{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,1,16]],"date-time":"2026-01-16T02:01:50Z","timestamp":1768528910635,"version":"3.49.0"},"posted":{"date-parts":[[2025,11,13]]},"group-title":"Biophysics","reference-count":70,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,11,13]],"date-time":"2025-11-13T00:00:00Z","timestamp":1762992000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"name":"Behavior of HIV in Viral Environments (B-HIVE) from NIAID of NIH","award":["U54 AI170855"],"award-info":[{"award-number":["U54 AI170855"]}]},{"name":"NIH","award":["1S10OD028655-01"],"award-info":[{"award-number":["1S10OD028655-01"]}]},{"name":"NSF","award":["OAC-1818253"],"award-info":[{"award-number":["OAC-1818253"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,11,13]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>The HIV-1 restriction factor, hSERINC3, functions as a lipid scramblase, translocating lipids across the bilayer in reconstituted proteoliposomes and the viral envelope. Phosphatidylserine(PS) scrambling and exposure at the outer leaflet are recognized to play important roles in several biological processes. To understand the mechanistic basis for hSERINC3-mediated PS lipid scrambling at atomistic resolution, we implemented the transition-tempered metadynamics (TTMetaD) enhanced sampling method. Our simulations sampled close-to-open hSERINC3 conformational transition during PS scrambling and demonstrated that while other non-ATP-dependent lipid transporters with similar architecture transport lipid following a \u201ctrap-and-flip\u201d mechanism, hSERINC3 adopts a \u201ccredit card\u201d mechanism of lipid scrambling and does not follow the classical \u201calternating access\u201d mechanism. Notably, we observe unfolding of the H8 NTD, consistent with the cryo-EM density map of WT-hSERINC3, mediates PS scrambling. A cluster of hydrophilic residues in the hSERINC3 central cavity, forming central gates and interacting with the PS headgroup, stabilizes the intermediate state of inner-groove scrambling and is also observed in the AlphaFold2 model of hSERINC5 that exhibits the highest viral restriction activity. Surprisingly, our simulations reveal distinct pathways for lipid translocation and pathway-dependent alterations of hSERINC3 central cavity, providing direct evidence for a non-canonical, closed-state out-of-groove PS scrambling in a complex membrane environment.<\/jats:p>","DOI":"10.1101\/2025.11.13.688349","type":"posted-content","created":{"date-parts":[[2025,11,14]],"date-time":"2025-11-14T04:40:22Z","timestamp":1763095222000},"source":"Crossref","is-referenced-by-count":0,"title":["Mechanism of Phosphatidylserine Lipid Scrambling by Human SERINC3, an HIV-1 Restriction Factor"],"prefix":"10.64898","author":[{"given":"Puja","family":"Banerjee","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mark","family":"Yeager","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Gregory A.","family":"Voth","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"key":"2025111701001215000_2025.11.13.688349v2.1","doi-asserted-by":"crossref","first-page":"1321","DOI":"10.1038\/s41589-020-00688-0","article-title":"Structural and functional consequences of reversible lipid asymmetry in living membranes","volume":"16","year":"2020","journal-title":"Nature chemical biology"},{"key":"2025111701001215000_2025.11.13.688349v2.2","doi-asserted-by":"publisher","DOI":"10.1038\/s41580-023-00604-z"},{"key":"2025111701001215000_2025.11.13.688349v2.3","doi-asserted-by":"publisher","DOI":"10.1002\/iub.515"},{"key":"2025111701001215000_2025.11.13.688349v2.4","doi-asserted-by":"crossref","first-page":"39","DOI":"10.1146\/annurev-biophys-090821-083030","article-title":"Phospholipid scrambling by G protein\u2013coupled receptors","volume":"51","year":"2022","journal-title":"Annual review of biophysics"},{"key":"2025111701001215000_2025.11.13.688349v2.5","doi-asserted-by":"publisher","DOI":"10.1038\/cdd.2016.7"},{"key":"2025111701001215000_2025.11.13.688349v2.6","doi-asserted-by":"publisher","DOI":"10.1126\/science.1236758"},{"key":"2025111701001215000_2025.11.13.688349v2.7","doi-asserted-by":"publisher","DOI":"10.1038\/s41556-019-0300-y"},{"key":"2025111701001215000_2025.11.13.688349v2.8","doi-asserted-by":"crossref","first-page":"2595","DOI":"10.1021\/acs.jpcb.3c06222","article-title":"Cooperative membrane binding of HIV-1 matrix proteins","volume":"128","year":"2024","journal-title":"The Journal of Physical Chemistry B"},{"key":"2025111701001215000_2025.11.13.688349v2.9","doi-asserted-by":"crossref","first-page":"389","DOI":"10.1016\/j.bpj.2024.01.006","article-title":"Molecular dynamics simulations of HIV-1 matrix-membrane interactions at different stages of viral maturation","volume":"123","year":"2024","journal-title":"Biophysical Journal"},{"key":"2025111701001215000_2025.11.13.688349v2.10","doi-asserted-by":"crossref","first-page":"42","DOI":"10.1016\/j.bpj.2023.11.017","article-title":"Conformational transitions of the HIV-1 Gag polyprotein upon multimerization and gRNA binding","volume":"123","year":"2024","journal-title":"Biophysical Journal"},{"key":"2025111701001215000_2025.11.13.688349v2.11","doi-asserted-by":"publisher","DOI":"10.1038\/nature13984"},{"key":"2025111701001215000_2025.11.13.688349v2.12","doi-asserted-by":"publisher","DOI":"10.1083\/jcb.202103105"},{"key":"2025111701001215000_2025.11.13.688349v2.13","doi-asserted-by":"publisher","DOI":"10.1016\/j.str.2017.11.020"},{"key":"2025111701001215000_2025.11.13.688349v2.14","doi-asserted-by":"publisher","DOI":"10.1016\/j.str.2022.05.010"},{"key":"2025111701001215000_2025.11.13.688349v2.15","doi-asserted-by":"publisher","DOI":"10.1016\/j.str.2024.01.012"},{"key":"2025111701001215000_2025.11.13.688349v2.16","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1607574113"},{"key":"2025111701001215000_2025.11.13.688349v2.17","doi-asserted-by":"publisher","DOI":"10.1038\/nature14953"},{"key":"2025111701001215000_2025.11.13.688349v2.18","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.28671"},{"key":"2025111701001215000_2025.11.13.688349v2.19","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1904210116"},{"key":"2025111701001215000_2025.11.13.688349v2.20","doi-asserted-by":"crossref","unstructured":"Smith, H.E. , Harrison-Rawn, T. , Zheng, W. , Ballesteros, A. & Sotomayor, M . 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