Detection of Peptides, Proteins, and Drugs That Selectively Interact With Protein Targets

Targeting of interacting proteins or peptides to small sequence motifs. (A) Differential interaction of Cdc42 effectors as detected by the Dual Bait procedure. Two Cdc42-interacting proteins, FBP17 (Fuchs et al. 2001) and SPEC1 (Pirone et al. 2000) were isolated by a yeast two-hybrid screen with LexA-Cdc42-L28. In this screen, 10⁷diploids arising from an interaction mating (Finley and Brent 1994)-based screen yielded 25 positive colonies showing interactions with LexA-Cdc42 L28. Counterscreening against cI fused to activated Cdc42 lacking the insert region (Cdc42 L28-Δ8) revealed two clones, both containing the same cDNA, showing differential binding. All yeast shown contain both baits, and AD-fusions as indicated at left. Beside AD-fused FBP17 (specific for Cdc42-L28) and SPEC1 (bound both forms equivalently), AD-Pak1, which binds both forms of Cdc42, and AD-Pak1 LL, which cannot bind either form of Cdc42 (Sells et al. 1997), are included as controls. Results with growth and colorimetric reporters for Cdc42-L28-Δ8 [gusA (X-Gluc) and LYS2, first and third lines] and LexA-Cdc42-L28 [lacZ (X-Gal) andLEU2, second and fourth lines], are shown. (B) Selection of peptides specifically targeting HEF1-DLVD. A LexA-HEF1(DLVD) bait was used to identify interacting peptide aptamers, in collaboration with R. Finley. Selected peptides were rescreened in parallel against LexA-HEF1(DVLD) or cI-HEF1(DLVA), differing from the original bait by only a D363A substitution. Shown are peptides that interact with both DLVD and DLVA variants (DLVD = DLVA; activates all four reporters; representative sequence HHASTPRRESPGIMSPL); or with DLVD but not DLVA (DLVD>>DLVA; activates lacZ andLEU2 only; representative sequence, SGKFGEALPGWLSSACWCFG), and a nonspecific control peptide (negative; activates no reporters; representative sequence, EQLKYNRFWPWQWWGGRRLR). (C) Confirmation that peptides interact with HEF1 in an in vitrosystem, using pulldowns of endogenous HEF1 from cell lysates with the three GST-fused peptides from B, or with GST only. (Top) Levels of GST peptide or GST only in reaction; (bottom) associated HEF1.