Hartmut Oschkinat

Structure of a protein determined by solid-state magic-angle-spinning NMR spectroscopy

The determination of a representative set of protein structures is a chief aim in structural genomics. Solid-state NMR may have a crucial role in structural investigations of those proteins that do not easily form crystals or are not accessible to solution NMR, such as amyloid systems or membrane proteins. Here we present a protein structure determined by solid-state magic-angle-spinning (MAS) NMR. Almost complete 13C and 15N resonance assignments for a micro-crystalline preparation of the α-spectrin Src-homology 3 (SH3) domain formed the basis for the extraction of a set of distance restraints. These restraints were derived from proton-driven spin diffusion (PDSD) spectra of biosynthetically site-directed, labelled samples obtained from bacteria grown using [1,3-13C]glycerol or [2-13C]glycerol as carbon sources. This allowed the observation of long-range distance correlations up to ∼7 Å. The calculated global fold of the α-spectrin SH3 domain is based on 286 inter-residue 13C–13C and six 15N–15N restraints, all self-consistently obtained by solid-state MAS NMR. This MAS NMR procedure should be widely applicable to small membrane proteins that can be expressed in bacteria.

Perspectives on NMR in drug discovery: a technique comes of age

In the past decade, the potential of harnessing the ability of nuclear magnetic resonance (NMR) spectroscopy to monitor intermolecular interactions as a tool for drug discovery has been increasingly appreciated in academia and industry. In this Perspective, we highlight some of the major applications of NMR in drug discovery, focusing on hit and lead generation, and provide a critical analysis of its current and potential utility.

Backbone and Side-Chain 13C and 15N Signal Assignments of the α-Spectrin SH3 Domain by Magic Angle Spinning Solid-State NMR at 17.6 Tesla

The backbone and side‐chain 13C and 15N signals of a solid 62‐residue (u‐13C,15N)‐labelled protein containing the α‐spectrin SH3 domain were assigned by two‐dimensional (2D) magic angle spinning (MAS) 15N–13C and 13C–13C dipolar correlation spectroscopy at 17.6 T. The side‐chain signal sets of the individual amino acids were identified by 2D 13C–13C proton‐driven spin diffusion and dipolar recoupling experiments. Correlations to the respective backbone nitrogen signals were established by 2D NCACX (CX=any carbon atom) experiments, which contain a proton–nitrogen and a nitrogen–carbon cross‐polarisation step followed by a carbon–carbon homonuclear transfer unit. Interresidue correlations leading to sequence‐specific assignments were obtained from 2D NCOCX experiments. The assignment is nearly complete for the SH3 domain residues 7–61, while the signals of the N‐ and C‐terminal residues 1–6 and 62, respectively, outside the domain boundaries are not detected in our MAS spectra. The resolution observed in these spectra raises expectations that receptor‐bound protein ligands and slightly larger proteins (up to 20 kDa) can be readily assigned in the near future by using three‐dimensional versions of the applied or analogous techniques.

Solid-state NMR and SAXS studies provide a structural basis for the activation of αB-crystallin oligomers

The small heat shock protein αB-crystallin (αB) contributes to cellular protection against stress. For decades, high-resolution structural studies on oligomeric αB have been confounded by its polydisperse nature. Here, we present a structural basis of oligomer assembly and activation of the chaperone using solid-state NMR and small-angle X-ray scattering (SAXS). The basic building block is a curved dimer, with an angle of ∼121° between the planes of the β-sandwich formed by α-crystallin domains. The highly conserved IXI motif covers a substrate binding site at pH 7.5. We observe a pH-dependent modulation of the interaction of the IXI motif with β4 and β8, consistent with a pH-dependent regulation of the chaperone function. N-terminal region residues Ser59-Trp60-Phe61 are involved in intermolecular interaction with β3. Intermolecular restraints from NMR and volumetric restraints from SAXS were combined to calculate a model of a 24-subunit αB oligomer with tetrahedral symmetry.

General structural motifs of amyloid protofilaments

Human CA150, a transcriptional activator, binds to and is co-deposited with huntingtin during Huntingtons disease. The second WW domain of CA150 is a three-stranded β-sheet that folds in vitro in microseconds and forms amyloid fibers under physiological conditions. We found from exhaustive alanine scanning studies that fibrillation of this WW domain begins from its denatured conformations, and we identified a subset of residues critical for fibril formation. We used high-resolution magic-angle-spinning NMR studies on site-specific isotopically labeled fibrils to identify abundant long-range interactions between side chains. The distribution of critical residues identified by the alanine scanning and NMR spectroscopy, along with the electron microscopy data, revealed the protofilament repeat unit: a 26-residue nonnative β-hairpin. The structure we report has similarities to the hairpin formed by the Aβ(1–40) protofilament, yet also contains closely packed side-chains in a “steric zipper” arrangement found in the cross-β spine formed from small peptides from the Sup35 prion protein. Fibrillation of unrelated amyloidogenic sequences shows the common feature of zippered repeat units that act as templates for fiber elongation.

AhR sensing of bacterial pigments regulates antibacterial defence

The aryl hydrocarbon receptor (AhR) is a highly conserved ligand-dependent transcription factor that senses environmental toxins and endogenous ligands, thereby inducing detoxifying enzymes and modulating immune cell differentiation and responses. We hypothesized that AhR evolved to sense not only environmental pollutants but also microbial insults. We characterized bacterial pigmented virulence factors, namely the phenazines from Pseudomonas aeruginosa and the naphthoquinone phthiocol from Mycobacterium tuberculosis, as ligands of AhR. Upon ligand binding, AhR activation leads to virulence factor degradation and regulated cytokine and chemokine production. The relevance of AhR to host defence is underlined by heightened susceptibility of AhR-deficient mice to both P. aeruginosa and M. tuberculosis. Thus, we demonstrate that AhR senses distinct bacterial virulence factors and controls antibacterial responses, supporting a previously unidentified role for AhR as an intracellular pattern recognition receptor, and identify bacterial pigments as a new class of pathogen-associated molecular patterns.

N-terminal domain of αB-crystallin provides a conformational switch for multimerization and structural heterogeneity

The small heat shock protein (sHSP) αB-crystallin (αB) plays a key role in the cellular protection system against stress. For decades, high-resolution structural studies on heterogeneous sHSPs have been confounded by the polydisperse nature of αB oligomers. We present an atomic-level model of full-length αB as a symmetric 24-subunit multimer based on solid-state NMR, small-angle X-ray scattering (SAXS), and EM data. The model builds on our recently reported structure of the homodimeric α-crystallin domain (ACD) and C-terminal IXI motif in the context of the multimer. A hierarchy of interactions contributes to build multimers of varying sizes: Interactions between two ACDs define a dimer, three dimers connected by their C-terminal regions define a hexameric unit, and variable interactions involving the N-terminal region define higher-order multimers. Within a multimer, N-terminal regions exist in multiple environments, contributing to the heterogeneity observed by NMR. Analysis of SAXS data allows determination of a heterogeneity parameter for this type of system. A mechanism of multimerization into higher-order asymmetric oligomers via the addition of up to six dimeric units to a 24-mer is proposed. The proposed asymmetric multimers explain the homogeneous appearance of αB in negative-stain EM images and the known dynamic exchange of αB subunits. The model of αB provides a structural basis for understanding known disease-associated missense mutations and makes predictions concerning substrate binding and the reported fibrilogenesis of αB.

Dynamic nuclear polarization of deuterated proteins.

Magic-angle spinning nuclear magnetic resonance (MAS NMR) spectroscopy has evolved as a robust and widely applicable technique for investigating the structure and dynamics of biological systems.[1–3] It is in fact rapidly becoming an indispensable tool in structural biology studies of amyloid,[4, 5] nanocrystalline,[6, 7] and membrane proteins.[8] However, it is clear that the low sensitivity of MAS experiments to directly detected 13C and 15N signals limits the utility of the approach, particularly when working with systems which are difficult to obtain in large quantities. This limit provides the impetus to develop methods to enhance the sensitivity of MAS experiments, the availability of which will undoubtedly broaden the applicability of the technique. Remarkable progress towards this goal has been achieved by incorporating high-frequency dynamic nuclear polarization (DNP) into the MAS NMR technique.[9–17] The DNP method exploits the microwave-driven transfer of polarization from a paramagnetic center, such as nitroxide free radical, to the nuclear spins, and has been demonstrated to produce uniformly polarized macromolecular samples. In principle signal enhancements, e = (γe/γI) ≈ 660 can be obtained for 1H and recently signal enhancements of e = 100–200 were observed in model compounds. However, in applications of DNP to MAS spectra of biological systems, including studies of lysozyme,[18] and bacteriorhodopsin,[16, 19, 20] the enhancements have been smaller, e = 40–50. An exception is the amyloidogenic peptide GNNQQNY7–13 which forms nanocrystals for which the proton T1 time is long and e ≈ 100.[21]

Secondary chemical shifts in immobilized peptides and proteins: a qualitative basis for structure refinement under magic angle spinning.

Resonance assignments recently obtained on immobilized polypeptides and a membrane protein aggregate under Magic Angle Spinning are compared to random coil values in the liquid state. The resulting chemical shift differences (secondary chemical shifts) are evaluated in light of the backbone torsion angle ψ previously reported using X-ray crystallography. In all cases, a remarkable correlation is found suggesting that the concept of secondary chemical shifts, well established in the liquid state, can be of similar importance in the context of multiple-labelled polypeptides studied under MAS conditions.

Rapid Proton-Detected NMR Assignment for Proteins with Fast Magic Angle Spinning

Using a set of six 1H-detected triple-resonance NMR experiments, we establish a method for sequence-specific backbone resonance assignment of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectra of 5–30 kDa proteins. The approach relies on perdeuteration, amide 2H/1H exchange, high magnetic fields, and high-spinning frequencies (ωr/2π ≥ 60 kHz) and yields high-quality NMR data, enabling the use of automated analysis. The method is validated with five examples of proteins in different condensed states, including two microcrystalline proteins, a sedimented virus capsid, and two membrane-embedded systems. In comparison to contemporary 13C/15N-based methods, this approach facilitates and accelerates the MAS NMR assignment process, shortening the spectral acquisition times and enabling the use of unsupervised state-of-the-art computational data analysis protocols originally developed for solution NMR.