Fig. 3: Validation of MP3-seq with Bcl-2 proteins.
From: Massively parallel measurement of protein–protein interactions by sequencing using MP3-seq
a, Colored crystal structure of Bcl-2 and its designed BH3-binding inhibitor49 (Protein Data Bank 5JSN). The binder is blue and Bcl-2 is colored according to the domains. b, BH3-binding domain annotations of the six human Bcl-2 homologs measured21,49, with the experimental truncation used by Alpha-seq and that in this work shown. c, MP3-seq P-LFCs for inhibitor and Bcl-2 interactions calculated from two biological replicates. Yellow boxes highlight intended on-target interactions. d, Correlations with Kd measurements from biolayer interferometry. P-LFCs are presented as the P-LFC ± SE and Kd values are presented as the mean ± s.d. Only PPIs within biolayer interferometry detection limits were used (n = 43). e, MP3-seq P-LFC distributions for interactions that were detected (n = 43) and undetected (n = 11) by biolayer interferometry because of instrument detection limits (Kd ≥ 25 mM). f, Pairwise Alpha-seq distributions. g, Batched Alpha-seq distributions. For all box plots in panels e–g, the whiskers are at ±1.5 times the interquartile range, while the boxes have border lines showing the first quartile, median and third quartile, with all data points for both sets shown in a swarm overlay. One-tailed independent t-tests (H1: µdetected > µundetected) were used to compare the detected and undetected measurements. ****P < 0.0001 and *P < 0.05; NS, not significant (P ≥ 0.05). Both MP3-seq P-LFC and pairwise Alpha-seq were significant for µdetected > µundetected (P = 1.858 × 10−5 and P = 0.0307, respectively), while batched Alpha-seq was not significant (P = 0.0904). h, Correlation of PUMA peptide and Mcl-1 (t) PPI (n = 13) P-LFCs from two biological replicates with PUMA peptide and full Mcl-1 average Kd measurements50. P-LFCs are presented as the P-LFC ± SE and Kd values are presented as the mean ± s.d.
