A lanthipeptide library used to identify a protein-protein interaction inhibitor

Abstract

In this article we describe the production and screening of a genetically encoded library of 10⁶ lanthipeptides in Escherichia coli using the substrate-tolerant lanthipeptide synthetase ProcM. This plasmid-encoded library was combined with a bacterial reverse two-hybrid system for the interaction of the HIV p6 protein with the UEV domain of the human TSG101 protein, which is a critical protein-protein interaction for HIV budding from infected cells. Using this approach, we identified an inhibitor of this interaction from the lanthipeptide library, whose activity was verified in vitro and in cell-based virus-like particle-budding assays. Given the variety of lanthipeptide backbone scaffolds that may be produced with ProcM, this method may be used for the generation of genetically encoded libraries of natural product-like lanthipeptides containing substantial structural diversity. Such libraries may be combined with any cell-based assay to identify lanthipeptides with new biological activities.

Figure 1. Representative illustration of lanthipeptide biosynthesis

(a) Two Ser residues in the ribosomally synthesized linear precursor peptide ProcA2.8 are dehydrated by ProcM to generate two dehydroalanine (Dha) residues. The cyclization domain of ProcM then catalyzes the regioselective addition of two thiols of Cys residues to the Dha residues to generate modified ProcA2.8 (mProcA2.8). (b) Chemical structures showing the products of the dehydration and cyclization processes. (c) Generic structure demonstrating the randomization of the residues within the two rings of mProcA2.8 (X = D, F, H, I, L, N, V, or Y). The sequence of the leader peptide is also depicted. In all panels, structures derived from Ser are in red and from Cys in blue.

Figure 2. Identifying a lanthipeptide inhibitor of the p6–UEV PPI

(a) Crystal structure of the PTAP peptide (PEPTAPPEE; pink) bound to UEV (purple); PDB ID = 3OBU. (b) Top, IPTG-induced expression of the 434-UEV and P22-p6 fusion proteins results in a functional repressor that inhibits expression of the reporter genes; HIS3 and KanR are required for survival on selective media. Bottom, a lanthipeptide inhibitor of the p6–UEV interaction relieves the repression of the reporter genes and confers a growth advantage on selective media. (c) Drop spotting 10-fold serial dilutions of the p6–UEV RTHS transformed with the plasmid encoding XY3-3 in the absence of IPTG and arabinose (top row); expression of neither the repressors nor XY3-3 is induced, and full growth is observed. Upon adding IPTG (30 μM; middle row), a functional repressor prevents expression of His3/KanR, resulting in inhibited cell growth. Upon addition of IPTG (30 μM) and arabinose (65 μM; bottom row), the repressor and XY3-3 are produced; XY3-3 disrupts the p6–UEV interaction, preventing formation of a functional repressor and providing a growth advantage. All plates were incubated at 37 °C for 58 h. (d) Structure of XY3-3; residues in set positions are in blue, while randomized positions are in pink.

Figure 3. Assessing the activity of XY3-3 in vitro

(a–c) ELISA data showing that XY3-3 disrupts the p6–UEV interaction with an IC₅₀ of 3.6 ± 0.3 μM (a), peptide 2 disrupts the p6–UEV interaction with an IC₅₀ of 2.7 ± 0.6 μM (b), and peptide 3 disrupts the p6–UEV interaction with an IC₅₀ of 1.9 ± 0.8 μM (c). (d) UEV selectively pulls down a fluorescent-labeled XY3-3 whereas p6 does not, indicating that XY3-3 binds to UEV. Lines indicate the mean, n=5. (e,f) MST analysis reveals that XY3-3 binds to UEV with a K_d of 16.0 ± 1.1 μM (e) and peptide 3 binds to UEV with a K_d of 4.0 ± 3.3 μM (f). (g–i) FP analysis reveals that a FITC-tagged derivative of peptide 3 binds to UEV with a K_d of 5.5 ± 0.5 μM (g), a fluorescein-tagged PTAP nonapeptide binds to UEV with a K_d of 16.6 ± 0.2 μM (h), and a competition FP assay demonstrates that full length p6 displaces peptide 3 from UEV with an IC₅₀ of 23.9 μM (i). Errors on K_d and IC₅₀ values are the standard error of the mean (S.E.M.) given by regression analysis from three independent experiments.

Figure 4. Cellular activity of XY3-3-Tat

(a) Cytotoxicity assay of XY3-3-Tat in HEK293T cells shows that the compound is toxic to cells as doses above 1 μM. Lines indicate the mean, n=3. (b) Virus-like particle budding assay in HEK293T cells shows that a 100 nM dose of XY3-3-Tat inhibits virus-like particle production by ∼65%. For uncropped blots see Supplementary Figure 18. (c) EGFR downregulation assay in HeLa cells shows XY3-3-Tat inhibits degradation of EGFR, in line with its mode of action of binding to UEV. For uncropped blots see Supplementary Figure 19.