Tracking a complete voltage-sensor cycle with metal-ion bridges.

Voltage-gated ion channels open and close in response to changes in membrane potential, thereby enabling electrical signaling in excitable cells. The voltage sensitivity is conferred through four voltage-sensor domains (VSDs) where positively charged residues in the fourth transmembrane segment (S4) sense the potential. While an open state is known from the Kv1.2/2.1 X-ray structure, the conformational changes underlying voltage sensing have not been resolved. We present 20 additional interactions in one open and four different closed conformations based on metal-ion bridges between all four segments of the VSD in the voltage-gated Shaker K channel. A subset of the experimental constraints was used to generate Rosetta models of the conformations that were subjected to molecular simulation and tested against the remaining constraints. This achieves a detailed model of intermediate conformations during VSD gating. The results provide molecular insight into the transition, suggesting that S4 slides at least 12 Å along its axis to open the channel with a 3(10) helix region present that moves in sequence in S4 in order to occupy the same position in space opposite F290 from open through the three first closed states.

The conserved phenylalanine in the K+ channel voltage-sensor domain creates a barrier with unidirectional effects.

Voltage-gated ion channels are crucial for regulation of electric activity of excitable tissues such as nerve cells, and play important roles in many diseases. During activation, the charged S4 segment in the voltage sensor domain translates across a hydrophobic core forming a barrier for the gating charges. This barrier is critical for channel function, and a conserved phenylalanine in segment S2 has previously been identified to be highly sensitive to substitutions. Here, we have studied the kinetics of K(v)1-type potassium channels (Shaker and K(v)1.2/2.1 chimera) through site-directed mutagenesis, electrophysiology, and molecular simulations. The F290L mutation in Shaker (F233L in K(v)1.2/2.1) accelerates channel closure by at least a factor 50, although opening is unaffected. Free energy profiles with the hydrophobic neighbors of F233 mutated to alanine indicate that the open state with the fourth arginine in S4 above the hydrophobic core is destabilized by ∼17 kJ/mol compared to the first closed intermediate. This significantly lowers the barrier of the first deactivation step, although the last step of activation is unaffected. Simulations of wild-type F233 show that the phenyl ring always rotates toward the extracellular side both for activation and deactivation, which appears to help stabilize a well-defined open state.

310-helix conformation facilitates the transition of a voltage sensor S4 segment toward the down state.

The activation of voltage-gated ion channels is controlled by the S4 helix, with arginines every third residue. The x-ray structures are believed to reflect an open-inactivated state, and models propose combinations of translation, rotation, and tilt to reach the resting state. Recently, experiments and simulations have independently observed occurrence of 3(10)-helix in S4. This suggests S4 might make a transition from α- to 3(10)-helix in the gating process. Here, we show 3(10)-helix structure between Q1 and R3 in the S4 segment of a voltage sensor appears to facilitate the early stage of the motion toward a down state. We use multiple microsecond-steered molecular simulations to calculate the work required for translating S4 both as α-helix and transformed to 3(10)-helix. The barrier appears to be caused by salt-bridge reformation simultaneous to R4 passing the F233 hydrophobic lock, and it is almost a factor-two lower with 3(10)-helix. The latter facilitates translation because R2/R3 line up to face E183/E226, which reduces the requirement to rotate S4. This is also reflected in a lower root mean-square deviation distortion of the rest of the voltage sensor. This supports the 3(10) hypothesis, and could explain some of the differences between the open-inactivated- versus activated-states.

The free energy barrier for arginine gating charge translation is altered by mutations in the voltage sensor domain.

The gating of voltage-gated ion channels is controlled by the arginine-rich S4 helix of the voltage-sensor domain moving in response to an external potential. Recent studies have suggested that S4 moves in three to four steps to open the conducting pore, thus visiting several intermediate conformations during gating. However, the exact conformational changes are not known in detail. For instance, it has been suggested that there is a local rotation in the helix corresponding to short segments of a 3(10)-helix moving along S4 during opening and closing. Here, we have explored the energetics of the transition between the fully open state (based on the X-ray structure) and the first intermediate state towards channel closing (C1), modeled from experimental constraints. We show that conformations within 3 Å of the X-ray structure are obtained in simulations starting from the C1 model, and directly observe the previously suggested sliding 3(10)-helix region in S4. Through systematic free energy calculations, we show that the C1 state is a stable intermediate conformation and determine free energy profiles for moving between the states without constraints. Mutations indicate several residues in a narrow hydrophobic band in the voltage sensor contribute to the barrier between the open and C1 states, with F233 in the S2 helix having the largest influence. Substitution for smaller amino acids reduces the transition cost, while introduction of a larger ring increases it, largely confirming experimental activation shift results. There is a systematic correlation between the local aromatic ring rotation, the arginine barrier crossing, and the corresponding relative free energy. In particular, it appears to be more advantageous for the F233 side chain to rotate towards the extracellular side when arginines cross the hydrophobic region.

Accuracy analysis of multiple structure alignments.

Protein structure alignment methods are essential for many different challenges in protein science, such as the determination of relations between proteins in the fold space or the analysis and prediction of their biological function. A number of different pairwise and multiple structure alignment (MStA) programs have been developed and provided to the community. Prior knowledge of the expected alignment accuracy is desirable for the user of such tools. To retrieve an estimate of the performance of current structure alignment methods, we compiled a test suite taken from literature and the SISYPHUS database consisting of proteins that are difficult to align. Subsequently, different MStA programs were evaluated regarding alignment correctness and general limitations. The analysis shows that there are large differences in the success between the methods in terms of applicability and correctness. The latter ranges from 44 to 75% correct core positions. Taking only the best method result per test case this number increases to 84%. We conclude that the methods available are applicable to difficult cases, but also that there is still room for improvements in both, practicability and alignment correctness. An approach that combines the currently available methods supported by a proper score would be useful. Until then, a user should not rely on just a single program.