The allosteric gating mechanism of the MthK channel

Figures

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  Figure 1.. The dominant allosteric motion that RCK dimer undergoes during state transition. (A) A functional MthK channel requires eight RCK domains to form an octameric gating ring on the intracellular side of the pore (PDB ID: 6UX4). There are three Ca2+-binding sites in each RCK domain; thus, a gating ring of the MthK channel contains a total of 24 Ca2+-binding sites. (B) RCK-dimer structures projected onto the second and third modes. Each point (or structure) is colored according to its PDB ID. The open and closed RCK dimers mainly differ in composition of the second eigenvector, C2. Two RCK dimers (PDB ID: 2OGU, 2AEM) are interpreted as intermediate during the allosteric transition. The ring-forming RCK dimers are highlighted by gray shadowing. (C) Visualization of the second eigenvector characterizing the dominant motion that the RCK dimer undergoes during closed-to-open state transition.

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  Figure 2.. The specific bilobed architecture transmits and amplifies the Ca2+-binding-induced conformational changes. (A) The mobility profile of the RCK dimer. Left: the average cumulative RMSD of each residue is plotted. Right: the RCK-dimer structure is colored according to each residue's average RMSD, with immobile residues (R206-E210, D226, Q227, I229, I182, S235) shown as spheres. Hydrogen bonds between immobile residues are shown as yellow dashed lines, whereas the black dashed lines represent the rotation axes of N-lobes. (B) Alternative stereochemical Ca2+-binding modes between closed (blue color, PDB ID: 2FY8) and open (yellow color, PDB ID: 2AEF) states at Site 1. The bound Ca2+ ion (wheat) and Ca2+-chelating water molecules (red) are shown as spheres. At Site 1, the swinging-back of E212 and the relative displacement of F232 upon Ca2+ binding are indicated. (C) Helix αF is tightly associated with the neighboring N-lobe by hydrophobic interactions and the ‘bolted interaction’, a hydrogen bond between R206 and S242. (D) ITC experiments to measure the Ca2+-binding affinities of the MthK-gating ring mutants. The total heat exchanged during each injection is fitted to a single-site…

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  Figure 3.. Intra-dimer cooperativity. (A) Open-state RCK dimer (PDB ID: 1LNQ) showing the disruption of the ionic lock (details in the inset). (B) Closed-state RCK dimer (PDB ID: 2FY8) showing intra-dimer ionic lock (details in the inset) that stabilizes the dimer in the closed state. (C) Single-channel traces of wild-type and mutant MthK channels without the addition of internal [Ca2+]. Membrane voltage was –100 mV so that channel openings cause a downward current. (D) and (E) Left: alternative stereochemical Ca2+-binding modes between closed (yellow color, PDB ID: 2FY8) and open (blue color, PDB ID: 3RBZ) at Site 2 (D) and Site 3 (E). Right: schematic drawings illustrating the allosteric coupling between different Ca2+-binding sites in an RCK dimer. The closed state is colored light blue and dark blue for two subunits, while the open state is shown as dashed white graphics. The RCK dimer is shown in closed (left), open (middle) and overlap (right) states. The C-lobes are shown as ovals, with helices αFs and αGs as rectangles. Two N-lobes are removed for clarity. (D) E266 is shown, while Ca2+ ions appear as red spheres, respectively. The rotation of helices αFs and N-lobes arising from…

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  Figure 4.. Inter-dimer cooperativity. (A) Left: the conformer plot of gating-ring structures. All structures are projected onto the second and third modes. The open and closed gating rings are separated along the second dimension, with a previously determined partially open gating ring (PDB ID: 2FY8) in between. The open rings are widely distributed along the third dimension, while the closed rings are clustered around zero. Right: conformational ensemble obtained from interpolating along the third modes. All structures, with adjacent RCK dimers colored differently, are superimposed with one subunit. This suggests that open-state gating rings display higher dynamics than closed-state gating rings with respect to the inter-dimer arrangement. (B) and (C) Side views of the closed state (PDB ID: 2FY8) showing the inter-dimer ionic lock (details in the inset) that stabilizes the assembly interface in the closed state (B) and the open-state gating ring (PDB ID: 1LNQ) showing the disruption of the ionic lock (details in the inset) (C). (D) Single-channel traces of wild-type and mutant MthK channels with or without the addition of internal [Ca2+]. Membrane voltage was –100 mV so that channel…