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Recent investigations of MIF and MIF\u20102 have shown them to possess N\u2010to\u2010C\u2010terminal allosteric crosstalk, but despite the similarity of this \u201callosteric pathway,\u201d its regulation of MIF and MIF\u20102 is not identical. Thus, structure alone does not preserve the precise allosteric mechanism, and additional residues that modulate MIF and MIF\u20102 function must be characterized. Cysteines have been identified as allosteric switches for the same biochemical functions of MIF, and small molecules targeting its N\u2010terminal enzymatic site have affected the structure of three proximal cysteines. Ebselen is a compound that forms covalent selenylsulfide bonds with MIF cysteines and is hypothesized to destabilize and dissociate the MIF trimer into monomers. Ebselen\u2010bound MIF also displays little\u2010to\u2010no catalysis or biological signaling. However, it is unclear whether Ebselen similarly affects the MIF\u20102 paralog, despite MIF\u20102 containing two related cysteines (MIF contains three). We used mutagenesis, nuclear magnetic resonance, molecular dynamics simulations, in vitro and in vivo biochemistry to investigate the mechanism of Ebselen as a modulator of MIF\u20102 via its cysteines. Our findings suggest that Ebselen partially disrupts the MIF\u20102 homotrimer, though the overall population of such a structure is &lt;35%, even on the timescale of many hours. Ebselen does attenuate the biological functions of MIF\u20102, and solution structural biology captures the conformational transitions preceding the destabilized MIF\u20102 trimer.<\/jats:p>","DOI":"10.1002\/pro.70344","type":"journal-article","created":{"date-parts":[[2025,10,16]],"date-time":"2025-10-16T13:33:47Z","timestamp":1760621627000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Atomistic modulation of\n                    <scp>MIF<\/scp>\n                    \u20102 structure, catalysis, and biological signaling via cysteine residues and a small molecule, Ebselen"],"prefix":"10.1002","volume":"34","author":[{"given":"Vinnie","family":"Widjaja","sequence":"first","affiliation":[{"name":"Department of Molecular Biology, Cell Biology, &amp; Biochemistry Brown University  Providence Rhode Island USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sirena M.","family":"D'Orazio","sequence":"additional","affiliation":[{"name":"Department of Molecular Biology, Cell Biology, &amp; Biochemistry Brown University  Providence Rhode Island USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Pragnya","family":"Das","sequence":"additional","affiliation":[{"name":"Division of Neonatology, Department of Pediatrics Cooper University Hospital  Camden New Jersey USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Divya T.","family":"Rajendran","sequence":"additional","affiliation":[{"name":"Division of Neonatology, Department of Pediatrics Cooper University Hospital  Camden New Jersey USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xander","family":"Takada","sequence":"additional","affiliation":[{"name":"Division of Neonatology, Department of Pediatrics Cooper University Hospital  Camden New Jersey USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yuanjun","family":"Shi","sequence":"additional","affiliation":[{"name":"Department of Chemistry Yale University  New Haven Connecticut USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Iz","family":"Varghese","sequence":"additional","affiliation":[{"name":"Department of Molecular Biology, Cell Biology, &amp; Biochemistry Brown University  Providence Rhode Island USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yannie","family":"Lam","sequence":"additional","affiliation":[{"name":"Department of Molecular Biology, Cell Biology, &amp; Biochemistry Brown University  Providence Rhode Island USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nicholas","family":"DaSilva","sequence":"additional","affiliation":[{"name":"Proteomics Core Facility Brown University  Providence Rhode Island USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4504-8038","authenticated-orcid":false,"given":"Jimin","family":"Wang","sequence":"additional","affiliation":[{"name":"Department of Chemistry Yale University  New Haven Connecticut USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Victor S.","family":"Batista","sequence":"additional","affiliation":[{"name":"Department of Chemistry Yale University  New Haven Connecticut USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Vineet","family":"Bhandari","sequence":"additional","affiliation":[{"name":"Division of Neonatology, Department of Pediatrics Cooper University Hospital  Camden New Jersey USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8878-5655","authenticated-orcid":false,"given":"George P.","family":"Lisi","sequence":"additional","affiliation":[{"name":"Department of Molecular Biology, Cell Biology, &amp; Biochemistry Brown University  Providence Rhode Island USA"},{"name":"Giuliani RNA Center Brown University  Providence Rhode Island USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2025,10,16]]},"reference":[{"key":"e_1_2_10_2_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.C500243200"},{"key":"e_1_2_10_3_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-018-04114-x"},{"key":"e_1_2_10_4_1","volume-title":"AMBER","author":"Case DA","year":"2025"},{"key":"e_1_2_10_5_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.jcim.3c01153"},{"key":"e_1_2_10_6_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jbc.2021.101061"},{"key":"e_1_2_10_7_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jbc.2023.104729"},{"key":"e_1_2_10_8_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1002716107"},{"key":"e_1_2_10_9_1","doi-asserted-by":"publisher","DOI":"10.4049\/jimmunol.181.4.2330"},{"key":"e_1_2_10_10_1","doi-asserted-by":"publisher","DOI":"10.1002\/biof.27"},{"issue":"6","key":"e_1_2_10_11_1","first-page":"337","article-title":"Bronchoalveolar lavage fluid differential cell count. 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