Quantitative and mutational analysis of soluble HIV-1 Vpu and calmodulin interactions

The HIV -1 Vpu protein aids viral adaptation by influencing host cell pathways via protein interactions. While Vpu is mainly found in plasma and endomembranes, we recently discovered a soluble form that forms a stable, equimolar complex with Ca ²⁺-bound calmodulin (Ca²⁺-CaM), potentially affecting Vpu’s cellular trafficking. Here, t o determine the binding affinity and identify regions of soluble Vpu involved in CaM binding, we used ensemble Förster 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 mutant with substitutions in Vpu’s helix 1. All Vpu’s variants were labeled at residue L42C, and 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 100–600 nM) revealed dissociation constants (Kd) and binding energies (∆G) for heterocomplexes. FL Vpu-Ca2+-CaM showed high stability (Kd ~40 nM, ∆G ~10.1 kcal/mol), while the truncated C -terminal region and V22A/W23Y mutant formed less stable complexes with Ca2+-CaM (Kd ~200 nM and 800 nM, ∆G ~9 kcal/mol and ~8.3 kcal/mol). This, a binding hot spot in Vpu’s CaM-binding motif in helix 1 was identified, which may control the stability of Vpu -Ca2+-CaM complex and Vpu ’s insertion in the membrane : We hypothesize that upon delivery to the membrane, the hydrophobic helix 1 of Vpu dissociates from Ca2+-CaM and inserts in the lipid bilayer; thereafter, CaM dissociates from Vpu facilitated by the reduced Vpu -Ca2+-CaM complex stability . The findings from this study advance our understanding of HIV-1 Vpu interactions with cellular components and may aid the development of antivirals. .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.15.718738doi: bioRxiv preprint 3

The h uman immunodeficiency virus 1 (HIV -1) is the causative agent of HIV/AIDS 1. HIV-1 employs advanced strategies to enter cells and regulate their functions 2-5. The understanding of HIV-1 pathogenesis requires insight into the virus's molecular mechanisms in infected cells. Our focus is on the HIV-1 encoded viral protein U (Vpu), which plays critical roles in the virus lifecycle through interacting with cellular membranes and proteins to control their function6-8. The protein is expressed in the infected cells and resides and functions in the membranes of trans-Golgi network, ER and plasma membrane 9, 10. It is one of the smallest HIV -1 proteins with molecular weight of 9 -16 kDa, single -pass α-helical type -I transmembrane protein. The amino acid (aa) sequence of Vpu consists of a luminal N -terminal domain ( residues 1–3), a highly hydrophobic transmembrane domain (residues 4–27, transmembrane helix 1 [TM helix 1]), and a C -terminal cytoplasmic tail encompassing two α-helices (residues 28–81) that are helix 2 and helix 3 interconnected by a linker region (Figure 1)11, 12. Through its activity, Vpu alters the integrity and natural abundance of many host proteins at the plasma membrane 13; it also antagonizes host proteins to prevent premat ure cell death 14 and promote efficient viral particle release 15, 16. Vpu achieves this by exhibiting substantial conformational flexibility, enabling it to interact with a range of host proteins —including the CD4 receptor , tetherin, MHC -I, and MHC -II. These interactions facilitate the degradation, altered localization, or regulatory modification (either down- or up-regulation) of these proteins1, 9, 17-19. We aim to understand how Vpu interacts with host calmodulin (CaM), which likely influences HIV-1 physiology in infected cells. Elevated CaM levels in HIV -1 infected cells suggest CaM’s key role in the viral life cycle 20, pointing to the significant role of CaM in the virus life cycle . Nearly all HIV -1 proteins have predicted non -canonical CaM -binding motifs, and direct interactions involving Gag, Nef, Tat, and others are reported21-25. These associations may facilitate protein trafficking, membrane insertion, regulate apoptosis, and contribute to CD4+ lymphocyte deterioration26-32. Previously, Vpu was thought to be solely a membrane protein 1, 8, 33, 34. Recent findings from our lab show Vpu also forms soluble homooligomers 35, 36. We next provided the first experimental evidence that the soluble Vpu interacts with Ca 2+-bound calmodulin (Ca 2+-CaM) in equimolar stoichiometry37, aligning with earlier CaM -binding sequence predictions in Vpu 25. While the .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.15.718738doi: bioRxiv preprint 4 putative CaM-binding motif was believed to span TM helix 1, the loop between helices 1 and 2, and part of helix 25, our further amino acid (aa) sequence analysis suggests helix 2 plays a more significant role, containing extensive imperfect CaM-binding motifs as seen in other proteins37-40. We further found that the C-terminal domain of Vpu (residues 28–81), which includes helices 2 and 3, interacts with Ca2+-CaM similarly to full -length Vpu ; therefore h elix 2 substantially contributes to the interaction with Ca2+-CaM37. Here, we report our results from the quantitative analysis of the association of soluble FL wild- type (WT) Vpu, FL Vpu with aa-s substitutions V22A and W23Y in the CaM-binding motif in helix 1 (which we refer to as FL Vpu -M) and a Vpu fragments containing the C-terminal region of WT Vpu (residues 28 -81 or 28 -78). The selected mutations, although did not change the hydrophobicity of this Vpu region, were sufficiently different than aa -s found in CaM -binding motifs, particularly in the 1-8-14 motif sequence FILVW40. We utilized ensemble Fӧster resonance energy transfer (e FRET) between labeled with cyanine 3 (Cy3) Vpu variants and labeled with cyanine 5 (Cy5) FL CaM in the presence of 1 mM CaCl2. We found that all Vpu variants interact with Ca2+-CaM. However, the strength of FL WT Vpu association with Ca2+-CaM was higher (Kd ~ 40 nM) than those for the truncated C-terminal of the WT Vpu ( Kd ~ 200 nM) and FL Vpu-M (Kd ~800 nM), which corresponded to binding energies (∆G) of ~-10.1 kcal/mol, ~-9.15 kcal/mol and ~-8.32 kcal/mol, respectively. Based on these findings, we concluded that the residues in helix 1 and helix 2 of Vpu ’s CaM -binding motif both contribute to the Vpu -Ca2+-CaM complex formation. The reduction of ~1-1.7 kcal/mol for Vpu lacking helix 1 or with substitutions in helix 1 suggests this segment of the CaM-binding motif participated in the heterocomplex stabilization, though most Vpu -CaM contacts occur in Vpu ’s helix 2 and the helix -1-helix 2 loop . This may relate to how soluble Vpu uses CaM for membrane trafficking.

1. The Vpu and CaM variants developed and utilized in this study In this study, we used five constructs of Vpu (Figure 1B). Three of them were tag-free: (i) FL WT Vpu, (ii) FL Vpu-M containing the mutations V22A/W23Y, (iii) Vpu C-terminal region (residues 28-78). The FL Vpu and FL Vpu-M were obtained after the removal of the N-terminal SUMO tag. The other three constructs carried SUMO tag fused to the N -terminus of the Vpu variants: (iv) .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.15.718738doi: bioRxiv preprint 5 SUMO-FL Vpu, and (v) SUMO-Vpu C-terminal region (residues 28-81). These last two constrcts are the same as those SUMO-tagged Vpu variant we used previously37. All Vpu variants contained a single cysteine mutation L42C (numbering in FL Vpu) used for fluorescent labeling. The introduced mutations V22A/W23Y did not destabilize the helical structure in this Vpu’s region (Figure S1). The CaM protein was the same as the one which we used earlier with a single cysteine residue at S39C37, which was labeled with Cy5 acceptor dye. Figure 1. The Vpu protein: (A) The structure of FL Vpu with helices 1, 2 and 3 designated . The earlier predicted CaM-binding motif is in magenta in the structure, and its amino acid sequence is shown below the structure. (B) The Vpu constructs used to test their binding to Ca2+-CaM. The helices 1 and 2 are underlined in black. The CaM -binding motif is in magenta. The cysteine residues used for labeling with Cy3 donor dye are highlighted in cyan. The original residues which were substituted for either alanine (A) and tyrosine in the V22A/W23Y mutant are bold. 2. The studied wild-type Vpu constructs bind Ca2+-CaM with nanomolar-range dissociation constants (Kd) and energies of ~-10 kcal/mol to ~9 kcal/mol. In this study we conducted quantitative analysis of the interaction between HIV-1 Vpu and human Ca2+-CaM, focusing on how different Vpu regions contribute to complex formation and stability. We compared the binding of WT FL Vpu (residues 20-50) with that of WT truncated Vpu (residues .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.15.718738doi: bioRxiv preprint 6 29-50) and further examined FL Vpu-M with the V22A/W23T mutations in helix 1 to assess the role of these residues in the association with Ca2+-CaM. 2.1. At concentrations above 100 nm, the Vpu constructs self-associate affecting their binding to Ca2+-CaM As we observed earlier , the truncated fragment of tag-free Vpu forms homo-oligomers at concentrations as low as 500 nm , and FL Vpu also self -associates into soluble oligomers 37. To evaluate how this Vpu homooligomerization affects the analysis Vpu -Ca2+-CaM complex formation, we probed the binding of tag-free FL Vpu, and tag-free Vpu C-terminal fragment to Ca2+-CaM. In series of experiments, we varied the concentrations of the Cy3-labled tag-free Vpu constructs (Cy3-Vpu) from 100 nM to 850 nM while the Cy5-labled Ca2+-CaM (Cy5-Ca2+-CaM) was at constant concentration of 500 nM. Fluorescent spectra for all combinations of Cy3-Vpu and Cy5-Ca2+-CaM were collected in the 555 nm to 800 nm range (Figure 3, left panels). Characteristic Cy3 emission peaks were observed at approximately 570 nm and 610 nm, along with a Cy5 acceptor emission peak at around 675 nm upon formation of the Cy3-Vpu variant–Cy5-Ca2+-CaM complex. The FRET efficiency (EFRET) for each sample was calculated using the equation41: 𝐸 = 1 − 𝐹𝐷𝐴/𝐹𝐷 (1) Where E is the FRET efficiency (EFRET), FD is the fluorescence intensity of the Cy3 donor at the maximum at about 610 nm without Cy5-Ca2+-CaM in the sample; and F DA is the fluorescence intensity at the same peak maximum for each sample in the presence of Cy5-Ca2+-CaM. Maximum EFRET was observed at 100 nM for WT FL and truncated Vpu. At higher concentrations of Vpu variants, EFRET decreased—especially for truncated Vpu C-terminal regions (Figure 1, right panels). This indicates that above 100 nM , Vpu homooligomerization limits the availability of monomers to bind Ca2+-CaM, reducing their interaction. We also conducted eFRET binding assay of the truncated C -terminal region of Vpu with Ca 2+- CaM at Vpu variants contraptions of 600 nM. However, the obtained Kds was in the range of ~ 3 µM (Figure S2), which reflects the self -oligomerization of Vpu variants, thus decreasing of the effective concertation of Vpu monomers forming a complex with Ca2+-CaM. .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.15.718738doi: bioRxiv preprint 7 Based on these results, we selected 100 nm for all next experiments to determine the dissociation constant Kd and ∆G of the Vpu monomer-Ca2+-CaM monomer heterocomplex, which is described in detail below. Figure 2. Normalized eFRET data (left) and FRET efficiencies (right) for of Cy3 -Vpu at increasing concentration from 100 nm to 850 nm and constant concentration of 500 nM of Cy5 - Ca2+-CaM: The data for FL Vpu are shown in the upper panels (A); and the data for the Vpu C - terminal fragment are shown in the lower panels (B). EFRET decrease upon increasing the Vpu variant concentration, suggesting inefficient Vpu-CaM binding due to the homooligomerization of Vpu. 2.2. FL WT Vpu and Vpu C-terminal region bind Ca2+-CaM with relatively high affinity forming a specific complex We utilized eFRET to detect the binding of Cy3-Vpu variants and Cy5-Ca2+-CaM. We studied the WT FL and Vpu C-terminal region with Ca2+-CaM with and without SUMO tag. All Vpu variants were at constant concentration of 100 nM, and series of samples with increasing Ca2+-CaM concentrations in the range from 100 nM to 600 nM were analyzed. The fluorescence spectra of .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.15.718738doi: bioRxiv preprint 8 Cy3-Vpu variants at 100 nM alone were also recorded and used as backgrounds, which were subtracted from the fluorescent spectra of Cy3 -Vpu variants in th e presence of increasing concentration of Cy5-Ca2+-CaM. As expected, as the Cy5 -Ca2+-CaM concertation increased, the intensity of the Cy3 -Vpu fluorescence peaks with maxima at ~570 nm and 610 nm decreased proportionally to the Vpu variant -Ca2+-CaM complexe s formation due to Cy3 -to-Cy5 energy transfer. Simultaneously, the intensity of Cy5 -Ca2+-CaM emission with a maximum at ~675 nm also increased reflecting the heterocomplex formation (Figure 3). Figure 3. eFRET data for 100 nM FL WT Vpu (upper panel) and 100 nM WT Vpu C -terminal (lower panel) labeled with Cy3 at residue L42C (numbering in FL Vpu) upon increasing the concentrations of Ca2+-CaM labeled with Cy5 at residue S39C. The Cy5-Ca2+-CaM concentration was varied from 100 nm to 600 nm. The fluorescent spectrum of Cy5-Vpu constructs without Cy5- Ca2+-CaM is shown in black. The red and blue arrows show the decrease of Cy3 emission at ~606nm-610 nm and increase of Cy5 emission at ~675 nm, respectively, which is a result of Vpu- Ca2+-CaM complex formation and Cy3-to-Cy5 energy transfer. .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.15.718738doi: bioRxiv preprint 9 We then used these eFRET data sets to estimate the dissociation constants and binding energies of the WT Vpu variants with Ca2+-CaM. To do so, we potted the change in the Cy3-Vpu variant intensity of the peak at ~610 nm (coinciding with Cy5 absorption) vs. Cy5 -Ca2+-CaM concentration (Figure 4 A, B, C, D, upper). Thereafter, using the method of quantitative FRET quenching42 we estimated the dissociation constants (Kd-s) for the complexes of WT FL and truncated Vpu variants with Ca 2+-CaM. The Kd-s were obtained by fitting the data in Figure 4 upper panels to the equation: 𝑌 = 𝑛 2 (𝐴 + 𝑋 + 𝐾𝑑 − √(𝐴 + 𝑋 + 𝐾𝑑)2 − 4𝐴𝑋 (2) Where, n is a constant related to FRET efficiency between donor (Vpu construct) and acceptor (CaM) – obtained from fittings, A is a constant concentration of donor (Cy3-Vpu), and X is the varying concentration of acceptor (Cy5-CaM). Further, we employed the FRET-based method (KD-FRET), which was used to quantify protein– protein interactions in bacterial cells, relaying on the change in EFRET43. For each series of samples of 100 nM FL Vpu or Vpu C -terminal region and increasing Cy5-Ca2+-CaM concentrations, the values of EFRET were calculated using Eq. (1) and plotted against the concentrations of Cy5-Ca2+- CaM (Figure 4 A, B, C, D, lower panels) . Again, the Kd-s were obtained by fitting these data to the equation: 𝐸 = 𝐸𝑚𝑎𝑥 ( [𝐷]+[𝐴]+ 𝐾𝑑− √([𝐷]+[𝐴]+ 𝐾𝑑)2−4[𝐷][𝐴] 2[𝐷] ) (3) Where Emax is the maximum FRET efficiency (obtained from fittings), [D] is the concentration of donor labeled protein (Vpu), [A] is the varying concentration of acceptor labeled protein (CaM) .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.15.718738doi: bioRxiv preprint 10 Figure 4. Analysis of the eFRET data in Figure 3 and estimated Kd-s for the binding of 100 n M Cy3-SUMO FL Vpu (A), 100 nM Cy3-FL Vpu (B), Cy3-SUMO Vpu C-terminal region (C), and Cy3- Vpu C-terminal region (D) upon increasing the concentration of Cy5-Ca2+-CaM in the range of 100 nM to 600 nM. The background-corrected eFRET spectra were used to estimate Kd. The

from fitting the Cy3 donor fluorescence decay at ~ 606 nm (method of Quantitative FRET Quenching) and fitting the FRET efficiencies (EFRET) (KD-FRET) to obtain Kd-s are shown in the upper and lower panels, respectively. The Kd values shown are in units of nM. There is a very good agreement between the Kd values derived using the two methods. All data are average of triplicate samples. Our fittings produced nearly method-independent Kd values for the same series of experiments. Thus, we obtained Kd of 39.07 ± 15.52 nM (quantitative FRET quenching) and 39.71 ± 15.63 nM (KD-FRET) for the binding of tag-free FL Vpu to Ca2+-CaM (Figure 4, B). These Kd values suggest that the FL Vpu has high affinity for Ca 2+-CaM. The calculated binding energy was ~10.1 kcal/mol, suggesting relatively stable complex formation44. The Kd for the tag-free Vpu C-terminal region-Ca2+-CaM complex increased to 226.9 ± 76.44 nM (quantitative FRET quenching ) and 267.21 ± 74.1 nM (KD-FRET) (Figure 4, D), which is about 5.7 times higher than the value for the FL Vpu, while ∆G was reduced by ~1 kcal/mol. Both the increased Kd and reduced ∆G suggest weaker binding of this Vpu variant to Ca2+-CaM compared to FL Vpu. Thus, our results indicate .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.15.718738doi: bioRxiv preprint 11 that the hydrophobic residues in the CaM-binding motif located in Vpu’s helix 1 CaM contribute sensible amount of free energy to the Vpu -Ca2+-CaM complex. Therefore, just the C -terminal region of Vpu has weakened interaction with Ca2+-CaM, as suggested by the calculated ∆G of ~8.35 kcal/ mol. These results agree with prior findings about HIV -1 MA protein -Ca2+-CaM complexes, which report ed a Kd of about 37 nm for FL CaM -binding motif in this protein and reduced binding for peptides containing only fragments of this motif45. We also noted that the EFRET for the Cy3-FL Vpu was about two times lower compared to that of Cy3-Vpu C-terminal fragment under the same concentrations of Cy5-Ca2+-CaM (Figures 3 and 4). This could be due to steric effect imposed by the FL Vpu or altered conformations of the proteins in the FL-Vpu- and Vpu C-terminus-Ca2+-CaM complexes, which results in an increased distance between the Cy3 donor and Cy5 acceptor in the FL Vpu-Ca2+-CaM complex46. Next, we aimed to determine the effect of SUMO tag on the Vpu -Ca2+-CaM binding affinities. Therefore, we determined the Kd and ΔG for the association of SUMO-FL WT Vpu and SUMO- WT Vpu C-terminal with Ca2+-CaM. We analyzed the FRET data for these complexes in the same way we did for SUMO tag-free Vpu variants, yielding Kd-s of ~113 nM and ~300nM and ΔG-s of ~-9.4 kcal/mol and ~-8.8 kcal/mol, respectively. Thus, the presence of SUMO tag indeed reduced the binding but by only 0.5 kcal/mol or less. Interestingly, the effect of SUMO tag on binding was more pronounced for the truncated fragment of Vpu vs. FL Vpu. The reason likely is that in the FL Vpu the SUMO tag is fused to the N-terminal and therefore, it is further away from the CaM- binding motif in Vpu, compared to Vpu C -terminal where SUMO is in close proximity to the binding site. 3. FL Vpu with V22A/W23Y mutations in the hydrophobic helix 1 has reduced affinity to Ca2+-CaM It was predicted that both hydrophobic (mostly located in helix 1) and positively charged residues (in the loop between helices 1 and 2 and in helix 2) in Vpu contribute to the interaction with CaM25, 37. Moreover, in this work we found that the truncated WT C-terminal region of Vpu has reduced binding to Ca 2+-CaM compared to WT FL Vpu, having an increased by a factor of ~5.7 Kd and reduced by ~ 1 kcal/mol ∆G, compared to WT FL Vpu. This points to the role of the hydrophobic residues in Vpu’s helix 1 in stabilizing the Vpu-Ca2+-CaM complex. To further test this .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.15.718738doi: bioRxiv preprint 12 hypothesis, we engineered a mutant V22A/W23Y (FL Vpu -M) with double substitution of residues in the imperfect 1-8-14 CaM-binding motif encompassing the residues IVVWS in Vpu’s helix 1 (Figure 1, B). We conducted the same eFRET-based assay and data analysis, as for all other Vpu variants (Figure 5). Strikingly, for this Vpu-M, we observed further decrease in its affinity to Ca2+-CaM, as the Kd increased to ~800 nm and ∆G decreased to ~ -8.32 kcal/mol, yielding a difference of 1.8 kcal/mol compared to FL WT Vpu. Thus, these results suggest the specific role of the V22 and W23 residues in Vpu -Ca2+-CaM interaction. The observed shift in Kd and ∆G is also like those caused by the truncation of Vpu’s helix 1, therefore confirming the involvement of the IVVWS motif. Figure 5. eFRET titration data and estimated Kd values for the binding of 100 nm Cy3-FL Vpu-M upon increasing the concentration of Cy5 -Ca2+-CaM in the range of 100 nm to 600 nm. The decrease of Cy3 emission at 606 nm and increase of EFRET upon increasing Ca 2+-CaM concentration and the corresponding fitted curves using the methods of Quantitative FRET Quenching and KD-FRET are plotted in the upper and lower panels, respectively. .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.15.718738doi: bioRxiv preprint 13 The Kd and ∆G values for the complexes of all Vpu constructs with Ca2+-CaM are summarized in Tables 1 and 2 and Figure 6. Table 1. The estimated Kd values for the complexes of all Vpu constructs used in this study with Ca2+-CaM. The mean value s ±standard deviation (SD) are shown. The data obtained used Quantitative FRET Quenching (Q-FRET Quenching) and KD-FRET are shown. Vpu construct Kd±SD, nM Q-FRET Quenching Kd±SD, nM KD-FRET FL WT Vpu 39.07 ± 15.52 39.1 ± 15.63 FL WT SUMO-Vpu 112.37 ± 8.04 113.09 ± 7.34 Vpu C-terminus 226.9 ± 76.44 267.21 ± 74.1 SUMO- Vpu C-terminus 351.26 ± 243.53 269.94 ± 141.74 FL Vpu-M 819.92 ± 177.06 778.19 ± 187.31 Table 2. The calculated binding energies ΔG-s for the complexes of all Vpu constructs used in this study with Ca 2+-CaM. The mean values ±standard deviation (SD) are shown. The data obtained used Quantitative FRET Quenching (Q-FRET Quenching) and KD-FRET are shown. Vpu construct ∆G±SD, kcal/mol Q-FRET Quenching ∆G±SD, kcal/mol KD-FRET FL WT Vpu -10.14 ± 0.24 −10.14 ± 0.24 FL WT SUMO-Vpu -9.45 ± 0.04 −9.44 ± 0.04 Vpu C-terminus -9.03 ± 0.20 −8.92 ± 0.16 SUMO- Vpu C-terminus -8.79 ± 0.41 −8.91 ± 0.31 FL Vpu-M -8.33 ± 0.13 −8.36 ± 0.14 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.15.718738doi: bioRxiv preprint 14 Figure 6. Dissociation constants (Kd) and binding energies (∆G) values for the binding of all Vpu constructs to Ca2+-CaM. The data obtained using both the Quantitative FRET Quenching (Q-FRET Quenching) and KD-FRET methods are shown.

In our previous studies, we uncovered that the assumed exclusively transmembrane HIV -1 Vpu protein can exist in a soluble form 35, 36, and later we revealed that th e soluble Vpu forms an equimolar complex with Ca 2+-CaM37, which we believe is linked to Vpu ’s trafficking to the membrane site. Indeed, the interactions of HIV-1 proteins seem to be critical for HIV-1 physiology in the infected cells. Besides Vpu, other HIV-1 proteins, e.g., the MA domain of Gag, Nef, Tat, etc. associate with CaM, and it is thought these interactions aid HIV -1 proteins ’ trafficking, insertion in the membrane, regulati on of cellular apoptosis and contribute to the CD4+ - lymphocytes deterioration 26-32. Notably, it was found that, compared to non -infected cells, the .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.15.718738doi: bioRxiv preprint 15 HIV-1 infected cells have higher CaM levels particularly in the membrane-bound organelle subcellular fraction20, suggesting that CaM may be used to traffic the synthesized in a soluble form HIV-1 proteins to the cellular location, including membranes, where they are active. These findings support the possible significant role of CaM in the HIV-1 life cycle. Additionally, CaM was linked to the replication and infectivity of other viruses, e.g., it interacts with Ebola matrix protein VP4047, highlighting the broader role of viral protein-CaM interactions. In this study, we aimed to gain deeper insight into how the HIV -1 Vpu protein binds with Ca2+ - CaM. Our previous work showed that Vpu variants form homooligomers 35-37, so we performed experiments to investigate the dissociation of these complexes. We found that at a concentration of 100 nM, all Vpu variants were mainly present in their monomeric form (Figur e 2). This led us to use this concentration in binding assays between Vpu variants and Ca 2+-CaM, under the assumption that only monomeric forms contribute to heterocomplex kinetics. This assumption was confirmed by a notable rise in effective Kd values at higher Vpu variant concentrations (600 nM), likely due to concentration-dependent homooligomerization. At levels above 100 nM, fewer Vpu monomers are available as more join homooligomers. Thus, our findings further support that Vpu variants interact with Ca2+-CaM as monomers in a equimolar ratio. Because of the observed homooligomerization of soluble Vpu in our prior35-37 and current studies, it is worth discussing what could be the origin of these oligomers. Besides, homooligomerization of the soluble form of other HIV -1 proteins that are directed to and function in the cellular membranes have also been observed. For example, it was found that the HIV-1 Nef protein forms homodimers and homotrimers in solution 48. Other study found that the HIV -1 MA alone forms trimer or higher order oligomer in solution 49. However, despite it was found that Nef and MA interact directly with Ca2+-CaM 22, 28, no indication that CaM-binds to either Nef or MA oligomer was reported. On the contrary, results from p rotein sedimentation and size exclusion chromatography (SEC) suggested that MA and CaM form a 1:1 complex 29. Therefore, the physiological significance of these HIV -1 proteins, i.e., Vpu , Nef and MA, homooligomers is not well understood. One possibility is that they are easily formed when isolated in isolated state in vitro; however, in the presence of a binding partner, e.g., Ca2+-CaM, their homooligomerization may be irrelevant or a transient state. One possibility could be that the soluble oligomers of HIV-1 proteins are biomacromolecular condensates formed to separate from the aqueous environment; they serve as a storage of these proteins when they do not engage in protein-protein or protein lipid .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.15.718738doi: bioRxiv preprint 16 interactions or could represent an uncharacterized functional state . Indeed, membraneless biomolecular condensates of HIV-1 proteins have been studied and discussed in the literature 50. The behavior of HIV-1 proteins thus adds to the much broader idea about protein clustering and liquid-liquid phase separations, which has been a topic of extensive research in the last years, as it is linked to both physiological and disease processes51, 52. Using an eFRET binding assay, we measured dissociation constants ( Kd) for Vpu variants (100 nM) with Ca2+-CaM across a concentration range from 100 nM to 600 nM. We found that FL WT Vpu forms a relatively stable complex with Kd of ~40 nM and ∆G of ~-10.1 kcal/mol. FL WT Vpu forms a stable complex ( Kd ~40 nM, ∆G ~-10.1 kcal/mol), while its truncated C-terminal region ( Kd ~200 nM, ∆G ~-9 kcal/mol) and FL Vpu -M with V22A/W23Y mutations (Kd ~800 nM, ∆G ~-8.3 kcal/mol) bind less tightly. These results indicate the IVVWS motif in helix 1 pays role of a hot spot in binding, stabilizing the Vpu-Ca2+-CaM complex; its absence weakens binding. This may relate to Vpu ’s transition from soluble to membrane -bound states, requiring helix 1 to unbind Ca 2+-CaM and inserts in the membrane. Thereafter, CaM is released as the complex destabilizes. This is plausible, as it was found earlier that the HIV-1 MA protein as well has two modes of interaction with Ca 2+-CaM that include the FL CaM -binding amino acid sequence (residues 8–43), but shorter regions of residues 11 -28 and 31 -46also bind with reduced affinity45, which might be a mechanism used by HIV-1 proteins. Our findings contribute to the understanding of how HIV-1 proteins interact with host CaM. The quantitative data which we provide will be useful in directing drug design to regulate the Vpu-host protein (CaM) interactions. The Kd and ∆G values for the Vpu -Ca2+-CaM complex provide a foundation for identifying drugs with stronger binding, as previously demonstrated 53, 54.

1. Protein designs, cloning, mutagenesis, expression, and purification The DNA-s encoding the SUMO -FL Vpu and SUMO -C-terminal region of Vpu with histidine tags were commercially synthesized and cloned in pET15b vector (GenScript, Inc.), as described 37. They contained the L42C mutation for labeling with a Cy3 donor. The peptide encompassing the Vpu residues 29-78 with a cysteine residue at position L42C (numbering in FL Vpu) was commercially synthesized (RS Synthesis). .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.15.718738doi: bioRxiv preprint 17 The CaM mutant S39C was produced as previously described 37. The SUMO -FL Vpu-M containing the V22A/W23Y mutations was generated using site -directed mutagenesis (GenScript, I nc.) The expression and purification procedure are fully described in Ishola et al. 202637. 2. Removing the SUMO tag The SUMO tag was removed through thrombin digestion: T he Nickel affinity–purified Vpu variants in a buffer of 20 mM Tris pH 8.0, 100 mM NaCl, 5% (w/v) glycerol, 1 mM DDM, and 100 M TCEP and 10 mM CaCl₂ was mixed with human thrombin (Millipore Sigma) at a ratio of 20 U/mg thrombin/1 mg Vpu protein. The reaction was allowed to proceed overnight at 22°C with gentle rotation. On the next day, the reaction mixture was incubated with Ni²⁺-NTA resin pre -equilibrated in binding of 20 mM Tris , pH 7.4, 150 mM NaCl, 1 mM CaCl₂, 5% (w/v) glycerol, 1 mM β-DDM, and 100 M TCEP. Binding was performed for 1 hour at 4°C with gentle mixing. Thereafter, the flow-through containing the SUMO tag and thrombin was discarded; the resin with bound Vpu protein was washed with 5 resin volumes of the same buffer. Next, the Vpu variant was eluted using 320 mM Imidazole. Afterwards, the Imidazole was removed from the Vpu proteins, and they were concentrated in centrifuge concentrators with 3 kDa MWCO at 4 ºC. The high degree of thrombin removal was confirmed using SDS-PAGE and Western Blotting (WB). 3. Labeling of the proteins with Cy3 donor and Cy5 acceptor Initially, the β-DDM was removed from the FL WT Vpu and FL Vpu-M (with and without SUMO tag) by washing the proteins with a buffer containing 20 mM Tris pH 7.4, 150 mM NaCl, 1 mM CaCl 2, 50 µM TCEP, and 5% glycerol. The Vpu C-terminal region (with and without SUMO tag) was also in this buffer. Buffer exchange and β-DDM removal was performed using several dilution and concentration steps at 4 °C to ensure complete replacement of the initial buffers. The final concent rations of all protein constructs were determined using a NanoDropTM One spectrophotometer. Thereafter, all these buffer-exchanged proteins were labeled with the cysteine-specific donor cyanine3 - maleimide (Cy3) at a 1:5 protein -to-dye molar ratio and incub ated for 3.5 h at 22 °C under constant agitation. Thereafter, the samples were placed at 4 °C and then incubated overnight under constant agitation. During the incubation, the samples were wrapped in aluminum foil to protect the fluorophores from light exposure, ensuring labeling occurred in a dark environment. On the next day, the unreacted Cy3 was removed by passing the protein/dyes mixtures 2 times through the NAP 5 column (Cytiva). The Cy3-labled Vpu variants were in a final buffer of 20 mM Tris pH 7.4, 150 mM NaCl, 1 mM CaCl2, 50 µM TCEP, and 5% glycerol, which was used in all FRET experiments. Ca2+-CaM was labeled using the same protocol but with the cysteine -specific cyanine5-maleimide (Cy5) acceptor instead of Cy3 donor, using the previously described protocol37. .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.15.718738doi: bioRxiv preprint 18 The details of the procedure for handling the Vpu C -terminal peptide were described in detail in Ishola et al. 2026 37. 4. Quantification of Cy3/Cy5 labeling efficiency The degree of labeling (labeling efficiency) is the molar ratio of bound dye concentration to protein concentration after removal of unreacted dye 55. The dye and protein concentrations were quantified using the protein and labels function of the NanoDrop TM One spectrophotometer. The parameters for Cy3 - maleimide and Cy5 -maleimide were entered into the system, and the calculations were performed as described by Chedda et al. (2023) [2]. For Cy3 -maleimide, dye concentration was determined from A55 0 using an extinction coefficient of 150,000 M⁻¹ cm⁻¹. For Cy5-maleimide, dye concentration was determined from A646 using an extinction coefficient of 250,000 M⁻¹ cm⁻¹. Protein concentration was calculated from the absorbance at 280 nm after correcting for dye absorbance at 280 nm using the dye -specific correction factors (CF280 = 0.09 for Cy3 and 0.04 for Cy5). Thus, protein and dye concentrations were calculated according to: [Protein] = 𝐴280 − (𝐶𝐹280 × 𝐴dye) 𝜀280,protein [Dye] = 𝐴dye 𝜀dye Labeling efficiency was then expressed as the molar dye-to-protein ratio: 𝐷/𝑃 = [Dye] [Protein] For the Cy3 -labeled Vpu C -terminal peptide, the protein extinction coefficient at 280 nm was 𝜀280,protein = 8604M⁻¹ cm⁻¹; therefore, the working equations were: [Cy3] = 𝐴555 150,000 [Protein] = 𝐴280 − (0.09 × 𝐴555) 8,604 𝐷/𝑃 = [Cy3] [Protein] .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.15.718738doi: bioRxiv preprint 19 Accordingly, the labeling efficiencies reported in Table 1 represent mol dye per mol protein rather than an arbitrary instrument-generated ratio. Vpu construct Labeling efficiency Cy3 SUMO-FLVpu 0.74 FL Vpu 0.5 SUMO-Vpu C-terminal region 0.2 Vpu C-terminal region 0.2 FL Vpu-M (FL Vpu M1) 0.39 Cy5 Calmodulin 0.55 5. Ensemble FRET experiments and data analysis The following samples were prepared for eFRET experiments: (a) Samples containing increasing concentration (100, 200, 400, 600, and 850 nM) of tag-free Cy3-labeled FL Vpu and Vpu C-terminal region mixed with constant 500 nM Cy5 -labeled CaM. The FRET data were collected using an FS5 spectrofluorometer (Edinburgh Instrument) with the donor excitation at 550 nm (1 nm excitation and 2 nm emission bandwidth), and emission recorded from 555 to 800 nm (b) Samples containing a mixture of 100 nM SUMO -FL Vpu, FL Vpu, SUMO-Vpu C-terminal fragment, Vpu C-terminal fragment, FL Vpu-M were titrated with increasing concentration of Ca2+-CaM at 100 nM, 200 nM, 350 nM, 450 nM, and 600 nM. The FRET data were collected using an FS5 spectrofluorometer (Edinburgh Instrument) with the donor excitation at 550 nm (3 nm excitation and emission bandwidth), and emission recorded from 555 to 800 nm All the samples were prepared in triplicate and in a cuvette ( Brand), and the incubation was done at room temperature in a dark room for 10 minutes to allow protein interaction. Two methods were used to determine the Kd for the Vpu variant-Ca2+-CaM complexes formation. The first

(Quantitative FRET Quenching) involves using a non -linear regression model to fit data sets of ∆Emm at the donor maximum wavelength and the total concentrations of CaM as described by Jiang et al. 56 and the second method (KD-FRET) involves calculating the FRET efficiency using the donor quenching equation according to Shresta et al.57 and then fitting the FRET efficiency data sets against the concentration .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.15.718738doi: bioRxiv preprint 20 of CaM in a non-linear regression model using a quadratic binding equation as described by Yi et al., 2025 58. The R 2 fitting goodness factor for all cases was equal or greater tha n 0.9. All the graph plotting and fitting to determine the Kd values were performed using Origin Pro 2023 (Origin Lab, Northampton, MA). AUTHOR CONTRIBUTION AO—experimental design, experiment, data analysis and interpretation, figures, writing the experimental procedures; EH— experimental design, experiment, data analysis and interpretation, figures, writing the experimental procedures; MMI —experiment; NR —experiment; ASD — experiment; ERG —conception, experimental design, data analysis and interpr etation, figures, writing the manuscript, supervision, finds acquisition.

This work was supported by the Gilead Research Scholars in HIV Award to ERG. We thank Juan Camilo Rueda Amador for help with initial experiments. ERG thanks Dr. Peter Borbat for fruitful discussions that led to the conception of this work. .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.15.718738doi: bioRxiv preprint 21 1. Khan, N; Geiger, JD. Role of Viral Protein U (Vpu) in HIV-1 Infection and Pathogenesis. Viruses. (2021), 13 (8). DOI: 10.3390/v13081466 2. Attwood, MM; Schioth, HB. Characterization of Five Transmembrane Proteins: With Focus on the Tweety, Sideroflexin, and YIP1 Domain Families. Front Cell Dev Biol. (2021), 9, 708754. DOI: 10.3389/fcell.2021.708754 3. Altfeld, M; Gale, M, Jr. Innate immunity against HIV-1 infection. Nat Immunol. (2015), 16 (6), 554-562. DOI: 10.1038/ni.3157 4. Hladik, F; McElrath, MJ. Setting the stage: host invasion by HIV. Nat Rev Immunol. (2008), 8 (6), 447-457. DOI: 10.1038/nri2302 5. Ramdas, P; Sahu, AK; Mishra, T; Bhardwaj, V; Chande, A. From Entry to Egress: Strategic Exploitation of the Cellular Processes by HIV-1. Front Microbiol. (2020), 11, 559792. DOI: 10.3389/fmicb.2020.559792 6. Dube, M; Bego, MG; Paquay, C; Cohen, EA. Modulation of HIV-1-host interaction: role of the Vpu accessory protein. Retrovirology. (2010), 7, 114. DOI: 10.1186/1742-4690-7-114 7. Langer, S; Hammer, C; Hopfensperger, K; Klein, L; Hotter, D; De Jesus, PD; Herbert, KM; Pache, L; Smith, N; van der Merwe, JA; et al. HIV-1 Vpu is a potent transcriptional suppressor of NF-kappaB-elicited antiviral immune responses. Elife. (2019), 8. DOI: 10.7554/eLife.41930 8. Strebel, K; Klimkait, T; Martin, MA. A novel gene of HIV-1, vpu, and its 16-kilodalton product. Science. (1988), 241 (4870), 1221-1223. DOI: 10.1126/science.3261888 9. Gonzalez, ME. Vpu Protein: The Viroporin Encoded by HIV-1. Viruses. (2015), 7 (8), 4352- 4368. DOI: 10.3390/v7082824 10. Hussain, A; Das, SR; Tanwar, C; Jameel, S. Oligomerization of the human immunodeficiency virus type 1 (HIV-1) Vpu protein--a genetic, biochemical and biophysical analysis. Virol J. (2007), 4, 81. DOI: 10.1186/1743-422X-4-81 11. Cohen, EA; Terwilliger, EF; Sodroski, JG; Haseltine, WA. Identification of a protein encoded by the vpu gene of HIV-1. Nature. (1988), 334 (6182), 532-534. 12. Strebel, K; Klimkait, T; Martin, MA. A novel gene of HIV-1, vpu, and its 16-kilodalton product. Science. (1988), 241 (4870), 1221-1223. 13. Cong, L; Sugden, SM; Leclair, P; Lim, CJ; Pham, TN; Cohen, ÉA. HIV-1 Vpu promotes phagocytosis of infected CD4+ T cells by macrophages through downregulation of CD47. Mbio. (2021), 12 (4), e01920-01921. 14. Wildum, S; Schindler, M; Münch, J; Kirchhoff, F. Contribution of Vpu, Env, and Nef to CD4 down-modulation and resistance of human immunodeficiency virus type 1-infected T cells to superinfection. Journal of virology. (2006), 80 (16), 8047-8059. .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.15.718738doi: bioRxiv preprint 22 15. Neil, SJ; Zang, T; Bieniasz, PD. Tetherin inhibits retrovirus release and is antagonized by HIV-1 Vpu. Nature. (2008), 451 (7177), 425-430. 16. Van Damme, N; Goff, D; Katsura, C; Jorgenson, RL; Mitchell, R; Johnson, MC; Stephens, EB; Guatelli, J. The interferon-induced protein BST-2 restricts HIV-1 release and is downregulated from the cell surface by the viral Vpu protein. Cell host & microbe. (2008), 3 (4), 245-252. 17. Willey, R; Maldarelli, F; Martin, MA; Strebel, K. Human immunodeficiency virus type 1 Vpu protein induces rapid degradation of CD4. Journal of virology. (1992), 66 (12), 7193-7200. 18. González, ME. Vpu protein: The viroporin encoded by HIV-1. Viruses. (2015), 7 (8), 4352- 4368. 19. McNatt, MW; Zang, T; Bieniasz, PD. Vpu binds directly to tetherin and displaces it from nascent virions. PLoS Pathog. (2013), 9 (4), e1003299. DOI: 10.1371/journal.ppat.1003299 20. Radding, W; Pan, ZQ; Hunter, E; Johnston, P; Williams, JP; McDonald, JM. Expression of HIV-1 envelope glycoprotein alters cellular calmodulin. Biochem Biophys Res Commun. (1996), 218 (1), 192-197. DOI: 10.1006/bbrc.1996.0034 21. Alaimo, A; Alberdi, A; Gomis-Perez, C; Fernandez-Orth, J; Gomez-Posada, JC; Areso, P; Villarroel, A. Cooperativity between calmodulin-binding sites in Kv7.2 channels. J Cell Sci. (2013), 126 (Pt 1), 244-253. DOI: 10.1242/jcs.114082 22. Hayashi, N; Matsubara, M; Jinbo, Y; Titani, K; Izumi, Y; Matsushima, N. Nef of HIV-1 interacts directly with calcium-bound calmodulin. Protein Sci. (2002), 11 (3), 529-537. DOI: 10.1110/ps.23702 23. Kurokawa, H; Osawa, M; Kurihara, H; Katayama, N; Tokumitsu, H; Swindells, MB; Kainosho, M; Ikura, M. Target-induced conformational adaptation of calmodulin revealed by the crystal structure of a complex with nematode Ca(2+)/calmodulin-dependent kinase kinase peptide. J Mol Biol. (2001), 312 (1), 59-68. DOI: 10.1006/jmbi.2001.4822 24. O'Neil, KT; DeGrado, WF. How calmodulin binds its targets: sequence independent recognition of amphiphilic alpha-helices. Trends Biochem Sci. (1990), 15 (2), 59-64. DOI: 10.1016/0968-0004(90)90177-d 25. McQueen, P; Donald, LJ; Vo, TN; Nguyen, DH; Griffiths, H; Shojania, S; Standing, KG; O'Neil, JD. Tat peptide-calmodulin binding studies and bioinformatics of HIV-1 protein- calmodulin interactions. Proteins. (2011), 79 (7), 2233-2246. DOI: 10.1002/prot.23048 26. Miller, MA; Mietzner, TA; Cloyd, MW; Robey, WG; Montelaro, RC. Identification of a calmodulin-binding and inhibitory peptide domain in the HIV-1 transmembrane glycoprotein. AIDS Res Hum Retroviruses. (1993), 9 (11), 1057-1066. DOI: 10.1089/aid.1993.9.1057 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.15.718738doi: bioRxiv preprint 23 27. Sham, SW; McDonald, JM; Micoli, KJ; Krishna, NR. Solution structure of a calmodulin- binding domain in the carboxy-terminal region of HIV type 1 gp160. AIDS Res Hum Retroviruses. (2008), 24 (4), 607-616. DOI: 10.1089/aid.2007.0202 28. Radding, W; Williams, JP; McKenna, MA; Tummala, R; Hunter, E; Tytler, EM; McDonald, JM. Calmodulin and HIV type 1: interactions with Gag and Gag products. AIDS Res Hum Retroviruses. (2000), 16 (15), 1519-1525. DOI: 10.1089/088922200750006047 29. Ghanam, RH; Fernandez, TF; Fledderman, EL; Saad, JS. Binding of calmodulin to the HIV-1 matrix protein triggers myristate exposure. J Biol Chem. (2010), 285 (53), 41911-41920. DOI: 10.1074/jbc.M110.179093 30. Vlach, J; Samal, AB; Saad, JS. Solution structure of calmodulin bound to the binding domain of the HIV-1 matrix protein. J Biol Chem. (2014), 289 (12), 8697-8705. DOI: 10.1074/jbc.M113.543694 31. Dick, A; Cocklin, S. Subtype Differences in the Interaction of HIV-1 Matrix with Calmodulin: Implications for Biological Functions. Biomolecules. (2021), 11 (9). DOI: 10.3390/biom11091294 32. Pan, G; Zhou, T; Radding, W; Saag, MS; Mountz, JD; McDonald, JM. Calmodulin antagonists inhibit apoptosis of CD4+ T-cells from patients with AIDS. Immunopharmacology. (1998), 40 (2), 91-103. DOI: 10.1016/s0162-3109(98)00018-6 33. Sharpe, S; Yau, WM; Tycko, R. Structure and dynamics of the HIV-1 Vpu transmembrane domain revealed by solid-state NMR with magic-angle spinning. Biochemistry. (2006), 45 (3), 918-933. DOI: 10.1021/bi051766k 34. Zhang, H; Lin, EC; Das, BB; Tian, Y; Opella, SJ. Structural determination of virus protein U from HIV-1 by NMR in membrane environments. Biochim Biophys Acta. (2015), 1848 (11 Pt A), 3007-3018. DOI: 10.1016/j.bbamem.2015.09.008 35. Majeed, S; Adetuyi, O; Borbat, PP; Majharul Islam, M; Ishola, O; Zhao, B; Georgieva, ER. Insights into the oligomeric structure of the HIV-1 Vpu protein. J Struct Biol. (2023), 215 (1), 107943. DOI: 10.1016/j.jsb.2023.107943 36. Majeed, S; Dang, L; Islam, MM; Ishola, O; Borbat, PP; Ludtke, SJ; Georgieva, ER. HIV-1 Vpu protein forms stable oligomers in aqueous solution via its transmembrane domain self- association. Sci Rep. (2023), 13 (1), 14691. DOI: 10.1038/s41598-023-41873-0 37. Ishola, O; Islam, MM; Hadadianpour, E; Borbat, PP; Ogunbowale, A; Amador, JCR; Georgieva, ER. The soluble state of the HIV-1 Vpu protein forms a complex with Ca2+- calmodulin. Protein Science. (2026), 35, e70487. DOI: doi: https://doi.org/10.1002/pro.70487Digital 38. O'Day, DH; Huber, RJ. Calmodulin binding proteins and neuroinflammation in multiple neurodegenerative diseases. BMC Neurosci. (2022), 23 (1), 10. DOI: 10.1186/s12868-022- 00695-y .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.15.718738doi: bioRxiv preprint 24 39. Osawa, M; Tokumitsu, H; Swindells, MB; Kurihara, H; Orita, M; Shibanuma, T; Furuya, T; Ikura, M. A novel target recognition revealed by calmodulin in complex with Ca2+-calmodulin- dependent kinase kinase. Nat Struct Biol. (1999), 6 (9), 819-824. DOI: 10.1038/12271 40. Rhoads, AR; Friedberg, F. Sequence motifs for calmodulin recognition. FASEB J. (1997), 11 (5), 331-340. DOI: 10.1096/fasebj.11.5.9141499 41. Shrestha, D; Jenei, A; Nagy, P; Vereb, G; Szollosi, J. Understanding FRET as a research tool for cellular studies. Int J Mol Sci. (2015), 16 (4), 6718-6756. DOI: 10.3390/ijms16046718 42. Jiang, L; Xiong, Z; Song, Y; Lu, Y; Chen, Y; Schultz, JS; Li, J; Liao, J. Protein-Protein Affinity Determination by Quantitative FRET Quenching. Sci Rep. (2019), 9 (1), 2050. DOI: 10.1038/s41598-018-35535-9 43. Yi, S; Kim, E; Yang, S; Kim, G; Bae, DW; Son, SY; Jeong, BG; Ji, JS; Lee, HH; Hahn, JS; et al. Direct Quantification of Protein-Protein Interactions in Living Bacterial Cells. Adv Sci (Weinh). (2025), 12 (19), e2414777. DOI: 10.1002/advs.202414777 44. Day, ES; Cote, SM; Whitty, A. Binding efficiency of protein-protein complexes. Biochemistry. (2012), 51 (45), 9124-9136. DOI: 10.1021/bi301039t 45. Samal, AB; Ghanam, RH; Fernandez, TF; Monroe, EB; Saad, JS. NMR, biophysical, and biochemical studies reveal the minimal Calmodulin binding domain of the HIV-1 matrix protein. J Biol Chem. (2011), 286 (38), 33533-33543. DOI: 10.1074/jbc.M111.273623 46. Osad'ko, IS. Dependence of FRET efficiency on distance in single donor-acceptor pairs. J Chem Phys. (2015), 142 (12), 125102. DOI: 10.1063/1.4915279 47. Han, Z; Harty, RN. Influence of calcium/calmodulin on budding of Ebola VLPs: implications for the involvement of the Ras/Raf/MEK/ERK pathway. Virus Genes. (2007), 35 (3), 511-520. DOI: 10.1007/s11262-007-0125-9 48. Arold, S; Hoh, F; Domergue, S; Birck, C; Delsuc, MA; Jullien, M; Dumas, C. Characterization and molecular basis of the oligomeric structure of HIV-1 nef protein. Protein Sci. (2000), 9 (6), 1137-1148. DOI: 10.1110/ps.9.6.1137 49. Banerjee, P; Monje-Galvan, V; Voth, GA. Cooperative Membrane Binding of HIV-1 Matrix Proteins. J Phys Chem B. (2024), 128 (11), 2595-2606. DOI: 10.1021/acs.jpcb.3c06222 50. Monette, A; Niu, M; Nijhoff Asser, M; Gorelick, RJ; Mouland, AJ. Scaffolding viral protein NC nucleates phase separation of the HIV-1 biomolecular condensate. Cell Rep. (2022), 40 (8), 111251. DOI: 10.1016/j.celrep.2022.111251 51. Choi, S; Lee, JM; Kim, KK. Biomolecular condensates: molecular structure, biological functions, diseases, and therapeutic targets. Mol Biomed. (2025), 6 (1), 99. DOI: 10.1186/s43556-025-00350-y .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.15.718738doi: bioRxiv preprint 25 52. Banani, SF; Lee, HO; Hyman, AA; Rosen, MK. Biomolecular condensates: organizers of cellular biochemistry. Nat Rev Mol Cell Biol. (2017), 18 (5), 285-298. DOI: 10.1038/nrm.2017.7 53. Corbi-Verge, C; Kim, PM. Motif mediated protein-protein interactions as drug targets. Cell Commun Signal. (2016), 14, 8. DOI: 10.1186/s12964-016-0131-4 54. Caporale, A; Adorinni, S; Lamba, D; Saviano, M. Peptide-Protein Interactions: From Drug Design to Supramolecular Biomaterials. Molecules. (2021), 26 (5). DOI: 10.3390/molecules26051219 55. Chadda, R; Lee, T; Mahoney-Kruszka, R; Kelley, EG; Bernhardt, N; Sandal, P; Robertson, JL. A thermodynamic analysis of CLC transporter dimerization in lipid bilayers. Proceedings of the National Academy of Sciences. (2023), 120 (41), e2305100120. 56. Jiang, L; Xiong, Z; Song, Y; Lu, Y; Chen, Y; Schultz, JS; Li, J; Liao, J. Protein–protein affinity determination by quantitative FRET quenching. Scientific reports. (2019), 9 (1), 2050. 57. Shrestha, D; Jenei, A; Nagy, P; Vereb, G; Szöllősi, J. Understanding FRET as a research tool for cellular studies. International journal of molecular sciences. (2015), 16 (4), 6718-6756. 58. Yi, S; Kim, E; Yang, S; Kim, G; Bae, DW; Son, SY; Jeong, BG; Ji, JS; Lee, HH; Hahn, JS. Direct quantification of protein–protein interactions in living bacterial cells. Advanced Science. (2025), 12 (19), 2414777. .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted April 16, 2026. ; https://doi.org/10.64898/2026.04.15.718738doi: bioRxiv preprint