Cryo-EM structural observation on the interaction of the bacterial energy-coupling factor transporter with perfluoroalkyl substances

Cryo-EM structural observation on the interaction of the bacterial energy-coupling factor transporter with perfluoroalkyl substances | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Cryo-EM structural observation on the interaction of the bacterial energy-coupling factor transporter with perfluoroalkyl substances Yanzheng Gao, Zeming Wang, Hui Li, Chao Qin, James Tiedje This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8140096/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The movement of exogenous chemicals through cell membranes using transport proteins and causing functional impairments to organisms is an important issue of molecular ecology. Per- and polyfluoroalkyl substances (PFAS) receives intensive concern by their global contamination and a limited understanding of their biological effects. Energy-coupling factor (ECF) transporters are critical to the absorption of vital micronutrients by bacteria and archaea, playing a significant role in maintaining microecological health. Our study found perfluorooctane sulfonate (PFOS) as an inhibitor on folate ECF transporter through competitive binding with folate and conformational alterations in ATP-binding domains, resulting in reduced folate absorption and ATPase activity. Molecular dynamics simulations depicted the process of PFOS internalization into plasma membrane prior to contact with ECF transporter. Using cryogenic electron microscopy (cryo-EM), we observed the conformational alterations caused by PFOS. The interaction mechanism between PFOS and folate ECF transporter was finally revealed though the combined approaches of cryo-EM and molecular simulation and elucidated as three critical pathway: membrane perturbation, competitive binding, and conformational changes. These findings reveal the functional impairments of exogenous chemicals on ECF transporters and advance the knowledge of ecotoxicity of PFAS. Earth and environmental sciences/Ecology/Molecular ecology Biological sciences/Microbiology/Environmental microbiology/Water microbiology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Main Referred to as “forever chemicals”, the presence and biological damages of per- and polyfluoroalkyl substances (PFAS) in the environment have gained global attention 1 , 2 . Over the past several decades, PFAS have grown to over 14,000 specific chemicals that are documented in U.S. Environmental Protection Agency’s CompTox Chemicals Dashboard and have been utilized in hundreds of various applications, such as aqueous fire-fighting foam, textile impregnation, and electroplating 3 . Their strong carbon-fluorine bonds provide exceptional chemical and thermal stability, cooperating with oxygen-containing functional groups to endow amphiphilic properties, leading to their widespread persistence in the environment and bioaccumulation in living organisms 1 , 3 . Between 1951 and 2015, PFAS emissions are estimated at 2,610–21,400 tons 4 . The aquatic environment is the most important source and sink of PFAS, and numerous studies confirm global presence of PFAS in surface and groundwater, with common concentrations ranging from tens of ng/L to hundreds of µg/L 5,6 , even reaching several mg/L in extremely polluted wastewater 7 , 8 . These findings have raised significant concerns about their ecotoxicology and ecological risks associated with environmental exposure to PFAS. Epidemiological studies have linked PFAS exposure to various health issues, including hepatopathy, nephropathy, reproductive and developmental issues, endocrine disruption, and cancer 9 . Animal experiments have confirmed the similar adverse effects 1 , 10 . Although intensive investigations have been conducted on the mechanisms of PFAS action and potential control measures, understanding the biological damages of PFAS at a molecular level remains limited due to the diversity of chemical classes and unknown sensitive biological targets. The anionic and amphiphilic nature of PFAS suggests that they can behave like phospholipids and proteinaceous substances rather than neutral and hydrophobic organic contaminants 11 , 12 . This characteristic promotes their enrichment in blood, liver, and other tissues through strong interactions with proteins, rather than the storage in lipid domains 13 – 15 . Similarities between PFAS and endogenous fatty acids are often cited to describe their behaviors within organisms 16 – 19 . Based on this assumption, membrane partitioning has been incorporated into bioconcentration models for elucidation of high bioaccumulation potential of PFAS 19 , 20 , and the interaction between PFAS and membrane phospholipids has received more attention in toxicity assessments, especially, regarding the activities of membrane-bound proteins 9 . Currently, the bioaccumulation models emphasize the contributions of PFAS interactions with proteins in evaluation of their distribution, accumulation and half-life in organisms 21 , 22 . The related in silico , in vitro , and in vivo experiments have demonstrated the close relationship between PFAS bioaccumulation and protein content. For example, PFAS are found to be strongly associated with serum albumin, which highly affects the migration, distribution, and accumulation within living organisms 23 , 24 . PFAS also perform high binding affinities to liver fatty acid binding proteins, which directly affects the redistribution of PFAS between liver and blood and causes the competitive displacement with fatty acids 18 , 25 , 26 . Moreover, organic anion transporter proteins in kidney, such as OAT1, OAT3 and Oatp1a1, are found to mediate cellular PFAS uptake due to their high affinities, supporting the renal elimination of PFAS 27 , 28 . Despite such progress, a comprehensive evaluation on PFAS-protein interactions remain insufficient understanding at molecular levels, which limits the prediction of their biological toxicities. Transport proteins in plasma membranes are a critical component in impacting the movement of exogenous chemicals through cell membranes and causing functional impairments to organisms. ATP-binding cassette (ABC) transporters, comprising two transmembrane domains and two cytoplasmic ATP-binding domains, facilitate the transmembrane transport of various substances such as nutrients, lipids, cholesterol, steroids, and drugs 29 – 33 . These transporters are indispensable for cellular nutrients transport in humans, animals, plants, and microorganisms, which potentially impacts the entire ecological system's health 34 , 35 . Previous studies have explored the transport mechanism of drugs and nutrients through ABC transporters 36 , and the up/down-regulation of gene expression in ABC transporters by PFAS, such as ABCA1, ABCB1, ABCB11, and ABCG2 transporters 37 , but the influence of chemicals on ABC transporters has not been fully elucidated. As a subgroup of ABC transporters superfamily, energy-coupling factor (ECF) transporters present in about 50% of prokaryotes 38 . The ECF transporters are essential to absorption of vital micronutrients, such as water-soluble vitamins 39 , 40 and metal ions 41 , 42 , by bacteria and archaea. Similar with other ABC transporters, ECF transporters consist of two cytosolic ATPase subunits (EcfA and EcfA') and two membrane-embedded components, including a scaffold protein (EcfT) and a substrate-translocating subunit (S-component) 40 . During the transport cycles, these transporters move substrates bound to the S-component into the cytoplasm, and return to their initial outward-facing state with the assistance of ATP hydrolysis 43 – 45 . Currently, no study (to date) has reported whether ECF transporters might transport harmful substances e.g., PFAS into cell or the contaminants could disrupt the transport functionality. In this study, perfluorooctane sulfonate (PFOS) was selected to investigate the interaction with folate ECF transporter (ECF-FolT2) from Lactobacillus brevis at molecular scale. PFOS is one of the most representative PFAS and has a molecular weight similar to folate (Extended Data Fig. 1 ). The ATPase activity and folate transport of ECF-FolT2 were analyzed under the exposure to PFOS in aqueous system. Cryogenic electron microscopy (cryo-EM) was employed to investigate structural alterations in the ECF-FolT2. We also revealed the interaction mechanism of PFOS with ECF-FolT2 aided by molecular simulations. Results PFOS inhibit the regular functions of ECF-FolT2 Reconstituting ECF-FolT2 into proteoliposomes was utilized to mimic the natural settings of cellular membranes and measure folate uptake by ECF-FolT2 under PFOS stress, while minimizing interference from other cellular components and activities. The preparation process of proteoliposomes is illustrated in Fig. 1 a and detailed in the Methods Section. Like previous studies, to ensure detectable outcomes under the laboratory condition, ECF-FolT2 concentration was selected at 100 mg/L, which was higher than that in many physiological settings 43 , 46 . Correspondingly, PFOS concentration was also set at mg/L levels to maintain the ratio of protein to PFOS in line with the actual situation. During 20-min reaction, apparent folate uptake occurred in the proteoliposome/ATP/Mg 2+ system (Extended Data Fig. 1 c), confirming that both ECF-FolT2 and ATP are essential components involved in this process. Upon exposure to PFOS, the quantity and rate of folate uptake by ECF-proteoliposomes were markedly reduced (Fig. 1 b). The results showed that the amount of folate transported into liposomes decreased from 11.5 to 10.9, 9.85, 8.01, and 7.51 pmol/µg protein at the PFOS concentration of 0, 20, 40, 60, and 80 µmol/L. ATPase activity assay of ECF-FolT2 showed the apparent functional impairment of ATPase activity. Experiment controls (free of PFOS) revealed that ECF-FolT2 could hydrolyze ATP with an activity of 3.4 U/mg protein (Extended Data Fig. 1 d). As PFOS concentration increased to 20, 40, 60, and 80 µmol/L, the corresponding ATPase activities dropped to 2.7, 2.4, 2.2 and 1.7 U/mg protein (Fig. 1 c). The maximum inhibition rate of ATPase activity reached 51.3% at PFOS concentration at 80 µmol/L. These findings clearly demonstrate that PFOS can disrupt the functional performance of ECF-FolT2. Competitive binding between PFOS and folate MST was employed to estimate the K D ; the smaller K D values reflect the stronger affinity. PFOS exhibited a strong affinity to ECF-FolT2 with a K D value of 568 µmol/L, but was weaker than folate ( K D = 16.6 µmol/L) (Fig. 1 e). The competitive binding assay using MST is shown in Fig. 1 d and f. PFOS binding to ECF-FolT2 in the presence or absence of folate both showed the upward trend as PFOS concentration increased. But in the presence of folate, the signal-to-noise ratio of the data was too low to meet the requirements for fitting a curve, which could be considered as non-binding to PFOS. The presence of folate significantly suppressed PFOS binding with ECF-FolT2. The results suggest that PFOS and folate compete for the same binding sites on ECF-FolT2, which is likely responsible for the observed reduction in folate uptake in ECF-proteoliposomes in the presence of PFOS. Interaction of PFOS with plasma membrane PFOS could effectively cause the decline of ECF-FolT2 functions in which PFOS penetration into the plasma membrane could be the critical initial step to damage ECF-FolT2. To do so, 200-ns molecular dynamics (MD) simulation of PFOS interaction with plasma membrane was performed to examine the perturbation mechanism. The plasma membrane model comprises the structures of 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoethanolamine (POPE), L- α -phosphatidylglycerol (POPG), and 1-palmitoyl-2-oleoyl- sn -glycero-3-cardiolipin (POCL1) at a molar ratio of 75:25:5 47 . Figure 2 a presents the initial and final snapshots illustrating the interaction between the plasma membrane and PFOS, and the time evolutions of the Z-coordinate of PFOS is shown in Extended Data Fig. 2 . During 200-ns MD simulations, the hydrophobic perfluoroalkyl group of PFOS inserted into plasma membrane in a nearly vertical orientation, and fully entered the plasma membrane at 98,440 ps. PFOS molecule was deeply internalized into the plasma membrane structures at the final stage of calculation. Partial density profiles show that the integration of PFOS in plasma membrane caused an outward expansion of POPE, POPG and POCL1 molecules and an inward convergence of water molecules (Fig. 2 b), suggesting an increase in intermolecular distance and permeability of plasma membrane. Regarding the interface with the highest lipid density as the boundary of plasma membrane, the initial thickness of plasma membrane was 3.56 nm, and increased to 3.92 nm after the internalization of PFOS (Fig. 2 c). Energetic analysis over 200-ns MD simulations revealed that PFOS exhibited high electrostatic and van der Waals interaction energies with plasma membrane (Fig. 2 d). The average electrostatic and van der Waals interaction energies for PFOS are − 86.5 and − 169.6 kJ/mol, respectively. At the initial stage of incorporation, the exclusion by water and electrostatic interaction of PFOS headgroup with plasma membrane facilitate the contact of hydrophobic perfluoroalkyl groups with plasma membrane surfaces. Then, van der Waals and electrostatic interactions synergistically drive PFOS molecules to penetrate the plasma membrane. The van der Waals interaction energy with hydrophobic inner layers of the membrane bilayer increases dramatically and eventually surpasses the electrostatic interaction energy. The overall process potentially impacts the permeability of cellular membranes. Structural alterations in ECF-FolT2 Cryo-EM was employed to examine the structural changes of ECF-FolT2 by exposure to PFOS. Approximately 400,650 particles obtained from micrographs were classified and overlapped according to their rotation angles to create 47 high-quality 2D movies which show mushroom-like protein single particles in various angles (Fig. 3 a). Using 3D classification and reconstruction, we produced the final reconstruction with a resolution of 3.93 Å (Fig. 3 b). As shown in Fig. 3 c, the protein was segmented into four subunits: EcfA, EcfA', EcfT, and FolT, and the right two images show the distribution of ECF-FolT2 surface potential. FolT2 is embedded in EcfT forming the transmembrane section. The X-shape coupling helices with positive charges in EcfT constitute the interface between EcfT–EcfS subcomplex and EcfA–EcfA' subcomplex. EcfA and EcfA' subunits compose the cytosolic bottom sections. All the detailed structural information of ECF-FolT2 affected by PFOS can be clearly discerned though cryo-EM structures. Compared to the native structure of ECF-FolT2 (PDB ID: 4HUQ) 43 , significant conformational alterations were found after interacting with PFOS (Fig. 3 d). In the structure alignment, the central area remained mainly green (native ECF-FolT2), while the periphery area is mainly covered by violet (this work), and spatial positions of many α-helixes occurred significant shifts, highlighting a tendency toward outward reversal. While the secondary structure analysis of ECF-FolT2 using circular dichroism spectroscopy revealed the minimal changes after interacting with PFOS (Extended Data Fig. 3 ), the structural alignment examined by cryo-EM analysis provides a clear evidence of changes in the tertiary structures. EcfA and EcfA' structures play a pivotal role in the functionality of ATPase. Alignment of PFOS-affected ECF-FolT2 with ATP-bound ECF-FolT2 (PDB ID: 5D3M) 48 provided more information about changes in functional domains (Fig. 4 a). PFOS-affected ECF-FolT2 also revealed an “eversion” state compared to ATP-bound ECF-FolT2, accompanied with significant shifts of many α-helixes. The enlarged images exhibit the details of ATP-binding domains in which protein structures originally responsible for ATP docking in EcfA and EcfA' show the varying degrees of displacement. Surface visualization of the protein demonstrates that severe deformation has distorted the surface structure of the binding pocket for ATP (Fig. 4 b). The volume and aperture reduction of the ATP-binding pocket could conceivably impair ATP recognition of ECF-FolT2. Moreover, alignment with ECF-FolT2 from Lactobacillus delbrueckii bound to ATP and ADP (PDB ID: 8BMP) 46 also confirmed deformation in the ATP/ADP-binding pocket (Extended Data Fig. 4 ). Significant structural changes can be observed both in the regions combining ATP and ADP in EcfA and EcfA', respectively, not conducive to the utilization of ATP and the release of ADP. All these results underscore the significant impact of PFOS on the tertiary structures of ECF-FolT2, particularly in regions crucial to ATP binding, thereby potentially disrupting its functionality. Binding interaction between ECF-FolT2 and PFOS The binding interaction between ECF-FolT2 and PFOS was analyzed using molecular docking to disclose the interaction mode and binding interfaces. As illustrated in Fig. 5 a, the optimal binding site for PFOS is located at FolT2, the folate transport subunit of ECF-FolT2. The docking energy with − 8.5 kcal/mol suggest that interaction is spontaneous. Figure 5 b shows the gradient isosurfaces representing force distributions with scatter plots summarizing the interaction strength. The results suggest that electrostatic and van der Waals forces contribute to the ECF-PFOS bindings. To further dissect these forces, energy decomposition analysis was conducted using the sobEDA method based on dispersion-corrected density functional theory (DFT) 49 . As illustrated in Fig. 5 c, the interaction energies, including the total interaction energy (Δ E int ), electrostatic energy (Δ E els ), exchange repulsion (Δ E xrep ), orbital interaction energy (Δ E orb ), and Coulomb correlation energy (Δ E C ), are−32.67,−24.65, 57.47,−13.27, and−52.22 kcal/mol, respectively. Δ E C dominates the attraction between ECF-FolT2 and PFOS. A quantitative analysis of molecular surface of PFOS was also performed to determine its contribution to intermolecular energies. The render maps and bar charts are depicted in Extended Data Fig. 5 for electrostatic surface potential (ESP) distribution of PFOS. The negative ESP values are predominantly located on the side containing oxygen functional group (sulfonic acid group), which could promote the generation of hydrogen bond with the electron-withdrawing groups in ECF-FolT2. These results support the experimental observation of strong interaction between PFOS and ECF-FolT2. Discussion A mechanistic scheme to elucidate the interaction modes between PFOS and ECF transporter is depicted in Fig. 6 , in which three key findings are outline: (a) membrane perturbation—PFOS insertion causes outward expansion of the plasma membrane structures, (b) competitive binding—PFOS occupies folate-binding sites, thereby interfering with substrate recognition, and (c) conformational changes—PFOS induces structural alterations in ECF-FolT2, particularly in ATP-binding pockets, and impairs its function. The position of ECF transporters in membranes and their toppling and expulsion mechanism involved in substrate transport could be interfered at different stages in the presence of PFOS (to be discussed below). PFOS penetration into the plasma membrane represents the critical initial step preceding protein binding. The ECF transporter is embedded within the plasma membrane, and half of the transporter is located beneath the cytoplasm plasma membrane, as confirmed by cryo-EM (Fig. 3 c). MD simulations demonstrate that PFOS could easily integrate into the plasma membrane (Fig. 2 a and Extended Data Fig. 2 ). During the initial stage of incorporation, the exclusion of PFOS from water and electrostatic interaction between PFOS and the headgroups of plasma membrane facilitate the contact of hydrophobic perfluoroalkyl groups with the membrane surfaces; this process is dominated by the Coulombic interaction energy. As the internalization occurs, van der Waals and electrostatic interactions synergistically drive PFOS molecules to move deep within the plasma membrane. The Lennard-Jones interaction energy, reflecting interactions with the hydrophobic inner layers of the plasma membrane bilayer, increases dramatically and eventually surpasses the contribution from Coulombic interaction energy (Fig. 2 d). The overall process is evidenced by the outward expansion of membranes and inward convergence of water molecules; these changes potentially impact the permeability of cellar membranes. The potential increase in cell membrane permeability will further raise the risk of PFAS exposure. The ECF transporter provides an illustrative example of how such interactions could disrupt membrane-associated protein function, offering insights into the broader implications of PFAS exposure to cellular integrity. The absorption of substrates is a fundamental function of the ECF transporter. As the binding and translocation domains, S-components could control the specific substrate for transportation 48 . For instance, FolT2-carrying ECF transporter only takes up folate, in general. However, when FolT2 binds with other substrates such as PFOS, folate uptake could be hindered due to the competition for the same binding sites. While the direct evidence of PFOS uptake through ECF transporters or the proportion of active transport process contributing to PFOS transmembrane movement has yet been obtained, it is confirmed by MST that folate transport is inhibited via competitive binding with PFOS chemicals. Especially when transitioning to the periplasm-facing state, FolT2 is more likely to interact with PFOS. The binding of PFOS with FolT2 could occur spontaneously, driven by intermolecular interaction energies, which are revealed though energy decomposition analysis (Fig. 5 c and Supplementary Table 1). The Δ E els , representing classical electrostatic interaction energy, provides strong attractive forces between PFOS and the protein. Exchange energy (Δ E x ) represents primarily the exchange function and potentially incorporate in Hartree-Fock exchange components, and Pauli repulsion energy (Δ E rep ) accounts for energy increase due to the Pauli exclusion principle between electrons of PFOS and ECF-FolT2. These two energies combine to form the exchange-repulsion effect (Δ E xrep ), which is the only positive value here representing the repulsive interactions. Δ E orb arises from energy changes due to intramolecular electron polarization and intermolecular charge transfer. The components of DFT correlation energy (Δ E DFTc ) and dispersion correction (Δ E dc ) collectively constitute Δ E C , which primarily represents van der Waals forces. Obviously, anionic characteristics are beneficial for the attraction between PFOS and proteins. The decreased energy from ATP decomposition might be another reason for the reduced folate uptake. ECF and other ABC transporters can switch between open and closed conformations using the energy from ATP-hydrolysis 46 , 50 , 51 . A few studies reported that some antibiotics could block ATPase activity of ABC transporter (TarGH) by binding to the transmembrane domain 52 . In this study, cryo-EM analysis revealed the alterations in tertiary structures of the two ATPase subunits in ECF-FolT2, located specifically in the surface structures of ATP-binding pocket (Fig. 4 and Extended Data Fig. 4 ). In the structural alignment with original ECF-FolT2 or ATP-bound ECF-FolT2, the ECF-FolT2 interacted with PFOS consistently showed an outward rotational posture. Structural comparison with ECF-FolT2 from Lactobacillus delbrueckii , which shares high homology and identical functionality with the ECF-FolT2 in this study, also demonstrated notable changes in both ATP- and ADP-binding pockets. Although PFOS may primarily bind with FolT2 subunit, the interaction could affect the whole protein and induce structural changes in EcfA and EcfA'. The observed conformational changes suggest that the ATP binding and utilization capability of ECF-FolT2 are compromised. The energy deficiency resulting from decreased ATPase activity would hinder folate transportation. These findings underscore that exposure to PFOS could affect functional integrity. Several studies have shown the inhibitory effects of PFAS on energy consumption of cells, but most of studies simply attribute this phenomenon to the shortage of ATP and down-regulation of gene expression related to ATP synthase 53 – 56 . This study elucidates a new potential impact that the injured proteins associated with ATPase activity such as ECF transporters could cause the reduction in ATP energies for cell activities. Considering the similarities in structures and functions of proteins in ABC superfamily, the damage on ATP-binding domains may extend to many transporters from other ABC families, and ultimately manifested as an energy depletion of the cell. ECF transporter family is a class of proteins widespread in about 50% of prokaryotes 38 , the adverse impacts of PFOS on substrate uptake and ATPase activity are likely to occur in other ECF transporters or even proteins from other ABC families. The negative charge and hydrophobic nature of PFAS confer strong protein-binding affinity through electrostatic and van der Waals interactions, which may be the reason for their high bioaccumulation in blood and visceral organs. However, the data on proteins in other ABC families, particularly the transporters related to humans, are limited. 37 More research to additional ABC transporter families helps to better understand their interactions with hazardous substances, and potential health risks associated with PFAS exposure. Many assays of measuring chemical toxicity quantify the physiological responses and attempt to elucidate the damages at the cellular levels; the general description of cytotoxicity fails to interpret the specific action mechanism. Traditional in vitro experiments, while valuable on action mechanisms, are frequently constrained by instrumental limitations or well-controlled experiments. Our application of cryo-EM represents an innovative protocol to measure the molecular-level impacts of PFOS on functional proteins and invokes a clear elucidation of conformation change. This combination approach not only contributes to understanding of PFAS impact to protein in structural biology, but also offers compelling evidence for elucidating interaction mechanism of PFAS with proteins. The approach has the potential to reveal the molecular toxicology of more typical hazardous substances. Conclusions In summary, this study utilized the combined approaches of cryo-EM and molecular simulation to investigate the interaction between PFOS and ECF-FolT2. PFOS can penetrate cell membranes, accompanied with the increase in the space between plasma membrane components and the expansion of plasma membrane volume; these changes can further enhance the invasion of PFOS. After the penetration, PFOS compete with folate for binding in the pocket of FolT2 subunit. PFOS can also induce conformational changes in ECF-FolT2, ultimately reducing ATPase activity of the transporter. Given the widespread presence of ECF transporters in organisms, these results present a significant threat to ECF transporters and potentially other proteins in the ABC family. Their interaction with PFAS may pose a broader threat to other cellular functions related to ABC family, with various toxicities and diseases potentially arising from similar molecular mechanisms. Understanding the interactions of PFAS with ECF transporters not only enhances knowledge of their adverse biological effects but also supports efforts to mitigate their impact. This study informs the development of the new research on molecular toxicology by PFAS and molecular effect targeting membrane transport proteins, offering opportunities for advancements in environmental toxicology. Methods Chemicals All chemicals used in the experiments were reagent-grade. Potassium perfluorooctane sulfonate (PFOS-K, linear, ≥ 98.0% purity), E. coli polar extract, and extruder set with block were purchased from Sigma-Aldrich (Shanghai, China). Isopropyl-β-ᴅ-thiogalactopyranoside (IPTG, 99% purity), folate (97% purity) and lecithin from egg yolk (> 98% purity) were obtained from Macklin (Shanghai, China). Folic Acid ELISA Kit (96T) was purchased from Meimian (Yancheng, China). Mg 2+ –ATPase assay kit (100T) was bought from Solarbio (Beijing, China). Extended Data Fig. 1 shows the chemical structural formulas of PFOS and folate. Protein expression and purification For gene construction of ECF-FolT2, we prepared two expression plasmids containing EcfA, EcfA', EcfT and FolT2 (Supplementary Fig. 8). Genes of EcfA (GI:122269078) and EcfA' (GI:122269077) were subcloned into pETDuet-1 vectors after the two T7 promoters, respectively, while EcfT (GI:122269079) and FolT2 (GI:116333470) were introduced into pRSFDuet-1 vectors by the same way with a tag of six histidine residues at the N terminus of EcfT. Supplementary Table 2 listed the primer sequence information during gene construction for ECF-FolT2. pETDuet-EcfA-EcfA' and pRSFDuet-EcfT-FolT2 were co-transformed into Escherichia coli C43 (DE3) cells, which performed in the over-expression of membrane proteins than general BL21 (DE3) cells 57 , 58 . The cells were incubated in Luria broth with 100 mg/L ampicillin and 50 mg/L kanamycin at 37 ℃, and reached the stage with an optical density at 600 nm (OD 600 ) of 0.6 ~ 0.8, and the expression of ECF-FolT2 was induced by 1 mmol/L isopropyl-β-ᴅ-thiogalactopyranoside (IPTG) for 4 h. The cells were collected by centrifugation at 8,000 rpm for 15 min, and resuspended in 25 mmol/L Tris-HCl (pH 8.0) with 150 mmol/L NaCl. The cell membrane fraction was obtained by ultrasonication and gradient centrifugation, and cracked in 25 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl and 1% (w/v) n -dodecyl-β-ᴅ-maltopyranoside (DDM, Macklin) for 2 h at 4 ℃ to extract membrane proteins. After centrifugation at 13,000 rpm for 1 h, the supernatant was loaded onto Ni-NTA affinity column and the proteins were adsorbed by the column. The column was washed with 25 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 0.04% (w/v) DDM and 20 mmol/L imidazole to remove the impurities, and ECF-FolT2 was eluted by 25 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 0.04% (w/v) DDM and 250 mmol/L imidazole. The eluent was further purified by size-exclusion chromatography (SEC) with a Superdex 200 Increase 10/300 GL column on an ÄKTA avant (Cytiva) with mobile phase of 25 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl and 0.04% (w/v) DDM (Extended Data Fig. 9a). The peak fraction containing ECF-FolT2 was collected and concentrated to about 10 mg/mL by using a 100-kDa molecular weight cutoff centrifugal concentrator. The ECF-FolT2 was identified through sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the 4 clear bands on the gel correspond to EcfA', EcfA, EcfT, and FolT2, respectively (Extended Data Fig. 9b). Reconstitution of ECF-FolT2 into liposomes The protein reconstitution into liposomes was performed as described by Geertsma et al . 59 with some modifications. E. coli polar lipid extract (Avanti Polar Lipids) and lecithin (from egg yolk, Macklin) at a ratio of 3:1 (w/w) were dissolved in a mixture of methanol and chloroform (3:1, v/v) at 50 mg/mL. After dried by nitrogen flow, the lipid film was resuspended in the buffer of 150 mmol/L NaCl and 25 mmol/L Tris-HCl (pH 8.0), and reached a concentration of 20 mg/mL. After ten cycles of freezing-thawing and eleven times of extrusion through 400 nm polycarbonate filters (Millipore), homogenous sized liposomes were obtained. The purified ECF-FolT2 (from the above experiment) was mixed with liposomes at a ratio of 1:50 (w/w). Subsequently, 3% (w/v) of Triton X-100 was added to destabilize liposomes and incubated for 2 h at 4 ℃. Bio-Beads SM-2 polystyrene beads (Bio-Rad) of 40 mg/mL was added into the suspension every two hours to remove Triton X-100. After the third addition, the mixture was incubated overnight at 4 ℃ with gentle agitation. Then another 2 h of gentle agitation with the last addition of 40 mg/mL Bio-Beads SM-2 was conducted. The mixture was filtered out through a 200-mesh sieve to remove the beads. One cycle of freezing-thawing and eleven times of extrusion through 400 nm polycarbonate filters would make the proteoliposomes have a homogenous size distribution. Proteoliposomes were centrifuged at 13,000 rpm for 30 min and resuspended into 150 mmol/L NaCl and 25 mmol/L Tris-HCl (pH 8.0) at a protein concentration of 2 mg/mL. Folate uptake assay with proteoliposomes under PFOS exposure Proteoliposomes supplemented with 5 mmol/L ATP and 5 mmol/L Mg 2+ , and followed by flash-freezing and thawing for three times, in order to enclose these substances into proteoliposomes. The proteoliposomes were extruded eleven times through a polycarbonate filter with 400-nm pore size. The proteoliposomes centrifuged at 13,000 rpm for 30 min to separate from the remaining external ATP and Mg 2+ . Folate uptake assay was conducted at 25 ℃ in 200 µL solution system, composed of 100 mg/L ECF-FolT2 (proteoliposomes), 2 µmol/L folate, 0 ~ 40 mg/L PFOS and 25 mM Tris-HCl (pH 8.0). At the indicated reaction time, an aliquot of sample was taken and high-speed centrifuged in 30 s to settle down proteoliposomes. The unabsorbed folate in supernatant was measured through Folic Acid ELISA Kit (Meimian). All samples were prepared in triplicates. ATPase activity assay of ECF-FolT2 under PFOS exposure ATPase activity of ECF-FolT2 was measured using a Mg 2+ –ATPase assay kit (Solarbio). Briefly, 100 mg/L of ECF-FolT2 was added into 100 µL solution containing 1 mmol/L MgCl 2 , 0.75 mmol/L ATP and 0 ~ 40 mg/L PFOS. After 30-min reaction at 25 ℃, a quencher (25 µL) from kit was quickly blended in the mixture and incubated for 10 min at 37 ℃. Then 20 µL of the processed samples was transferred into a new tube, and mixed with 200 µL of chromogenic solution. After placing in a water bath at 40 ℃ for 10 min, the samples were measured by microplate reader at 660 nm. All samples were prepared in triplicate. Microscale thermophoresis measurement To measure the thermophoresis changes of target protein molecules, microscale thermophoresis (MST) technology was used to evaluate the binding affinity between target protein and ligand. 90 µL of ECF-FolT2 solution (1 µmol/L) was mixed with 90 µL NTA dye (100 nM) and incubated for 30 min at room temperature, then centrifuged at 15,000 g for 4 min, and the supernatant was transferred to a new tube. Ligand (folate and PFOS) solutions of 1 mmol/L were diluted by gradient to a range from 1 × 10 –8 to 1 × 10 –2 mol/L. Ligand solutions of 10 µL with different concentrations were mixed with 10 µL NTA-labeled ECF-FolT2 solutions respectively. After incubation for 15 min at room temperature, the samples were taken with capillary tubes for the relative fluorescence measurement. MO-Affinity analysis software was used to analyze the data and calculate equilibrium dissociation constants ( K D ) by fitting the experimental data to equation: $$\:\frac{\text{∆}\text{F}}{\text{∆}{\text{F}}_{\text{max}}}\text{=}\frac{\left[\text{ECF}\right]\text{+}\left[\text{ligand}\right]\text{+}{\text{K}}_{\text{D}}\text{−}\sqrt{{\left(\left[\text{ECF}\right]\text{+}\left[\text{ligand}\right]\text{+}{\text{K}}_{\text{D}}\right)}^{\text{2}}\text{−4}\text{×}\left[\text{ECF}\right]\text{+}\left[\text{ligand}\right]}}{\text{2}\text{×}\left[\text{ECF}\right]}$$ 1 where Δ F is change in relative fluorescence of ECF-FolT2 induced by folate or PFOS, [ECF] is the concentration of ECF-FolT2, and [ligand] is the added concentrations of folate or PFOS. As for competitive binding assay, the NTA-labeled ECF-FolT2 was supplemented with folate at a concentration of 400 µM, which was about 40 times of its K D . The mixtures (10 µL) were mixed with a series of PFOS dilutions (10 µL) with concentrations ranging from 1 × 10 –8 to 1 × 10 –2 mol/L respectively. The competition of PFOS with folate for the binding sites was evaluated by comparing the fluorescence intensity changes between the samples of folate-binding to ECF-FolT2 and those controls without added folate. Each group of sample was conducted with three measurements. Cryo-EM sample preparation and data acquisition The entire processes of data collection, processing, and model construction were completed by Shanxi Academy of Advanced Research and Innovation. Briefly, the purified ECF-FolT2 was mixed with PFOS at a molar ratio of 1:20. The mixture was applied at a volume of 2.5 µL onto grid (Quantifoil Au R1.2/1.3 300-mesh) and blotted with filter paper for 4 s using Vitrobot Mark IV chamber (Thermo Fisher Scientific) at 10 ℃ with 100% humidity and subsequently plunged frozen in liquid ethane-propane mixture cooled by liquid nitrogen. The grid was loaded to a 200-keV Talos F200C G2 cryo-EM (Thermo Fisher Scientific), and the datasets of ECF-FolT2 affected by PFOS in selected grid regions were aligned, summed and dose weighted on Warp software 60 . A total of 9,207 cryo-EM micrographs were collected and conducted on cryoSPARC software 61 for patching contrast transfer function (CTF) estimation. The single particles of ECF-FolT2 were auto-picked by blob picking and performed 2D classification for 2D-template generation and excluding the obvious junks. The particles went through heterogenous refinement with seven ab-initio reconstruction. The particles from the best class were extracted for non-uniform refinement and Bayesian polishing on software Relion 62 . Processed particles were re-imported into cryoSPARC for non-uniform refinement, and resulted in a final reconstruction of 3.93 Å. The particles refined into one complete protein structure in which the head section was enveloped by detergent micelles (Extended Data Fig. 10a). These detergent micelles were further removed, and the density maps of ECF-FolT2 structure are visualized from side, front, and top perspectives in Extended Data Fig. 10b. Molecular simulations Molecular dynamics simulation The interactions between PFOS and plasma membrane were conducted through molecular dynamics (MD) simulation. Phospholipid bilayer model (80 × 80 Å) contained POPE, POPG, and POCL1 with a molar ratio of 75:25:5 was generated on CHARMM-GUI ( https://charmm-gui.org/ ) 63 . PFOS molecules were manually added into the membrane system using VMD software 64 , where PFOS was placed to the center position of the plasma membrane surface as an initial state. Topology files of POPE, POPG, POCL1 and PFOS for CHARMM36 were generated based on the implementation in GROMACS. In order to simulate the water-membrane interface, TIP3P model water molecules and sodium ions were used to fill the grid box under periodic boundary conditions. MD simulations were performed with a time length of 200 ns with the setting of temperature at 298 k and pressure of 1 atm. Other molecular simulations After optimizing the molecular geometries by Gaussian 16 software using the HF/3-21G basis sets, posture of PFOS with the lowest energy was obtained though Multiwfn 65 and used as object for all molecular simulations. Multiwfn was used for quantitative analysis of the molecular surface properties of PFOS and its electrostatic potential. The electrostatic potential distribution on the PFOS molecular surface was rendered using VMD. AutoDock Vina 66 , 67 was conducted to simulate the primary binding site of PFOS on ECF. And the docking results were input LigPlot + v.2.2 to establish the 2D interaction diagrams between PFOS and ECF-FolT2. Energy decomposition analysis between PFOS and interacting residues was conducted using sobEDA method. This method is based on Gaussian quantum chemistry program and Multiwfn wave function analysis code. The combined structure of interacting residues and PFOS was used as the object. All execution commands and details were incorporated into a script in sh format file. An xyz format file contained the coordinates of the entire system. Atomic number, net charge, and spin multiplicity of each fragment were defined by txt format file. A gjf format file was used as a template file for the script to generate input files to Gaussian. Under the 6-311 + G(2d,p) basis set, the total interaction energy (Δ E int ) between PFOS and interacting residues was calculated, and further decomposed into electrostatic energy (Δ E els ), exchange (Δ E x ), Pauli repulsion (Δ E rep ), exchange repulsion (Δ E xrep = Δ E x + Δ E rep ), orbital (Δ E orb ), DFT correlation (Δ E DFTc ), dispersion correction (Δ E dc ), and Coulomb correlation (Δ E c = Δ E DFTc + Δ E dc ). Declarations Competing interests All other authors declare they have no competing interests. Author contributions Conceptualization: Z.W., Y.G. Methodology: Z.W., C.Q. Investigation: Z.W., C.Q. Visualization: Z.W., C.Q. Funding acquisition: Y.G. Project administration: Y.G. Supervision: H.L., Y.G. Writing – original draft: Z.W. Writing – review & editing: H.L., Y.G., J.M.T. Acknowledgments This work was supported by the National Natural Science Foundation of China (grant no. 42430703, U22A20590), the National Key Research and Development Program of China (grant no. 2023YFC3708100, 2023YFE0110800). Data Availability All substantial data to support the results in this paper are contained within it and its Supplementary Information. The cryo-EM map has been deposited in the Electron Microscopy Data Bank (EMDB) under accession code EMD-62647 (ECF-FolT2 affected by PFOS). The coordinates have been deposited in the RCSB Protein Data Bank (PDB) under accession code 9KYM (ECF-FolT2 affected by PFOS). The sequences of ECF-FolT2 are available in the following links: EcfA (UniProt: Q03PY5 residues 2 to 279); EcfA' (UniProt: Q03PY6 residues 2 to 290); EcfT (UniProt: Q03PY7 residues 1 to 266); FolT2 (UniProt: Q03PY7 residues 1 to 177). Source data are saved as Source Data File, and available with this paper. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8140096","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":550692647,"identity":"a3a74010-83f2-41d7-9824-f64809070621","order_by":0,"name":"Yanzheng 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University","correspondingAuthor":false,"prefix":"","firstName":"James","middleName":"","lastName":"Tiedje","suffix":""}],"badges":[],"createdAt":"2025-11-18 02:41:02","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8140096/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8140096/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":97325115,"identity":"ecb11e06-988c-4f75-a0e3-7bf6de03be83","added_by":"auto","created_at":"2025-12-03 08:34:08","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":150656,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe impact of PFOS on the functions of ECF-FolT2.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e, Strategies of the preparation of ECF-FolT2 in proteoliposomes. \u003cstrong\u003eb\u003c/strong\u003e, Folate uptake assay by proteoliposomes in the presence of PFOS. \u003cstrong\u003ec\u003c/strong\u003e, ATPase activity assay of ECF-FolT2 in the presence of PFOS. \u003cstrong\u003ed\u003c/strong\u003e, Experimental logic of competitive binding assay between PFOS and folate to ECF-FolT2. \u003cstrong\u003ee\u003c/strong\u003e, MST assays for the binding of folate and PFOS to ECF-FolT2. \u003cstrong\u003ef\u003c/strong\u003e, Binding affinity analysis of PFOS to ECF-FolT2 in the absence or presence of the bound folate.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8140096/v1/a6d8e3f408e2e25dacc43443.jpg"},{"id":97325113,"identity":"c1adcb78-c9a2-4e55-9425-ed2eb27697be","added_by":"auto","created_at":"2025-12-03 08:34:08","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":158861,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMolecular dynamics simulation of the interactions between plasma membranes and PFOS.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e, Time sequences of typical snapshots depicting PFOS positions in plasma membrane. \u003cstrong\u003eb\u003c/strong\u003e, Partial density profiles changes of water, POPE, POPG, and POCL1 molecules in plasma membrane system during 200-ns MD simulation. \u003cstrong\u003ec\u003c/strong\u003e, Changes in thickness of plasma membrane during 200-ns MD simulation. \u003cstrong\u003ed\u003c/strong\u003e, Time evolutions of the membrane interaction energy with PFOS.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8140096/v1/83ccd74df23ccf3c630a201e.jpg"},{"id":97325117,"identity":"7fde4f3c-03c2-4b76-9d2a-242636445a18","added_by":"auto","created_at":"2025-12-03 08:34:08","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":144798,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCryo-EM structures of ECF-FolT2 affected by PFOS.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e, 2D classifications of PFOS-affected ECF-FolT2 particles. \u003cstrong\u003eb\u003c/strong\u003e, The Gold-standard Fourier shell correlation (GSFSC) curves of the PFOS-affected ECF-FolT2 EM map. \u003cstrong\u003ec\u003c/strong\u003e, Structure of ECF-FolT2 affected by PFOS shown in ribbon representation (\u003cem\u003eleft\u003c/em\u003e) and in terms of surface potential (\u003cem\u003eright\u003c/em\u003e, red refers to negative charges, and blue indicates positive charges). The cyan shadow represents the plasma membrane region. \u003cstrong\u003ed\u003c/strong\u003e, Structure alignment of ECF-FolT2 affected by PFOS (this work, \u003cem\u003eviolet\u003c/em\u003e) and the original ECF-FolT2 (PDB ID: 4HUQ, \u003cem\u003egreen\u003c/em\u003e), and the conformational changes are indicated with red arrows.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8140096/v1/38caba66a0b3f32a145ca1e3.jpg"},{"id":97325118,"identity":"2595356b-c52c-49bd-8440-65da6388a13c","added_by":"auto","created_at":"2025-12-03 08:34:08","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":159216,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStructure alignment of ECF-FolT2 (this work, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eviolet\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e) affected by PFOS and ECF-FolT2 (PDB ID: 5D3M, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ecyans\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e) bound to ATP (\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ered\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e). a\u003c/strong\u003e, ECF-FolT2s are shown as sticks, and the conformational changes are indicated with red arrows. \u003cstrong\u003eb\u003c/strong\u003e, ECF-FolT2s are shown as surface.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8140096/v1/f45706e3b6fa385067f13090.jpg"},{"id":97369889,"identity":"1f9409e9-92fc-4de5-ac57-32a9df68c1fb","added_by":"auto","created_at":"2025-12-03 16:26:01","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":123110,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMolecular docking and weak interaction analysis between ECF-FolT2 and PFOS.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e, Binding site of PFOS on ECF-FolT2 by molecular docking. \u003cstrong\u003eb\u003c/strong\u003e, Models illustrating the gradient isosurface of PFOS with key interfacial residues. Left figure is the gradient isosurfaces, where PFOS is represented by ball-and-stick models, residues are in licorice models, and the color blocks sandwiched between the two are isosurfaces. Right figure is the plots of δg \u003cem\u003evs\u003c/em\u003e. the product of the sign (λ\u003csub\u003e2\u003c/sub\u003e) and ρ; sign(λ\u003csub\u003e2\u003c/sub\u003e)ρ (–0.08 ~ 0.08 a.u.) represents the color gradient range from blue to red. \u003cstrong\u003ec\u003c/strong\u003e, Energy decomposition analysis between PFOS and residues of ECF-FolT2.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8140096/v1/fbf4c52ad74e13b95b0501a2.jpg"},{"id":97369088,"identity":"0d263265-1460-4099-b555-355fe23a31f7","added_by":"auto","created_at":"2025-12-03 16:23:37","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":107845,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic illustration for the mechanism of PFOS on ECF-FolT2.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e, membrane perturbation. \u003cstrong\u003eb\u003c/strong\u003e, competitive binding. \u003cstrong\u003ec\u003c/strong\u003e, conformational change.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8140096/v1/60738c1ec78e162a87c5979d.jpg"},{"id":97664660,"identity":"303d6c75-5331-425d-9ab8-69c83a4c0676","added_by":"auto","created_at":"2025-12-08 09:12:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1840510,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8140096/v1/d99bea14-ef06-47e7-b34e-dc1d0a59ec18.pdf"},{"id":97369578,"identity":"98788943-dc13-4241-8abf-ec72b5cee5b2","added_by":"auto","created_at":"2025-12-03 16:25:11","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":31091,"visible":true,"origin":"","legend":"Supplementary Information","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-8140096/v1/8882b106bf6d43d9e9fab75a.docx"},{"id":97325119,"identity":"cc526ce4-b2bf-43b3-a46b-9bcc13d74df6","added_by":"auto","created_at":"2025-12-03 08:34:09","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2083313,"visible":true,"origin":"","legend":"","description":"","filename":"ExtendedDataFig.docx","url":"https://assets-eu.researchsquare.com/files/rs-8140096/v1/1677bee12c1e1fa8eeae3ec1.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Cryo-EM structural observation on the interaction of the bacterial energy-coupling factor transporter with perfluoroalkyl substances","fulltext":[{"header":"Main","content":"\u003cp\u003eReferred to as \u0026ldquo;forever chemicals\u0026rdquo;, the presence and biological damages of per- and polyfluoroalkyl substances (PFAS) in the environment have gained global attention\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Over the past several decades, PFAS have grown to over 14,000 specific chemicals that are documented in U.S. Environmental Protection Agency\u0026rsquo;s CompTox Chemicals Dashboard and have been utilized in hundreds of various applications, such as aqueous fire-fighting foam, textile impregnation, and electroplating\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Their strong carbon-fluorine bonds provide exceptional chemical and thermal stability, cooperating with oxygen-containing functional groups to endow amphiphilic properties, leading to their widespread persistence in the environment and bioaccumulation in living organisms\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Between 1951 and 2015, PFAS emissions are estimated at 2,610\u0026ndash;21,400 tons\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. The aquatic environment is the most important source and sink of PFAS, and numerous studies confirm global presence of PFAS in surface and groundwater, with common concentrations ranging from tens of ng/L to hundreds of \u0026micro;g/L\u003csup\u003e5,6\u003c/sup\u003e, even reaching several mg/L in extremely polluted wastewater\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. These findings have raised significant concerns about their ecotoxicology and ecological risks associated with environmental exposure to PFAS. Epidemiological studies have linked PFAS exposure to various health issues, including hepatopathy, nephropathy, reproductive and developmental issues, endocrine disruption, and cancer \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Animal experiments have confirmed the similar adverse effects\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Although intensive investigations have been conducted on the mechanisms of PFAS action and potential control measures, understanding the biological damages of PFAS at a molecular level remains limited due to the diversity of chemical classes and unknown sensitive biological targets.\u003c/p\u003e\u003cp\u003eThe anionic and amphiphilic nature of PFAS suggests that they can behave like phospholipids and proteinaceous substances rather than neutral and hydrophobic organic contaminants\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. This characteristic promotes their enrichment in blood, liver, and other tissues through strong interactions with proteins, rather than the storage in lipid domains\u003csup\u003e\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Similarities between PFAS and endogenous fatty acids are often cited to describe their behaviors within organisms\u003csup\u003e\u003cspan additionalcitationids=\"CR17 CR18\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Based on this assumption, membrane partitioning has been incorporated into bioconcentration models for elucidation of high bioaccumulation potential of PFAS\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, and the interaction between PFAS and membrane phospholipids has received more attention in toxicity assessments, especially, regarding the activities of membrane-bound proteins\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Currently, the bioaccumulation models emphasize the contributions of PFAS interactions with proteins in evaluation of their distribution, accumulation and half-life in organisms\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. The related \u003cem\u003ein silico\u003c/em\u003e, \u003cem\u003ein vitro\u003c/em\u003e, and \u003cem\u003ein vivo\u003c/em\u003e experiments have demonstrated the close relationship between PFAS bioaccumulation and protein content. For example, PFAS are found to be strongly associated with serum albumin, which highly affects the migration, distribution, and accumulation within living organisms\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. PFAS also perform high binding affinities to liver fatty acid binding proteins, which directly affects the redistribution of PFAS between liver and blood and causes the competitive displacement with fatty acids\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Moreover, organic anion transporter proteins in kidney, such as OAT1, OAT3 and Oatp1a1, are found to mediate cellular PFAS uptake due to their high affinities, supporting the renal elimination of PFAS\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Despite such progress, a comprehensive evaluation on PFAS-protein interactions remain insufficient understanding at molecular levels, which limits the prediction of their biological toxicities.\u003c/p\u003e\u003cp\u003eTransport proteins in plasma membranes are a critical component in impacting the movement of exogenous chemicals through cell membranes and causing functional impairments to organisms. ATP-binding cassette (ABC) transporters, comprising two transmembrane domains and two cytoplasmic ATP-binding domains, facilitate the transmembrane transport of various substances such as nutrients, lipids, cholesterol, steroids, and drugs\u003csup\u003e\u003cspan additionalcitationids=\"CR30 CR31 CR32\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. These transporters are indispensable for cellular nutrients transport in humans, animals, plants, and microorganisms, which potentially impacts the entire ecological system's health\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Previous studies have explored the transport mechanism of drugs and nutrients through ABC transporters\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, and the up/down-regulation of gene expression in ABC transporters by PFAS, such as ABCA1, ABCB1, ABCB11, and ABCG2 transporters\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e, but the influence of chemicals on ABC transporters has not been fully elucidated. As a subgroup of ABC transporters superfamily, energy-coupling factor (ECF) transporters present in about 50% of prokaryotes\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. The ECF transporters are essential to absorption of vital micronutrients, such as water-soluble vitamins\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e and metal ions\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e, by bacteria and archaea. Similar with other ABC transporters, ECF transporters consist of two cytosolic ATPase subunits (EcfA and EcfA') and two membrane-embedded components, including a scaffold protein (EcfT) and a substrate-translocating subunit (S-component)\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. During the transport cycles, these transporters move substrates bound to the S-component into the cytoplasm, and return to their initial outward-facing state with the assistance of ATP hydrolysis\u003csup\u003e\u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Currently, no study (to date) has reported whether ECF transporters might transport harmful substances e.g., PFAS into cell or the contaminants could disrupt the transport functionality.\u003c/p\u003e\u003cp\u003eIn this study, perfluorooctane sulfonate (PFOS) was selected to investigate the interaction with folate ECF transporter (ECF-FolT2) from \u003cem\u003eLactobacillus brevis\u003c/em\u003e at molecular scale. PFOS is one of the most representative PFAS and has a molecular weight similar to folate (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The ATPase activity and folate transport of ECF-FolT2 were analyzed under the exposure to PFOS in aqueous system. Cryogenic electron microscopy (cryo-EM) was employed to investigate structural alterations in the ECF-FolT2. We also revealed the interaction mechanism of PFOS with ECF-FolT2 aided by molecular simulations.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003ePFOS inhibit the regular functions of ECF-FolT2\u003c/h2\u003e\u003cp\u003eReconstituting ECF-FolT2 into proteoliposomes was utilized to mimic the natural settings of cellular membranes and measure folate uptake by ECF-FolT2 under PFOS stress, while minimizing interference from other cellular components and activities. The preparation process of proteoliposomes is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea and detailed in the Methods Section. Like previous studies, to ensure detectable outcomes under the laboratory condition, ECF-FolT2 concentration was selected at 100 mg/L, which was higher than that in many physiological settings\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Correspondingly, PFOS concentration was also set at mg/L levels to maintain the ratio of protein to PFOS in line with the actual situation. During 20-min reaction, apparent folate uptake occurred in the proteoliposome/ATP/Mg\u003csup\u003e2+\u003c/sup\u003e system (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec), confirming that both ECF-FolT2 and ATP are essential components involved in this process. Upon exposure to PFOS, the quantity and rate of folate uptake by ECF-proteoliposomes were markedly reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). The results showed that the amount of folate transported into liposomes decreased from 11.5 to 10.9, 9.85, 8.01, and 7.51 pmol/\u0026micro;g protein at the PFOS concentration of 0, 20, 40, 60, and 80 \u0026micro;mol/L.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eATPase activity assay of ECF-FolT2 showed the apparent functional impairment of ATPase activity. Experiment controls (free of PFOS) revealed that ECF-FolT2 could hydrolyze ATP with an activity of 3.4 U/mg protein (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). As PFOS concentration increased to 20, 40, 60, and 80 \u0026micro;mol/L, the corresponding ATPase activities dropped to 2.7, 2.4, 2.2 and 1.7 U/mg protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). The maximum inhibition rate of ATPase activity reached 51.3% at PFOS concentration at 80 \u0026micro;mol/L. These findings clearly demonstrate that PFOS can disrupt the functional performance of ECF-FolT2.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCompetitive binding between PFOS and folate\u003c/h3\u003e\n\u003cp\u003eMST was employed to estimate the \u003cem\u003eK\u003c/em\u003e\u003csub\u003eD\u003c/sub\u003e; the smaller \u003cem\u003eK\u003c/em\u003e\u003csub\u003eD\u003c/sub\u003e values reflect the stronger affinity. PFOS exhibited a strong affinity to ECF-FolT2 with a \u003cem\u003eK\u003c/em\u003e\u003csub\u003eD\u003c/sub\u003e value of 568 \u0026micro;mol/L, but was weaker than folate (\u003cem\u003eK\u003c/em\u003e\u003csub\u003eD\u003c/sub\u003e = 16.6 \u0026micro;mol/L) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). The competitive binding assay using MST is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed and f. PFOS binding to ECF-FolT2 in the presence or absence of folate both showed the upward trend as PFOS concentration increased. But in the presence of folate, the signal-to-noise ratio of the data was too low to meet the requirements for fitting a curve, which could be considered as non-binding to PFOS. The presence of folate significantly suppressed PFOS binding with ECF-FolT2. The results suggest that PFOS and folate compete for the same binding sites on ECF-FolT2, which is likely responsible for the observed reduction in folate uptake in ECF-proteoliposomes in the presence of PFOS.\u003c/p\u003e\n\u003ch3\u003eInteraction of PFOS with plasma membrane\u003c/h3\u003e\n\u003cp\u003ePFOS could effectively cause the decline of ECF-FolT2 functions in which PFOS penetration into the plasma membrane could be the critical initial step to damage ECF-FolT2. To do so, 200-ns molecular dynamics (MD) simulation of PFOS interaction with plasma membrane was performed to examine the perturbation mechanism. The plasma membrane model comprises the structures of 1-palmitoyl-2-oleoyl-\u003cem\u003esn\u003c/em\u003e-glycero-3-phosphoethanolamine (POPE), L-\u003cem\u003eα\u003c/em\u003e-phosphatidylglycerol (POPG), and 1-palmitoyl-2-oleoyl-\u003cem\u003esn\u003c/em\u003e-glycero-3-cardiolipin (POCL1) at a molar ratio of 75:25:5\u003csup\u003e47\u003c/sup\u003e. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea presents the initial and final snapshots illustrating the interaction between the plasma membrane and PFOS, and the time evolutions of the Z-coordinate of PFOS is shown in Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. During 200-ns MD simulations, the hydrophobic perfluoroalkyl group of PFOS inserted into plasma membrane in a nearly vertical orientation, and fully entered the plasma membrane at 98,440 ps. PFOS molecule was deeply internalized into the plasma membrane structures at the final stage of calculation. Partial density profiles show that the integration of PFOS in plasma membrane caused an outward expansion of POPE, POPG and POCL1 molecules and an inward convergence of water molecules (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), suggesting an increase in intermolecular distance and permeability of plasma membrane. Regarding the interface with the highest lipid density as the boundary of plasma membrane, the initial thickness of plasma membrane was 3.56 nm, and increased to 3.92 nm after the internalization of PFOS (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eEnergetic analysis over 200-ns MD simulations revealed that PFOS exhibited high electrostatic and van der Waals interaction energies with plasma membrane (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). The average electrostatic and van der Waals interaction energies for PFOS are \u0026minus;\u0026thinsp;86.5 and \u0026minus;\u0026thinsp;169.6 kJ/mol, respectively. At the initial stage of incorporation, the exclusion by water and electrostatic interaction of PFOS headgroup with plasma membrane facilitate the contact of hydrophobic perfluoroalkyl groups with plasma membrane surfaces. Then, van der Waals and electrostatic interactions synergistically drive PFOS molecules to penetrate the plasma membrane. The van der Waals interaction energy with hydrophobic inner layers of the membrane bilayer increases dramatically and eventually surpasses the electrostatic interaction energy. The overall process potentially impacts the permeability of cellular membranes.\u003c/p\u003e\n\u003ch3\u003eStructural alterations in ECF-FolT2\u003c/h3\u003e\n\u003cp\u003eCryo-EM was employed to examine the structural changes of ECF-FolT2 by exposure to PFOS. Approximately 400,650 particles obtained from micrographs were classified and overlapped according to their rotation angles to create 47 high-quality 2D movies which show mushroom-like protein single particles in various angles (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Using 3D classification and reconstruction, we produced the final reconstruction with a resolution of 3.93 \u0026Aring; (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, the protein was segmented into four subunits: EcfA, EcfA', EcfT, and FolT, and the right two images show the distribution of ECF-FolT2 surface potential. FolT2 is embedded in EcfT forming the transmembrane section. The X-shape coupling helices with positive charges in EcfT constitute the interface between EcfT\u0026ndash;EcfS subcomplex and EcfA\u0026ndash;EcfA' subcomplex. EcfA and EcfA' subunits compose the cytosolic bottom sections. All the detailed structural information of ECF-FolT2 affected by PFOS can be clearly discerned though cryo-EM structures. Compared to the native structure of ECF-FolT2 (PDB ID: 4HUQ)\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e, significant conformational alterations were found after interacting with PFOS (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). In the structure alignment, the central area remained mainly \u003cem\u003egreen\u003c/em\u003e (native ECF-FolT2), while the periphery area is mainly covered by \u003cem\u003eviolet\u003c/em\u003e (this work), and spatial positions of many α-helixes occurred significant shifts, highlighting a tendency toward outward reversal. While the secondary structure analysis of ECF-FolT2 using circular dichroism spectroscopy revealed the minimal changes after interacting with PFOS (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), the structural alignment examined by cryo-EM analysis provides a clear evidence of changes in the tertiary structures.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eEcfA and EcfA' structures play a pivotal role in the functionality of ATPase. Alignment of PFOS-affected ECF-FolT2 with ATP-bound ECF-FolT2 (PDB ID: 5D3M)\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e provided more information about changes in functional domains (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). PFOS-affected ECF-FolT2 also revealed an \u0026ldquo;eversion\u0026rdquo; state compared to ATP-bound ECF-FolT2, accompanied with significant shifts of many α-helixes. The enlarged images exhibit the details of ATP-binding domains in which protein structures originally responsible for ATP docking in EcfA and EcfA' show the varying degrees of displacement. Surface visualization of the protein demonstrates that severe deformation has distorted the surface structure of the binding pocket for ATP (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). The volume and aperture reduction of the ATP-binding pocket could conceivably impair ATP recognition of ECF-FolT2. Moreover, alignment with ECF-FolT2 from \u003cem\u003eLactobacillus delbrueckii\u003c/em\u003e bound to ATP and ADP (PDB ID: 8BMP)\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e also confirmed deformation in the ATP/ADP-binding pocket (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Significant structural changes can be observed both in the regions combining ATP and ADP in EcfA and EcfA', respectively, not conducive to the utilization of ATP and the release of ADP. All these results underscore the significant impact of PFOS on the tertiary structures of ECF-FolT2, particularly in regions crucial to ATP binding, thereby potentially disrupting its functionality.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eBinding interaction between ECF-FolT2 and PFOS\u003c/h3\u003e\n\u003cp\u003eThe binding interaction between ECF-FolT2 and PFOS was analyzed using molecular docking to disclose the interaction mode and binding interfaces. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, the optimal binding site for PFOS is located at FolT2, the folate transport subunit of ECF-FolT2. The docking energy with \u0026minus;\u0026thinsp;8.5 kcal/mol suggest that interaction is spontaneous. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb shows the gradient isosurfaces representing force distributions with scatter plots summarizing the interaction strength. The results suggest that electrostatic and van der Waals forces contribute to the ECF-PFOS bindings. To further dissect these forces, energy decomposition analysis was conducted using the sobEDA method based on dispersion-corrected density functional theory (DFT)\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, the interaction energies, including the total interaction energy (Δ\u003cem\u003eE\u003c/em\u003e\u003csub\u003eint\u003c/sub\u003e), electrostatic energy (Δ\u003cem\u003eE\u003c/em\u003e\u003csub\u003eels\u003c/sub\u003e), exchange repulsion (Δ\u003cem\u003eE\u003c/em\u003e\u003csub\u003exrep\u003c/sub\u003e), orbital interaction energy (Δ\u003cem\u003eE\u003c/em\u003e\u003csub\u003eorb\u003c/sub\u003e), and Coulomb correlation energy (Δ\u003cem\u003eE\u003c/em\u003e\u003csub\u003eC\u003c/sub\u003e), are\u0026minus;32.67,\u0026minus;24.65, 57.47,\u0026minus;13.27, and\u0026minus;52.22 kcal/mol, respectively. Δ\u003cem\u003eE\u003c/em\u003e\u003csub\u003eC\u003c/sub\u003e dominates the attraction between ECF-FolT2 and PFOS. A quantitative analysis of molecular surface of PFOS was also performed to determine its contribution to intermolecular energies. The render maps and bar charts are depicted in Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e for electrostatic surface potential (ESP) distribution of PFOS. The negative ESP values are predominantly located on the side containing oxygen functional group (sulfonic acid group), which could promote the generation of hydrogen bond with the electron-withdrawing groups in ECF-FolT2. These results support the experimental observation of strong interaction between PFOS and ECF-FolT2.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eA mechanistic scheme to elucidate the interaction modes between PFOS and ECF transporter is depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, in which three key findings are outline: (a) membrane perturbation\u0026mdash;PFOS insertion causes outward expansion of the plasma membrane structures, (b) competitive binding\u0026mdash;PFOS occupies folate-binding sites, thereby interfering with substrate recognition, and (c) conformational changes\u0026mdash;PFOS induces structural alterations in ECF-FolT2, particularly in ATP-binding pockets, and impairs its function. The position of ECF transporters in membranes and their toppling and expulsion mechanism involved in substrate transport could be interfered at different stages in the presence of PFOS (to be discussed below).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ePFOS penetration into the plasma membrane represents the critical initial step preceding protein binding. The ECF transporter is embedded within the plasma membrane, and half of the transporter is located beneath the cytoplasm plasma membrane, as confirmed by cryo-EM (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). MD simulations demonstrate that PFOS could easily integrate into the plasma membrane (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). During the initial stage of incorporation, the exclusion of PFOS from water and electrostatic interaction between PFOS and the headgroups of plasma membrane facilitate the contact of hydrophobic perfluoroalkyl groups with the membrane surfaces; this process is dominated by the Coulombic interaction energy. As the internalization occurs, van der Waals and electrostatic interactions synergistically drive PFOS molecules to move deep within the plasma membrane. The Lennard-Jones interaction energy, reflecting interactions with the hydrophobic inner layers of the plasma membrane bilayer, increases dramatically and eventually surpasses the contribution from Coulombic interaction energy (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). The overall process is evidenced by the outward expansion of membranes and inward convergence of water molecules; these changes potentially impact the permeability of cellar membranes. The potential increase in cell membrane permeability will further raise the risk of PFAS exposure. The ECF transporter provides an illustrative example of how such interactions could disrupt membrane-associated protein function, offering insights into the broader implications of PFAS exposure to cellular integrity.\u003c/p\u003e\u003cp\u003eThe absorption of substrates is a fundamental function of the ECF transporter. As the binding and translocation domains, S-components could control the specific substrate for transportation\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. For instance, FolT2-carrying ECF transporter only takes up folate, in general. However, when FolT2 binds with other substrates such as PFOS, folate uptake could be hindered due to the competition for the same binding sites. While the direct evidence of PFOS uptake through ECF transporters or the proportion of active transport process contributing to PFOS transmembrane movement has yet been obtained, it is confirmed by MST that folate transport is inhibited \u003cem\u003evia\u003c/em\u003e competitive binding with PFOS chemicals. Especially when transitioning to the periplasm-facing state, FolT2 is more likely to interact with PFOS. The binding of PFOS with FolT2 could occur spontaneously, driven by intermolecular interaction energies, which are revealed though energy decomposition analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec and Supplementary Table\u0026nbsp;1). The Δ\u003cem\u003eE\u003c/em\u003e\u003csub\u003eels\u003c/sub\u003e, representing classical electrostatic interaction energy, provides strong attractive forces between PFOS and the protein. Exchange energy (Δ\u003cem\u003eE\u003c/em\u003e\u003csub\u003ex\u003c/sub\u003e) represents primarily the exchange function and potentially incorporate in Hartree-Fock exchange components, and Pauli repulsion energy (Δ\u003cem\u003eE\u003c/em\u003e\u003csub\u003erep\u003c/sub\u003e) accounts for energy increase due to the Pauli exclusion principle between electrons of PFOS and ECF-FolT2. These two energies combine to form the exchange-repulsion effect (Δ\u003cem\u003eE\u003c/em\u003e\u003csub\u003exrep\u003c/sub\u003e), which is the only positive value here representing the repulsive interactions. Δ\u003cem\u003eE\u003c/em\u003e\u003csub\u003eorb\u003c/sub\u003e arises from energy changes due to intramolecular electron polarization and intermolecular charge transfer. The components of DFT correlation energy (Δ\u003cem\u003eE\u003c/em\u003e\u003csub\u003eDFTc\u003c/sub\u003e) and dispersion correction (Δ\u003cem\u003eE\u003c/em\u003e\u003csub\u003edc\u003c/sub\u003e) collectively constitute Δ\u003cem\u003eE\u003c/em\u003e\u003csub\u003eC\u003c/sub\u003e, which primarily represents van der Waals forces. Obviously, anionic characteristics are beneficial for the attraction between PFOS and proteins.\u003c/p\u003e\u003cp\u003eThe decreased energy from ATP decomposition might be another reason for the reduced folate uptake. ECF and other ABC transporters can switch between open and closed conformations using the energy from ATP-hydrolysis\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. A few studies reported that some antibiotics could block ATPase activity of ABC transporter (TarGH) by binding to the transmembrane domain\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. In this study, cryo-EM analysis revealed the alterations in tertiary structures of the two ATPase subunits in ECF-FolT2, located specifically in the surface structures of ATP-binding pocket (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In the structural alignment with original ECF-FolT2 or ATP-bound ECF-FolT2, the ECF-FolT2 interacted with PFOS consistently showed an outward rotational posture. Structural comparison with ECF-FolT2 from \u003cem\u003eLactobacillus delbrueckii\u003c/em\u003e, which shares high homology and identical functionality with the ECF-FolT2 in this study, also demonstrated notable changes in both ATP- and ADP-binding pockets. Although PFOS may primarily bind with FolT2 subunit, the interaction could affect the whole protein and induce structural changes in EcfA and EcfA'. The observed conformational changes suggest that the ATP binding and utilization capability of ECF-FolT2 are compromised. The energy deficiency resulting from decreased ATPase activity would hinder folate transportation. These findings underscore that exposure to PFOS could affect functional integrity. Several studies have shown the inhibitory effects of PFAS on energy consumption of cells, but most of studies simply attribute this phenomenon to the shortage of ATP and down-regulation of gene expression related to ATP synthase\u003csup\u003e\u003cspan additionalcitationids=\"CR54 CR55\" citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. This study elucidates a new potential impact that the injured proteins associated with ATPase activity such as ECF transporters could cause the reduction in ATP energies for cell activities. Considering the similarities in structures and functions of proteins in ABC superfamily, the damage on ATP-binding domains may extend to many transporters from other ABC families, and ultimately manifested as an energy depletion of the cell.\u003c/p\u003e\u003cp\u003eECF transporter family is a class of proteins widespread in about 50% of prokaryotes\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, the adverse impacts of PFOS on substrate uptake and ATPase activity are likely to occur in other ECF transporters or even proteins from other ABC families. The negative charge and hydrophobic nature of PFAS confer strong protein-binding affinity through electrostatic and van der Waals interactions, which may be the reason for their high bioaccumulation in blood and visceral organs. However, the data on proteins in other ABC families, particularly the transporters related to humans, are limited.\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e More research to additional ABC transporter families helps to better understand their interactions with hazardous substances, and potential health risks associated with PFAS exposure.\u003c/p\u003e\u003cp\u003eMany assays of measuring chemical toxicity quantify the physiological responses and attempt to elucidate the damages at the cellular levels; the general description of cytotoxicity fails to interpret the specific action mechanism. Traditional \u003cem\u003ein vitro\u003c/em\u003e experiments, while valuable on action mechanisms, are frequently constrained by instrumental limitations or well-controlled experiments. Our application of cryo-EM represents an innovative protocol to measure the molecular-level impacts of PFOS on functional proteins and invokes a clear elucidation of conformation change. This combination approach not only contributes to understanding of PFAS impact to protein in structural biology, but also offers compelling evidence for elucidating interaction mechanism of PFAS with proteins. The approach has the potential to reveal the molecular toxicology of more typical hazardous substances.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, this study utilized the combined approaches of cryo-EM and molecular simulation to investigate the interaction between PFOS and ECF-FolT2. PFOS can penetrate cell membranes, accompanied with the increase in the space between plasma membrane components and the expansion of plasma membrane volume; these changes can further enhance the invasion of PFOS. After the penetration, PFOS compete with folate for binding in the pocket of FolT2 subunit. PFOS can also induce conformational changes in ECF-FolT2, ultimately reducing ATPase activity of the transporter. Given the widespread presence of ECF transporters in organisms, these results present a significant threat to ECF transporters and potentially other proteins in the ABC family. Their interaction with PFAS may pose a broader threat to other cellular functions related to ABC family, with various toxicities and diseases potentially arising from similar molecular mechanisms. Understanding the interactions of PFAS with ECF transporters not only enhances knowledge of their adverse biological effects but also supports efforts to mitigate their impact. This study informs the development of the new research on molecular toxicology by PFAS and molecular effect targeting membrane transport proteins, offering opportunities for advancements in environmental toxicology.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cb\u003eChemicals\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAll chemicals used in the experiments were reagent-grade. Potassium perfluorooctane sulfonate (PFOS-K, linear, ≥ 98.0% purity), \u003cem\u003eE. coli\u003c/em\u003e polar extract, and extruder set with block were purchased from Sigma-Aldrich (Shanghai, China). Isopropyl-β-ᴅ-thiogalactopyranoside (IPTG, 99% purity), folate (97% purity) and lecithin from egg yolk (\u0026gt; 98% purity) were obtained from Macklin (Shanghai, China). Folic Acid ELISA Kit (96T) was purchased from Meimian (Yancheng, China). Mg\u003csup\u003e2+\u003c/sup\u003e–ATPase assay kit (100T) was bought from Solarbio (Beijing, China). Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the chemical structural formulas of PFOS and folate.\u003c/p\u003e\u003cp\u003e\u003cb\u003eProtein expression and purification\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFor gene construction of ECF-FolT2, we prepared two expression plasmids containing EcfA, EcfA', EcfT and FolT2 (Supplementary Fig.\u0026nbsp;8). Genes of EcfA (GI:122269078) and EcfA' (GI:122269077) were subcloned into pETDuet-1 vectors after the two T7 promoters, respectively, while EcfT (GI:122269079) and FolT2 (GI:116333470) were introduced into pRSFDuet-1 vectors by the same way with a tag of six histidine residues at the N terminus of EcfT. Supplementary Table\u0026nbsp;2 listed the primer sequence information during gene construction for ECF-FolT2. pETDuet-EcfA-EcfA' and pRSFDuet-EcfT-FolT2 were co-transformed into \u003cem\u003eEscherichia coli\u003c/em\u003e C43 (DE3) cells, which performed in the over-expression of membrane proteins than general BL21 (DE3) cells\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e,\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. The cells were incubated in Luria broth with 100 mg/L ampicillin and 50 mg/L kanamycin at 37 ℃, and reached the stage with an optical density at 600 nm (OD\u003csub\u003e600\u003c/sub\u003e) of 0.6 ~ 0.8, and the expression of ECF-FolT2 was induced by 1 mmol/L isopropyl-β-ᴅ-thiogalactopyranoside (IPTG) for 4 h. The cells were collected by centrifugation at 8,000 rpm for 15 min, and resuspended in 25 mmol/L Tris-HCl (pH 8.0) with 150 mmol/L NaCl. The cell membrane fraction was obtained by ultrasonication and gradient centrifugation, and cracked in 25 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl and 1% (w/v) \u003cem\u003en\u003c/em\u003e-dodecyl-β-ᴅ-maltopyranoside (DDM, Macklin) for 2 h at 4 ℃ to extract membrane proteins. After centrifugation at 13,000 rpm for 1 h, the supernatant was loaded onto Ni-NTA affinity column and the proteins were adsorbed by the column. The column was washed with 25 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 0.04% (w/v) DDM and 20 mmol/L imidazole to remove the impurities, and ECF-FolT2 was eluted by 25 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 0.04% (w/v) DDM and 250 mmol/L imidazole. The eluent was further purified by size-exclusion chromatography (SEC) with a Superdex 200 Increase 10/300 GL column on an ÄKTA avant (Cytiva) with mobile phase of 25 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl and 0.04% (w/v) DDM (Extended Data Fig.\u0026nbsp;9a). The peak fraction containing ECF-FolT2 was collected and concentrated to about 10 mg/mL by using a 100-kDa molecular weight cutoff centrifugal concentrator. The ECF-FolT2 was identified through sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the 4 clear bands on the gel correspond to EcfA', EcfA, EcfT, and FolT2, respectively (Extended Data Fig.\u0026nbsp;9b).\u003c/p\u003e\u003cp\u003e\u003cb\u003eReconstitution of ECF-FolT2 into liposomes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe protein reconstitution into liposomes was performed as described by Geertsma \u003cem\u003eet al\u003c/em\u003e.\u003csup\u003e59\u003c/sup\u003e with some modifications. \u003cem\u003eE. coli\u003c/em\u003e polar lipid extract (Avanti Polar Lipids) and lecithin (from egg yolk, Macklin) at a ratio of 3:1 (w/w) were dissolved in a mixture of methanol and chloroform (3:1, v/v) at 50 mg/mL. After dried by nitrogen flow, the lipid film was resuspended in the buffer of 150 mmol/L NaCl and 25 mmol/L Tris-HCl (pH 8.0), and reached a concentration of 20 mg/mL. After ten cycles of freezing-thawing and eleven times of extrusion through 400 nm polycarbonate filters (Millipore), homogenous sized liposomes were obtained. The purified ECF-FolT2 (from the above experiment) was mixed with liposomes at a ratio of 1:50 (w/w). Subsequently, 3% (w/v) of Triton X-100 was added to destabilize liposomes and incubated for 2 h at 4 ℃. Bio-Beads SM-2 polystyrene beads (Bio-Rad) of 40 mg/mL was added into the suspension every two hours to remove Triton X-100. After the third addition, the mixture was incubated overnight at 4 ℃ with gentle agitation. Then another 2 h of gentle agitation with the last addition of 40 mg/mL Bio-Beads SM-2 was conducted. The mixture was filtered out through a 200-mesh sieve to remove the beads. One cycle of freezing-thawing and eleven times of extrusion through 400 nm polycarbonate filters would make the proteoliposomes have a homogenous size distribution. Proteoliposomes were centrifuged at 13,000 rpm for 30 min and resuspended into 150 mmol/L NaCl and 25 mmol/L Tris-HCl (pH 8.0) at a protein concentration of 2 mg/mL.\u003c/p\u003e\u003ch3\u003eFolate uptake assay with proteoliposomes under PFOS exposure\u003c/h3\u003e\u003cp\u003eProteoliposomes supplemented with 5 mmol/L ATP and 5 mmol/L Mg\u003csup\u003e2+\u003c/sup\u003e, and followed by flash-freezing and thawing for three times, in order to enclose these substances into proteoliposomes. The proteoliposomes were extruded eleven times through a polycarbonate filter with 400-nm pore size. The proteoliposomes centrifuged at 13,000 rpm for 30 min to separate from the remaining external ATP and Mg\u003csup\u003e2+\u003c/sup\u003e. Folate uptake assay was conducted at 25 ℃ in 200 µL solution system, composed of 100 mg/L ECF-FolT2 (proteoliposomes), 2 µmol/L folate, 0 ~ 40 mg/L PFOS and 25 mM Tris-HCl (pH 8.0). At the indicated reaction time, an aliquot of sample was taken and high-speed centrifuged in 30 s to settle down proteoliposomes. The unabsorbed folate in supernatant was measured through Folic Acid ELISA Kit (Meimian). All samples were prepared in triplicates.\u003c/p\u003e\u003ch2\u003eATPase activity assay of ECF-FolT2 under PFOS exposure\u003c/h2\u003e\u003cp\u003eATPase activity of ECF-FolT2 was measured using a Mg\u003csup\u003e2+\u003c/sup\u003e–ATPase assay kit (Solarbio). Briefly, 100 mg/L of ECF-FolT2 was added into 100 µL solution containing 1 mmol/L MgCl\u003csub\u003e2\u003c/sub\u003e, 0.75 mmol/L ATP and 0 ~ 40 mg/L PFOS. After 30-min reaction at 25 ℃, a quencher (25 µL) from kit was quickly blended in the mixture and incubated for 10 min at 37 ℃. Then 20 µL of the processed samples was transferred into a new tube, and mixed with 200 µL of chromogenic solution. After placing in a water bath at 40 ℃ for 10 min, the samples were measured by microplate reader at 660 nm. All samples were prepared in triplicate.\u003c/p\u003e\u003ch2\u003eMicroscale thermophoresis measurement\u003c/h2\u003e\u003cp\u003eTo measure the thermophoresis changes of target protein molecules, microscale thermophoresis (MST) technology was used to evaluate the binding affinity between target protein and ligand. 90 µL of ECF-FolT2 solution (1 µmol/L) was mixed with 90 µL NTA dye (100 nM) and incubated for 30 min at room temperature, then centrifuged at 15,000 \u003cem\u003eg\u003c/em\u003e for 4 min, and the supernatant was transferred to a new tube. Ligand (folate and PFOS) solutions of 1 mmol/L were diluted by gradient to a range from 1 × 10\u003csup\u003e–8\u003c/sup\u003e to 1 × 10\u003csup\u003e–2\u003c/sup\u003e mol/L. Ligand solutions of 10 µL with different concentrations were mixed with 10 µL NTA-labeled ECF-FolT2 solutions respectively. After incubation for 15 min at room temperature, the samples were taken with capillary tubes for the relative fluorescence measurement. MO-Affinity analysis software was used to analyze the data and calculate equilibrium dissociation constants (\u003cem\u003eK\u003c/em\u003e\u003csub\u003eD\u003c/sub\u003e) by fitting the experimental data to equation:\u003c/p\u003e\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:\\frac{\\text{∆}\\text{F}}{\\text{∆}{\\text{F}}_{\\text{max}}}\\text{=}\\frac{\\left[\\text{ECF}\\right]\\text{+}\\left[\\text{ligand}\\right]\\text{+}{\\text{K}}_{\\text{D}}\\text{−}\\sqrt{{\\left(\\left[\\text{ECF}\\right]\\text{+}\\left[\\text{ligand}\\right]\\text{+}{\\text{K}}_{\\text{D}}\\right)}^{\\text{2}}\\text{−4}\\text{×}\\left[\\text{ECF}\\right]\\text{+}\\left[\\text{ligand}\\right]}}{\\text{2}\\text{×}\\left[\\text{ECF}\\right]}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003cp\u003ewhere Δ\u003cem\u003eF\u003c/em\u003e is change in relative fluorescence of ECF-FolT2 induced by folate or PFOS, [ECF] is the concentration of ECF-FolT2, and [ligand] is the added concentrations of folate or PFOS.\u003c/p\u003e\u003cp\u003eAs for competitive binding assay, the NTA-labeled ECF-FolT2 was supplemented with folate at a concentration of 400 µM, which was about 40 times of its \u003cem\u003eK\u003c/em\u003e\u003csub\u003eD\u003c/sub\u003e. The mixtures (10 µL) were mixed with a series of PFOS dilutions (10 µL) with concentrations ranging from 1 × 10\u003csup\u003e–8\u003c/sup\u003e to 1 × 10\u003csup\u003e–2\u003c/sup\u003e mol/L respectively. The competition of PFOS with folate for the binding sites was evaluated by comparing the fluorescence intensity changes between the samples of folate-binding to ECF-FolT2 and those controls without added folate. Each group of sample was conducted with three measurements.\u003c/p\u003e\u003ch2\u003eCryo-EM sample preparation and data acquisition\u003c/h2\u003e\u003cp\u003eThe entire processes of data collection, processing, and model construction were completed by Shanxi Academy of Advanced Research and Innovation. Briefly, the purified ECF-FolT2 was mixed with PFOS at a molar ratio of 1:20. The mixture was applied at a volume of 2.5 µL onto grid (Quantifoil Au R1.2/1.3 300-mesh) and blotted with filter paper for 4 s using Vitrobot Mark IV chamber (Thermo Fisher Scientific) at 10 ℃ with 100% humidity and subsequently plunged frozen in liquid ethane-propane mixture cooled by liquid nitrogen. The grid was loaded to a 200-keV Talos F200C G2 cryo-EM (Thermo Fisher Scientific), and the datasets of ECF-FolT2 affected by PFOS in selected grid regions were aligned, summed and dose weighted on Warp software\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. A total of 9,207 cryo-EM micrographs were collected and conducted on cryoSPARC software\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e for patching contrast transfer function (CTF) estimation. The single particles of ECF-FolT2 were auto-picked by blob picking and performed 2D classification for 2D-template generation and excluding the obvious junks. The particles went through heterogenous refinement with seven ab-initio reconstruction. The particles from the best class were extracted for non-uniform refinement and Bayesian polishing on software Relion\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. Processed particles were re-imported into cryoSPARC for non-uniform refinement, and resulted in a final reconstruction of 3.93 Å. The particles refined into one complete protein structure in which the head section was enveloped by detergent micelles (Extended Data Fig.\u0026nbsp;10a). These detergent micelles were further removed, and the density maps of ECF-FolT2 structure are visualized from side, front, and top perspectives in Extended Data Fig.\u0026nbsp;10b.\u003c/p\u003e\u003ch2\u003eMolecular simulations\u003c/h2\u003e\u003ch2\u003eMolecular dynamics simulation\u003c/h2\u003e\u003cp\u003eThe interactions between PFOS and plasma membrane were conducted through molecular dynamics (MD) simulation. Phospholipid bilayer model (80 × 80 Å) contained POPE, POPG, and POCL1 with a molar ratio of 75:25:5 was generated on CHARMM-GUI (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://charmm-gui.org/\u003c/span\u003e\u003cspan address=\"https://charmm-gui.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)\u003csup\u003e63\u003c/sup\u003e. PFOS molecules were manually added into the membrane system using VMD software\u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e, where PFOS was placed to the center position of the plasma membrane surface as an initial state. Topology files of POPE, POPG, POCL1 and PFOS for CHARMM36 were generated based on the implementation in GROMACS. In order to simulate the water-membrane interface, TIP3P model water molecules and sodium ions were used to fill the grid box under periodic boundary conditions. MD simulations were performed with a time length of 200 ns with the setting of temperature at 298 k and pressure of 1 atm.\u003c/p\u003e\u003ch2\u003eOther molecular simulations\u003c/h2\u003e\u003cp\u003eAfter optimizing the molecular geometries by Gaussian 16 software using the HF/3-21G basis sets, posture of PFOS with the lowest energy was obtained though Multiwfn\u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e and used as object for all molecular simulations. Multiwfn was used for quantitative analysis of the molecular surface properties of PFOS and its electrostatic potential. The electrostatic potential distribution on the PFOS molecular surface was rendered using VMD. AutoDock Vina\u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e,\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e was conducted to simulate the primary binding site of PFOS on ECF. And the docking results were input LigPlot\u003csup\u003e+\u003c/sup\u003e v.2.2 to establish the 2D interaction diagrams between PFOS and ECF-FolT2. Energy decomposition analysis between PFOS and interacting residues was conducted using sobEDA method. This method is based on Gaussian quantum chemistry program and Multiwfn wave function analysis code. The combined structure of interacting residues and PFOS was used as the object. All execution commands and details were incorporated into a script in sh format file. An xyz format file contained the coordinates of the entire system. Atomic number, net charge, and spin multiplicity of each fragment were defined by txt format file. A gjf format file was used as a template file for the script to generate input files to Gaussian. Under the 6-311 + G(2d,p) basis set, the total interaction energy (Δ\u003cem\u003eE\u003c/em\u003e\u003csub\u003eint\u003c/sub\u003e) between PFOS and interacting residues was calculated, and further decomposed into electrostatic energy (Δ\u003cem\u003eE\u003c/em\u003e\u003csub\u003eels\u003c/sub\u003e), exchange (Δ\u003cem\u003eE\u003c/em\u003e\u003csub\u003ex\u003c/sub\u003e), Pauli repulsion (Δ\u003cem\u003eE\u003c/em\u003e\u003csub\u003erep\u003c/sub\u003e), exchange repulsion (Δ\u003cem\u003eE\u003c/em\u003e\u003csub\u003exrep\u003c/sub\u003e = Δ\u003cem\u003eE\u003c/em\u003e\u003csub\u003ex\u003c/sub\u003e + Δ\u003cem\u003eE\u003c/em\u003e\u003csub\u003erep\u003c/sub\u003e), orbital (Δ\u003cem\u003eE\u003c/em\u003e\u003csub\u003eorb\u003c/sub\u003e), DFT correlation (Δ\u003cem\u003eE\u003c/em\u003e\u003csub\u003eDFTc\u003c/sub\u003e), dispersion correction (Δ\u003cem\u003eE\u003c/em\u003e\u003csub\u003edc\u003c/sub\u003e), and Coulomb correlation (Δ\u003cem\u003eE\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e = Δ\u003cem\u003eE\u003c/em\u003e\u003csub\u003eDFTc\u003c/sub\u003e + Δ\u003cem\u003eE\u003c/em\u003e\u003csub\u003edc\u003c/sub\u003e).\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eAll other authors declare they have no competing interests.\u003c/p\u003e\n\u003ch2\u003eAuthor contributions\u003c/h2\u003e\n\u003cp\u003eConceptualization: Z.W., Y.G. Methodology: Z.W., C.Q. Investigation: Z.W., C.Q. Visualization: Z.W., C.Q. Funding acquisition: Y.G. Project administration: Y.G. Supervision: H.L., Y.G. Writing – original draft: Z.W. Writing – review \u0026amp; editing: H.L., Y.G., J.M.T.\u003c/p\u003e\n\u003ch2\u003eAcknowledgments\u003c/h2\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (grant no. 42430703, U22A20590), the National Key Research and Development Program of China (grant no. 2023YFC3708100, 2023YFE0110800).\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eAll substantial data to support the results in this paper are contained within it and its Supplementary Information. The cryo-EM map has been deposited in the Electron Microscopy Data Bank (EMDB) under accession code EMD-62647 (ECF-FolT2 affected by PFOS). The coordinates have been deposited in the RCSB Protein Data Bank (PDB) under accession code 9KYM (ECF-FolT2 affected by PFOS). The sequences of ECF-FolT2 are available in the following links: EcfA (UniProt: Q03PY5 residues 2 to 279); EcfA' (UniProt: Q03PY6 residues 2 to 290); EcfT (UniProt: Q03PY7 residues 1 to 266); FolT2 (UniProt: Q03PY7 residues 1 to 177). Source data are saved as Source Data File, and available with this paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eEvich MG et al (2022) Per- and polyfluoroalkyl substances in the environment. Science 375:512\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHan M et al (2025) Sources and transport of per- and polyfluoroalkyl substance (PFAS) in agricultural soil\u0026ndash;plant systems. N Contam 1:e005\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGluege J et al (2020) An overview of the uses of per- and polyfluoroalkyl substances (PFAS). 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J Comput Chem 31:455\u0026ndash;461\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEberhardt J, Santos-Martins D, Tillack AF, Forli S AutoDock Vina 1.2.0: New Docking Methods, Expanded Force Field, and, Bindings P (2021) \u003cem\u003eJ. Chem Inf. Model.\u003c/em\u003e 61, 3891\u0026ndash;3898\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8140096/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8140096/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe movement of exogenous chemicals through cell membranes using transport proteins and causing functional impairments to organisms is an important issue of molecular ecology. Per- and polyfluoroalkyl substances (PFAS) receives intensive concern by their global contamination and a limited understanding of their biological effects. Energy-coupling factor (ECF) transporters are critical to the absorption of vital micronutrients by bacteria and archaea, playing a significant role in maintaining microecological health. Our study found perfluorooctane sulfonate (PFOS) as an inhibitor on folate ECF transporter through competitive binding with folate and conformational alterations in ATP-binding domains, resulting in reduced folate absorption and ATPase activity. Molecular dynamics simulations depicted the process of PFOS internalization into plasma membrane prior to contact with ECF transporter. Using cryogenic electron microscopy (cryo-EM), we observed the conformational alterations caused by PFOS. The interaction mechanism between PFOS and folate ECF transporter was finally revealed though the combined approaches of cryo-EM and molecular simulation and elucidated as three critical pathway: membrane perturbation, competitive binding, and conformational changes. These findings reveal the functional impairments of exogenous chemicals on ECF transporters and advance the knowledge of ecotoxicity of PFAS.\u003c/p\u003e","manuscriptTitle":"Cryo-EM structural observation on the interaction of the bacterial energy-coupling factor transporter with perfluoroalkyl substances","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-03 08:34:04","doi":"10.21203/rs.3.rs-8140096/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fe23426c-f035-45ad-8895-95916197c0dd","owner":[],"postedDate":"December 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":58595818,"name":"Earth and environmental sciences/Ecology/Molecular ecology"},{"id":58595819,"name":"Biological sciences/Microbiology/Environmental microbiology/Water microbiology"}],"tags":[],"updatedAt":"2026-05-11T13:13:42+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-03 08:34:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8140096","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8140096","identity":"rs-8140096","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}