Karin Giller

Structure of the Human Voltage-Dependent Anion Channel.

The voltage-dependent anion channel (VDAC), also known as mitochondrial porin, is the most abundant protein in the mitochondrial outer membrane (MOM). VDAC is the channel known to guide the metabolic flux across the MOM and plays a key role in mitochondrially induced apoptosis. Here, we present the 3D structure of human VDAC1, which was solved conjointly by NMR spectroscopy and x-ray crystallography. Human VDAC1 (hVDAC1) adopts a β-barrel architecture composed of 19 β-strands with an α-helix located horizontally midway within the pore. Bioinformatic analysis indicates that this channel architecture is common to all VDAC proteins and is adopted by the general import pore TOM40 of mammals, which is also located in the MOM.

Toxin-induced conformational changes in a potassium channel revealed by solid-state NMR.

The active site of potassium (K+) channels catalyses the transport of K+ ions across the plasma membrane—similar to the catalytic function of the active site of an enzyme—and is inhibited by toxins from scorpion venom. On the basis of the conserved structures of K+ pore regions and scorpion toxins, detailed structures for the K+ channel–scorpion toxin binding interface have been proposed. In these models and in previous solution-state nuclear magnetic resonance (NMR) studies using detergent-solubilized membrane proteins, scorpion toxins were docked to the extracellular entrance of the K+ channel pore assuming rigid, preformed binding sites. Using high-resolution solid-state NMR spectroscopy, here we show that high-affinity binding of the scorpion toxin kaliotoxin to a chimaeric K+ channel (KcsA-Kv1.3) is associated with significant structural rearrangements in both molecules. Our approach involves a combined analysis of chemical shifts and proton–proton distances and demonstrates that solid-state NMR is a sensitive method for analysing the structure of a membrane protein–inhibitor complex. We propose that structural flexibility of the K+ channel and the toxin represents an important determinant for the high specificity of toxin–K+ channel interactions.

Pre-fibrillar α-synuclein variants with impaired β-structure increase neurotoxicity in Parkinson's disease models

The relation of α‐synuclein (αS) aggregation to Parkinsons disease (PD) has long been recognized, but the mechanism of toxicity, the pathogenic species and its molecular properties are yet to be identified. To obtain insight into the function different aggregated αS species have in neurotoxicity in vivo, we generated αS variants by a structure‐based rational design. Biophysical analysis revealed that the αS mutants have a reduced fibrillization propensity, but form increased amounts of soluble oligomers. To assess their biological response in vivo, we studied the effects of the biophysically defined pre‐fibrillar αS mutants after expression in tissue culture cells, in mammalian neurons and in PD model organisms, such as Caenorhabditis elegans and Drosophila melanogaster. The results show a striking correlation between αS aggregates with impaired β‐structure, neuronal toxicity and behavioural defects, and they establish a tight link between the biophysical properties of multimeric αS species and their in vivo function.

Atomic model of the type III secretion system needle

Pathogenic bacteria using a type III secretion system (T3SS) to manipulate host cells cause many different infections including Shigella dysentery, typhoid fever, enterohaemorrhagic colitis and bubonic plague. An essential part of the T3SS is a hollow needle-like protein filament through which effector proteins are injected into eukaryotic host cells. Currently, the three-dimensional structure of the needle is unknown because it is not amenable to X-ray crystallography and solution NMR, as a result of its inherent non-crystallinity and insolubility. Cryo-electron microscopy combined with crystal or solution NMR subunit structures has recently provided a powerful hybrid approach for studying supramolecular assemblies, resulting in low-resolution and medium-resolution models. However, such approaches cannot deliver atomic details, especially of the crucial subunit–subunit interfaces, because of the limited cryo-electron microscopic resolution obtained in these studies. Here we report an alternative approach combining recombinant wild-type needle production, solid-state NMR, electron microscopy and Rosetta modelling to reveal the supramolecular interfaces and ultimately the complete atomic structure of the Salmonella typhimurium T3SS needle. We show that the 80-residue subunits form a right-handed helical assembly with roughly 11 subunits per two turns, similar to that of the flagellar filament of S. typhimurium. In contrast to established models of the needle in which the amino terminus of the protein subunit was assumed to be α-helical and positioned inside the needle, our model reveals an extended amino-terminal domain that is positioned on the surface of the needle, while the highly conserved carboxy terminus points towards the lumen.

Structure of the mitochondrial translocator protein in complex with a diagnostic ligand.

Translocation in Injury The translocator protein TSPO is essential for the import of cholesterol and porphyrins into mitochondria. TSPO expression increases in areas of brain injury and during neuroinflammation and, thus, has diagnostic and therapeutic implications. Jaremko et al. (p. 1363) used nuclear magnetic resonance spectroscopy to determine the high-resolution structure of the 18- kilodalton mammalian TSPO with the ligand PK11195, which stabilized the structure and resolved the conformation as a tight bundle of five helices. A ligand stabilizes a mammalian mitochondrial cholesterol transporter, allowing high-resolution structural analysis. The 18-kilodalton translocator protein TSPO is found in mitochondrial membranes and mediates the import of cholesterol and porphyrins into mitochondria. In line with the role of TSPO in mitochondrial function, TSPO ligands are used for a variety of diagnostic and therapeutic applications in animals and humans. We present the three-dimensional high-resolution structure of mammalian TSPO reconstituted in detergent micelles in complex with its high-affinity ligand PK11195. The TSPO-PK11195 structure is described by a tight bundle of five transmembrane α helices that form a hydrophobic pocket accepting PK11195. Ligand-induced stabilization of the structure of TSPO suggests a molecular mechanism for the stimulation of cholesterol transport into mitochondria.

A structural link between inactivation and block of a K+ channel.

Gating the ion-permeation pathway in K+ channels requires conformational changes in activation and inactivation gates. Here we have investigated the structural alterations associated with pH-dependent inactivation gating of the KcsA-Kv1.3 K+ channel using solid-state NMR spectroscopy in direct reference to electrophysiological and pharmacological experiments. Transition of the KcsA-Kv1.3 K+ channel from a closed state at pH 7.5 to an inactivated state at pH 4.0 revealed distinct structural changes within the pore, correlated with activation-gate opening and inactivation-gate closing. In the inactivated K+ channel, the selectivity filter adopts a nonconductive structure that was also induced by binding of a pore-blocking tetraphenylporphyrin derivative. The results establish a structural link between inactivation and block of a K+ channel in a membrane setting.

Coupling of activation and inactivation gate in a K+-channel: potassium and ligand sensitivity

Potassium (K+)‐channel gating is choreographed by a complex interplay between external stimuli, K+ concentration and lipidic environment. We combined solid‐state NMR and electrophysiological experiments on a chimeric KcsA–Kv1.3 channel to delineate K+, pH and blocker effects on channel structure and function in a membrane setting. Our data show that pH‐induced activation is correlated with protonation of glutamate residues at or near the activation gate. Moreover, K+ and channel blockers distinctly affect the open probability of both the inactivation gate comprising the selectivity filter of the channel and the activation gate. The results indicate that the two gates are coupled and that effects of the permeant K+ ion on the inactivation gate modulate activation‐gate opening. Our data suggest a mechanism for controlling coordinated and sequential opening and closing of activation and inactivation gates in the K+‐channel pore.

Solid-State NMR Spectroscopy Applied to a Chimeric Potassium Channel in Lipid Bilayers

We show that solid-state NMR can be used to investigate the structure and dynamics of a chimeric potassium channel, KcsA-Kv1.3, in lipid bilayers. Sequential resonance assignments were obtained using a combination of (15)N- (13)C and (13)C- (13)C correlation experiments conducted on fully labeled and reverse-labeled as well as C-terminally truncated samples. Comparison of our results with those from X-ray crystallography and solution-state NMR in micelles on the closely related KcsA K (+) channel provides insight into the mechanism of ion channel selectivity and underlines the important role of the lipid environment for membrane protein structure and function.

13C spin dilution for simplified and complete solid-state NMR resonance assignment of insoluble biological assemblies.

A strategy for simplified and complete resonance assignment of insoluble and noncrystalline proteins by solid-state NMR (ssNMR) spectroscopy is presented. Proteins produced with [1-(13)C]- or [2-(13)C]glucose are very sparsely labeled, and the resulting 2D ssNMR spectra exhibit smaller line widths (by a factor of ∼2 relative to uniformly labeled proteins) and contain a reduced number of cross-peaks. This allows for an accelerated and straightforward resonance assignment without the necessity of time-consuming 3D spectroscopy or sophisticated pulse sequences. The strategy aims at complete backbone and side-chain resonance assignments based on bidirectional sequential walks. The approach was successfully demonstrated with the de novo assignment of the Type Three Secretion System PrgI needle protein. Using a limited set of simple 2D experiments, we report a 97% complete resonance assignment of the backbone and side-chain (13)C atoms.

Kinetics of Conformational Sampling in Ubiquitin

Molecular recognition plays a central role in many biological processes. For enzymatic reactions and slow protein–protein recognition events, turn-over rates and on-rates in the millisecond-to-second time scale have been connected to internal protein dynamics detected with atomic resolution by NMR spectroscopy, and in particular conformational sampling could be established as a mechanism for enzyme–substrate and protein–protein recognition. Recent theoretical studies indicate that faster rates of conformational interconversion in the microsecond time scale might limit on-rates for protein–protein recognition. However experimental proofs were lacking so far, mainly because such rates could not be determined accurately enough and kinetic experiments in the microsecond time range are difficult to perform. Nevertheless, for proteins and TAR-RNA, recent studies based on residual dipolar couplings (RDCs) and other NMR spectroscopy techniques have detected substantial internal dynamics in a time window from the rotational correlation time tc (one-digit nanoseconds) to approximately 50 ms, called the supra-tc window in the following. However, the exact rates of internal dynamics within this four orders of magnitude wide time window could not be determined. Supra-tc dynamics in ubiquitin [9] and TAR-RNA could be connected to the conformational sampling required for molecular recognition. While the amplitudes of motions have been indirectly detected by RDCs and characterized in great detail, it has so far been impossible to directly observe these motions and to determine the exact rate of these supra-tc motions. In contrast, conformational sampling in enzymes occurs on a time scale that is 100 to 1000 times slower than supra-tc dynamics and therefore NMR relaxation dispersion (RD) techniques have been able to establish the functional link to enzyme kinetics with atomic resolution at physiological conditions. 5] However, for technical reasons, RD is not sensitive to motion faster than approximately 50 ms (RD window) and therefore does not access motion in the supra-tc window at room temperature. Here we determine the rate of interconversion between conformers of free ubiquitin by a combination of NMR RD experiments in super-cooled solution and dielectric relaxation spectroscopy (DR). Furthermore, we corroborate the motional amplitudes in the RDC-derived ensembles quantitatively with the observed amplitudes of RD and DR. The methods utilized herein can be used to directly study protein dynamics in a time range that was previously inaccessible. Significant motional amplitude in the supra-tc window has been observed using RDC measurements, and was connected to the conformational sampling for a protein in the ground