Relevance of Lysine Snorkeling in the Outer Transmembrane Domain of Small Viral Potassium Ion Channels

Transmembrane domains (TMDs) are often flanked by Lys or Arg because they keep their aliphatic parts in the bilayer and their charged groups in the polar interface. Here we examine the relevance of this so-called “snorkeling” of a cationic amino acid, which is conserved in the outer TMD of small viral K+ channels. Experimentally, snorkeling activity is not mandatory for KcvPBCV-1 because K29 can be replaced by most of the natural amino acids without any corruption of function. Two similar channels, KcvATCV-1 and KcvMT325, lack a cytosolic N-terminus, and neutralization of their equivalent cationic amino acids inhibits their function. To understand the variable importance of the cationic amino acids, we reanalyzed molecular dynamics simulations of KcvPBCV-1 and N-terminally truncated mutants; the truncated mutants mimic KcvATCV-1 and KcvMT325. Structures were analyzed with respect to membrane positioning in relation to the orientation of K29. The results indicate that the architecture of the protein (including the selectivity filter) is only weakly dependent on TMD length and protonation of K29. The penetration depth of Lys in a given protonation state is independent of the TMD architecture, which leads to a distortion of shorter proteins. The data imply that snorkeling can be important for K+ channels; however, its significance depends on the architecture of the entire TMD. The observation that the most severe N-terminal truncation causes the outer TMD to move toward the cytosolic side suggests that snorkeling becomes more relevant if TMDs are not stabilized in the membrane by other domains

Relevance of Lysine Snorkeling in the Outer Transmembrane Domain of Small Viral Potassium Ion Channels

Transmembrane domains (TMDs) are often flanked by Lys or Arg because they keep their aliphatic parts in the bilayer and their charged groups in the polar interface. Here we examine the relevance of this so-called “snorkeling” of a cationic amino acid, which is conserved in the outer TMD of small viral K+ channels. Experimentally, snorkeling activity is not mandatory for KcvPBCV-1 because K29 can be replaced by most of the natural amino acids without any corruption of function. Two similar channels, KcvATCV-1 and KcvMT325, lack a cytosolic N-terminus, and neutralization of their equivalent cationic amino acids inhibits their function. To understand the variable importance of the cationic amino acids, we reanalyzed molecular dynamics simulations of KcvPBCV-1 and N-terminally truncated mutants; the truncated mutants mimic KcvATCV-1 and KcvMT325. Structures were analyzed with respect to membrane positioning in relation to the orientation of K29. The results indicate that the architecture of the protein (including the selectivity filter) is only weakly dependent on TMD length and protonation of K29. The penetration depth of Lys in a given protonation state is independent of the TMD architecture, which leads to a distortion of shorter proteins. The data imply that snorkeling can be important for K+ channels; however, its significance depends on the architecture of the entire TMD. The observation that the most severe N-terminal truncation causes the outer TMD to move toward the cytosolic side suggests that snorkeling becomes more relevant if TMDs are not stabilized in the membrane by other domains

Computational study of the Kcv potassium channel

The K+ channel Kcv from Paramecium bursaria chlorella virus is the smallest known functional K+ channel. As a minimal working model, this K+ channel protein can be considered close to being prototypical in order to understand basic channel design principles and to gain insight into fundamental transport mechanisms. The objective of this work was the computational study of the impact of various mutations of the Kcv N-Terminus on the Kcv function. By the mean of molecular dynamics simulations of K+ channel models in explicit membrane and explicit solvent, the structure, dynamics, and thermodynamics of alternate model systems was examined on the atomic level. In addition to the wildtype, a hyperactive point mutant (KcvP13A) and two inactive deletion mutants (KcvDN8 and KcvDN14) were examined. The protonation state of a key amino acid (Lys29) was also exhaustingly studied. As a working hypothesis it was assumed that analog topology results in analog functionality. Hence, Kcv homology models were generated as well as KirBac1.1 X-ray structure models adapted in analogy to Kcv. A new method was developed in order to extract reasonable and symmetric expectation structures from very long trajectories. These structures can be compared to structures determined by structural biological methods and can be used as an input for proceeding with advanced methods, like e.g. the Poisson-Boltzmann theory or the 3D-RISM integral equation theory. Latter method was used in order to determin the ionic distribution around the protein. Most important results from this thesis are: 1. A workflow was developed that allows the creation of plausible K+ channel homology models. The quality of such a model is good enough to exhibit a full ion transition cycle during simulation. 2. The interaction of positively charged amino acids in the N-terminal helix with the C-terminus results in mutant dependent saltbridge patterns. Weakening of this interaction correlates with dysfuntion. 3. The N-terminus can act as a steric barrier for the transition of K+ ions and, thus, may contribute to the gating mechanism. 4. The protonation state of Lys29 is crutial for the transition of K+ ions and, thus, may also contribute to the gating mechanism. 5. Funtional analogy between distantly related K+ channels is apparent. 6. The reason for the hyperactivity of the point mutant P13A was not determined satisfactorily. Further investigations are necessary in order to generate more plausible models

Salt bridges in the miniature viral channel Kcv are important for function.

The viral potassium channel Kcv comprises only 94 amino acids, which represent the pore module of more complex K(+) channels. As for Kir-type channels, Kcv also has a short N-terminal helix exposed to the cytoplasm, upstream of the first transmembrane domain. Here we show that this helix is relevant for Kcv function. The presence of charged amino acids, which form dynamic inter- and intra-subunit salt bridges is crucial. Electrophysiological measurements, yeast rescue experiments and molecular dynamics simulations show that mutants in which the critical salt bridge formation is impaired have no or reduced channel activity. We conclude that these salt bridges destabilise the complexation of K(+) ions by negative charges on the inner transmembrane domain at the entrance into the cavity. This feature facilitates a continuous and coordinated transfer of ions between the cavity and the cytoplasm for channels without the canonical bundle crossing

Model development for the viral Kcv potassium channel.

A computational model for the open state of the short viral Kcv potassium channel was created and tested based on homology modeling and extensive molecular-dynamics simulation in a membrane environment. Particular attention was paid to the structure of the highly flexible N-terminal region and to the protonation state of membrane-exposed lysine residues. Data from various experimental sources, NMR spectroscopy, and electrophysiology, as well as results from three-dimensional reference interaction site model integral equation theory were taken into account to select the most reasonable model among possible variants. The final model exhibits spontaneous ion transitions across the complete pore, with and without application of an external field. The nonequilibrium transport events could be induced reproducibly without abnormally large driving potential and without the need to place ions artificially at certain key positions along the transition path. The transport mechanism through the filter region corresponds to the classic view of single-file motion, which in our case is coupled to frequent exchange of ions between the innermost filter position and the cavity