Figure S3.

Spike residue D484 and TMEM106B residues M210 and F213 enhance spike-TMEM106B binding, related to Figure 3

(A) Differences in hydrogen-deuterium exchange (ΔHDX) between the spike S1 subunit of the Belgium/GHB-03021 isolate alone and when in the presence of excess TMEM106B. Negative values indicate protection and positive values deprotection from exchange in the presence of TMEM106B. The threshold of significance calculated with 98% CI at ±0.42 Da is indicated with a dashed gray line. Peptides are arranged from the N to C terminus according to their peptide center residue. See Table S3 and Data S1 for HDX data tables and deuterium uptake plots.

(B) Biolayer interferometry results of S1 binding to immobilized TMEM106B. Data are represented as the dependence of the observed rate constant on S1 concentration for S1^E484 (Wuhan-Hu-1) and S1^D484 (Belgium/GHB-03021).

(C) Thermodynamic parameters for S1^E484 and S1^D484 subunit binding to immobilized TMEM106B. For each variant, k_on was determined from the slope of the plot of k_obs against (S1) for the association phase, k_off was obtained from the intercept of the plot of k_obs against (S1) for the association phase, K_D kinetic was calculated as k_off/k_on, and K_D equilibrium was calculated from the plot of the amplitude versus (S1).

(D) Biolayer interferometry traces of 6 μM S1^D484 (blue) and 6 μM S1^D484 premixed with 8 μM ACE2 (green) binding to immobilized TMEM106B (luminal domain).

(E) NCI-H1975 monoclonal TMEM106B knockout cells transduced with cDNA encoding wild-type (WT) TMEM106B or TMEM106B containing single amino acid changes, infected with SARS-CoV-2 Belgium/GHB-03021/2020. Cells were stained for dsRNA after 24 h. Infected cells were quantified by high-content imaging analysis (n = 8 wells examined over two independent experiments). Data were log-transformed and analyzed using one-way ANOVA with Dunnett’s multiple comparison test, comparing each condition with WT TMEM106B.

(F) Biolayer interferometry traces of 13.5 μM S1^D484 binding to immobilized wild-type TMEM106B (blue) or mutant TMEM106B^M210A/F213A (red).

(G) NCI-H1975 monoclonal TMEM106B knockout cells transduced with cDNA encoding WT or mutant TMEM106B and stained with anti-TMEM106B (Ab09) and DAPI. Representative images are shown, scale bars, 10 μm.

(H) WT or TMEM106B^KO NCI-H1975 cells transduced with cDNA encoding human, mouse (mus musculus), hamster (Mesocricetus auratus), or African green monkey (Chlorocebus sabaeus) TMEM106B and infected with SARS-CoV-2 Belgium/GHB-03021/2020. Cell viability was determined by MTS assay after 3 days (n = 6 wells examined over two independent experiments). Data were analyzed using one-way ANOVA with Dunnett’s multiple comparison test, comparing each condition with TMEM106B^KO cells.

(E and G) Data are mean ± SEM. ^∗∗∗∗p < 0.0001; ^∗∗0.001 < p < 0.01; ^∗0.01 < p < 0.05; ns, not significant.