Hinge-bending motions in the pore domain of a bacterial voltage-gated sodium channel

Abstract

Computational methods and experimental data are used to provide structural models for NaChBac, the homo-tetrameric voltage-gated sodium channel from the bacterium Bacillus halodurans, with a closed and partially open pore domain. Molecular dynamic (MD) simulations on membrane-bound homo-tetrameric NaChBac structures, each comprising six helical transmembrane segments (labeled S1 through S6), reveal that the shape of the lumen, which is defined by the bundle of four alpha-helical S6 segments, is modulated by hinge bending motions around the S6 glycine residues. Mutation of these glycine residues into proline and alanine affects, respectively, the structure and conformational flexibility of the S6 bundle. In the closed channel conformation, a cluster of stacked phenylalanine residues from the four S6 helices hinders diffusion of water molecules and Na(+) ions. Activation of the voltage sensor domains causes destabilization of the aforementioned cluster of phenylalanines, leading to a more open structure. The conformational change involving the phenylalanine cluster promotes a kink in S6, suggesting that channel gating likely results from the combined action of hinge-bending motions of the S6 bundle and concerted reorientation of the aromatic phenylalanine side-chains.

Figure 1

Structure of the selectivity filter with two solvated Na⁺ ions: (A) upper view from the outer vestibule; (B) side-view. Main chain groups from residues Thr189, Leu190 and Glu191 and the side-chain of Glu191 plus water molecules are shown in stick representation, while Na⁺ ions are rendered as yellow spheres.

Figure 2

Shown are the patterns of salt-bridges in the voltage-sensing domain (VSD) of NaChBac for selected conformations of the channel: (A) closed; (B) early-activated-open; and (C) activated-partially-open (see text). The C-alpha atoms of the arginines and the acidic residues of the VSD are shown as blue and red spheres, respectively. The thickness of the black cylinders reflects the relative occurrence of each salt-bridge along the MD simulation trajectory.

Figure 3

Root mean square deviation (RMSD) of the backbone atoms from the initial structure employed in the MD simulation plotted as a function of time for: (A) closed; (B) early-activated-open; and (C) activated-partially-open conformations, respectively (see text). The RMSD is shown separately for different regions of the channel: entire channel (black), voltage-sensing domains (blue), pore domain (red), and selectivity filter (green).

Figure 4

Conformations of the NaChBac pore domain along the activation pathway as determined in the present study, with only the S6-helix bundle shown for clarity. Upper panels: (A, B) space-filling representation of the channel lumen for the closed and activated-partially-open conformations, respectively. Phenyl groups Phe224 and Phe227 are shown in blue and cyan, respectively. Bottom panels: (C, D) cartoon representation of the S6-helix bundle highlighting the hinge-bending motions around residues G219 and G229 (red spheres) involving the three helical regions (blue, red, and orange) for the closed and activated-partially-open conformations, respectively.

Figure 5

Probability distributions of S6 kink angles around G219 (A) and G229 (B) for the activated-partially-open (black) and closed (blue) conformations, along with results for the mutants G219A (red) and G219P (green). The angles are measured between the helical segments adjacent to the glycine residues: segments 210–218 and 220–228 for G219 and 218–228 and 230–240 for G229.

Figure 6

Shown is a superposition of structures of the NaChBac S4–S5 linker for closed (red), early-activated-open (blue) and activated-partially-open (green) conformations (see text), highlighting the displacement of the linker along the direction normal to the membrane.

Figure 7

Shown are the positions of water oxygen atoms projected onto the membrane normal (Z). Blue shading highlights the portion of the MD trajectory sampled before an external electrostatic field was applied. Under the electric field, the frequency of wetting/dewetting transitions at the hydrophobic gate (located approximately between -7 Å and -15 Å) increases dramatically.