Phage-assisted evolution of botulinum neurotoxin proteases with reprogrammed specificity

Abstract

Although bespoke, sequence-specific proteases have the potential to advance biotechnology and medicine, generation of proteases with tailor-made cleavage specificities remains a major challenge. We developed a phage-assisted protease evolution system with simultaneous positive and negative selection and applied it to three botulinum neurotoxin (BoNT) light-chain proteases. We evolved BoNT/X protease into separate variants that preferentially cleave vesicle-associated membrane protein 4 (VAMP4) and Ykt6, evolved BoNT/F protease to selectively cleave the non-native substrate VAMP7, and evolved BoNT/E protease to cleave phosphatase and tensin homolog (PTEN) but not any natural BoNT protease substrate in neurons. The evolved proteases display large changes in specificity (218- to >11,000,000-fold) and can retain their ability to form holotoxins that self-deliver into primary neurons. These findings establish a versatile platform for reprogramming proteases to selectively cleave new targets of therapeutic interest.

Figure 1.

(A) Schematic of the PACE/PANCE dual positive and negative selection for proteolysis. (B) Summary of the strategy for stepwise evolution of a Ykt6-selective and a VAMP4-selective protease from promiscuous wild-type BoNT/X LC protease. (C) Four major substrates of wild-type BoNT/X protease, with the cleavage position indicated by the blue wedge. Positions in VAMP4, VAMP5, and Ykt6 identical to VAMP1 are in black, while positions in VAMP5 and Ykt6 identical to VAMP4 are in grey. (D) Selected Ykt6-selective and VAMP4-selective BoNT/X protease variants. (E-G) Representative kinetic traces showing cleavage activity of wild-type BoNT/X protease, X(4130)B1 protease, and X(5101)B1 protease on the seven major BoNT/X substrates. Reactions in (E-G) were performed at 37 °C with 2.5 nM protease and 5 μM substrate.

Figure 2.

(A) Top: Superposition of the 1.8-Å resolution x-ray crystal structure of evolved X(4130)B1 (light blue) with the wild-type BoNT/X LC (grey, PDB 6F4E). Mutated residues in X(4130)B1 are shown in purple, with the active site zinc shown as a black sphere. Bottom: Superposition of a BoNT/F LC-bound VAMP2-mimic (orange, from PDB 3FIE) and the crystal structure of X(4130)B1 (light blue), showing the close proximity of the P3 Arg (orange) to E166 and I167 (purple). The active site zinc is shown as a black sphere. (B) Evaluation of X(4130)B1 and X(5010)B1 activity on full-length substrates in human HEK293T cell lysates by immunoblot. Actin served as a loading control. (C) Evolved protease delivery and activity as chimeric BoNT holotoxins (53) added to cultured rat cortical neurons. Neurons were treated with the indicated concentration of holotoxin for 16 h. Cleavage on endogenous VAMP2, VAMP4, and Ykt6 was detected by immunoblot on neuron lysates.

Figure 3.

(A) Top: Summary of the strategy for stepwise evolution of a VAMP7-cleaving protease from BoNT/F LC protease, followed by dual positive and negative selection for evolving VAMP7 selectivity from promiscuous variants. Bottom: substrates used during the evolution of a VAMP7-selective protease from a VAMP1-cleaving protease. The orange wedge shows the wild-type BoNT/F cleavage site. The predicted (hollow) and actual (filled) purple wedges show VAMP7 cleavage sites of the evolved F(3230)A3 protease. (B) Selected variants from evolution of VAMP7-selective BoNT/F proteases. Mutations in grey enriched during negative selection. (C) Apparent protease activity by luciferase assay for selected clones from various stages of BoNT/F evolution. (D) Comparison of wild-type BoNT/F protease (left) and evolved F(3230)A3 protease (right) activity on VAMP1 and VAMP7. Assays were performed using proteases containing an N-terminal MBP tag under standard assay conditions at 37 °C with 5 μM substrate and either 2.5 nM BoNT/F or 50 nM F(3230)A3.

Figure 4.

(A) Strategy for stepwise evolution of a PTEN-cleaving protease from wild-type SNAP-25-cleaving BoNT/E LC protease. (B) Protease cleavage substrates used during the evolution of a PTEN-selective protease. (C) Selected variants from the evolution of a PTEN-selective BoNT/E protease. Mutations in green enriched during the positive selection, and mutations in grey enriched during negative selection. (D) Apparent protease activity by luciferase assay of clones from BoNT/E evolution. dBoNT/F is an inactive BoNT/F protease mutant. (E) Comparison of wild-type BoNT/E LC (left) and E(4130)A2 protease (right) activity on SNAP25 and PTEN. Assays were performed under standard conditions at 37 °C using 5 μM substrate and 10 nM protease. (F) Evaluation of full-length PTEN cleavage in cells. FLAG-tagged full-length PTEN was co-expressed with either HA-tagged E(4130)A2 or HA-tagged PH-E(4130)A2 in HEK293 cells. PH-E(4130)A2 contains an N-terminal pleckstrin homology (PH) domain fused to E(4130)A2. Cell lysates were analyzed by immunoblot, detecting PTEN (via FLAG-tag) and E(4130)A2 (via HA tag). Actin served as a loading control. Numbers indicate the ratio of BoNT/LC plasmid:PTEN substrate plasmid. LC, light-chain proteases. (G) PTEN and SNARE protein cleavage in cultured rat cortical neurons transduced with lentivirus expressing RFP (negative control), PH-E(4130)A2 protease, or the PH-E(4130)A2(L166A) protease. Neuron lysates were analyzed by immunoblot, detecting endogenous PTEN, SNAP-25, VAMP2, and Syntaxin 1. Actin served as a loading control. The asterisk marks the minor cleavage of SNAP-25 by PH-E(4130)A2 detected with long exposure time (Long expo.), which is not observed for PH-E(4130)A2(L166A).