Mapping protein selectivity landscapes using multi-target selective screening and next-generation sequencing of combinatorial libraries

Abstract

Characterizing the binding selectivity landscape of interacting proteins is crucial both for elucidating the underlying mechanisms of their interaction and for developing selective inhibitors. However, current mapping methods are laborious and cannot provide a sufficiently comprehensive description of the landscape. Here, we introduce a novel and efficient strategy for comprehensively mapping the binding landscape of proteins using a combination of experimental multi-target selective library screening and in silico next-generation sequencing analysis. We map the binding landscape of a non-selective trypsin inhibitor, the amyloid protein precursor inhibitor (APPI), to each of the four human serine proteases (kallikrein-6, mesotrypsin, and anionic and cationic trypsins). We then use this map to dissect and improve the affinity and selectivity of APPI variants toward each of the four proteases. Our strategy can be used as a platform for the development of a new generation of target-selective probes and therapeutic agents based on selective protein-protein interactions.

Fig. 1

Yeast-surface display of APPI-3M. a Schematic drawing of the pairwise selective screen using the YSD system. A naive library of mutated APPI-3M variants was displayed on the yeast cell surface and presented to pairs of proteases. Each protease in the pair (denoted A or B) was labeled with a different fluorescent dye—Alexa Fluor-650 or Alexa Fluor-488 (represented by green and blue stars, respectively). b Pairwise selective screen. Flow cytometry sorting was used to screen the library to isolate APPI-3M variants, with enhanced selectivity toward each of the four serine proteases (Meso: mesotrypsin; KLK6; Anionic: anionic trypsin; Cationic: cationic trypsin). In each sort, two variant populations were collected inside the black gates, yielding sorted library populations of protease-selective APPI-3M variants. Green and blue colors represent a high and low cell densities, respectively

Fig. 2

Single mutation selectivity landscape of APPI-3M. The colors in the heatmaps indicate the enrichment ratio (defined as the frequency of a certain mutation in the sorted library divided by its frequency in the naive library) and represent the effect of a single-amino acid substitution on APPI-3M selectivity toward one serine protease (a: mesotrypsin, b: KLK6, c: cationic trypsin, and d: anionic trypsin) versus the other three. The different colors of the heatmaps correspond to the scale shown on the right of each panel and indicate log2 of the enrichment ratio [yellow and red: positive (increased selectivity); green: negative (decreased selectivity)]. The position of the substituted amino acid is shown on the X axis and the substituting amino acid is shown on the Y axis. Meso: mesotrypsin; Cationic: cationic trypsin; Anionic: anionic trypsin. See Supplementary Figure 3 for further details

Fig. 3

Double mutation selectivity landscape of APPI-3M. Heatmaps demonstrating the effect of double-amino acid mutations in APPI-3M on the selectivity toward KLK6 versus the three other serine proteases. a The effect of different pairs of mutated residues on selectivity is illustrated by the colors of the heatmaps (red = increased selectivity, enrichment ratio >1; blue = decreased selectivity, enrichment ratio <1). The contribution of each double mutation to selectivity was summed and the maps demonstrate the overall effect. The X and Y axes indicate the position of the substituted amino acid residues. See Supplementary Figure 4 for further details. b The effect of different amino acid mutations at residues 11 and 17 of APPI-3M on its selectivity toward KLK6, illustrated by the colors in the heatmaps. The X axis indicates amino acids mutated at residue 17 and the Y axis indicates amino acids mutated at residue 11. c Crystal structure of APPI-3M (PDB ID: 5C67). Cartoon representation of APPI-3M illustrating the positions of correlated residues Thr-11 and Gly-17 (red) within the APPI binding loop (green, positions 11–18). The APPI scaffold is shown in gray