Olaf Pongs

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.

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.

Active sites in Escherichia coli ribosomes

The model of active sites in E. coli ribosome illustrated in the figure is based on the presently available experimental results. It is far from being complete and should not be overinterpreted as an accurate topographical model. More data on the functional role of ribosomal components and on the topography of the subunits can be expected in the near future and will add to the knowledge on the active sites in ribosomes.

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.

Structural Determinants of Specific Lipid Binding to Potassium Channels

We have investigated specific lipid binding to the pore domain of potassium channels KcsA and chimeric KcsA-Kv1.3 on the structural and functional level using extensive coarse-grained and atomistic molecular dynamics simulations, solid-state NMR, and single channel measurements. We show that, while KcsA activity is critically modulated by the specific and cooperative binding of anionic nonannular lipids close to the channels selectivity filter, the influence of nonannular lipid binding on KcsA-Kv1.3 is much reduced. The diminished impact of specific lipid binding on KcsA-Kv1.3 results from a point-mutation at the corresponding nonannular lipid binding site leading to a salt-bridge between adjacent KcsA-Kv1.3 subunits, which is conserved in many voltage-gated potassium channels and prevents strong nonannular lipid binding to the pore domain. Our findings elucidate how protein-lipid and protein-protein interactions modulate K(+) channel activity. The combination of MD, NMR, and functional studies as shown here may help to dissect the structural and dynamical processes that are critical for the functioning of larger membrane proteins, including Kv channels in a membrane setting.

The codon binding site of the Escherichia coli ribosome as studied with a chemically reactive A-U-G analog

5′-{4-(bromo[2- 14 C]acetamido)phenylphospho}-adenylyl-(3′-5′)-uridylyl-(3′-5′)-guanosine was reacted with 70 S ribosomes and 30 S subunits. The yield of this reaction after repurification of the ribosomes varied from 15 to 70% depending on the nature of the ribosome and the reaction conditions. The reaction products were characterized by standard polyacrylamide gel electrophoresis and finally identified by autoradiography of Ouchterlony double-diffusion tests. The A-U-G affinity label reacts in freshly prepared active ribosomes with proteins S18 and S21 in a 9:1 ratio. Exposure of ribosomes to low temperatures leads to an additional labeling of protein S4, the ram gene product. The same proteins are also labeled in 30 S subunits by the A-U-G analog. Using slowly frozen and thawed 30 S subunits, the yield of the affinity labeling reaction was markedly increased up to 60%. The proteins mainly labeled were now proteins S4 and S12, the strA gene products. Label was also detected in protein S11 and, to a very small extent, in protein S13. Functional analysis showed that covalent linkage of A-U-G to proteins S18 or S4 programs ribosomes for IF2 (initiation factor 2)-dependent initiator-tRNA binding in a puromycin-resistant site, while linkage to protein S12 programs ribosomes for IF2-dependent initiator-tRNA binding to a puromycin-sensitive site. For the binding of fMet-tRNA Met f into the puromycin-sensitive site, the GTP analog guanylyl-(β,γ-methylene)-diphosphonate cannot substitute for GTP. Our results are compatible with two possible models. o (1) There are two independent codon binding sites whose accessibility, however, depends on the conformation of the 30 S subunit. (2) There is only one codon binding site which can alternatively exist in two conformations, one directing tRNA into the donor site (initiation) and the other directing tRNA into the acceptor site (elongation) of the peptidyl transferase center of the ribosome. Our experiments could not demonstrate the simultaneous binding of more than one codon per ribosome. This may be due to the structure of the modified A-U-G analog.

The inactivation behaviour of voltage-gated K-channels may be determined by association of α- and β-subunits

Abstract Voltage-gated K-channels of the Shaker related subfamily have two subunits, membrane integrated α- and peripheral β-subunits. α-Subunits may assemble as tetramers and form in in vitro expression systems functional K-channels. β-Subunits cannot form channels by themselves. Life for α-subunits, the rat nervous system apparently expresses a family of β-subunit proteins. We have demonstrated that one rat K-channel β-subunit, Kvβ1, contains an inactivating domain. Upon association of α- and Kvβ1-subunits, delayed-rectifier type K-channels are converted to rapidly inactivating A-type K-channels. The β-subunit inactivation domain acts via a ball and chain type mechanism previously proposed for N-type inactivation of α-subunits. The association of α- and β-subunits endows the nervous system with an unprecedented flexibility and diversity of K-channels which may play an important role in the regulation of nervous excitability.

Protein dynamics detected in a membrane-embedded potassium channel using two-dimensional solid-state NMR spectroscopy.

We report longitudinal (15)N relaxation rates derived from two-dimensional ((15)N, (13)C) chemical shift correlation experiments obtained under magic angle spinning for the potassium channel KcsA-Kv1.3 reconstituted in multilamellar vesicles. Thus, we demonstrate that solid-state NMR can be used to probe residue-specific backbone dynamics in a membrane-embedded protein. Enhanced backbone mobility was detected for two glycine residues within the selectivity filter that are highly conserved in potassium channels and that are of core relevance to the filter structure and ion selectivity.

High-Resolution 3D Structure Determination of Kaliotoxin by Solid-State NMR Spectroscopy

High-resolution solid-state NMR spectroscopy can provide structural information of proteins that cannot be studied by X-ray crystallography or solution NMR spectroscopy. Here we demonstrate that it is possible to determine a protein structure by solid-state NMR to a resolution comparable to that by solution NMR. Using an iterative assignment and structure calculation protocol, a large number of distance restraints was extracted from 1H/1H mixing experiments recorded on a single uniformly labeled sample under magic angle spinning conditions. The calculated structure has a coordinate precision of 0.6 Å and 1.3 Å for the backbone and side chain heavy atoms, respectively, and deviates from the structure observed in solution. The approach is expected to be applicable to larger systems enabling the determination of high-resolution structures of amyloid or membrane proteins.

A site accessible to extracellular TEA+ and K+ influences intracellular Mg2+ block of cloned potassium channels.

The members of the RCK family of cloned voltage-dependent K+ channels are quite homologous in primary structure, but they are highly diverse in functional properties. RCK4 channels differ from RCK1 and RCK2 channels in inactivation and permeation properties, the sensitivity to external TEA, and to current modulation by external K+ ions. Here we show several other interesting differences: While RCK1 and RCK2 are blocked in a voltage and concentration dependent manner by internal Mg2+ ions, RCK4 is only weakly blocked at very high potentials. The single-channel current-voltage relations of RCK4 are rather linear while RCK2 exhibits an inwardly rectifying single-channel current in symmetrical K+ solutions. The deactivation of the channels, measured by tail current protocols, is faster in RCK4 by a factor of two compared with RCK2. In a search for the structural motif responsible for these differences, point mutants creating homology between RCK2 and RCK4 in the pore region were tested. The single-point mutant K533Y in the background of RCK4 conferred the properties of Mg2+ block, tail current kinetics, and inward ion permeation of RCK2 to RCK4. This mutant was previously shown to be responsible for the alterations in external TEA sensitivity and channel regulation by external K+ ions. Thus, this residue is expected to be located at the external side of the pore entrance. The data are consistent with the idea that the mutation alters the channel occupancy by K+ and thereby indirectly affects internal Mg2+ block and channel closing.