Ishola Olamide, Islam Md Majharul, Hadadianpour Elaheh, Borbat Peter P, Georgieva Elka R
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409.
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853.
bioRxiv. 2025 Jun 17:2025.06.12.658902. doi: 10.1101/2025.06.12.658902.
The HIV-1-encoded membrane protein Vpu plays key roles in virus lifecycle. Our lab recently revealed a soluble form of Vpu, and we strived to determine its possible physiological function. Here, we provide solid experimental proof that soluble Vpu interacts with Ca-bound calmodulin (Ca-CaM). A putative CaM-binding motif in Vpu was predicted, but there was no experimental evidence of the Vpu-CaM association. We applied double electron electron-resonance (DEER) and protein spin labeling to detect the soluble Vpu-CaM complex. We found that soluble full-length and truncated C-terminal region of Vpu directly interact with Ca-CaM. DEER results from the spin-labeled double cysteine mutant S39C/A103C of CaM showed that upon association with Vpu Ca-CaM adopts a more closed conformation compared to those in the absence of Vpu. This restructuring is in agreement with previously observed Ca-CaM association with cellular and other HIV-1 proteins. Our results indicate that soluble Vpu and CaM form an equimolar complex. DEER results from doubly spin-labeled at residues Q36C/I61C in Vpu C-terminal region suggest that Vpu's helices 2 and 3 move away from each other to facilitated CaM binding. These observations tell that under physiological conditions the soluble Vpu-CaM complex may provide Vpu with a trafficking pathway to membrane destination.
人类免疫缺陷病毒1型（HIV-1）编码的膜蛋白Vpu在病毒生命周期中发挥关键作用。我们实验室最近发现了一种可溶性形式的Vpu，并努力确定其可能的生理功能。在此，我们提供了确凿的实验证据，证明可溶性Vpu与钙结合钙调蛋白（Ca-CaM）相互作用。预测了Vpu中一个假定的CaM结合基序，但没有Vpu-CaM关联的实验证据。我们应用双电子顺磁共振（DEER）和蛋白质自旋标记来检测可溶性Vpu-CaM复合物。我们发现，可溶性全长Vpu及其截短的C末端区域直接与Ca-CaM相互作用。对CaM的自旋标记双半胱氨酸突变体S39C/A103C进行DEER实验的结果表明，与Vpu结合后，Ca-CaM与不存在Vpu时相比，构象更加封闭。这种结构重组与之前观察到的Ca-CaM与细胞及其他HIV-1蛋白的关联一致。我们的结果表明，可溶性Vpu和CaM形成了等摩尔复合物。对Vpu C末端区域的Q36C/I61C残基进行双自旋标记的DEER实验结果表明，Vpu的螺旋2和螺旋3相互远离，以促进CaM结合。这些观察结果表明，在生理条件下，可溶性Vpu-CaM复合物可能为Vpu提供一条通向膜靶点的运输途径。
