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Evidence has accumulated that both Htt and hPrp40A are modulated by the intracellular Ca<jats:sup>2+<\/jats:sup> sensor calmodulin (CaM). Here we report characterization of the interaction of human CM with the third FF domain (FF<jats:sub>3<\/jats:sub>) of hPrp40A using calorimetric, fluorescence and structural approaches. Homology modeling, differential scanning calorimetry and small angle X\u2010ray scattering (SAXS) data show FF<jats:sub>3<\/jats:sub> forms a folded globular domain. CaM was found to bind FF<jats:sub>3<\/jats:sub> in a Ca<jats:sup>2+<\/jats:sup>\u2010dependent manner with a 1:1 stoichiometry and a dissociation constant (<jats:italic>K<\/jats:italic><jats:sub>d<\/jats:sub>) of 25\u2009\u00b1\u20093\u00a0\u03bcM at 25\u00b0C. NMR studies showed that both domains of CaM are engaged in binding and SAXS analysis of the FF<jats:sub>3<\/jats:sub>\u2010CaM complex revealed CaM occupies an extended configuration. Analysis of the FF<jats:sub>3<\/jats:sub> sequence showed that the anchors for CaM binding must be buried in its hydrophobic core, suggesting that binding to CaM requires unfolding of FF<jats:sub>3<\/jats:sub>. Trp anchors were proposed based on sequence analysis and confirmed by intrinsic Trp fluorescence of FF<jats:sub>3<\/jats:sub> upon binding of CaM and substantial reductions in affinity for Trp\u2010Ala FF<jats:sub>3<\/jats:sub> mutants. The consensus model of the complex showed that binding to CaM binding occurs to an extended, non\u2010globular state of the FF<jats:sub>3<\/jats:sub>, consistent with coupling to transient unfolding of the domain. The implications of these results are discussed in the context of the complex interplay of Ca<jats:sup>2+<\/jats:sup> signaling and Ca<jats:sup>2+<\/jats:sup> sensor proteins in modulating Prp40A\u2010Htt function.<\/jats:p>","DOI":"10.1002\/pro.4606","type":"journal-article","created":{"date-parts":[[2023,2,22]],"date-time":"2023-02-22T09:10:48Z","timestamp":1677057048000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Binding by calmodulin is coupled to transient unfolding of the third <scp>FF<\/scp> domain of <scp>Prp40A<\/scp>"],"prefix":"10.1002","volume":"32","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9470-6666","authenticated-orcid":false,"given":"A.","family":"D\u00edaz Casas","sequence":"first","affiliation":[{"name":"Department of Biochemistry Vanderbilt University  Nashville Tennessee USA"},{"name":"Center for Structural Biology Vanderbilt University  Nashville Tennessee USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"J. J.","family":"Cordoba","sequence":"additional","affiliation":[{"name":"Department of Biochemistry Vanderbilt University  Nashville Tennessee USA"},{"name":"Center for Structural Biology Vanderbilt University  Nashville Tennessee USA"},{"name":"Chemical and Physical Biology Graduate Program Vanderbilt University  Nashville Tennessee USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"B. J.","family":"Ferrer","sequence":"additional","affiliation":[{"name":"Department of Biochemistry Vanderbilt University  Nashville Tennessee USA"},{"name":"Center for Structural Biology Vanderbilt University  Nashville Tennessee USA"},{"name":"Chemical and Physical Biology Graduate Program Vanderbilt University  Nashville Tennessee USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"S.","family":"Balakrishnan","sequence":"additional","affiliation":[{"name":"Department of Biochemistry Vanderbilt University  Nashville Tennessee USA"},{"name":"Center for Structural Biology Vanderbilt University  Nashville Tennessee USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"J. E.","family":"Wurm","sequence":"additional","affiliation":[{"name":"Chemical and Physical Biology Graduate Program Vanderbilt University  Nashville Tennessee USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"B.","family":"Pastrana\u2010R\u00edos","sequence":"additional","affiliation":[{"name":"Department of Chemistry University of Puerto Rico, Mayag\u00fcez Campus  Mayag\u00fcez Puerto Rico USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2180-0790","authenticated-orcid":false,"given":"W. 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