-dependent α-helical conformations and tetrameric structure of the wild-type (WT) channel but has significantly altered protonation and tautomeric equilibria at H37. At high pH, the H37 structure is shifted toward the π tautomer and less cationic tetrads, consistent with faster forward deprotonation to the C terminus. At low pH, the mutant channel contains more cationic tetrads than the WT channel, consistent with faster reverse protonation from the C terminus.15N NMR spectra allow the extraction of four H37 pKas and show that the pKas are more clustered in the mutant channel compared to WT M2. Moreover, binding of the antiviral drug, amantadine, at the N-terminal pore at low pH did not convert all histidines to the neutral state, as seen in WT M2, but left half of all histidines cationic, unambiguously demonstrating C-terminal protonation of H37 in the mutant. These results indicate that asymmetric conduction in WT M2 is due to W41 inhibition of C-terminal acid activation by H37. When Trp is replaced by Phe, protons can be transferred to H37 bidirectionally with distinct rate constants. Keywords: magic-angle-spinning NMR; tautomeric equilibrium; proton dissociation equilibrium; ion channels; gatingNational Institutes of Health (U.S.) (Grant GM088204

Structure and dynamics of influenza M2 proton channels from solid-state NMR

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemistry, February, 2021Cataloged from the official PDF of thesis.Includes bibliographical references.The A and B strains of influenza virus places a substantial burden on health, causing around 30 million illnesses, several hundred thousand hospitalizations, and a few tens of thousands of deaths every year in the United States alone. Developing novel antiviral and vaccines against influenza requires understanding the proteins employed by these infectious virions. The matrix-2 protein (M2) is an essential viral protein that conducts protons in the endosomes of infected host cells and induces membrane curvature to facilitate virus budding. M2's proton channel activity is encapsulated by a transmembrane domain (TM) that is targeted by the FDA-approved antiviral drugs amantadine and rimantadine to inhibit viral replication. Proton conduction by M2's TM is mediated by a His-xxx-Trp motif conserved in the otherwise disparate M2 from influenza A (AM2) and influenza B (BM2) strains.The histidine selects for protons and activates the channel at low pH, while the tryptophan is responsible for gating and unidirectional conduction from the N-terminus (outside) to the C-terminus (inside). M2 conducts protons across lipid bilayers at a moderate rate of ca. 10-1000 s⁻¹. AM2 is well characterized and several high-resolution structures in a variety of membrane and membrane-mimetic environments are available, yet the mechanism of gating and the rate-limiting step of proton conduction are unknown. A gating-deficient mutant was utilized to determine that asymmetric conduction in AM2 is due to tryptophan blocking activation of histidine from the C-terminal side under low pH. Further, in phospholipid bilayers, AM2 shows two discrete conformations that interconvert on the proton conduction timescale, providing the first experimental evidence for a transporter-like mechanism.In contrast to AM2, BM2 is relatively poorly studied and no high-resolution structures in membranes is available. BM2 shares little sequence homology with AM2, is not inhibited by the antiviral drugs targeting influenza, and conducts protons faster but more bidirectionally than AM2. The first high-resolution structures of membrane-embedded BM2 in the closed and open states are reported. In contrast to the transporter-like motion of AM2, BM2 undergoes a subtler channel-like scissor opening motion, that allows for more efficient proton conduction at the expense of some bidirectional current.by Venkata Shiva Mandala.Ph. D.Ph.D. Massachusetts Institute of Technology, Department of Chemistr

High-sensitivity protein solid-state NMR spectroscopy

© 2019 Elsevier Ltd The sensitivity of solid-state nuclear magnetic resonance (SSNMR) spectroscopy for structural biology is significantly increased by 1H detection under fast magic-angle spinning (MAS) and by dynamic nuclear polarization (DNP) from electron spins to nuclear spins. The former allows studies of the structure and dynamics of small quantities of proteins under physiological conditions, while the latter permits studies of large biomolecular complexes in lipid membranes and cells, protein intermediates, and protein conformational distributions. We highlight recent applications of these two emerging SSNMR technologies and point out areas for future development

Determination of long-range distances by fast magic-angle-spinning radiofrequency-driven 19 F-19 F dipolar recoupling NMR

Nanometer-range distances are important for restraining the three-dimensional structure and oligomeric assembly of proteins and other biological molecules. Solid-state NMR determination of protein structures typically utilizes 13C-13C and 13C-15N distance restraints, which can only be measured up to ∼7 Å because of the low gyromagnetic ratios of these nuclear spins. To extend the distance reach of NMR, one can harvest the power of 19F, whose large gyromagnetic ratio in principle allows distances up to 2 nm to be measured. However, 19F possesses large chemical shift anisotropies (CSAs) as well as large isotropic chemical shift dispersions, which pose challenges to dipolar coupling measurements. Here, we demonstrate 19F-19F distance measurements at high magnetic fields under fast magic-angle spinning (MAS) using radiofrequency-driven dipolar recoupling (RFDR). We show that 19F-19F cross-peaks for distances up to 1 nm can be readily observed in two-dimensional 19F-19F correlation spectra using less than 5 ms of RFDR mixing. This efficient 19F-19F dipolar recoupling is achieved using practically accessible MAS frequencies of 15-55 kHz, moderate 19F radio frequency field strengths, and no 1H decoupling. Experiments and simulations show that the fastest polarization transfer for aromatic fluorines with the highest distance accuracy is achieved using either fast MAS (e.g., 60 kHz) with large pulse duty cycles (>50%) or slow MAS with strong 19F pulses. Fast MAS considerably reduces relaxation losses during the RFDR π-pulse train, making finite-pulse RFDR under fast-MAS the method of choice. Under intermediate MAS frequencies (25-40 kHz) and intermediate pulse duty cycles (15-30%), the 19F CSA tensor orientation has a quantifiable effect on the polarization transfer rate; thus, the RFDR buildup curves encode both distance and orientation information. At fast MAS, the impact of CSA orientation is minimized, allowing pure distance restraints to be extracted. We further investigate how relayed transfer and dipolar truncation in multifluorine environments affect polarization transfer. This fast-MAS 19F RFDR approach is complementary to 19F spin diffusion for distance measurements and will be the method of choice under high-field fast-MAS conditions that are increasingly important for protein structure determination by solid-state NMR.German National Academy of Sciences (grant no. LPDS-2017-14)NIH (grant no. GM088204

Structure and dynamics of membrane proteins from solid-state NMR

Solid-state nuclear magnetic resonance (SSNMR) spectroscopy elucidates membrane protein structures and dynamics in atomic detail to yield mechanistic insights. By interrogating membrane proteins in phospholipid bilayers that closely resemble biological membranes, SSNMR spectroscopists have revealed ion conduction mechanisms, substrate transport dynamics, and oligomeric interfaces of seven-transmembrane helix proteins. Research has also identified conformational plasticity underlying virus-cell membrane fusions by complex protein machineries, and β-sheet folding and assembly by amyloidogenic proteins bound to lipid membranes. These studies collectively show that membrane proteins exhibit extensive structural plasticity to carry out their functions. Because of the inherent dependence of NMR frequencies on molecular orientations and the sensitivity of NMR frequencies to dynamical processes on timescales from nanoseconds to seconds, SSNMR spectroscopy is ideally suited to elucidate such structural plasticity, local and global conformational dynamics, protein-lipid and protein-ligand interactions, and protonation states of polar residues. New sensitivity-enhancement techniques, resolution enhancement by ultrahigh magnetic fields, and the advent of 3D and 4D correlation NMR techniques are increasingly aiding these mechanistically important structural studies

The peptide hormone glucagon forms amyloid fibrils with two coexisting β-strand conformations

Glucagon and insulin maintain blood glucose homeostasis and are used to treat hypoglycemia and hyperglycemia, respectively, in patients with diabetes. Whereas insulin is stable for weeks in its solution formulation, glucagon fibrillizes rapidly at the acidic pH required for solubility and is therefore formulated as a lyophilized powder that is reconstituted in an acidic solution immediately before use. Here we use solid-state NMR to determine the atomic-resolution structure of fibrils of synthetic human glucagon grown at pharmaceutically relevant low pH. Unexpectedly, two sets of chemical shifts are observed, indicating the coexistence of two β-strand conformations. The two conformations have distinct water accessibilities and intermolecular contacts, indicating that they alternate and hydrogen bond in an antiparallel fashion along the fibril axis. Two antiparallel β-sheets assemble with symmetric homodimer cross sections. This amyloid structure is stabilized by numerous aromatic, cation-π, polar and hydrophobic interactions, suggesting mutagenesis approaches to inhibit fibrillization could improve this important drug.National Institutes of Health (U.S.) (Grant AG059661)National Institutes of Health (U.S.). Ruth L. Kirschstein National Research Service Award (1F31AI133989

Water orientation and dynamics in the closed and open influenza B virus M2 proton channels

© 2021, The Author(s). The influenza B M2 protein forms a water-filled tetrameric channel to conduct protons across the lipid membrane. To understand how channel water mediates proton transport, we have investigated the water orientation and dynamics using solid-state NMR spectroscopy and molecular dynamics (MD) simulations. 13C-detected water 1H NMR relaxation times indicate that water has faster rotational motion in the low-pH open channel than in the high-pH closed channel. Despite this faster dynamics, the open-channel water shows higher orientational order, as manifested by larger motionally-averaged 1H chemical shift anisotropies. MD simulations indicate that this order is induced by the cationic proton-selective histidine at low pH. Furthermore, the water network has fewer hydrogen-bonding bottlenecks in the open state than in the closed state. Thus, faster dynamics and higher orientational order of water molecules in the open channel establish the water network structure that is necessary for proton hopping

Structure and dynamics of the drug-bound bacterial transporter EmrE in lipid bilayers

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.NIH (Grants GM066976

In vitro 0N4R tau fibrils contain a monomorphic β-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 [subscript 13]C and [subscript 15]N chemical shifts was observed for residues in the four repeats, indicating a single β-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 β-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.NIH (grant nos. AG059661 and AG002132