Ligand Type Name 6 Crystallization-grade After 447-52D After V3 cocktail Receptor CD4 Resonance Units 5 1 5 1 5 1 Broadly neutralizing antibodies 2G12 VRC26.9 Resonance Units Resonance Units 3 1 15 1 5 VRC1 Resonance Units 3 1 5 1 5 1 5 1 Supplementary Data Set 1 Characterization of purified BG55 SOSIP.664 after negative selection with V3 antibodies. BG55 SOSIP.664 antigenicity before (left panel) and after (middle and right panel) negative selection with V3 antibodies by SPR. CD4- Ig or antibodies were captured on an anti-fc surface, and a nm solution of BG55 SOSIP.664 was used to measure binding. Nature Structural & Molecular Biology: doi:1.138/nsmb.351
a ECL units Antigenicity of purified BG55 SOSIP.664 and mutants by MSD-ECLIA 1. X 1 6 7.5 X 1 5 5. X 1 5 2.5 X 1 5 1. X 1 6 7.5 X 1 5 5. X 1 5 2.5 X 1 5 1. X 1 6 7.5 X 1 5 5. X 1 5 2.5 X 1 5 BG55 SOSIP.664 1-1 1 1 1 1-1 Y191W 1 1 1 1-1 Q432P A433P 1C 433C 1 1 1 [Trimer], µg/ml 1-1 1 1 1 1-1 1 1 1 $ b VRC1 b12 F15 1.5e 17b 17b+sCD4 PGT121 PGT128 2G12 PGT145 VRC26.9 447-52D 447-52D+sCD4 PGT151 3522 8ANC195 Temporal Stability of SOSIP, I1C A433C and A433P Retained quaternary structure integrity (fraction) 1..8.6 SOSIP 1C 433C A433P c Antigenicity of purified BG55 SOSIP.664 and I1C A433C in absence and presence of CD4 by MSD-ECLIA d Antigenicity of purified BG55 SOSIP.664 and I1C A433C by ELISA ECL units 1. X 1 6 7.5 X 1 5 5. X 1 5 2.5 X 1 5 1. X 1 6 7.5 X 1 5 5. X 1 5 2.5 X 1 5 BG55 SOSIP.664 1C 433C -scd4 +scd4 -scd4 +scd4 OD (45 nm) 3 2 1 3 2 1 BG55 SOSIP.664 1C-433C $ VRC1 b12 F15 1.5e 17b 17b+sCD4 PGT121 PGT128 2G12 PGT145 VRC26.9 447-52D e 1. X 1 6 7.5 X 1 5 5. X 1 5 2.5 X 1 5 1-1 1 1 1 1-1 1 1 1 1-1 1 1 1 1-1 1 1 1 [Trimer], µg/ml Affinity and kinetics of interaction of BG55 SOSIP.664 and 1C 433C by Surface Plasmon Resonance 3 2 1.1.1.1.1 1 1.1.1.1 1 1 [Antibodies], µg/ml PGT145 PGT151 VRC1 35O22 PGT128 VRC26 8ANC195 447-52D+sCD4 BG55 SOSIP.664 1C 433C f Affinity and kinetics of interaction of BG55 SOSIP.664 and 1C 433C by Biolayer interferometry VRC26 PGT145 VRC1 35O22 PGT151 b12 F15 17b 447-52D BG55 SOSIP.664 1C 433C Supplementary Data Set 2 Antigenicity of purified BG55 SOSIP.664 and selected variants. (a) Antigenicity of BG55 SOSIP.664 and mutants by MSD-ECLIA. Plots show binding of neutralizing (green), non- or weakly neutralizing antibodies (magenta) and antibodies in presence of CD4 (yellow) to BG55 SOSIP.664 and mutants. (b) Temporal stability of BG55 SOSIP.664, A433P and I1C A433C. (c) Comparison of antigenicity of BG55 SOSIP.664 and 1C 433C in absence and presence of soluble, 2-domain CD4 by MSD-ECLIA. Antibodies are color coded as in (a). (d) Antigenicity of BG55 SOSIP.664 and 1C 433C assessed by ELISA. Antibodies are color coded as in (a). (e) Binding of BG55 SOSIP.664 and 1C 433C measured by Surface Plasmon Resonance (SPR) and (f) by Biolayer Interferometry (BLI). Supplementary Table 5 lists kinetics and affinity parameters obtained from SPR and BLI measurements. Supplementary Table 6 lists pairwise comparison statistics between the four methods (MSD-ECLIA, ELISA, SPR and BLI) described above. Nature Structural & Molecular Biology: doi:1.138/nsmb.351
a 8 6 Conformational triggering of co-receptor site: 17b detection (-/+ CD4) BG55 SOSIP.664 BG55 SOSIP.664 Q432P BG55 SOSIP.664 A433P BG55 SOSIP.664 I1C A433C +scd4 -scd4 1 1 1 1 b Enhanced CD4-induction of P313W mutant:17b detection (+scd4) BG55 SOSIP.664 1 3 1 5 Conformational triggering of HR2-site: C34 detection (-/+ CD4) BG55 SOSIP.664 BG55 SOSIP.664 Q432P BG55 SOSIP.664 A433P BG55 SOSIP.664 I1C A433C +scd4 +scd4 -scd4 -scd4 1 1 1 1 BG55 SOSIP.664 P313W 1 3 c Affinity and Kinetics of 17b binding to activated trimer BG55 SOSIP.664 BG55.SOSIP.664 Q432P BG55.SOSIP.664 A433P BG55.SOSIP.664 I1C A433C Response Units 5 nm to 31.25 nm 5 nm to 31.25 nm d Time course of CD4 activation of BG55 SOSIP.664 374 detection 17b detection P313W BG55 SOSIP.664 I1C A433C P313W BG55 SOSIP.664 I1C A433C Time (8 min -1 hr) Supplementary Data Set 3 CD4-induced activation of HIV-1 Env. (a) SPR analysis of (top panel) co-receptor site exposure and (bottom panel) HR2-site exposure upon CD4 activation. (b) CD4-induced binding of SOSIP and P313W mutant to 17b. Trimer samples at concentrations from nm to 2.5 nm in 2-fold dilutions were combined with nm scd4 and injected on a RU 17b IgG surface. (c) SPR single-cycle kinetics analysis of 17b Fab binding to soluble trimers activated with scd4. Under conditions of constant 5 nm scd4 flow, 17b Fab was injected at incrementally in 2-fold dilutions from 25 nm to 1.5 nm on trimer captured on a 2G12 chip. A433P and I1C A433C were further subjected to 17b Fab concentrations ranging from 5 nm to 31.25 nm (insets). (d) Time-dependent increase in exposure of V3 epitope (374 detection) and bridging sheet (17b detection) in SOSIP trimers on incubation with scd4. Nature Structural & Molecular Biology: doi:1.138/nsmb.351
a b c Molecule BG55 SOSIP.664 N322 BG55 SOSIP.664 1C 433C CD4 d1d2 CD4 d1d4 Mass (19,994 ± 225 Da) x 3 = 329,982 ± 675 Da (19,6 ± 884 Da) x 3 = 327,18 ± 2652 Da,329 ± 131 Da 44,629 ± 167 Da sum of residuals (1-12 ) Fitted stoichiometry CD4:SOSIP d CD4 D1-D2 e CD4 D1-D4 Fit A1 BG55SOSIP.664: Fit A2 BG55SOSIP.664: 1 SOSIP with1 bound CD4 Fit A3 BG55SOSIP.664: 1 SOSIP with 2 bound CD4 Fit A4 BG55SOSIP.664: 1 SOSIP with 3 bound CD4 Fit A1 BG55SOSIP.664: Fit A2 BG55SOSIP.664: 1 bound SOSIP Fit A3 BG55SOSIP.664: 2 bound SOSIP Fit A4 BG55SOSIP.664: 3 bound SOSIP Fit B1 SOSIP 1C 433C : Fit B2 SOSIP 1C 433C : 1 SOSIP 1C 433C Fit B3 SOSIP 1C 433C : 1 SOSIP 1C 433C with 2 bound CD4 Fit B4 SOSIP 1C 433C : 1 SOSIP 1C 433C with 3 bound CD4 Fit B1 SOSIP 1C 433C : Fit B2 SOSIP 1C 433C : 1 SOSIP 1C 433C Fit B3 SOSIP 1C 433C : 1 SOSIP 1C 433C with 2 bound CD4 Fit B4 SOSIP 1C 433C : 1 SOSIP 1C 433C with 3 bound CD4 Supplementary Data Set 4 Analysis of stoichiometry of CD4-binding to BG55 SOSIP.664 or the DS variant, BG55 SOSIP.664 1C 433C. (a) Masses determined for each molecule by MALDI-TOF mass spectrometry. For the BG55 SOSIP molecules, monomer masses were determined, but trimeric masses were used in the analytical ultracentrifugation fitting. (b) Dependence of fit quality as a function of stoichiometry is shown for ultracentrifugation runs performed with four CD4 D1D2 molecules for each trimer. The residual errors are plotted for each. For wild-type trimer, as expected the best fit is at stoichiometry of three CD4 D1D2 molecules per trimer. For the DS mutant, BG55 SOSIP, the residual errors are lowest at 1:1 stoichiometry and fail to fit higher numbers of bound CD4 molecules, indicating 1 CD4 molecule bound per DS trimer. (c) Same as (b), but using CD4 D1- D4, which has a larger mass. Identical results were obtained with best fit at three CD4 D1D4 per trimer for wild-type, and one CD4 D1D4 for the DS mutant. Fitting of the raw sedimentation equilibrium data and residual errors of the fits are shown in panels (d) and (e) for CD4 D1D2 and CD4 D1D4, respectively. Calculations shown were made using the program HeteroAnalysis (http://www.biotech.uconn.edu/auf). Similar trends were observed in fits with the program SEDPHAT (Vistica et al.). Nature Structural & Molecular Biology: doi:1.138/nsmb.351
Relative Light Units 2 1 6 1 1 6 5 1 5 Time BG55 (s) BG55 Q432P BG55 A433P BG55 I1C A433C 2 1 6 2 1 6 2 1 6 1 1 6 5 1 5 1 1 6 5 1 5 1 1 1 1 1 1 1 1 1 1 1 1 Dilution Dilution Dilution Dilution 1 1 6 5 1 5 Supplementary Data Set 5 Virus entry. Ability of HIV-1 BG55 and mutants to enter CD4+CCR5+ cells. TZM-bl cells were infected with pseudoviruses bearing the indicated Env construct. The cells expressed luciferase upon infection; luciferase activity measured in Relative Light Units. Nature Structural & Molecular Biology: doi:1.138/nsmb.351
Supplementary Data Set 6 Uptake plots for all observable peptides for ligand-free BG55 SOSIP.664 (WT) (blue), WT+sCD4 (red), ligand-free DS-SOSIP (1C 433C) (cyan), and DS-SOSIP+sCD4 (purple). Error bars show the standard deviation from duplicate measurements. Nature Structural & Molecular Biology: doi:1.138/nsmb.351