Structural and functional properties of a plant NRAMP-related aluminum transporter.

The transport of transition metal ions by members of the SLC11/NRAMP family constitutes a ubiquitous mechanism for the uptake of Fe2+ and Mn2+ across all kingdoms of life. Despite the strong conservation of the family, two of its branches have evolved a distinct substrate preference with one mediating Mg2+ uptake in prokaryotes and another the transport of Al3+ into plant cells. Our previous work on the SLC11 transporter from Eggerthella lenta revealed the basis for its Mg2+ selectivity (Ramanadane et al., 2022). Here, we have addressed the structural and functional properties of a putative Al3+ transporter from Setaria italica. We show that the protein transports diverse divalent metal ions and binds the trivalent ions Al3+ and Ga3+, which are both presumable substrates. Its cryo-electron microscopy (cryo-EM) structure displays an occluded conformation that is closer to an inward- than an outward-facing state, with a binding site that is remodeled to accommodate the increased charge density of its transported substrate.

Structural and functional properties of a magnesium transporter of the SLC11/NRAMP family.

Members of the ubiquitous SLC11/NRAMP family catalyze the uptake of divalent transition metal ions into cells. They have evolved to efficiently select these trace elements from a large pool of Ca2+ and Mg2+, which are both orders of magnitude more abundant, and to concentrate them in the cytoplasm aided by the cotransport of H+ serving as energy source. In the present study, we have characterized a member of a distant clade of the family found in prokaryotes, termed NRMTs, that were proposed to function as transporters of Mg2+. The protein transports Mg2+ and Mn2+ but not Ca2+ by a mechanism that is not coupled to H+. Structures determined by cryo-EM and X-ray crystallography revealed a generally similar protein architecture compared to classical NRAMPs, with a restructured ion binding site whose increased volume provides suitable interactions with ions that likely have retained much of their hydration shell.

Molecular dissection of multiphase inactivation of the bacterial sodium channel NaVAb.

Homotetrameric bacterial voltage-gated sodium channels share major biophysical features with their more complex eukaryotic counterparts, including a slow-inactivation mechanism that reduces ion-conductance activity during prolonged depolarization through conformational changes in the pore. The bacterial sodium channel NaVAb activates at very negative membrane potentials and inactivates through a multiphase slow-inactivation mechanism. Early voltage-dependent inactivation during one depolarization is followed by late use-dependent inactivation during repetitive depolarization. Mutations that change the molecular volume of Thr206 in the pore-lining S6 segment can enhance or strongly block early voltage-dependent inactivation, suggesting that this residue serves as a molecular hub controlling the coupling of activation to inactivation. In contrast, truncation of the C-terminal tail enhances the early phase of inactivation yet completely blocks late use-dependent inactivation. Determination of the structure of a C-terminal tail truncation mutant and molecular modeling of conformational changes at Thr206 and the S6 activation gate led to a two-step model of these gating processes. First, bending of the S6 segment, local protein interactions dependent on the size of Thr206, and exchange of hydrogen-bonding partners at the level of Thr206 trigger pore opening followed by the early phase of voltage-dependent inactivation. Thereafter, conformational changes in the C-terminal tail lead to late use-dependent inactivation. These results have important implications for the sequence of conformational changes that lead to multiphase inactivation of NaVAb and other sodium channels.

Structures of closed and open states of a voltage-gated sodium channel.

Bacterial voltage-gated sodium channels (BacNavs) serve as models of their vertebrate counterparts. BacNavs contain conserved voltage-sensing and pore-forming domains, but they are homotetramers of four identical subunits, rather than pseudotetramers of four homologous domains. Here, we present structures of two NaVAb mutants that capture tightly closed and open states at a resolution of 2.8-3.2 Å. Introduction of two humanizing mutations in the S6 segment (NaVAb/FY: T206F and V213Y) generates a persistently closed form of the activation gate in which the intracellular ends of the four S6 segments are drawn tightly together to block ion permeation completely. This construct also revealed the complete structure of the four-helix bundle that forms the C-terminal domain. In contrast, truncation of the C-terminal 40 residues in NavAb/1-226 captures the activation gate in an open conformation, revealing the open state of a BacNav with intact voltage sensors. Comparing these structures illustrates the full range of motion of the activation gate, from closed with its orifice fully occluded to open with an orifice of ∼10 Å. Molecular dynamics and free-energy simulations confirm designation of NaVAb/1-226 as an open state that allows permeation of hydrated Na+, and these results also support a hydrophobic gating mechanism for control of ion permeation. These two structures allow completion of a closed-open-inactivated conformational cycle in a single voltage-gated sodium channel and give insight into the structural basis for state-dependent binding of sodium channel-blocking drugs.