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Our recent studies revealed soluble Vpu oligomers, prompting further investigation into their interactions with cellular proteins. Notably, Vpu may form a complex with calmodulin (CaM) due to its putative CaM\u2010binding motif; however, experimental proof of this association is unavailable. Here, we present definitive experimental evidence that the soluble Vpu complex interacts in vitro with calcium\u2010bound CaM (Ca\n                    <jats:sup>2+<\/jats:sup>\n                    \u2010CaM), its active form. Using double electron\u2013electron resonance (DEER) spectroscopy and protein spin labeling, we detected the formation of a soluble Vpu\u2013Ca\n                    <jats:sup>2+<\/jats:sup>\n                    \u2010CaM complex. Both the full\u2010length (FL) and truncated C\u2010terminal regions of Vpu bind Ca\n                    <jats:sup>2+<\/jats:sup>\n                    \u2010CaM. DEER experiments on a spin\u2010labeled CaM cysteine mutant S39C\/A103C revealed that, upon association with Vpu, Ca\n                    <jats:sup>2+<\/jats:sup>\n                    \u2010CaM undergoes a transition from an open to a more closed conformation, consistent with previous reports of Ca\n                    <jats:sup>2+<\/jats:sup>\n                    \u2010CaM interactions with other proteins. Furthermore, we observed that the binding of Vpu to Ca\n                    <jats:sup>2+<\/jats:sup>\n                    \u2010CaM leads to dissociation of soluble Vpu oligomers, as evidenced by a reduction in DEER modulation depth for FL Vpu spin\u2010labeled at residue L42C. FRET analysis with a fluorescently labeled C\u2010terminal cysteine mutant of Vpu confirmed this result. Like FL Vpu, the Vpu C\u2010terminal region forms soluble homooligomers that dissociate upon binding to Ca\n                    <jats:sup>2+<\/jats:sup>\n                    \u2010CaM. Collectively, our results suggest that soluble Vpu and Ca\n                    <jats:sup>2+<\/jats:sup>\n                    \u2010CaM form an equimolar complex. DEER analysis of the Vpu C\u2010terminal region spin\u2010labeled at residues Q36C\/I61C demonstrated that Vpu undergoes significant conformational changes to facilitate Ca\n                    <jats:sup>2+<\/jats:sup>\n                    \u2010CaM binding. These findings could be relevant to Vpu\u2013CaM interactions under physiological conditions.\n                  <\/jats:p>","DOI":"10.1002\/pro.70487","type":"journal-article","created":{"date-parts":[[2026,1,23]],"date-time":"2026-01-23T13:29:11Z","timestamp":1769174951000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["The soluble state of the\n                    <scp>HIV<\/scp>\n                    \u20101 Vpu protein forms a complex with Ca\n                    <sup>2+<\/sup>\n                    \u2010calmodulin"],"prefix":"10.1002","volume":"35","author":[{"given":"Olamide","family":"Ishola","sequence":"first","affiliation":[{"name":"Department of Chemistry and Biochemistry Texas Tech University  Lubbock Texas USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Md Majharul","family":"Islam","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biochemistry Texas Tech University  Lubbock Texas USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Elaheh","family":"Hadadianpour","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biochemistry Texas Tech University  Lubbock Texas USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Peter P.","family":"Borbat","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Chemical Biology Cornell University  Ithaca New York USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Adeyemi","family":"Ogunbowale","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biochemistry Texas Tech University  Lubbock Texas USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Juan C. 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