The soluble state of the HIV-1 Vpu protein forms a complex with Ca ²⁺ -calmodulin

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 (Ca ²⁺ -CaM) its active form. Using double electron electron-resonance (DEER) spectroscopy and protein spin labeling, we detected the formation of a soluble Vpu–Ca ²⁺ -CaM complex. Both the full-length (FL) and truncated C-terminal regions of Vpu bind Ca ²⁺ -CaM. DEER experiments on a spin-labeled CaM cysteine mutant S39C/A103C revealed that, upon association with Vpu, Ca ²⁺ -CaM undergoes a transition from an open to a more closed conformation, consistent with previous reports of Ca ²⁺ -CaM interactions with other proteins.

Furthermore, we observed that the binding of Vpu to Ca ²⁺ -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 Ca ²⁺ -CaM. Collectively, our results suggest that soluble Vpu and Ca ²⁺ -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 Ca ²⁺ -CaM binding. These findings could be relevant to Vpu-CaM interactions under physiological conditions.