Quantitative and amino acid sequence analysis of soluble HIV-1 Vpu and calmodulin interactions

Ogunbowale, A.; Hadadianpour, E.; Ishola, O.; Islam, M. M.; Ramos, N.; Saffarian Delkhosh, A.; Georgieva, E. R. · biophysics · 2026-09-07 · 原文

DOI：10.64898/2026.04.15.718738作者：7 位

HIV-1 Vpu supports viral adaptation through host-protein interactions. Although mainly membrane-associated, we recently identified a soluble Vpu form that forms a stable complex with Ca2+-bound calmodulin (Ca2+-CaM), potentially influencing Vpu trafficking. Here, to determine the binding affinity and identify regions of soluble Vpu involved in CaM binding, we used ensemble Forster Resonance Energy Transfer (eFRET). We tested Cy3-labeled full-length (FL) Vpu, a C-terminal fragment (helices 2 and 3), and a Cy3-labeled FL Vpu V22A/W23Y/I33N mutant with substitutions of key residues in Vpu helix 1 and helices 1-to-2 loop having a role in the interaction with Ca2+-CaM. All Vpu variants were labeled at residue L42C. Ca2+-CaM was tagged with Cy5 at residue S39C. eFRET analysis of 100 nM Cy3-Vpu variants mixed with Cy5-Ca2+-CaM (in the range 100 nM-2.5 microM) revealed heterocomplexes formation with dissociation constants (Kd) and binding free energies (deltaG). FL Vpu-Ca2+-CaM showed highest stability (Kd ~74 nM, deltaG ~-9.7 kcal/mol), while the truncated C-terminal region and V22A/W23Y/I33N mutant formed weaker complexes with Ca2+-CaM (Kd ~182 nM and 800 nM, deltaG ~-9.2 kcal/mol and ~-

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1. 人话版

HIV-1 Vpu supports viral adaptation through host-protein interactions.

Although mainly membrane-associated, we recently identified a soluble Vpu form that forms a stable complex with Ca2+-bound calmodulin (Ca2+-CaM), potentially influencing Vpu trafficking.

2. 领域脉络

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3. 机制拆解

Here, to determine the binding affinity and identify regions of soluble Vpu involved in CaM binding, we used ensemble Forster Resonance Energy Transfer (eFRET).

4. 证据与数字

We tested Cy3-labeled full-length (FL) Vpu, a C-terminal fragment (helices 2 and 3), and a Cy3-labeled FL Vpu V22A/W23Y/I33N mutant with substitutions of key residues in Vpu helix 1 and helices 1-to-2 loop having a role in the interaction with Ca2+-CaM.

All Vpu variants were labeled at residue L42C.

Ca2+-CaM was tagged with Cy5 at residue S39C.

5. 反例与边界

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6. 跨领域连接与意外收获

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