The Hong group develops and applies magic-angle-spinning solid-state NMR spectroscopy to elucidate the structure and dynamics of biological macromolecules. We develop multidimensional correlation techniques involving 1H, 13C, 15N, 19F, 2H, and 31P spins to measure protein conformation, inter-atomic distances, and geometry of molecular motions. These methods are driven by important problems in biological chemistry. We are currently studying three classes of biomolecular systems: virus membrane proteins, amyloid fibrils, and plant cell walls. Structural information from our spectroscopic studies provide atomic insights into the mechanisms of influenza virus uncoating and budding, virus-cell membrane fusion, self-assembled structures of amyloid fibrils in diseases and functions, and three-dimensional structures of the polysaccharide networks in plant cell walls.

Influenza virus M2 proteins
            Ion channels and transporters provide conduits for the passage of ions and polar compounds across the hydrophobic barrier of lipid membranes. The influenza M2 protein is a low-pH activated proton channel, which acidifies the endosomally trapped virion to cause virus uncoating. Since 2010 it was discovered that M2 also mediates membrane scission in the last step of virus budding and release. The proton-channel function is mostly carried out by the transmembrane domain (TMD) of the protein, whereas the membrane-scission function is conducted by an amphipathic helix C-terminal to the TMD. We investigate the structure and dynamics of M2 bound to phospholipid bilayers to elucidate the mechanisms of proton conduction and membrane scission. The M2 protein of influenza A viruses, which are responsible for all flu pandemics in history, is also the target of one of two classes of antiviral drugs, but currently circulating seasonal flu viruses have evolved drug-resistant mutations. Influenza B virus M2 is so far not druggable, thus structure elucidation of AM2 and BM2 is important for drug development.

            Using solid-state NMR, we determine the three-dimensional structures and oligomeric assembly of AM2 and BM2 in lipid bilayers, elucidate proton-transfer equilibria (pKa’s) and kinetics (Fig. 1A), investigate how protein sidechain motions and global motions mediate proton transfer (Fig. 1B), elucidate the drug-binding equilibria (Fig. 1C), and investigate the structures of protein-cholesterol complex (Fig. 1D) and protein-protein complexes. This wealth of information is obtained from chemical shifts, long-range intermolecular distances, and motionally averaged NMR spectra.

Figure 1. Influenza M2 structure and dynamics investigated using solid-state NMR. (A) Influenza BM2 has a distinct TMD sequence from AM2, and contains two histidine residues in the TMD. 15N NMR spectra show that the proton dissociation constants (pKa’s) of the proton-selective His19 differ significantly from the pKa’s of the corresponding histidine in AM2 due to the influence of the peripheral histidine residue, His27 1,2. (B) 2D 13C-13C correlation spectra reveal millisecond-timescale motions of the TMD at high temperature, demonstrating that the rate-limiting step of proton conduction is a conformational change between a closed and open form of the four-helix bundle 3.  (C) The antiviral drug amantadine binds M2 constructs containing the intact cytoplasmic domain, but does not bind a shorter construct comprising only the TMD and an amphipathic helix. This anomalous loss of drug binding can be attributed to the curvature-inducing function of the amphipathic helix4. (D) Cholesterol-complexed structure of M2 to mediate membrane scission during virus budding. The structure is determined using 13C-19F distance measurements between the protein and cholesterol 5.
Viral Fusion Proteins
            Membrane curvature is essential to many biological processes such as endocytosis, vesicle trafficking, and cell division. Proteins can sense, stabilize, and induce membrane curvature. An important class of membrane proteins that induce membrane curvature is viral fusion proteins, which merge the virus envelope and the target membrane to enable virus entry into cells. They accomplish this task by undergoing complex conformational rearrangements, as seen in X-ray crystal structures of water-soluble ectodomains of these proteins (Fig. 2A). These protein conformational changes presumably lower the free energy barriers for membrane dehydration and membrane structural changes from the lamellar state to hemifused intermediates to the final fused state. However, this conceptual framework excludes two key hydrophobic domains: the N-terminal fusion peptide (FP) domain and the C-terminal transmembrane domain (TMD), which play important roles in destabilizing the lamellar structures of the two lipid membranes.
            Using solid-state NMR spectroscopy and complementary techniques such as small-angle X-ray scattering (SAXS), we investigate the conformations and oligomeric structures of the hydrophobic FP and TMD of viral fusion proteins in biologically relevant lipid membranes. Our studies of the parainfluenza virus fusion protein indicate that both the FP and the TMD have membrane-dependent structures (Fig. 2B), and the β-sheet conformation is responsible for generating saddle-splay curvature to the membrane. The latter is manifested in 31P NMR spectra (Fig. 2C) and SAXS data. We also measure water-lipid and water-protein interactions to obtain information about membrane dehydration during fusion. By coupling these protein structure measurements with membrane morphology experiments, we obtain comprehensive information about the protein and membrane structural changes along the fusion pathway (Fig. 2D). We are also investigating the structure of the HIV fusion protein, gp41.

Figure 2. Solid-state NMR studies of virus-cell fusion. (A) Virus-cell fusion model. The fusion protein undergoes a series of conformational changes to transition from a compact prefusion structure to a membrane-bound post-fusion structure. The N-terminal fusion peptide is encapsulated in the globular head in the prefusion state but inserts into the target cell membrane in the extended intermediate state, while the C-terminal TMD is anchored in the viral envelope. Hairpin formation by the protein ectodomain pulls the cell membrane and the virus envelope together, causing putative membrane intermediates such as the hemifusion diaphragm. Action of the FP and TMD eventually causes full merger of the two membranes.  (B) Conformations of the parainfluenza virus 5 TMD in different lipid membranes determined by 2D 13C-13C correlation NMR spectra 6,7. The TMD is α-helical in POPC membranes, adopts a mixed helix/sheet structure in DOPC/DOPG membranes, and becomes predominantly β-sheet in negative-curvature POPE membranes. These membrane-dependent conformations suggest that the local lipid composition of the membrane has significant influence on the site of virus-cell fusion. (C) 31P NMR spectra show that the PIV5 F TMD converts the DOPE membrane from the hexagonal phase to a cubic phase, as manifested by a strong isotropic peak 6.  (D) Model of the membrane-dependent conformations of the FP and TMD of the PIV5 fusion protein, likely in a hemifusion intermediate. Local enrichment of phosphatidylethanolamine lipids causes β-strand conformations in both the FP and TMD, which in turn cause negative Gaussian curvature and dehydration to the membrane. 
Amyloid Fibrils
            Amyloids are highly aggregated β-sheet structures formed by a large number of peptides and proteins. They are involved in many neurodegenerative disorders but are also used by organisms for a variety of biological functions. We are investigating the structures and assemblies of several amyloids involved in Alzheimer’s disease, formed from peptide hormones, and designed for catalysis. Our structural studies focus on addressing how amino acid sequences dictate the three-dimensional fold, the origin of molecular structural polymorphism, how water impacts fibril formation (Fig. 3A), and how metal ions stabilize amyloid fibrils (Fig. 3B).

Figure 3. Amyloid fibril structure and hydration investigated by solid-state NMR. (A) Water-edited 13C NMR of Aβ(1-40) fibrils proves the presence of a central water-filled pore in the three-fold symmetric fibril. Measurement of water 1H relaxation times indicates that the hydration water of the Aβ fibril is dynamically heterogeneous 8.  (B) Zinc-bound structure of a catalytic amyloid peptide containing two histidines 9. Measurement of histidine sidechain chemical shifts and rotameric structure indicate that the parallel-in-register β-sheets are stabilized by a Zn2+ at each strand-strand interface. Each histidine coordinates one and two Zn2+ ions at a 1 : 1 ratio. This coordination stoichiometry leaves one free imidazole nitrogen per peptide available to bind water or hydroxide ions to catalyze ester hydrolysis.
Plant Cell Walls
            Plant cell walls provide mechanical strength to plant cells while at the same time allowing plants to grow rapidly. Plant cell walls primarily contain three types of polysaccharides: cellulose, hemicellulose, and pectins (Fig. 4A). Although the chemical composition of plant cell walls is relatively well known, the three-dimensional architecture and the dynamics of cell wall polysaccharides have long been elusive due to the lack of high-resolution structural techniques to characterize this insoluble and disordered material.
            We are pioneering the application of multidimensional solid-state NMR to elucidate the structures and dynamics of the polysaccharides of intact primary cell walls. By enriching whole plants with 13C, we are able to employ 2D and 3D correlation MAS NMR techniques to detect and resolve the signals of the complex mixture of polysaccharides in intact cell walls (Fig. 4B), and determine their spatial contacts and mobilities. By using sensitivity-enhancing dynamic nuclear polarization (DNP) and paramagnetic relaxation enhancement NMR techniques, we elucidate how polysaccharides interact with proteins to loosen the cell walls during growth (Fig. 4C). Using model plants of both dicot (e.g. Arabidopsis thaliana) and grass (e.g. Brachypodium distachyon and Zea mays) families, we have shown that cellulose, hemicellulose and pectins form a single three-dimensional network (Fig. 4D) instead of two separate networks, thus revising the long-held view of the plant cell wall structure. With our collaborators, we also investigate the structural polymorphism of cellulose microfibrils, hydration of wall polysaccharides, interactions of cellulose with matrix polysaccharides, and the effects of genetic mutations on cell-wall structure.

Figure 4. Solid-state NMR studies of the structure and dynamics of plant cell walls. (A) Cartoon representation of the primary cell walls of dicots, in which cellulose microfibrils act as the scaffold of the cell wall, surrounded by pectins and hemicelluloses. (B) Representative 2D 13C-13C correlation spectra of primary cell walls of Arabidopsis. High magnetic fields enhance spectral resolution and allow resonance assignment, thus permitting the detection of intermolecular contacts between different polysaccharides 10,11. (C) Expansin binding to cellulose in plant cell walls 12. By measuring expansin-to-polysaccharide 1H spin diffusion under DNP, we showed for the first time that expansin’s binding target in the cell wall is cellulose rather than matrix polysaccharides. Molecular dynamics simulation shows how expansin may dock onto the cellulose microfibril. (D) Schematic of the single-network model of the dicot primary wall based on our solid-state NMR data 13,14.
Solid-state NMR techniques for structure determination
            Driven by our interest in answering important biological questions, we continue to expand the capability of solid-state NMR spectroscopy. We have a long-standing interest in increasing the distance reach of NMR. Using nuclear spins with high gyromagnetic ratios such as 19F and 1H, and exploiting multi-spin effects, we are pushing the distance upper limit of NMR to ~2 nm (Fig. 5A). This capability opens up many possibilities for determining the oligomeric structures and global conformational changes of proteins, and for determining the structures of protein-lipid, protein-carbohydrate and other macromolecular complexes. To determine the amplitude of molecular motion, we develop anisotropic-isotropic correlation experiments involving both dipolar interactions and 2H quadrupolar interactions (Fig. 5B) under fast MAS and in high magnetic fields.

Figure 5. Solid-state NMR methods for distance measurements and dynamics studies. (A) 19F-19F dipolar coupling measurements by spin exchange and dipolar recoupling now can probe distances to 1.8 nm. (B) 2H-13C correlation experiments to investigate small-amplitude motions under fast MAS 15.

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