Magic-angle spinning NMR structure of Opa60 in lipid bilayers.

Here we report the structure of Opa60 in lipid bilayers using proton-detected magic-angle spinning nuclear magnetic resonance (MAS NMR). Preparations including near-native oligosaccharide lipids reveal a consistent picture of a stable transmembrane beta barrel with a minor increase in the structured region as compared with the previously reported detergent structure. The large variable loops known to interact with host proteins could not be detected, confirming their dynamic nature even in a lipid bilayer environment. The structure provides a starting point for investigation of the functional role of Opa60 in gonococcal infection, which is understood to involve interaction with host proteins. At the same time, it demonstrates the recent advances in proton-detected methodology for membrane protein structure determination at atomic resolution by MAS NMR.

Real-time tracking of drug binding to influenza A M2 reveals a high energy barrier.

The drug Rimantadine binds to two different sites in the M2 protein from influenza A, a peripheral site and a pore site that is the primary site of efficacy. It remained enigmatic that pore binding did not occur in certain detergent micelles, and in particular incomplete binding was observed in a mixture of lipids selected to match the viral membrane. Here we show that two effects are responsible, namely changes in the protein upon pore binding that prevented detergent solubilization, and slow binding kinetics in the lipid samples. Using 55-100 kHz magic-angle spinning NMR, we characterize kinetics of drug binding in three different lipid environments: DPhPC, DPhPC with cholesterol and viral mimetic membrane lipid bilayers. Slow pharmacological binding kinetics allowed the characterization of spectral changes associated with non-specific binding to the protein periphery in the kinetically trapped pore-apo state. Resonance assignments were determined from a set of proton-detected 3D spectra. Chemical shift changes associated with functional binding in the pore of M2 were tracked in real time in order to estimate the activation energy. The binding kinetics are affected by pH and the lipid environment and in particular cholesterol. We found that the imidazole-imidazole hydrogen bond at residue histidine 37 is a stable feature of the protein across several lipid compositions. Pore binding breaks the imidazole-imidazole hydrogen bond and limits solubilization in DHPC detergent.

Direct Detection of Bound Ammonium Ions in the Selectivity Filter of Ion Channels by Solid-State NMR.

The flow of ions across cell membranes facilitated by ion channels is an important function for all living cells. Despite the huge amount of structural data provided by crystallography, elucidating the exact interactions between the selectivity filter atoms and bound ions is challenging. Here, we detect bound 15N-labeled ammonium ions as a mimic for potassium ions in ion channels using solid-state NMR under near-native conditions. The non-selective ion channel NaK showed two ammonium peaks corresponding to its two ion binding sites, while its potassium-selective mutant NaK2K that has a signature potassium-selective selectivity filter with four ion binding sites gave rise to four ammonium peaks. Ions bound in specific ion binding sites were identified based on magnetization transfer between the ions and carbon atoms in the selectivity filters. Magnetization transfer between bound ions and water molecules revealed that only one out of four ions in the selectivity filter of NaK2K is in close contact with water, which is in agreement with the direct knock-on ion conduction mechanism where ions are conducted through the channel by means of direct interactions without water molecules in between. Interestingly, the potassium-selective ion channels investigated here (NaK2K and, additionally, KcsA-Kv1.3) showed remarkably different chemical shifts for their bound ions, despite having identical amino acid sequences and crystal structures of their selectivity filters. Molecular dynamics simulations show similar ion binding and conduction behavior between ammonium and potassium ions and identify the origin of the differences between the investigated potassium channels.

Structure and Gating Behavior of the Human Integral Membrane Protein VDAC1 in a Lipid Bilayer.

The voltage-dependent anion channel (VDAC), the most abundant protein in the outer mitochondrial membrane, is responsible for the transport of all ions and metabolites into and out of mitochondria. Larger than any of the ß-barrel structures determined to date by magic-angle spinning (MAS) NMR, but smaller than the size limit of cryo-electron microscopy (cryo-EM), VDAC1's 31 kDa size has long been a bottleneck in determining its structure in a near-native lipid bilayer environment. Using a single two-dimensional (2D) crystalline sample of human VDAC1 in lipids, we applied proton-detected fast magic-angle spinning NMR spectroscopy to determine the arrangement of ß strands. Combining these data with long-range restraints from a spin-labeled sample, chemical shift-based secondary structure prediction, and previous MAS NMR and atomic force microscopy (AFM) data, we determined the channel's structure at a 2.2 Å root-mean-square deviation (RMSD). The structure, a 19-stranded ß-barrel, with an N-terminal α-helix in the pore is in agreement with previous data in detergent, which was questioned due to the potential for the detergent to perturb the protein's functional structure. Using a quintuple mutant implementing the channel's closed state, we found that dynamics are a key element in the protein's gating behavior, as channel closure leads to the destabilization of not only the C-terminal barrel residues but also the α2 helix. We showed that cholesterol, previously shown to reduce the frequency of channel closure, stabilizes the barrel relative to the N-terminal helix. Furthermore, we observed channel closure through steric blockage by a drug shown to selectively bind to the channel, the Bcl2-antisense oligonucleotide G3139.

Backbone Torsion Angle Determination Using Proton Detected Magic-Angle Spinning Nuclear Magnetic Resonance.

Protein torsion angles define the backbone secondary structure of proteins. Magic-angle spinning (MAS) NMR methods using carbon detection have been developed to measure torsion angles by determining the relative orientation between two anisotropic interactions─dipolar coupling or chemical shift anisotropy. Here we report a new proton-detection based method to determine the backbone torsion angle by recoupling NH and CH dipolar couplings within the HCANH pulse sequence, for protonated or partly deuterated samples. We demonstrate the efficiency and precision of the method with microcrystalline chicken α spectrin SH3 protein and the influenza A matrix 2 (M2) membrane protein, using 55 or 90 kHz MAS. For M2, pseudo-4D data detect a turn between transmembrane and amphipathic helices.

Insights into the molecular mechanism of amyloid filament formation: Segmental folding of α-synuclein on lipid membranes.

Recent advances in the structural biology of disease-relevant α-synuclein fibrils have revealed a variety of structures, yet little is known about the process of fibril aggregate formation. Characterization of intermediate species that form during aggregation is crucial; however, this has proven very challenging because of their transient nature, heterogeneity, and low population. Here, we investigate the aggregation of α-synuclein bound to negatively charged phospholipid small unilamellar vesicles. Through a combination of kinetic and structural studies, we identify key time points in the aggregation process that enable targeted isolation of prefibrillar and early fibrillar intermediates. By using solid-state nuclear magnetic resonance, we show the gradual buildup of structural features in an α-synuclein fibril filament, revealing a segmental folding process. We identify distinct membrane-binding domains in α-synuclein aggregates, and the combined data are used to present a comprehensive mechanism of the folding of α-synuclein on lipid membranes.

A ß-barrel for oil transport through lipid membranes: Dynamic NMR structures of AlkL.

The protein AlkL is known to increase permeability of the outer membrane of bacteria for hydrophobic molecules, yet the mechanism of transport has not been determined. Differing crystal and NMR structures of homologous proteins resulted in a controversy regarding the degree of structure and the role of long extracellular loops. Here we solve this controversy by determining the de novo NMR structure in near-native lipid bilayers, and by accessing structural dynamics relevant to hydrophobic substrate permeation through molecular-dynamics simulations and by characteristic NMR relaxation parameters. Dynamic lateral exit sites large enough to accommodate substrates such as carvone or octane occur through restructuring of a barrel extension formed by the extracellular loops.