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bioRxiv · 10.1101/2025.06.12.658902

The soluble HIV-1 Vpu protein interacts with calmodulin in a Ca2+-dependent manner

Ishola, O.·Islam, M. M.·Hadadianpour, E.·Borbat, P. P.·Georgieva, E. R.
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Abstract

The HIV-1 Vpu membrane protein is crucial to the virus lifecycle. 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-binding motif; however, experimental proof of this association remained unavailable. Here, we present definitive experimental evidence that the soluble Vpu complex interacts in vitro with calcium-bound CaM (Ca2+-CaM) its active form. Using double electron electron-resonance (DEER) spectroscopy and protein spin labeling, we detected the formation of a soluble Vpu-Ca2+-CaM complex. Both the full-length (FL) and truncated C-terminal regions of Vpu bind Ca2+-CaM. DEER experiments on a spin-labeled CaM cysteine mutant S39C/A103C revealed that, upon association with Vpu, Ca2+-CaM undergoes a transition from an open to a more closed conformation, consistent with previous reports of Ca2+-CaM interactions with other proteins. Furthermore, we observed that the binding of Vpu to Ca2+-CaM leads to dissociation of soluble Vpu oligomers, as evidenced by a reduction in DEER modulation depth for FL Vpu spin-labeled at residue L42C. FRET analysis with a fluorescently labeled C-terminal cysteine mutant of Vpu confirmed this result. Like FL Vpu, the Vpu C-terminal region forms soluble homooligomers that dissociate upon binding to Ca2+-CaM. Collectively, our results suggest that soluble Vpu and Ca2+-CaM form an equimolar complex. DEER analysis of Vpu C-terminal region spin-labeled at residues Q36C/I61C demonstrated that Vpu undergoes significant conformational changes to facilitate Ca2+-CaM binding. These findings could be relevant to Vpu-CaM interactions under physiological conditions.

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Ishola, O., Islam, M. M., Hadadianpour, E., Borbat, P. P., Georgieva, E. R.. 2025-06-17. The soluble HIV-1 Vpu protein interacts with calmodulin in a Ca2+-dependent manner. https://doi.org/10.1101/2025.06.12.658902

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