Dimeric Assembly Research Articles

Reconstitution of SPO11-dependent double-strand break formation.

Homologous meiotic recombination starts with DNA double-strand breaks (DSBs) generated by SPO11 protein 1 . SPO11 is critical for meiosis in most species but the DSBs it makes are also dangerous because of their mutagenic 2 and gametocidal 3 potential, so cells must foster SPO11's beneficial functions while minimizing its risks 4 . SPO11 mechanism and regulation remain poorly understood. Here we report reconstitution of DNA cleavage in vitro with purified recombinant mouse SPO11 bound to its essential partner TOP6BL. Similar to their yeast orthologs 5,6 , SPO11- TOP6BL complexes are monomeric (1:1) in solution and bind tightly to DNA. Unlike in yeast, however, dimeric (2:2) assemblies of mouse SPO11-TOP6BL cleave DNA to form covalent 5' attachments requiring SPO11 active site residues, divalent metal ions, and SPO11 dimerization. Surprisingly, SPO11 can also manifest topoisomerase activity by relaxing supercoils and resealing DNA that it has nicked. Structure modeling with AlphaFold3 7 illuminates the protein-DNA interface and suggests that DNA is bent prior to cleavage. Deep sequencing of in vitro cleavage products reveals a rotationally symmetric base composition bias that partially explains DSB site preferences in vivo. Cleavage is inefficient on complex DNA substrates, partly because SPO11 is readily trapped in DSB-incompetent (presumably monomeric) binding states that exchange slowly. However, cleavage is improved by using substrates that favor DSB-competent dimer assembly, or by fusing SPO11 to an artificial dimerization module. Our results inform a model in which intrinsically feeble dimerization restrains SPO11 activity in vivo, making it exquisitely dependent on accessory proteins that focus and control DSB formation so that it happens only at the right time and the right places.

Domain architecture of the Mycobacterium tuberculosis MabR (Rv2242), a member of the PucR transcription factor family

MabR (Rv2242), a PucR-type transcription factor, plays a crucial role in regulating mycolic acid biosynthesis in Mycobacterium tuberculosis. To understand its regulatory mechanisms, we determined the crystal structures of its N-terminal and C-terminal domains. The N-terminal domain adopts a globin-like fold, while the C-terminal domain comprises an α/β GGDEF domain and an all-α effector domain with a helix-turn-helix DNA-binding motif. This unique domain combination is specific to Actinomycetes. Biochemical and computational studies suggest that full-length MabR forms both dimeric and tetrameric assemblies in solution. Structural analysis revealed two distinct dimerization interfaces within the N- and C-terminal domains, further supporting a tetrameric organization. These findings provide valuable insights into the domain architecture, oligomeric state, and potential regulatory mechanisms of MabR.

Anion Recognition-Directed Supramolecular Catalysis with Functional Macrocycles and Molecular Cages.

ConspectusThe development of supramolecular chemistry has provided a variety of host molecules and noncovalent tools for boosting catalytic processes, stimulating the emergence and advance of supramolecular catalysis, among which macrocyclic and cage-like compounds have attracted great attention due to their possession of an enzyme-mimetic cavity and recognition ability. While the privileged scaffolds such as crown ethers, cyclodextrins, cucurbiturils, calixarenes, and metal-coordinated cages have been widely used, their skeletons usually do not contain a directional binding site; binding and activation mainly rely on cation-associated interactions or hydrophobic effects. In this context, the recent advance of anion supramolecular chemistry has drawn our attention to developing an anion recognition-directed approach by using tailor-made functionalized macrocycles and cages. Anions are important widely existing species in both biological and chemical systems and play an important role in regulating the structure and function of enzymes. We envisioned that by taking advantage of anions, including their rich variety, diverse geometry, and multiple interaction sites, the sophisticated cooperation of multiple noncovalent interactions can be manipulated in a confined cavity for directing efficient and selective catalysis.Following this concept, we initiated our study by introducing typical thiourea H-bonding groups to design and synthesize a series of bis-thiourea macrocycles, especially chiral macrocycles, by incorporating chiral linkers. Taking advantage of the obtained strong, cooperative anion binding, a macrocycle-enabled counteranion trapping strategy was developed, which afforded greatly enhanced catalytic efficiency and excellent stereocontrol in acid-catalyzing reactions. Furthermore, inspired by sulfate-induced macrocyclic dimerization assembly, we built a substrate-induced assembly system, enabling an induced-fit cooperative activation network for efficient and enantioselective catalysis. In addition, anion recognition-driven chirality gearing with a more sophisticated trithiourea cage was revealed, which could provide a basis for implementing anion-triggered allosteric catalysis within the induced helical space. Not limited to hydrogen bonding, the emerging anion-π interactions were largely exploited. A series of triazine-based prism cages containing three V-shaped electron-deficient π-cavities were constructed, and their anion-π binding properties were studied. Based on this system, cooperative anion-π activation was established for driving highly efficient and selective catalysis, which paved a way to push anion-π interactions toward more practical and useful catalyst design.These results demonstrated that the anion-recognition direction can serve as a powerful, versatile approach for boosting highly efficient and selective supramolecular catalysis. It is feasible not only for employing exogenous anions (e.g., counteranion) as a handle but also for recognition and regulation of anionic active intermediates/transition states, from use in conventional H-bonding to emerging anion-π recognition.

Molecular mechanism of IgE-mediated FcεRI activation.

Allergic diseases, affecting over a quarter of individuals in industrialized countries, have become significant public health concerns1,2. The high-affinity Fc receptor for IgE (FcεRI), mainly present on mast cells and basophils, plays a crucial role in allergic diseases3-5. Monomeric IgE binding to FcεRI regulates mast cell survival, differentiation, and maturation6-8. However, the underlying molecular mechanism remains unclear. Here we demonstrate that, prior to IgE binding, FcεRI mostly exists as a homo-dimer on human mast cell membrane. The structure of human FcεRI confirms the dimeric organization, with each promoter comprising one α subunit, one β subunit, and two γ subunits. The transmembrane helices of the α subunits form a layered arrangement with those of the γ and β subunits. The dimeric interface is mediated by a four-helix bundle of the α and γ subunits at the intracellular juxtamembrane region. Cholesterol-like molecules embedded within the transmembrane domain may stabilize the dimeric assembly. Upon IgE binding, the dimeric FcεRI dissociates into two protomers, each binding to an IgE molecule. Importantly, this process elicits transcriptional activation of Egr1/3 and Ccl2 in rat basophils, which can be attenuated by inhibiting the FcεRI dimer-to-monomer transition. Collectively, our study unveils the mechanism of antigen-independent, IgE-mediated FcεRI activation.

Cryo-electron microscopy structure of the di-domain core of Mycobacterium tuberculosis polyketide synthase 13, essential for mycobacterial mycolic acid synthesis.

Mycobacteria are known for their complex cell wall, which comprises layers of peptidoglycan, polysaccharides and unusual fatty acids known as mycolic acids that form their unique outer membrane. Polyketide synthase 13 (Pks13) of Mycobacterium tuberculosis, the bacterial organism causing tuberculosis, catalyses the last step of mycolic acid synthesis prior to export to and assembly in the cell wall. Due to its essentiality, Pks13 is a target for several novel anti-tubercular inhibitors, but its 3D structure and catalytic reaction mechanism remain to be fully elucidated. Here, we report the molecular structure of the catalytic core domains of M. tuberculosis Pks13 (Mt-Pks13), determined by transmission cryo-electron microscopy (cryoEM) to a resolution of 3.4 Å. We observed a homodimeric assembly comprising the ketoacyl synthase (KS) domain at the centre, mediating dimerization, and the acyltransferase (AT) domains protruding in opposite directions from the central KS domain dimer. In addition to the KS-AT di-domains, the cryoEM map includes features not covered by the di-domain structural model that we predicted to contain a dimeric domain similar to dehydratases, yet likely lacking catalytic function. Analytical ultracentrifugation data indicate a pH-dependent equilibrium between monomeric and dimeric assembly states, while comparison with the previously determined structures of M. smegmatis Pks13 indicates architectural flexibility. Combining the experimentally determined structure with modelling in AlphaFold2 suggests a structural scaffold with a relatively stable dimeric core, which combines with considerable conformational flexibility to facilitate the successive steps of the Claisen-type condensation reaction catalysed by Pks13.

Novel Anti-Trop2 Nanobodies Disrupt Receptor Dimerization and Inhibit Tumor Cell Growth.

Background: Trop2 (trophoblast cell-surface antigen 2) is overexpressed in multiple malignancies and is closely associated with poor prognosis, thus positioning it as a promising target for pan-cancer therapies. Despite the approval of Trop2-targeted antibody-drug conjugates (ADCs), challenges such as side effects, drug resistance, and limited efficacy persist. Recent studies have shown that the dimeric forms of Trop2 are crucial for its oncogenic functions, and the binding epitopes of existing Trop2-targeted drugs lie distant from the dimerization interface, potentially limiting their antitumor efficacy. Method: A well-established synthetic nanobody library was screened against Trop2-ECD. The identified nanobodies were extensively characterized, including their binding specificity and affinity, as well as their bioactivities in antigen-antibody endocytosis, cell proliferation, and the inhibition of Trop2 dimer assembly. Finally, ELISA based epitope analysis and AlphaFold 3 were employed to elucidate the binding modes of the nanobodies. Results: We identified two nanobodies, N14 and N152, which demonstrated high affinity and specificity for Trop2. Cell-based assays confirmed that N14 and N152 can facilitate receptor internalization and inhibit growth in Trop2-positive tumor cells. Epitope analysis uncovered that N14 and N152 are capable of binding with all three subdomains of Trop2-ECD and effectively disrupt Trop2 dimerization. Predictive modeling suggests that N14 and N152 likely target the epitopes at the interface of Trop2 cis-dimerization. The binding modality and mechanism of action demonstrated by N14 and N152 are unique among Trop2-targeted antibodies. Conclusions: we identified two novel nanobodies, N14 and N152, that specifically bind to Trop2. Importantly, these nanobodies exhibit significant anti-tumor efficacy and distinctive binding patterns, underscoring their potential as innovative Trop2-targeted therapeutics.

Insights into Transient Dimerisation of Carnitine/Acylcarnitine Carrier (SLC25A20) from Sarkosyl/PAGE, Cross-Linking Reagents, and Comparative Modelling Analysis.

The carnitine/acylcarnitine carrier (CAC) is a crucial protein for cellular energy metabolism, facilitating the exchange of acylcarnitines and free carnitine across the mitochondrial membrane, thereby enabling fatty acid β-oxidation and oxidative phosphorylation (OXPHOS). Although CAC has not been crystallised, structural insights are derived from the mitochondrial ADP/ATP carrier (AAC) structures in both cytosolic and matrix conformations. These structures underpin a single binding centre-gated pore mechanism, a common feature among mitochondrial carrier (MC) family members. The functional implications of this mechanism are well-supported, yet the structural organization of the CAC, particularly the formation of dimeric or oligomeric assemblies, remains contentious. Recent investigations employing biochemical techniques on purified and reconstituted CAC, alongside molecular modelling based on crystallographic AAC dimeric structures, suggest that CAC can indeed form dimers. Importantly, this dimerization does not alter the transport mechanism, a phenomenon observed in various other membrane transporters across different protein families. This observation aligns with the ping-pong kinetic model, where the dimeric form potentially facilitates efficient substrate translocation without necessitating mechanistic alterations. The presented findings thus contribute to a deeper understanding of CAC's functional dynamics and its structural parallels with other MC family members.

Structural and Magnetic Properties of Dimeric Capsule Assemblies Formed by Cyclic Trinuclear Complexes.

Cyclic trinuclear homo-metal complexes, [{Fe(L3+2Br)py}3] (1) and [{Mn(L3+2Br)}3(py)2 MeOH] (2), along with a hetero-metal complex, [FeMn2(L3+2H)3(DMF)3] (3), were synthesized using asymmetric ditopic ligands (H3L3+2H: 2-(2-hydroxyphenyl)-6-ol-5-(salicylideneamino)benzoxazole, H3L3+2Br: 2-(2-hydrox-5-bromoyphenyl)-6-ol-5-(5-bromosalicylideneamino)benzoxazole). The molecular structure of 1 is characterized by a tripod structure with three-fold symmetry, where an enantiomer pair forms a dimeric capsule with dimensions of approximately 3 × 1.6 × 1.6 nm3. Complexes 2 and 3, which lack three-fold symmetry, exhibit similar molecular structures to previously reported complexes with these ligands, but do not form a capsule structure. Magnetic measurements of 1-3 reveal the presence of significantly weak antiferromagnetic interactions between the metal ions.

DNA origami-templated gold nanorod dimer nanoantennas: enabling addressable optical hotspots for single cancer biomarker SERS detection.

The convergence of DNA origami and surface-enhanced Raman spectroscopy (SERS) has opened a new avenue in bioanalytical sciences, particularly in the detection of single-molecule proteins. This breakthrough has enabled the development of advanced sensor technologies for diagnostics. DNA origami offers a highly controllable framework for the precise positioning of nanostructures, resulting in superior SERS signal amplification. In our investigation, we have successfully designed and synthesized DNA origami-based gold nanorod monomer and dimer assemblies. Moreover, we have evaluated the potential of dimer assemblies for label-free detection of a single biomolecule, namely epidermal growth factor receptor (EGFR), a crucial biomarker in cancer research. Our findings have revealed that the significant Raman amplification generated by DNA origami-assembled gold nanorod dimer nanoantennas facilitates the label-free identification of Raman peaks of single proteins, which is a prime aim in biomedical diagnostics. The present work represents a significant advancement in leveraging plasmonic nanoantennas to realize single protein SERS for the detection of various cancer biomarkers with single-molecule sensitivity.

Cryo-EM reconstruction of oleate hydratase bound to a phospholipid membrane bilayer

Oleate hydratase (OhyA) is a bacterial peripheral membrane protein that catalyzes FAD-dependent water addition to membrane bilayer-embedded unsaturated fatty acids. The opportunistic pathogen Staphylococcus aureus uses OhyA to counteract the innate immune system and support colonization. Many Gram-positive and Gram-negative bacteria in the microbiome also encode OhyA. OhyA is a dimeric flavoenzyme whose carboxy terminus is identified as the membrane binding domain; however, understanding how OhyA binds to cellular membranes is not complete until the membrane-bound structure has been elucidated. All available OhyA structures depict the solution state of the protein outside its functional environment. Here, we employ liposomes to solve the cryo-electron microscopy structure of the functional unit: the OhyA•membrane complex. The protein maintains its structure upon membrane binding and slightly alters the curvature of the liposome surface. OhyA preferentially associates with 20–30 nm liposomes with multiple copies of OhyA dimers assembling on the liposome surface resulting in the formation of higher-order oligomers. Dimer assembly is cooperative and extends along a formed ridge of the liposome. We also solved an OhyA dimer of dimers structure that recapitulates the intermolecular interactions that stabilize the dimer assembly on the membrane bilayer as well as the crystal contacts in the lattice of the OhyA crystal structure. Our work enables visualization of the molecular trajectory of membrane binding for this important interfacial enzyme.

Binding of Linear Anions and Formation of Anion Encapsulated Dimeric Capsular Assembly by Cis‐5,15‐bis(3,5‐trifluoromethylphenyl)calix[4]pyrrole

AbstractCis‐5,15‐bis(3,5‐trifluoromethylphenyl)calix[4]pyrrole (1) forms stable 1/1 complexes with linear anions such as N3−, SCN− and NCO‐ with the highest affinity for NCO− anion in acetonitrile solution. Crystallization of receptor 1 with TBASCN and (TMA)2SO4 resulted in the formation of dimeric capsular assembly, {(TMA)2[12 ⋅ (SCN)2]}, in which two thiocyanate anions are encapsulated. On the other hand, 1 ⋅ OCN(TMA) and 1 ⋅ N3(TMA) complexes form one dimensional columnar structure in the solid state.

Conformational Dynamics and Molecular Characterization of Alsin MORN Monomer and Dimeric Assemblies.

Despite the ubiquity of membrane occupation recognition nexus (MORN) motifs across diverse species in both eukaryotic and prokaryotic organisms, these protein domains remain poorly characterized. Their significance is underscored in the context of the Alsin protein, implicated in the debilitating condition known as infantile-onset ascending hereditary spastic paralysis (IAHSP). Recent investigations have proposed that mutations within the Alsin MORN domain disrupt proper protein assembly, precluding the formation of the requisite tetrameric configuration essential for the protein's inherent biological activity. However, a comprehensive understanding of the relationship between the biological functions of Alsin and its three-dimensional molecular structure is hindered by the lack of available experimental structures. In this study, we employed and compared several protein structure prediction algorithms to identify a three-dimensional structure for the putative MORN of Alsin. Furthermore, inspired by experimental pieces of evidence from previous studies, we employed the developed models to predict and investigate two homo-dimeric assemblies, characterizing their stability. This study's insights into the three-dimensional structure of the Alsin MORN domain and the stability dynamics of its homo-dimeric assemblies suggest an antiparallel linear configuration stabilized by a noncovalent interaction network.

Structure, dimeric conformation, and coenzyme versatility of p-hydroxybenzoate hydroxylase from Arthrobacter sp. PAMC25564

p-Hydroxybenzoate hydroxylase (PHBH) catalyzes the ortho-hydroxylation of 4-hydroxybenzoate (4-HB) to protocatechuate (PCA). PHBHs are commonly known as homodimers, and the prediction of pyridine nucleotide binding and specificity remains an ongoing focus in this field. Therefore, our study aimed to determine the dimerization interface in AspPHBH from Arthrobacter sp. PAMC25564 and identify the canonical pyridine nucleotide-binding residues, along with coenzyme specificity, through site-directed mutagenesis. The results confirm a functional dimeric assembly from a tetramer that appeared in the crystallographic asymmetric unit identical to that established in previous studies. Furthermore, AspPHBH exhibits coenzyme versatility, utilizing both NADH and NADPH, with a preference for NADH. Rational engineering experiments demonstrated that targeted mutations in coenzyme surrounding residues profoundly impact NADPH binding, leading to nearly abrogated enzymatic activity compared to that of NADH. R50, R273, and S166 emerged as significant residues for NAD(P)H binding, having a near-fatal impact on NADPH binding compared to NADH. Likewise, the E44 residue plays a critical role in determining coenzyme specificity. Overall, our findings contribute to the fundamental understanding of the determinants of PHBH's active dimeric conformation, coenzyme binding and specificity holding promise for biotechnological advancements.

Bacterial histone HBb from Bdellovibrio bacteriovorus compacts DNA by bending.

Histones are essential for genome compaction and transcription regulation in eukaryotes, where they assemble into octamers to form the nucleosome core. In contrast, archaeal histones assemble into dimers that form hypernucleosomes upon DNA binding. Although histone homologs have been identified in bacteria recently, their DNA-binding characteristics remain largely unexplored. Our study reveals that the bacterial histone HBb (Bd0055) is indispensable for the survival of Bdellovibrio bacteriovorus, suggesting critical roles in DNA organization and gene regulation. By determining crystal structures of free and DNA-bound HBb, we unveil its distinctive dimeric assembly, diverging from those of eukaryotic and archaeal histones, while also elucidating how it binds and bends DNA through interaction interfaces reminiscent of eukaryotic and archaeal histones. Building on this, by employing various biophysical and biochemical approaches, we further substantiated the ability of HBb to bind and compact DNA by bending in a sequence-independent manner. Finally, using DNA affinity purification and sequencing, we reveal that HBb binds along the entire genomic DNA of B. bacteriovorus without sequence specificity. These distinct DNA-binding properties of bacterial histones, showcasing remarkable similarities yet significant differences from their archaeal and eukaryotic counterparts, highlight the diverse roles histones play in DNA organization across all domains of life.

Epsilon subunit of T-complex protein-1 from Leishmania donovani: A tetrameric chaperonin

The cytosolic T-complex protein-1 ring complex (TRiC), also referred as chaperonin containing TCP-1(CCT), comprising eight different subunits stacked in double toroidal rings, binds to around 10 % of newly synthesized polypeptides and facilitates their folding in ATP dependent manner. In Leishmania, among five subunits of TCP1 complex, identified either by transcriptome or by proteome analysis, only LdTCP1γ has been well characterized. It forms biologically active homo-oligomeric complex and plays role in protein folding and parasite survival. Lack of information regarding rest of the TCP1 subunits and its structural configuration laid down the necessity to study individual subunits and their role in parasite pathogenicity. The present study involves the cloning, expression and biochemical characterization of TCP1ε subunit (LdTCP1ε) of Leishmania donovani, the causative agent of visceral leishmaniasis. LdTCP1ε exhibited significant difference in primary structure as compared to LdTCP1γ and was evolutionary close to LdTCP1 zeta subunit. Recombinant protein (rLdTCP1ε) exhibited two major bands of 132 kDa and 240 kDa on native-PAGE that corresponds to the dimeric and tetrameric assembly of the epsilon subunit, which showed the chaperonin activity (ATPase and luciferase refolding activity). LdTCP1ε also displayed an increased expression upto 2.7- and 1.8-fold in the late log phase and stationary phase promastigotes and exhibited majorly vesicular localization. The study, thus for the first time, provides an insight for the presence of highly diverge but functionally active dimeric/tetrameric TCP1 epsilon subunit in Leishmania parasite.

A conserved N-terminal motif of CUL3 contributes to assembly and E3 ligase activity of CRL3KLHL22

The CUL3-RING E3 ubiquitin ligases (CRL3s) play an essential role in response to extracellular nutrition and stress stimuli. The ubiquitin ligase function of CRL3s is activated through dimerization. However, how and why such a dimeric assembly is required for its ligase activity remains elusive. Here, we report the cryo-EM structure of the dimeric CRL3KLHL22 complex and reveal a conserved N-terminal motif in CUL3 that contributes to the dimerization assembly and the E3 ligase activity of CRL3KLHL22. We show that deletion of the CUL3 N-terminal motif impairs dimeric assembly and the E3 ligase activity of both CRL3KLHL22 and several other CRL3s. In addition, we found that the dynamics of dimeric assembly of CRL3KLHL22 generates a variable ubiquitination zone, potentially facilitating substrate recognition and ubiquitination. These findings demonstrate that a CUL3 N-terminal motif participates in the assembly process and provide insights into the assembly and activation of CRL3s.

Mechanism of Ψ-Pro/C-degron recognition by the CRL2FEM1B ubiquitin ligase

The E3 ligase-degron interaction determines the specificity of the ubiquitin‒proteasome system. We recently discovered that FEM1B, a substrate receptor of Cullin 2-RING ligase (CRL2), recognizes C-degrons containing a C-terminal proline. By solving several cryo-EM structures of CRL2FEM1B bound to different C-degrons, we elucidate the dimeric assembly of the complex. Furthermore, we reveal distinct dimerization states of unmodified and neddylated CRL2FEM1B to uncover the NEDD8-mediated activation mechanism of CRL2FEM1B. Our research also indicates that, FEM1B utilizes a bipartite mechanism to recognize both the C-terminal proline and an upstream aromatic residue within the substrate. These structural findings, complemented by in vitro ubiquitination and in vivo cell-based assays, demonstrate that CRL2FEM1B-mediated polyubiquitination and subsequent protein turnover depend on both FEM1B-degron interactions and the dimerization state of the E3 ligase complex. Overall, this study deepens our molecular understanding of how Cullin-RING E3 ligase substrate selection mediates protein turnover.

Structure of the interleukin-5 receptor complex exemplifies the organizing principle of common beta cytokine signaling

Cytokines regulate immune responses by binding to cell surface receptors, including the common subunit beta (βc), which mediates signaling for GM-CSF, IL-3, and IL-5. Despite known roles in inflammation, the structural basis of IL-5 receptor activation remains unclear. We present the cryo-EM structure of the human IL-5 ternary receptor complex, revealing architectural principles for IL-5, GM-CSF, and IL-3. In mammalian cell culture, single-molecule imaging confirms hexameric IL-5 complex formation on cell surfaces. Engineered chimeric receptors show that IL-5 signaling, as well as IL-3 and GM-CSF, can occur through receptor heterodimerization, obviating the need for higher-order assemblies of βc dimers. These findings provide insights into IL-5 and βc receptor family signaling mechanisms, aiding in the development of therapies for diseases involving deranged βc signaling.

Cucurbit[8]uril-regulated face-to-face dimerization assembly enhanced photosensitization and photocatalysis

Cucurbit[8]uril-regulated face-to-face dimerization assembly enhanced photosensitization and photocatalysis

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.