Voltage-gated sodium channels (Navs) play essential roles in excitable tissues, with their activation and opening resulting in the initial phase of the action potential. The cycling of Navs through open, closed and inactivated states, and their closely choreographed relationships with the activities of other ion channels lead to exquisite control of intracellular ion concentrations in both prokaryotes and eukaryotes. Here we present the 2.45 Å resolution crystal structure of the complete NavMs prokaryotic sodium channel in a fully open conformation. A canonical activated conformation of the voltage sensor S4 helix, an open selectivity filter leading to an open activation gate at the intracellular membrane surface and the intracellular C-terminal domain are visible in the structure. It includes a heretofore unseen interaction motif between W77 of S3, the S4–S5 interdomain linker, and the C-terminus, which is associated with regulation of opening and closing of the intracellular gate.

Voltage-gated sodium channels (Navs) are responsible for the initiation of the action potential in excitable cells. Several prokaryotic sodium channels, most notably NavMs from Magnetococcus marinus and NavAb from Arcobacter butzleri, have been shown to be good models for human sodium channels based on their sequence homologies and high levels of functional similarities, including ion flux, and functional consequences of critical mutations. The complete full-length crystal structures of these prokaryotic sodium channels captured in different functional states have now revealed the molecular natures of changes associated with the gating process. These include the structures of the intracellular gate, the selectivity filter, the voltage sensors, the intra-membrane fenestrations, and the transmembrane (TM) pore. Here we have identified for the first time how changes in the fenestrations in the hydrophobic TM region associated with the opening of the intracellular gate could modulate the state-dependent ingress and binding of drugs in the TM cavity, in a way that could be exploited for rational drug design.

Cap-dependent initiation of translation is regulated by the interaction of the eukaryotic translation initiation factor 4E (eIF4E) with the 120-residue disordered eIF4E binding protein 2 (4E-BP2) in a phosphorylation-dependent manner. Previous NMR studies have shown that 4E-BP2 interacts with eIF4E at two distinct sites, via an a-helical structure at the canonical binding site and a disordered secondary binding site $20 residues away. Phosphorylation of the 4E-BP2 at 5 distinct sites decreases its binding affinity for the eIF4E by ca. 4000 times, partially due to the formation of a 4-stranded b-sheet that sequesters the canonical binding site. Single-molecule fluorescence resonance energy transfer (smFRET) studies of the 4E-BP2 between residues 32 and 91 show an increase in FRET efficiency upon binding to the eIF4E. This confirms that the 4E-BP2 adopts extended conformations in the bound state that wrap around eIF4E. Intermolecular smFRET with evanescent-field excitation was used to study the interaction between donor-labelled surface-immobilized eIF4E and acceptor-labelled free-diffusing 4E-BP2. FRET efficiency-time trajectories from thousands of individual molecules were used to derive the distribution of t ON and t OFF on-and offbinding times at the sub-ensemble level. Finally, the kinetic binding data is analyzed using several candidate models to gain an understanding of the physical mechanism of interaction in the 4E complex.