Structural and Biophysical Basis for PFAS Binding by Human Sterol Carrier Protein-2

Structural and Biophysical Basis for PFAS Binding by Human Sterol Carrier Protein-2 | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (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];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Structural and Biophysical Basis for PFAS Binding by Human Sterol Carrier Protein-2 View ORCID Profile Aaron S. Birchfield , View ORCID Profile Rachel L. Signorelli , Kyla T. Cang , View ORCID Profile César A. Ramírez-Sarmiento , View ORCID Profile Brian Fuglestad doi: https://doi.org/10.1101/2025.10.27.684906 Aaron S. Birchfield a Department of Chemistry, Virginia Commonwealth University , Richmond, VA 23284, U.S.A. Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Aaron S. Birchfield Rachel L. Signorelli a Department of Chemistry, Virginia Commonwealth University , Richmond, VA 23284, U.S.A. Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Rachel L. Signorelli Kyla T. Cang a Department of Chemistry, Virginia Commonwealth University , Richmond, VA 23284, U.S.A. Find this author on Google Scholar Find this author on PubMed Search for this author on this site César A. Ramírez-Sarmiento b Institute for Biological and Medical Engineering, Schools of Engineering, Medicine and Biological Sciences, Pontificia Universidad Católica de Chile , Santiago 7820436, Chile c ANID, Millennium Science Initiative Program, Millennium Institute for Integrative Biology (iBio) , Santiago 8331150, Chile Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for César A. Ramírez-Sarmiento For correspondence: fuglestadb{at}vcu.edu cesar.ramirez{at}uc.cl Brian Fuglestad a Department of Chemistry, Virginia Commonwealth University , Richmond, VA 23284, U.S.A. d The Center for Drug Discovery, Virginia Commonwealth University , Richmond, VA 23298, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Brian Fuglestad For correspondence: fuglestadb{at}vcu.edu cesar.ramirez{at}uc.cl Abstract Full Text Info/History Metrics Supplementary material Data/Code Preview PDF Abstract Per- and polyfluoroalkyl substances (PFAS) are harmful environmental contaminants that bioaccumulate in human tissues and are linked to adverse health outcomes. While PFAS are known to bind to a variety of lipid binding proteins (LBPs), such as human serum albumin and fatty acid-binding proteins (FABPs), the broader molecular basis for their biological distribution and breadth of protein binding in humans remains unanswered. We hypothesize that some distribution and persistence of PFAS in humans arises from a distributed network of lipid transfer proteins that collectively solubilize and transport these compounds. To support this hypothesis, we investigated the interaction between various PFAS and human sterol carrier protein 2 (SCP2), a promiscuous, structurally distinct LBP with no previously reported binding with PFAS. Using a combination of screening, fluorescence displacement assays, protein structure prediction of PFAS-SCP2 complexes, and NMR experiments, we demonstrate for the first time that SCP2 is a PFAS-binding protein. Our findings establish SCP2 as a new PFAS-interacting protein, providing insights into the residues participating in these interactions and further supporting the hypothesis that PFAS engage with a broad network of LBPs to facilitate their distribution and persistence in the human body. Introduction Per- and polyfluoroalkyl substances (PFAS) are persistent synthetic chemicals that have been widely used for decades and are widely detected in water, soil, and air. They bioaccumulate in the liver, kidney, and testes, among other tissues, and persist in serum. 1 – 3 Biomonitoring studies report measurable concentrations in >95% of the general population. 4 , 5 PFAS exposure has been linked to adverse health outcomes, including developmental toxicity, immune suppression, and metabolic disorders. 6 , 7 Despite ubiquitous human exposure and connection to negative health outcomes, little is known about cellular mechanisms of PFAS distribution. A growing body of evidence indicates that PFAS engage directly with proteins, raising critical questions about the molecular basis of their biological distribution, target proteins, and effects. To date, PFAS have been shown to bind diverse human proteins, most prominently serum albumin (HSA) and members of the fatty acid–binding protein (FABP) family, with additional interactions reported for peroxisome proliferator-activated receptor γ (PPARγ) and transthyretin. 8 – 11 A unifying feature among these proteins is the presence of large hydrophobic pockets that normally accommodate fatty acids or other lipids, offering plausible binding sites for PFAS engagement as lipid mimics in these and other lipid-binding proteins. High-resolution structural studies confirm these interactions. Recent crystal structures of FABP4 complexed with perfluorooctanoic acid (PFOA), perfluorodecanoic acid (PFDA), and perfluorohexadecanoic acid (PFHxDA) reveal that PFAS interact through a variety of binding modes (PDB ID: 9MP2, 9MIW, 9MIZ). 8 Structures of PFAS complexes with HSA (PDB ID: 7AAI), 12 FABP3 (PDB ID: 7FD7), 13 PPARγ (PDB ID: 8U57), 10 and transthyretin (PDB ID: 5JID) 11 further support this paradigm. Importantly, the replacement chemical GenX was found to bind to HSA (PDB ID: 7Z57). 14 These results demonstrate that protein binding is not limited to phased-out legacy compounds but extends to next-generation replacements, underscoring the urgent need for a framework to test and predict protein interactions across emerging chemistries. Reported binding affinities span from sub-micromolar to low millimolar, comparable to those of endogenous fatty acids. 8 , 9 , 15 , 16 Yet circulating PFAS concentrations in the general population are typically nanomolar, 17 – 19 and many emerging replacement PFAS (e.g., GenX, ADONA) have limited biomonitoring data altogether. However, the emerging trend of lipid-carrier proteins that bind to PFAS suggests that distribution and persistence in humans may, in part, arise from a distributed network of lipid-binding proteins (LBPs) that collectively solubilize and distribute PFAS. This framework offers a mechanistic basis for connecting protein binding to long-term health effects. Understanding bioaccumulation and distribution mechanisms may also guide the design of fluorochemicals with reduced protein binding and biological persistence. To provide initial evidence for this model, we extended the scope of known PFAS-interacting proteins to sterol carrier protein 2 (SCP2), a LBP that is structurally distinct from FABPs with no prior PFAS-interaction data. SCP2, also known as non-specific lipid-transfer protein, is a promiscuous lipid binder that is known to transport a range of lipids, including cholesterol, long-chain fatty acids, acyl-CoA esters, and bile acid intermediates. 20 – 22 Given its broad scope of lipid binding, demonstrating PFAS binding to SCP2 would support the hypothesis that PFAS engage a distributed network of LBPs, while comparison of binding trends, such as chain-length dependence of affinity, would begin to define the physicochemical features underlying this interaction. Here, we establish SCP2 as a new PFAS-binding protein, lay the foundation for a link between chemical features and protein binding, and provide further evidence for the broad propensity of lipid-binding proteins to engage with PFAS. Results and Discussion To broadly assess the PFAS chemical space for SCP2 interactions, we recombinantly expressed and purified the protein. Delipidation of SCP2 was confirmed using protein NMR (Supplementary Fig. S1). We then performed an initial fluorescence displacement screen using NBD-stearic acid (NBD-SA) as a competitive probe. This fatty-acid analog has been reported to bind SCP2 with high affinity (K d = 0.23-0.30 µM) 23 , 24 and, in our hands, exhibited a similar K d of 0.31 µM. Because most PFAS require a co-solvent for solubility, 8 assays were conducted in the presence of 5% ethanol or methanol, which produced only modest increases in the apparent K d of the NBD-SA probe (0.40 µM and 0.35 µM, respectively; Supplementary Fig. S2). While SCP2 is known to weakly bind ethanol, 23 , 24 the minimal effect on NBD-SA affinity indicates minimal interference. To account for these effects, all controls and assays contained matched co-solvent, and IC 50 values were converted to K i using the probe K d measured under the solvent condition of the assay. By observing NBD-SA displacement from SCP2, each PFAS was first screened at a fixed concentration of 10 µM across major classes, including perfluoroalkyl carboxylic acids (PFCAs), perfluoroalkyl sulfonates (PFSAs), fluorotelomer sulfonates and related derivatives, and perfluoroalkyl ether acids, along with a small set of structurally distinct PFAS (Supplementary Table S1) to identify potential binders. Fluorescence intensities were normalized, and compounds producing a reduction in signal to ≤0.75 (corresponding to ≥25% displacement of the NBD–SCP2 complex) were designated as hits ( Fig. 1 ). Compounds meeting this criterion were advanced to full competitive binding titrations to determine inhibition constants (K i ). Download figure Open in new tab Fig. 1. PFAS screening with SCP2. Competitive fluorescence displacement screen of SCP2 using NBD-SA as a probe and with 10 µM fixed concentration of PFAS. The dashed line indicates the threshold for selecting hits for further characterization. Notably, long-chain and very-long-chain PFCAs were shown to bind SCP2, and follow-up titrations revealed a clear chain-length dependence on affinity (C10–C16) ( Fig. 2A ). As chain length increased, binding affinity increased from PFDA (C10, K i = 9.5 ± 1.8 µM) to a maximum at PFTrDA (C13, K i = 0.41 ± 0.02 µM), then declined to PFHxDA (C16, K i = 8.4 ± 0.6 µM). This non-monotonic dependence suggests that extending the perfluoroalkyl chain enhances stabilization within the binding pocket up to an optimum, beyond which additional carbons reduce affinity, plausibly due to steric constraints. Similar chain-length optima have been reported for FABP1 and FABP4. 8 , 25 Additionally, perfluorooctanesulfonic acid (PFOS) was shown to bind SCP2 with a K i of 4.9 ± 0.3 µM (Supplementary Fig. S3 and Table S2). Interestingly, the corresponding carboxylate, PFOA, showed only minimal binding up to 200 µM (Supplementary Fig. S4), indicating that the sulfonate headgroup enables interactions not accessible to carboxylates of the same chain length. Together, these results highlight two determinants of SCP2–PFAS recognition: chain length, where increased hydrophobic contacts stabilize PFAS only above a threshold length (analogous to fatty acid binding, which occurs with C ≥10 but not shorter chains), 20 , 26 and headgroup chemistry, where sulfonates can promote binding even at shorter chain lengths where carboxylates fail. Download figure Open in new tab Fig. 2. Structure–activity relationships and deep learning generated co-folding models of PFAS bound to SCP2. (A) Affinity determination of PFAS identified as hits in the SCP2-PFAS screen, showing log K i values from follow-up titrations (PFCAs and PFOS). Dotted line indicates the upper concentration limit tested (200 µM converted to K i = 64 µM). (B) Boltz-2 models of SCP2 bound to long-chain PFCAs (C10–C16), illustrating placement of the perfluoroalkyl chain within the hydrophobic cavity and the carboxylate oriented toward the solvent-exposed portal. We unsuccessfully attempted to investigate structural determinates for PFCA binding to SCP2 by co-crystallography. This result aligns with the longstanding crystallization challenges of SCP2: the only available human SCP2 crystal structure is in complex with the peroxisomal import receptor PEX5, 27 complemented by a solution NMR structure. 28 To date, no mammalian ligand-bound SCP2 structures have been reported, underscoring a major gap in structural knowledge. Considering these limitations, we employed a recent deep-learning workflow (Boltz-2) 29 for co-folding prediction of SCP2–PFCA complexes (C10–C16) and analyzed their predicted binding orientations ( Fig. 2B ). Co-folding placed all PFCAs (C10–C16) within the canonical hydrophobic pocket of SCP2, 27 , 28 identified based on partial overlap with palmitate in the structure of holo-SCP2 from the yellow fever mosquito Aedes aegypti (PDB ID: 2KSI). 30 In the predicted PFAS complex structures, the perfluorinated tail extends into the binding cavity and the carboxylate is oriented toward the solvent-exposed portal ( Fig. 2B ). This geometry suggests that portal-flanking residues (Lys98, Lys100 and neighboring Asn104) can stabilize polar headgroups, while the fluorinated tails are accommodated by hydrophobic β-sheet and helical cavity walls. This model is consistent with mutagenesis studies that showed substitutions within the portal reduced long chain fatty acid (LFCA)/acyl-CoA binding, 31 , 32 and suggests that PFCAs bind at the LCFA binding site in a similar manner. In the absence of mammalian ligand-bound SCP2 structures, these models provide valuable structural information and align with known fatty-acid/FA-CoA binding modes. 22 , 33 Using two different sets of parameters for the Boltz-2 co-folding predictions, increasing the recycling steps and diffusion samples from 3 and 5 (default) to 10 and 25 (exhaustive) led to similar binding poses (Supplementary Fig. S5). The binding affinity determined by Boltz-2 did not correlate strongly with the experimental binding affinities determined by the fluorescence displacement screening assays (Supplementary Table S3). This is expected, as PFAS fall outside the chemical space represented in Boltz-2’s training data, which is dominated by drug-like, hydrocarbon scaffolds. 29 Per- and polyfluoroalkyl chains exhibit unique conformational and electrostatic properties, including the fluorine gauche effect, high chain rigidity, and atypical charge delocalization, that are not well captured by conventional force fields. 33 – 36 Regardless, confidence metrics for the predicted complex indicate that the predictions are reliable, based on the overall and protein-ligand interface predicted TM-scores (pTM > 0.939 [default], > 0.943 [exhaustive]; ipTM > 0.903 [default], > 0.916 [exhaustive]), average per-residue predicted local distance difference test (complex pLDDT > 0.854 [default], > 0.862 [exhaustive]; complex ipLDDT > 0.793 [default], > 0.811 [exhaustive]), and predicted distance error (complex PDE < 0.414 Å [default], < 0.413 Å [exhaustive]; iPDE < 0.628 Å [default], < 0.623 Å [exhaustive]) (Supplementary Table S4). In the absence of crystallization, we turned to solution NMR to qualitatively assess SCP2–PFAS interactions and verify plausibility of the predictive models. Chemical shift perturbation (CSP) mapping of PFCA binding to SCP2 was complicated by the required 5% ethanol co-solvent, whose presence partially mimicked the spectral changes of a liganded state. However, the overall perturbation pattern was consistent with the Boltz-2 models ( Fig. 2B ). The largest shifts occurred across the portal corridor, encompassing the β4–β5 region and adjacent α3–α5 helices, precisely where the headgroup and tail contacts were predicted in the models (Supplementary Figures S6-S11). To overcome the co-solvent limitation and obtain residue-level confirmation under fully aqueous conditions, we next examined PFOS, which is sufficiently soluble in buffer to allow titrations without the need for co-solvent (Supplementary Fig. S12). 37 At a 1:5 concentration of SCP2:PFOS, PFOS induced chemical-shift changes localized across the cavity and its portal, spanning the β4–β5 region and adjacent α3–α5 helices ( Fig. 3 ). The largest shifts (Leu81, Met84, Ala108, Met109, Lys110) support a model in which the sulfonate headgroup engages the basic portal, while the perfluoroalkyl tail occupies the central cavity, consistent with a Boltz-2 SCP2– PFOS model ( Fig. 3B , Fig. S13). These CSP clusters overlap with residues previously identified at the cavity wall/portal by NMR (including 16-doxylstearate PRE), 28 , 32 and coincide with function-critical portal residues from mutagenesis studies. 31 , 32 Altogether, the PFOS CSP data provide strong experimental support for the predictive models. These results suggest that PFCAs and PFOS use the same binding cavity as fatty acids in SCP2, with the sulfonate headgroup enabling interactions not accessible to chain-matched carboxylates. 21 , 38 Download figure Open in new tab Fig. 3. Structural analysis of SCP2–PFOS interactions. (A) NMR chemical shift perturbations (CSPs) of SCP2 upon PFOS titration, plotted versus residue number. Gold bars represent CSPs 0.05 ppm > 0.10 ppm, and red bars represent CSPs ≥ 0.10 ppm. Gray bars represent CSPs < 0.05, while black stars represent residues for which no CSPs were derived. (B) Boltz-2 model of SCP2–PFOS with CSP data from panel A mapped onto the structure (gray = 0.1 ppm, red = ≥ 0.10 ppm), highlighting perturbations across the portal corridor encompassing the β4–β5 region and adjacent α3–α5 helices (~73–110), with maxima at Leu81/Met84 and Ala108–Lys110. White residues indicate those that are unobserved due to lack of signal or overlap. The data support a binding mode in which the sulfonate headgroup engages the basic portal while the perfluoroalkyl tail is accommodated within the hydrophobic cavity. Conclusion The present work highlights SCP2 as a previously unknown PFAS-binding protein. Together with prior studies, this work further supports a distributed-carrier model in which multiple lipid-binding proteins contribute to low-occupancy, cumulative distribution and accumulation of PFAS. Conceptually, the results suggest a strategy in which a minimal lipid-binding protein panel (e.g., HSA, FABP1/4, SCP2) can be used to screen emerging PFAS to anticipate biomolecular interactions. In the longer term, such a strategy could inform alternative fluoro-chemistries with reduced protein binding and potentially diminished biological residence and distribution. Integrating PFAS–protein interaction data with toxicological and exposure studies will be key to translating molecular binding into predictive insight on health risk and safer chemical design. Acknowledgements This work was funded by the National Institutes of Health (NIH) grant R35GM147221 and Agencia Nacional de Investigación y Desarrollo (ANID) through Fondo Nacional de Desarrollo Científico y Tecnológico (FONDECYT 1240205) and the ANID Millennium Science Initiative Program (ICN17_022). Powered@NLHPC: This research was partially supported by the supercomputing infrastructure of the NLHPC (CCSS210001). We gratefully acknowledge Dr. Faik Musayev’s assistance with crystallography trials and technical assistance from Dr. Yun Qu. Funder Information Declared National Institutes of Health, https://ror.org/01cwqze88 , R35GM147221 Agencia Nacional de Investigación y Desarrollo , FONDECYT 1240205 Millennium Science Initiative, https://ror.org/01c080z51 , ICN17_022 Footnotes https://zenodo.org/records/17438460 References (1). ↵ Maxwell , D. L. ; Oluwayiose , O. A. ; Houle , E. ; Roth , K. ; Nowak , K. ; Sawant , S. ; Paskavitz , A. L. ; Liu , W. ; Gurdziel , K. ; Petriello , M. C. ; Richard Pilsner , J. Mixtures of Per- and Polyfluoroalkyl Substances (PFAS) Alter Sperm Methylation and Long-Term Reprogramming of Offspring Liver and Fat Transcriptome . Environ Int 2024 , 186 , 108577 . doi: 10.1016/j.envint.2024.108577 . OpenUrl CrossRef PubMed (2). Vujic , E. ; Ferguson , S. S. ; Brouwer , K. L. R. Effects of PFAS on Human Liver Transporters: Implications for Health Outcomes . 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