FRET Analysis of the Promiscuous yet Specific Interactions of the HIV-1 Vpu Transmembrane Domain  Gregory B. Cole, Sean E. Reichheld, Simon Sharpe  Biophysical Journal  Volume 113, Issue 9, Pages 1992-2003 (November 2017) DOI: 10.1016/j.bpj.2017.09.010 Copyright © 2017 Biophysical Society Terms and Conditions

Figure 1 Secondary structure of TMD peptides reconstituted into POPC liposomes. CD spectra were recorded for each peptide at a peptide-to-lipid ratio of 0.04 and are reported as MRE. All spectra were recorded at 25°C in 50 mM sodium phosphate buffer (pH 7.5). The peptide concentration was 55 μM in each case. To see this figure in color, go online. Biophysical Journal 2017 113, 1992-2003DOI: (10.1016/j.bpj.2017.09.010) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 2 Homooligomerization of Vpu and tetherin TM domain peptides. Shown here is the donor quenching of dansyl-labeled VpuTM (A), or TethTM (B) in the presence of increasing mole fractions of dabsyl-labeled acceptor peptide. The peptide-to-lipid ratio was maintained at 0.01 in each sample by addition of unlabeled peptide; the total concentration of POPC was 1 mM. All experiments were carried out in 50 mM sodium phosphate buffer (pH 7.5) and liposomes were incubated overnight before measurements. Data is fit to the equation FRET Eff = K[1−(1−X)n−1], where K is a constant and X is the acceptor mole ratio. The oligomer size, n, was equal to 2 for TethTM. An n of 3 was used for VpuTM, as this value resulted in the best fit of lipid titration experiments (Fig. S4), suggesting that this represents the average or most prevalent oligomeric species. Competition experiments with unlabeled peptide are shown for VpuTM (C) and TethTM (D). Each competition experiment consists of three samples: donor peptide only, 1:1 donor/acceptor, and 1:1:2 donor/acceptor unlabeled. Confidence intervals are reported as ns (p > 0.05), (∗p ≤ 0.05), (∗∗p ≤ 0.01), or (∗∗∗∗p ≤ 0.0001). Biophysical Journal 2017 113, 1992-2003DOI: (10.1016/j.bpj.2017.09.010) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 3 Heterooligomerization of VpuTM with its targets. Donor quenching of dansyl-labeled VpuTM is reported in the presence of increasing mole fractions of dabsyl-labeled target peptides (acceptor). The total peptide concentration was maintained at 10 μM by addition of unlabeled target peptide. Experiments were carried out in 50 mM sodium phosphate buffer (pH 7.5) and liposomes were incubated overnight before measurements. As in Fig. 2, experimental data was fit to FRET Eff = K[1−(1−X)n−1], where n is equal to 2 for NTB-A and PVR, and 3 TethTM. To see this figure in color, go online. Biophysical Journal 2017 113, 1992-2003DOI: (10.1016/j.bpj.2017.09.010) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 4 Competition experiments with unlabeled target peptides confirm the interactions between Vpu and target TMD peptides. Relative fluorescence yields are shown for samples containing donor-labeled Vpu with acceptor-labeled and/or -unlabeled (A) TethTM, (B) NTB-A TM, and (C) PVR TM. Negative controls using VpuTM RD are shown for (D) TethTM, (E) NTB-A TM, and (F) PVR TM. Competition experiments were completed using the same donor/acceptor-unlabeled ratios described in Fig. 2. Confidence intervals are reported as ns (p > 0.05), (∗p ≤ 0.05), (∗∗p ≤ 0.01), or (∗∗∗∗p ≤ 0.0001). Biophysical Journal 2017 113, 1992-2003DOI: (10.1016/j.bpj.2017.09.010) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 5 Target TM domain peptides compete for interaction with VpuTM. Unlabeled competition experiments were carried out using donor-labeled VpuTM in the presence of acceptor-labeled TethTM and equal molar amounts of each unlabeled target peptide. Competition experiments were completed under identical conditions to the VpuTM/TethTM homooligomer experiments (Fig. 4). Confidence intervals are reported as ns (p > 0.05) or ∗(p ≤ 0.05). Biophysical Journal 2017 113, 1992-2003DOI: (10.1016/j.bpj.2017.09.010) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 6 Thermodynamic analysis of Vpu TMD homo- and heterooligomeric assemblies. (A) Shown here is dilution of VpuTM (solid line) or TethTM (dashed line) in POPC liposomes, showing loss of FRET efficiency due to dissociation of the homooligomers. The TethTM data were fit to a monomer-dimer equilibrium using Eqs. 11, 12, and 13. VpuTM was modeled as a trimer using Eqs. 14 and 15. (B) Shown here is dilution of the VpuTM heterooligomers in POPC liposomes. Fit lines were generated from the Kd values calculated from Eqs. 16, 17, and 18 B. To see this figure in color, go online. Biophysical Journal 2017 113, 1992-2003DOI: (10.1016/j.bpj.2017.09.010) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 7 Thermodynamic and kinetic analysis of the VpuTM domain interactions with target TM domain peptides. Free energies (kcal mol−1) and dissociation constants are shown for homooligomeric and heterooligomeric interactions of Vpu and target transmembrane domain peptides. Parameters were calculated from the data shown in Fig. 6, A and B. Values in parentheses are for the contribution of individual monomers to the Vpu homotrimer, or the Vpu-tetherin heterotrimer. Biophysical Journal 2017 113, 1992-2003DOI: (10.1016/j.bpj.2017.09.010) Copyright © 2017 Biophysical Society Terms and Conditions