Structural Basis Research Articles

Structural basis of deoxynucleotide addition by HIV-1 RT during reverse transcription

Reverse transcription of the retroviral RNA genome into DNA is an integral step during HIV-1 replication. Despite a wealth of structural information on reverse transcriptase (RT), we lack insight into the intermediate states of DNA synthesis. Using catalytically active substrates, and a blot/diffusion cryo-electron microscopy approach, we capture 11 structures encompassing reactant, intermediate and product states of dATP addition by RT at 2.2 to 3.0 Å resolution. In the reactant state, dATP binding to RT-template/primer involves a single Mg2+ (site B) inducing formation of a negatively charged pocket where a second floating Mg2+ can bind (site A). During the intermediate state, the α-phosphate oxygen from a previously unobserved dATP conformer aligns with site A Mg2+ and the primer 3′-OH for nucleophilic attack. The product state, comprises two substrate conformations including an incorporated dAMP with the pyrophosphate leaving group coordinated by metal B and stabilized through H-bonds. Moreover, K220 mutants significantly impact the rate of dNTP incorporation by RT and HIV-1 replication capacity. This work sheds light into the dynamic components of a reaction that is central to HIV-1 replication.

Bioguided Identification of Polymethoxyflavones as Novel Vascular CaV1.2 Channel Blockers from Citrus Peel

The huge amount of citrus peel produced worldwide represents an economic burden for society. However, this agricultural by-product is a rich source of natural molecules, potentially endowed with interesting pharmacological activities. In this regard, we decided to investigate if the polymethoxyflavones contained in citrus peel waste could be exploited as novel vasorelaxant agents. A hydroalcoholic blond orange (Citrus sinensis) peel extract, obtained by ultrasonication, was partitioned in dichloromethane. Column chromatography allowed for the isolation of four polymethoxyflavones, namely, scutellarein tetramethyl ether, nobiletin, tangeretin, and sinensetin, identified by nuclear magnetic resonance (NMR) spectroscopy and UPLC-HRMS/MS and confirmed by multivariate curve resolution of NMR fractional spectra. The four molecules showed interesting in vitro vasorelaxant activity, at least, in part, due to the blockade of smooth muscle CaV1.2 channels. Molecular modeling and docking analysis elucidated the binding mode of the polymethoxyflavones at the homology model of the rat CaV1.2c subunit and provided the structural basis to rationalise the highest activity of scutellarein tetramethyl ether in the set and the dramatic effect of the additional methoxy group occurring in nobiletin and sinensetin. In conclusion, citrus peel can be considered a freely available, valuable source of vasoactive compounds worthy of pharmaceutical and/or nutraceutical exploitation.

3D and 2D-QSAR Studies on Natural Flavonoids for Nitric Oxide Production Inhibitory Activity

Background: Nitric oxide (NO), an important second messenger molecule, regulates numerous physiological responses, while excessive NO generates negative effects on the circulatory, nervous and immune systems. Recently, some natural flavonoids were reported to possess the capability of inhibiting LPS-induced NO production. To fully understand the nature of their own NO inhibitory activity, it is necessary to address the structural requirements of flavonoids as NO inhibitors. Objective: The objective of this work was to develop efficient QSAR models for predicting the NOinhibitory activity of new flavonoids and improving insights into the critical properties of the chemical structures that were required for the ideal NO production inhibitory activities. Methods: To provide insights into the structural basis of flavonoids as NO inhibitors, 3D quantitative structure-activity relationship (3D-QSAR) and 2D-QSAR models were developed on a dataset of 55 flavonoids using comparative molecular field analysis (CoMFA), comparative molecular similarity indices analysis (CoMSIA) and hologram quantitative structure-activity relationship (HQSAR) approaches. Results: The statistically significant models for CoMFA, CoMSIA and HQSAR resulted in crossvalidated coefficient (q2) values of 0.523, 0.572 and 0.639, non-cross-validated coefficient (r2) values of 0.793, 0.828 and 0.852, respectively. The robustness of these models was further affirmed using a test set of 18 compounds, which resulted in predictive correlation coefficients (r2 pred) of 0.968, 0.954 and 0.906. Furthermore, the models-derived contour maps were appraised for activity trends for the molecules analyzed. Conclusion: The 3D and 2D-QSAR models constructed in this paper were efficient in estimating the NO inhibitory activities of flavonoids and facilitating the design of flavonoid-derived NO production inhibitors.

Structural insights to the RRM-domain of the glycine-rich RNA-binding protein from Sorghum bicolor and its role in cold stress tolerance in E. coli

Sorghum bicolor Glycine-rich RNA-binding protein (SbGRBP), exhibit the ability to bind both single-stranded and double-stranded DNA. The expression of SbGRBP is regulated by heat stress, with the protein localizing to the nucleus and cytosol. The present study delves into the structure and ssDNA binding ability of its truncated version (SbGRBP1–119) which lacks glycine rich domain (GR). This protein has the ability to bind ssDNA Using Nuclear Magnetic Resonance (NMR) spectroscopy, we have revealed the secondary structure of SbGRBP1–119, highlighting the typical configuration of GRBPs with four β-sheets and two α-helices. Notably, we found two additional α-helices at the N-terminal region that seem to interact with ssDNA, a novel observation for GRBPs. Key residues crucial for ssDNA binding were identified, suggesting a specific interaction with the oligonucleotide sequence 5’-TTCTGG-3′. Preliminary assays hinted that SbGRBP1–119 might bolster E. coli resilience to cold stress, indicating a potential chaperone-like role under stress conditions. This study sheds light on the structural basis of SbGRBP1–119's interaction with nucleic acids, deepening our understanding about the role of GRBPs' in RNA metabolism and regulation.

Structural basis for the effects of Ser387 phosphorylation of MgcRacGAP on its GTPase-activating activities for CDC42 and RHOA

MgcRacGAP is a GTPase-activating protein (GAP) for the Rho family GTPases. During cytokinesis, MgcRacGAP localizes to the midbody, where it activates the GTPase activity of Rho family GTPases to facilitate cytokinesis. In the midbody, Aurora B phosphorylates Ser387 within the GAP domain of human MgcRacGAP, a modification that is suggested to influence GTPase preference. However, there are conflicting reports, with some studies indicating that Ser387 phosphorylation does not alter the GTPase preference of MgcRacGAP. This controversy highlights the need for a deeper understanding of the molecular interactions involved, which can be clarified through structural analyses. In the present study, we determined the crystal structures of the wild-type MgcRacGAP GAP domain complexed with CDC42•GDP•AlF4− and the S378D phosphomimetic mutant GAP domain fused with RHOA•GDP•AlF4−. Additionally, crystal structures of the GAP domains were determined for the S387D and S387A mutants. Our analysis revealed that neither GTPase binding nor S387D mutation affected the overall structure of the GAP domain. However, comparison of the CDC42•MgcRacGAP (wild-type) complex with the RHOA-MgcRacGAP(S378D) fusion protein structure indicated that the S387D mutation caused positional shifts in both CDC42 and RHOA relative to MgcRacGAP. These shifts reduced interactions with CDC42 more severely than those with RHOA. In fact, the S387D mutation decreased the GTPase-activating activity of MgcRacGAP toward CDC42, while its impact on RHOA was only moderate. This difference in the rate of activity reduction may play an important role in GTPase preference.

Evaluating the electronic and structural basis of carbon selenide-based quantum dots as photovoltaic design materials: A DFT and ML analysis

We present a new study on the design, discovery and space generation of carbon selenide based photovoltaic (PV) materials. By extending acceptors and leveraging density functional theory (DFT) and machine learning (ML) analysis, we discover new QDs with remarkable PV properties. We employ various ML models, to correlate the exciton binding energy (Eb) of 938 relevant compounds from literature with their molecular descriptors of structural features that influence their performance. Our study demonstrates the potential of ML approaches in streamlining the design and discovery of high-efficiency PV materials. Also the RDKit computed molecular descriptors correlates with PV parameters revealed maximum absorption (λmax) ranges of 509–531 nm, light harvesting efficiency (LHE) above 92 %, Open Circuit Voltage (Voc) of 0.22–0.45 V, and short Circuit (Jsc) currents of 37.92–42.75 mA/cm2. Their Predicted Power Conversion Efficiencies (PCE) using the Scharber method reaches upto 09–13 %. This study can pave the way for molecular descriptor-based design of new PV materials, promising a paradigm shift in the development of high-efficiency solar energy conversion technologies.

Conformational Plasticity of SpyCas9 Induced by AcrIIA4 and AcrIIA2: Insights from Molecular Dynamics Simulation

CRISPR-Cas9 systems constitute bacterial adaptive immune systems that protect against phage infections. Bacteriophages encode anti-CRISPR proteins (Acrs) that mitigate the bacterial immune response. However, the structural basis for their inhibitory actions from a molecular perspective remains elusive. In this study, through microsecond atomistic molecular dynamics simulations, we demonstrated the remarkable flexibility of Streptococcus pyogenes Cas9 (SpyCas9) and its conformational adaptability during interactions with AcrIIA4 and AcrIIA2. Specifically, we demonstrated that the binding of AcrIIA4 and AcrIIA2 to SpyCas9 induces a conformational rearrangement that causes spatial separation between the nuclease and cleavage sites, thus making the endonuclease inactive. This separation disrupts the transmission of signals between the protospacer adjacent motif recognition and nuclease domains, thereby impeding the efficient processing of double-stranded DNA. The simulation also reveals that AcrIIA4 and AcrIIA2 cause different structural variations of SpyCas9. Our research illuminates the precise mechanisms underlying the suppression of SpyCas9 by AcrIIA4 and AcrIIA2, thus presenting new possibilities for controlling genome editing with higher accuracy.

Structural Basis for the Pathogenicity of Parkin Catalytic Domain Mutants

Mutations in the E3 ubiquitin ligase parkin cause a familial form of Parkinson’s disease (PD). Parkin and the mitochondrial kinase PINK1 assure quality control of mitochondria through selective autophagy of mitochondria (mitophagy). Whereas numerous parkin mutations have been functionally and structurally characterized, several PD mutations found in the catalytic Rcat domain of parkin remain poorly understood. Here, we characterize two pathogenic Rcat mutants, T415N and P437L. We demonstrate that both mutants exhibit impaired activity using autoubiquitination and ubiquitin vinyl sulfone assays. We determine the minimal ubiquitin binding segment and show that both mutants display impaired binding of ubiquitin charged on the E2 enzyme. Finally, we use AlphaFold 3 to predict a model of the phospho-parkin:phospho-ubiquitin:ubiquitin-charged E2 complex. The model shows the repressor-element of parkin (REP) and the N-terminal residues of the catalytic domain form a helix to position ubiquitin for transfer from the E2 to parkin. Our results rationalize the pathogenicity of the parkin mutations and deepen our understanding of the active parkin-E2∼Ub complex.

Clustering of synaptic engram: Functional and structural basis of memory

Studies on memory engram have demonstrated how experience and learning can be allocated at a neuronal level for centuries. Recently emerging evidence narrowed down further to the synaptic connections and their patterned allocation on dendrites. Notably, groups of synapses within a specific range within dendrites known as ’synaptic clusters’ have been revealed in association with learning and memory. Previous investigations have shown that a variety of factors mediated by both presynaptic inputs and postsynaptic dendrites contribute to clustering. Here, we review the neural mechanism of synaptic clustering and its correlation with memory. We highlight the recent findings about the clustering of synaptic engrams and memory formation and discuss future directions.

Structural basis for full-length chemerin recognition and signaling through chemerin receptor 1

Chemerin, a chemotactic adipokine, plays essential roles in adipogenesis and inflammation. Serum chemerin concentration is closely associated with obesity and metabolism disorders. The mature form of chemerin (residues 21-157) acts primarily through chemerin receptor 1 (CMKLR1) for transmembrane signaling. As a result, CMKLR1 serves as a promising target for therapeutic intervention of immunometabolic diseases such as diabetes and multiple sclerosis. Here, we present a high-resolution cryo-EM structure of CMKLR1-Gi signaling complex bound to biologically active full-length chemerin. The mature chemerin shows binding features distinct from its C-terminal nonapeptide including interaction with both the extracellular loops (ECLs) and the N-terminus of CMKLR1. Combining results from functional assays, our studies demonstrate that chemerin interacts with CMKLR1 in a “two-site” mode similar to chemokine-chemokine receptor interactions, but acting as a “reverse chemokine” by inserting its C-terminus instead of the N-terminus as in the case of chemokines into the transmembrane binding pocket of CMKLR1. These structural insights are expected to help develop synthetic analogs with therapeutic potential.

High-resolution crystal structure of PD-1 in complex with retifanlimab, the FDA-approved immune checkpoint blocking antibody for treating Merkel cell carcinoma

Retifanlimab is a humanized monoclonal antibody that specifically targets programmed cell death protein 1 (PD-1), an essential immune checkpoint that modulates T-cell immune responses. Several anti-PD-1 antibodies have been market-approved, marking a significant advancement in the treatment of diverse tumor types by restoring the T-cell immune response. Recently, the US FDA approved retifanlimab for treating metastatic or recurrent locally advanced Merkel cell carcinoma. We present the crystal structure of PD-1 in complex with the retifanlimab Fab at a resolution of 1.54 Å to elucidate the structural basis for the mechanism of action of this antibody. This work clarifies the detailed interactions and conformational alterations that occur upon antibody binding. The epitope of retifanlimab partially overlaps with the ligand binding site, and its binding induced unique conformations of the flexible loops within PD-1, including BC, C'D, and FG loops, thereby optimizing interactions with the antibody. A thorough analysis of its interaction with PD-1 and other FDA-approved anti-PD-1 antibodies may provide valuable insights into the rational design of enhanced therapies to regulate immune responses in cancer treatment.

Structural basis of orientated asymmetry in a mGlu heterodimer

The structural basis for the allosteric interactions within G protein-coupled receptors (GPCRs) heterodimers remains largely unknown. The metabotropic glutamate (mGlu) receptors are complex dimeric GPCRs important for the fine tuning of many synapses. Heterodimeric mGlu receptors with specific allosteric properties have been identified in the brain. Here we report four cryo-electron microscopy structures of mGlu2-4 heterodimer in different states: an inactive state bound to antagonists, two intermediate states bound to either mGlu2 or mGlu4 agonist only and an active state bound to both glutamate and a mGlu4 positive allosteric modulator (PAM) in complex with Gi protein. In addition to revealing a unique PAM binding pocket among mGlu receptors, our data bring important information for the asymmetric activation of mGlu heterodimers. First, we show that agonist binding to a single subunit in the extracellular domain is not sufficient to stabilize an active dimer conformation. Single-molecule FRET data show that the monoliganded mGlu2-4 can be found in both intermediate states and an active one. Second, we provide a detailed view of the asymmetric interface in seven-transmembrane (7TM) domains and identified key residues within the mGlu2 7TM that limits its activation leaving mGlu4 as the only subunit activating G proteins.

Targeting histidinol-phosphate aminotransferase in Acinetobacter baumannii with salvianolic acid B: A structure-based approach to novel antibacterial strategies

Undoubtedly, Acinetobacter baumannii is a major ESKAPE pathogen that poses a significant threat to public health, causing severe nosocomial infections with high mortality rates in healthcare settings. Due to the rapid development of antibiotic resistance, only a limited number of antibiotics remain effective against infections caused by multidrug-resistant (MDR) Acinetobacter baumannii. The discovery of new class of antibiotic molecules still lags behind the rate of growing worldwide burden of antimicrobial resistance (AMR). To expedite the discovery of new therapeutic molecules, we have focused on HisC from A. baumannii (AbHisC), a crucial enzyme involved in the seventh step of histidine biosynthesis. This pathway is absent in humans. We have employed the advanced computational techniques to target this promising drug target. AbHisC was cloned, overexpressed, and purified. Three distinct sets of libraries containing ∼60,000 natural compounds from ZINC database were screened against AbHisC using Schrödinger's glide module software. Based on the docking score and glide energy, top 25 hits were further subjected to induced fit (IF) docking. Top four out of the twenty five compounds from IF docking were subjected to 100ns molecular dynamics simulations, and it was observed that salvianolic acid B (SA-B) (a naturally occurring compound) complex with AbHisC, was found to be extremely stable. The glide energy and docking score of SA-B were −88.59 kcal/mol and −10.4 kcal/mol. SA-B was also found to quench the intrinsic fluorescence of tyrosine indicating its binding to the target. The dissociation constant calculated using Surface Plasmon Resonance was found to be 3.4x10−9 M. Based on these results we can conclude that SA-B can serve as the potential inhibitor of AbHisC that can further form the basis of structure based drug design against this deadly pathogen.

The structural basis for high-affinity c-di-GMP binding to the GSPII-B domain of the traffic ATPase PilF from Thermus thermophilus

c-di-GMP is an important second messenger in bacteria regulating e. g. motility, biofilm formation, cell wall biosynthesis, infectivity and natural transformability. It binds to a multitude of intracellular receptors. This includes proteins containing general secretory pathway II (GSPII) domains such as the N-terminal domain of the Vibrio cholerae ATPase MshE (MshEN) which binds c-di-GMP with two copies of a 24-amino acids sequence motif. The traffic ATPase PilF from Thermus thermophilus is important for type IV pilus biogenesis, twitching motility, surface attachment and natural DNA-uptake and contains three consecutive homologous GPSII domains.We show that only two of these domains bind c-di-GMP and define the structural basis for the exceptional high affinity of the GSPII-B domain for c-di-GMP, which is 83fold higher than that of the prototypical MshEN domain. Our work establishes an extended consensus sequence for the c-di-GMP binding motif and highlights the role of hydrophobic residues for high-affinity recognition of c-di-GMP. Our structure is the first example for a c-di-GMP-binding domain not relying on arginine residues for ligand recognition. We also show that c-di-GMP binding induces local unwinding of an α-helical turn as well as subdomain reorientation to reinforce intermolecular contacts between c-di-GMP and the C-terminal subdomain. Abolishing c-di-GMP-binding to GSPII-B reduces twitching motility and surface attachment but not natural DNA-uptake.Overall, our work contributes to a better characterization of c-di-GMP binding in this class of effector domains, allows the prediction of high-affinity c-di-GMP-binding family members and advances our understanding of the importance of c-di-GMP binding for T4P-related functions.

Structural basis for C. elegans pairing center DNA binding specificity by the ZIM/HIM-8 family proteins

Pairing center (PC) on each chromosome of Caenorhabditis elegans is crucial for homolog pairing and initiating synapsis. Within each PC, clusters of 11/12 bp DNA motif recruit one of four paralogous meiosis-specific proteins: ZIM-1, ZIM-2, ZIM-3, or HIM-8. However, the mechanistic basis underlying the specificity of ZIM/HIM-8-PC DNA interaction remains elusive. Here, we describe crystal structures of HIM-8, ZIM-1 and ZIM-2 DNA binding domains (ZF1, ZF2 and CTD) in complex with their cognate PC DNA motifs, respectively. These structures demonstrated the ZF1-2-CTD folds as an integrated structural unit crucial for its DNA binding specificity. Base-specific DNA-contacting residues are exclusively distributed on ZF1-2 and highly conserved. Furthermore, the CTD potentially contributes to the conformational diversity of ZF1-2, imparting binding specificity to distinct PC DNA motifs. These findings shed light on the mechanism governing PC DNA motif recognition by ZIM/HIM-8 proteins, suggesting a co-evolution relationship between PC DNA motifs and ZF1-2-CTD in shaping the specific recognition.

The characterization and structural basis of a human broadly binding antibody to HBV core protein.

Hepatitis B virus (HBV) core protein (HBc) plays a crucial role in the virus life cycle, making it an important detection marker for HBV infection and a potential target for treatment. However, several commercially available monoclonal antibodies (mAbs) or polyclonal antibodies (pAbs) targeting HBc have certain limitations in detecting HBV across different genotypes in various biochemical assays, such as enzyme-linked immunosorbent assay, western blot, immunofluorescence assay, flow cytometry, and immune spot assay. In this study, we identified 12 human anti-HBc mAbs and evaluated their potential application in multiple biochemical assays. These mAbs mainly recognized the epitopes near residues 20-22 amino acids (a.a.) and 77-78 a.a. of HBc, demonstrating a broadly cross-genotypic activity. Notably, three Group II mAbs, named cAbA1, cAbD4, and cAbF9, displayed excellent capacities for the detection of HBV infection in all tested biochemical assays. Furthermore, we determined a 3.22 Å of cryo-electron microscopy (cryo-EM) structure of the fragment of antigen binding (Fab) of cAbD4 complexed with HBc dimer, which was the highest resolution of the structural model for Fab-HBc to date. Collectively, our findings provided excellent and reliable antibody candidates for live HBV detection and revealed the recognition mechanism and the detailed interaction information of a potent human anti-HBc mAb binding to the spike tips of HBc dimer.IMPORTANCEThe lack of excellent detection Abs for live hepatitis B virus (HBV) infection and high-resolution structures of the Ab-HBV core protein (HBc) complex largely limited the development of HBV-related research. This study reports a panel of anti-HBc monoclonal antibodies (mAbs) with excellent capacities for detecting HBV infection in multiple biochemical assays and determines a 3.22 Å of cryo-EM structure of HBc with a potent binding mAb. These findings provide excellent and reliable detecting tools for HBV-related research and promote the understanding of the recognition mechanism of anti-HBc mAbs to HBc particles.

Engineering Supramolecular [c2]Daisy Chains for Structural Hierarchy-Dependent Emission and Photoreactivity.

Organic photofunctional materials exhibit properties that are highly dependent on their structural hierarchy. The variability in intermolecular interactions and molecular packing in both monomeric and aggregated states complicates the controllability and predictability of their photophysical and photochemical properties. To address this challenge, we developed three luminescent supramolecular [c2]daisy chains as simplified models. The rigid and mutually embedded linkers between the host and guests facilitate the formation of [c2]daisy chains with balanced stability and dynamics. Additionally, the close and tunable π-π interactions between the luminescent units provide a structural basis for fluorescence modulation and topochemical photoreactions. We performed two sets of comparisons to assess luminescence and photoreactivity: one comparison involves molecules with and without crown ethers, and the other contrasting their behavior under UV excitation in solution (diluted and concentrated) versus in the aggregated and crystalline states. Specifically, in the crystalline state, [c2]daisy chains effectively stabilize molecular packing, leading to highly efficient dimer-dependent emission. This unique structure remains in both solution (c > 1 mM) and aggregated states, which can direct the reaction pathway toward rapid and efficient intermolecular photocycloaddition upon UV irradiation. However, in highly diluted solution (10 μM), [c2]daisy chains dissociate into monomers, which further undergo intramolecular photocyclization. This study provides new insights into employing supramolecular strategies for controllable molecular aggregation and the fine-tuning of photoreaction pathways and kinetics.

Defining barriers to food systems sustainability: a novel conceptual framework

The transformation of food systems emerges as a critical challenge necessitating a deep, holistic comprehension of the complex and multifaceted barriers that hinder progress towards sustainability. The existing literature is not consistent in identifying these barriers. Building upon existing work, this paper introduces a comprehensive, integrated, and interdisciplinary framework to dissect the nature and origins of the barriers to food system sustainability. Our framework categorizes these impediments into five domains: political economy, socio-technical, socio-cultural, biophysical, and socio-economic barriers, and highlights their intricate interplay and interconnected nature. We pinpoint the foundational role of political economy barriers as the cornerstone of a “system of barriers” that create or perpetuate unsustainability. This framework not only advances academic knowledge by providing a structured basis for analysing sustainability barriers but also serves as a practical tool for policymakers, researchers, and practitioners, to foster transdisciplinarity and develop targeted interventions. We call for further empirical research, emphasizing the need for comparative analyses, longitudinal studies, and the exploration of feedback loops and non-linear dynamics between barriers, to inform effective and sustainable food system transformation strategies.

Structural basis of chiral wrap and T-segment capture by Escherichia coli DNA gyrase

Type II topoisomerase DNA gyrase transduces the energy of ATP hydrolysis into the negative supercoiling of DNA. The postulated catalytic mechanism involves stabilization of a chiral DNA loop followed by the passage of the T-segment through the temporarily cleaved G-segment resulting in sign inversion. The molecular basis for this is poorly understood as the chiral loop has never been directly observed. We have obtained high-resolution cryoEM structures of Escherichia coli gyrase with chirally wrapped 217 bp DNA with and without the fluoroquinolone moxifloxacin (MFX). Each structure constrains a positively supercoiled figure-of-eight DNA loop stabilized by a GyrA β-pinwheel domain which has the structure of a flat disc. By comparing the catalytic site of the native drug-free and MFX-bound gyrase structures both of which contain a single metal ion, we demonstrate that the enzyme is observed in a native precatalytic state. Our data imply that T-segment trapping is not dependent on the dimerization of the ATPase domains which appears to only be possible after strand passage has taken place.

Structural mechanisms of human sodium-coupled high-affinity choline transporter CHT1

Mammalian sodium-coupled high-affinity choline transporter CHT1 uptakes choline in cholinergic neurons for acetylcholine synthesis and plays a critical role in cholinergic neurotransmission. Here, we present the high-resolution cryo-EM structures of human CHT1 in apo, substrate- and ion-bound, hemicholinium-3-inhibited, and ML352-inhibited states. These structures represent three distinct conformational states, elucidating the structural basis of the CHT1-mediated choline uptake mechanism. Three ion-binding sites, two for Na+ and one for Cl–, are unambiguously defined in the structures, demonstrating that both ions are indispensable cofactors for high-affinity choline-binding and are likely transported together with the substrate in a 2:1:1 stoichiometry. The two inhibitor-bound CHT1 structures reveal two distinct inhibitory mechanisms and provide a potential structural platform for designing therapeutic drugs to manipulate cholinergic neuron activity. Combined with the functional analysis, this study provides a comprehensive view of the structural mechanisms underlying substrate specificity, substrate/ion co-transport, and drug inhibition of a physiologically important symporter.