Structural insights into ion selectivity and transport mechanisms of Oryza sativa HKT2;1 and HKT2;2/1 transporters.

Plant high-affinity K+ transporters (HKTs) play a pivotal role in maintaining the balance of Na+ and K+ ions in plants, thereby influencing plant growth under K+-depleted conditions and enhancing tolerance to salinity stress. Here we report the cryo-electron microscopy structures of Oryza sativa HKT2;1 and HKT2;2/1 at overall resolutions of 2.5 Å and 2.3 Å, respectively. Both transporters adopt a dimeric assembly, with each protomer enclosing an ion permeation pathway. Comparison between the selectivity filters of the two transporters reveals the critical roles of Ser88/Gly88 and Val243/Gly243 in determining ion selectivity. A constriction site along the ion permeation pathway is identified, consisting of Glu114, Asn273, Pro392, Pro393, Arg525, Lys517 and the carboxy-terminal Trp530 from the neighbouring protomer. The linker between domains II and III adopts a stable loop structure oriented towards the constriction site, potentially participating in the gating process. Electrophysiological recordings, yeast complementation assays and molecular dynamics simulations corroborate the functional importance of these structural features. Our findings provide crucial insights into the ion selectivity and transport mechanisms of plant HKTs, offering valuable structural templates for developing new salinity-tolerant cultivars and strategies to increase crop yields.

Structural basis for Gemin5 decamer-mediated mRNA binding.

Gemin5 in the Survival Motor Neuron (SMN) complex serves as the RNA-binding protein to deliver small nuclear RNAs (snRNAs) to the small nuclear ribonucleoprotein Sm complex via its N-terminal WD40 domain. Additionally, the C-terminal region plays an important role in regulating RNA translation by directly binding to viral RNAs and cellular mRNAs. Here, we present the three-dimensional structure of the Gemin5 C-terminal region, which adopts a homodecamer architecture comprised of a dimer of pentamers. By structural analysis, mutagenesis, and RNA-binding assays, we find that the intact pentamer/decamer is critical for the Gemin5 C-terminal region to bind cognate RNA ligands and to regulate mRNA translation. The Gemin5 high-order architecture is assembled via pentamerization, allowing binding to RNA ligands in a coordinated manner. We propose a model depicting the regulatory role of Gemin5 in selective RNA binding and translation. Therefore, our work provides insights into the SMN complex-independent function of Gemin5.

High-resolution structures of human Nav1.7 reveal gating modulation through α-π helical transition of S6IV.

Nav1.7 represents a preeminent target for next-generation analgesics for its critical role in pain sensation. Here we report a 2.2-Å resolution cryo-EM structure of wild-type (WT) Nav1.7 complexed with the ß1 and ß2 subunits that reveals several previously indiscernible cytosolic segments. Reprocessing of the cryo-EM data for our reported structures of Nav1.7(E406K) bound to various toxins identifies two distinct conformations of S6IV, one composed of α helical turns only and the other containing a π helical turn in the middle. The structure of ligand-free Nav1.7(E406K), determined at 3.5-Å resolution, is identical to the WT channel, confirming that binding of Huwentoxin IV or Protoxin II to VSDII allosterically induces the α → π transition of S6IV. The local secondary structural shift leads to contraction of the intracellular gate, closure of the fenestration on the interface of repeats I and IV, and rearrangement of the binding site for the fast inactivation motif.

Structure of the human Meckel-Gruber protein Meckelin.

Mutations in the Meckelin gene account for most cases of the Meckel-Gruber syndrome, the most severe ciliopathy with a 100% mortality rate. Here, we report a 3.3-Å cryo­electron microscopy structure of human Meckelin (also known as TMEM67 and MKS3). The structure reveals a unique protein fold consisting of an unusual cysteine-rich domain that folds as an arch bridge stabilized by 11 pairs of disulfide bonds, a previously uncharacterized domain named ß sheet­rich domain, a previously unidentified seven-transmembrane fold wherein TM4 to TM6 are broken near the cytoplasmic surface of the membrane, and a coiled-coil domain placed below the transmembrane domain. Meckelin forms a stable homodimer with an extensive dimer interface. Our structure establishes a framework for dissecting the function and disease mechanisms of Meckelin.

Structural determination of human Nav1.4 and Nav1.7 using single particle cryo-electron microscopy.

Voltage-gated sodium (Nav) channels are responsible for the initiation and propagation of action potentials. Their abnormal functions are associated with numerous diseases, such as epilepsy, cardiac arrhythmia, and pain syndromes. Therefore, these channels represent important drug targets. Even in the post-resolution revolution era, a lack of structural information continues to impede structure-based drug discovery. The limiting factor for the structural determination of Nav channels using single particle cryo-electron microscopy (cryo-EM) resides in the generation of sufficient high-quality recombinant proteins. After extensive trials, we have been successful in determining a series of high-resolution structures of Nav channels, including NavPaS from American cockroach, Nav1.4 from electric eel, and human Nav1.1, Nav1.2, Nav1.4, Nav1.5, and Nav1.7, with distinct strategies. These structures established the framework for understanding the electromechanical coupling and disease mechanism of Nav channels, and for facilitating drug discovery. Here, we exemplify these methods with two specific cases, human Nav1.4 and Nav1.7, which may shed light on the structural determination of other membrane proteins.

Structural Basis for the Modulation of Human KCNQ4 by Small-Molecule Drugs.

Among the five KCNQ channels, also known as the Kv7 voltage-gated potassium (Kv) channels, KCNQ2-KCNQ5 control neuronal excitability. Dysfunctions of KCNQ2-KCNQ5 are associated with neurological disorders such as epilepsy, deafness, and neuropathic pain. Here, we report the cryoelectron microscopy (cryo-EM) structures of human KCNQ4 and its complexes with the opener retigabine or the blocker linopirdine at overall resolutions of 2.5, 3.1, and 3.3 Å, respectively. In all structures, a phosphatidylinositol 4,5-bisphosphate (PIP2) molecule inserts its head group into a cavity within each voltage-sensing domain (VSD), revealing an unobserved binding mode for PIP2. Retigabine nestles in each fenestration, inducing local shifts. Instead of staying within the central pore, linopirdine resides in a cytosolic cavity underneath the inner gate. Electrophysiological analyses of various mutants corroborated the structural observations. Our studies reveal the molecular basis for the modulatory mechanism of neuronal KCNQ channels and provide a framework for structure-facilitated drug discovery targeting these important channels.

Molecular basis for pore blockade of human Na+ channel Nav1.2 by the µ-conotoxin KIIIA.

The voltage-gated sodium channel Nav1.2 is responsible for the initiation and propagation of action potentials in the central nervous system. We report the cryo-electron microscopy structure of human Nav1.2 bound to a peptidic pore blocker, the µ-conotoxin KIIIA, in the presence of an auxiliary subunit, ß2, to an overall resolution of 3.0 angstroms. The immunoglobulin domain of ß2 interacts with the shoulder of the pore domain through a disulfide bond. The 16-residue KIIIA interacts with the extracellular segments in repeats I to III, placing Lys7 at the entrance to the selectivity filter. Many interacting residues are specific to Nav1.2, revealing a molecular basis for KIIIA specificity. The structure establishes a framework for the rational design of subtype-specific blockers for Nav channels.

Structures of human Nav1.7 channel in complex with auxiliary subunits and animal toxins.

Voltage-gated sodium channel Nav1.7 represents a promising target for pain relief. Here we report the cryo-electron microscopy structures of the human Nav1.7-ß1-ß2 complex bound to two combinations of pore blockers and gating modifier toxins (GMTs), tetrodotoxin with protoxin-II and saxitoxin with huwentoxin-IV, both determined at overall resolutions of 3.2 angstroms. The two structures are nearly identical except for minor shifts of voltage-sensing domain II (VSDII), whose S3-S4 linker accommodates the two GMTs in a similar manner. One additional protoxin-II sits on top of the S3-S4 linker in VSDIV The structures may represent an inactivated state with all four VSDs "up" and the intracellular gate closed. The structures illuminate the path toward mechanistic understanding of the function and disease of Nav1.7 and establish the foundation for structure-aided development of analgesics.

Structure of the human voltage-gated sodium channel Nav1.4 in complex with ß1.

Voltage-gated sodium (Nav) channels, which are responsible for action potential generation, are implicated in many human diseases. Despite decades of rigorous characterization, the lack of a structure of any human Nav channel has hampered mechanistic understanding. Here, we report the cryo-electron microscopy structure of the human Nav1.4-ß1 complex at 3.2-Å resolution. Accurate model building was made for the pore domain, the voltage-sensing domains, and the ß1 subunit, providing insight into the molecular basis for Na+ permeation and kinetic asymmetry of the four repeats. Structural analysis of reported functional residues and disease mutations corroborates an allosteric blocking mechanism for fast inactivation of Nav channels. The structure provides a path toward mechanistic investigation of Nav channels and drug discovery for Nav channelopathies.

Structural basis for the modulation of voltage-gated sodium channels by animal toxins.

Animal toxins that modulate the activity of voltage-gated sodium (Nav) channels are broadly divided into two categories-pore blockers and gating modifiers. The pore blockers tetrodotoxin (TTX) and saxitoxin (STX) are responsible for puffer fish and shellfish poisoning in humans, respectively. Here, we present structures of the insect Nav channel NavPaS bound to a gating modifier toxin Dc1a at 2.8 angstrom-resolution and in the presence of TTX or STX at 2.6-Å and 3.2-Å resolution, respectively. Dc1a inserts into the cleft between VSDII and the pore of NavPaS, making key contacts with both domains. The structures with bound TTX or STX reveal the molecular details for the specific blockade of Na+ access to the selectivity filter from the extracellular side by these guanidinium toxins. The structures shed light on structure-based development of Nav channel drugs.

Structure of the Nav1.4-ß1 Complex from Electric Eel.

Voltage-gated sodium (Nav) channels initiate and propagate action potentials. Here, we present the cryo-EM structure of EeNav1.4, the Nav channel from electric eel, in complex with the ß1 subunit at 4.0 Å resolution. The immunoglobulin domain of ß1 docks onto the extracellular L5I and L6IV loops of EeNav1.4 via extensive polar interactions, and the single transmembrane helix interacts with the third voltage-sensing domain (VSDIII). The VSDs exhibit "up" conformations, while the intracellular gate of the pore domain is kept open by a digitonin-like molecule. Structural comparison with closed NavPaS shows that the outward transfer of gating charges is coupled to the iris-like pore domain dilation through intricate force transmissions involving multiple channel segments. The IFM fast inactivation motif on the III-IV linker is plugged into the corner enclosed by the outer S4-S5 and inner S6 segments in repeats III and IV, suggesting a potential allosteric blocking mechanism for fast inactivation.

Structure of a eukaryotic voltage-gated sodium channel at near-atomic resolution.

Voltage-gated sodium (Nav) channels are responsible for the initiation and propagation of action potentials. They are associated with a variety of channelopathies and are targeted by multiple pharmaceutical drugs and natural toxins. Here, we report the cryogenic electron microscopy structure of a putative Nav channel from American cockroach (designated NavPaS) at 3.8 angstrom resolution. The voltage-sensing domains (VSDs) of the four repeats exhibit distinct conformations. The entrance to the asymmetric selectivity filter vestibule is guarded by heavily glycosylated and disulfide bond-stabilized extracellular loops. On the cytoplasmic side, a conserved amino-terminal domain is placed below VSDI, and a carboxy-terminal domain binds to the III-IV linker. The structure of NavPaS establishes an important foundation for understanding function and disease mechanism of Nav and related voltage-gated calcium channels.

Structural basis for the gating mechanism of the type 2 ryanodine receptor RyR2.

RyR2 is a high-conductance intracellular calcium (Ca2+) channel that controls the release of Ca2+ from the sarco(endo)plasmic reticulum of a variety of cells. Here, we report the structures of RyR2 from porcine heart in both the open and closed states at near-atomic resolutions determined using single-particle electron cryomicroscopy. Structural comparison reveals a breathing motion of the overall cytoplasmic region resulted from the interdomain movements of amino-terminal domains (NTDs), Helical domains, and Handle domains, whereas almost no intradomain shifts are observed in these armadillo repeats-containing domains. Outward rotations of the Central domains, which integrate the conformational changes of the cytoplasmic region, lead to the dilation of the cytoplasmic gate through coupled motions. Our structural and mutational characterizations provide important insights into the gating and disease mechanism of RyRs.