In this study we present a high resolution structure of the NaKNΔ19 channel in an open conformation. A comparison of this open state structure with that of the previously determined closed state shows that the region surrounding the selectivity filter, the top 1/3 of the pore at towards the extracellular side, remains static during channel gating. In contrast, major conformational changes occur in the inner helices at residue Gly87 just below the filter. The structure similarity between NaKNΔ19 and MthK pore points to similar pore opening mechanics in these two channels, specifically, utilization of a conserved glycine residue as a gating hinge to allow for inner helix bending. This glycine hinge has also been functionally shown to be important for gating in other tetrameric cation channels, such as voltage-gated K+
], although these channel pores may not open as wide as what is observed in MthK or NaK.
Even though the intra- and inter-subunit interactions in the open state of NaK appear to be less extensive than those in the closed state, mainly due to the disruption of the helix bundle crossing, a number of factors could contribute to the stabilization of the open state captured in the crystal. First, the M0 helices are absent in NaKNΔ19. In the closed NaK structure, four M0 helices form a cuff surrounding the bundle crossing and could prevent the inner helices from splaying open, a spatial hindrance absent in the truncated construct. Indeed, NaKNΔ19 does functionally display substantially higher ion conduction rates compared to the full length channel in 86
Rb flux assays[11
], which most likely stems from a higher channel open probability. Second, the use of detergents instead of lipids in the purification and crystallization procedures, in other words the absence of a lipid environment in the crystal, could allow for more conformational freedom for the channel. Finally, protein packing in the crystal could be a main factor contributing to the open state. Two kinds of inter-tetramer packing interactions were observed in the NaKNΔ19 crystal (Supplementary Fig. 2
). One is the anti-parallel hydrophobic packing between outer helices from two neighboring channel tetramers (Supplementary Fig. 2
, squared region). The other is hydrophilic contacts between the C-terminal end of the inner helix and the extracellular surface of a neighboring tetramer (Supplementary Fig 2
, circled regions). These hydrophilic contacts are predominantly hydrogen bonding in nature and some are mediated by water molecules. Since both kinds of inter-tetramer packing interactions are quite extensive, we believe this is the dominant force stabilizing NaKNΔ19 in the open conformation. Despite the possibility of structural distortion due to crystal packing, the fact that NaKNΔ19 and MthK, two different crystals of different proteins, share virtually the same structure is a clear indication that NaKNΔ19 does represent an open pore structure.
KcsA has been one of the most well studied K+
channels in regards to its gating properties, specifically the physical movement of its intracellular gate [16
]. The global conformational changes observed between the open and closed conformation structures of NaK match quite well with those observed in KcsA. In a recent study using single KcsA tetramers labeled with gold nano crystals, a global twisting motion of +/- 40° was observed for inner helices in channels undergoing gating transitions[24
]. Such a movement can be easily mapped on to the NaK structures. By using the Cα of Ala99 as an example, the movement of residues at the C-terminal part of inner helices between closed and open state results in a rotation of about 42° relative to the central axis (Supplementary Fig. 3a
), equivalent to the global twisting motion reported for the KcsA pore.
It is also important to note that some of the cross subunit (diagonal) distances calculated between residues in the open and closed conformation of NaK are quite different from those reported in a gating study on KcsA using Electron Paramagnetic Resonance (EPR) spectroscopic methods [17
]. For example, the distance between two spin-labeled Ala109s in KcsA shows a decrease of about 9 Å from closed to open states in the EPR measurement, whereas the diagonal distance between the Cβs of the equivalent residues (Lys97) in NaK show an increase of 5.5 Å from closed to open state based on our crystal structures. We believe this discrepancy arises mainly from contributions of the spin label arm length, which can extend up to 9 Å from the Cβ of the labeled residue and the paramagnetic center in the nitroxide moiety [25
], rather than from differences in gating mechanics. As shown in Supplementary Fig 3b
, in the closed state of NaK, Lys97 is positioned at the distal face of the inner helix relative to the central axis of the pore. A spin label at this position likely has the nitroxide moiety extended away from the central axis leading to an overestimation of the diagonal distance. On the other hand, Lys97 in the open state would be oriented more towards the ion conduction pathway as a consequence of inner helix bending and twisting and the spin label at this position will have the nitroxide moiety extending closer to the central axis leading to an underestimation of the diagonal distance. If we measure the diagonal distance between the amine atoms of Lys97s instead of Cßs as a simple approximation of the distance between the paramagnetic centers, a decrease of 7 Å is observed (from 29 Å in the closed state to 22 Å in the open state), which matches well with that observed in the EPR measurement of KcsA labeled at the equivalent residue (Ala109). Here we only consider one residue as an example instead of mapping and comparing every relevant NaK residue with corresponding KcsA residues in the EPR studies. The important conclusion that arises, however, is that the structure of KcsA in its open state would likely be similar to that of NaKNΔ19 or MthK and the structures of NaK and NaKNΔ19 represent the general structures of the tetrameric cation channel pore in closed and open states, respectively.