Steric complementarity directs sequence promiscuous leader binding in RiPP biosynthesis

Abstract

Enzymes that generate ribosomally synthesized and posttranslationally modified peptide (RiPP) natural products have garnered significant interest, given their ability to produce large libraries of chemically diverse scaffolds. Such RiPP biosynthetic enzymes are predicted to bind their corresponding peptide substrates through sequence-specific recognition of the leader sequence, which is removed after the installation of posttranslational modifications on the core sequence. The conservation of the leader sequence within a given RiPP class, in otherwise disparate precursor peptides, further supports the notion that strict sequence specificity is necessary for leader peptide engagement. Here, we demonstrate that leader binding by a biosynthetic enzyme in the lasso peptide class of RiPPs is directed by a minimal number of hydrophobic interactions. Biochemical and structural data illustrate how a single leader-binding domain can engage sequence-divergent leader peptides using a conserved motif that facilitates hydrophobic packing. The presence of this simple motif in noncognate peptides results in low micromolar affinity binding by binding domains from several different lasso biosynthetic systems. We also demonstrate that these observations likely extend to other RiPP biosynthetic classes. The portability of the binding motif opens avenues for the engineering of semisynthetic hybrid RiPP products.

Keywords: RiPPs; biochemistry; biosynthesis.

Fig. 1.

(A) The therbactin gene cluster of T. terrenum. (B) Proposed biosynthetic route for therbactin α and therbactin β. (C−F) MALDI-TOF-MS analysis of the TbiB1- and TbiB2-mediated protease reaction observing formation of (C) TbiAα leader: 2730.3 m/z; (D) TbiAα core: 3970.9 m/z; (E) TbiAβ leader: 2602.4 m/z; and (F) TbiAβ core: 4459.1 m/z.

Fig. 2.

(A) Sequence alignment of TbiAα and TbiAβ leader peptides. The (B) TbiAα (blue) and (C) TbiAβ T(-5)E (purple) leader peptides interact through steric complementarity through insertion of hydrophobic residues into corresponding pockets found in TbiB1. Simulated annealing omit map (contoured at 2σ above background) calculated with Fourier coefficients (F_obs − F_calc) with phases from the final models minus the coordinates of the leader peptides omitted prior to calculations.

Fig. 3.

(A) Weblogo analysis (45) of 400 aligned leader sequences demonstrates the conserved motif. (B) Sequences of the McjA peptide and the engineered McjAYxxP peptides. The 3 mutated residues in McjAYxxP are indicated in red. (C and D) Competitive binding assays for the (C) truncated McjA leader peptide and (D) truncated McjAYxxP leader peptide indicate that the orthogonal McjA leader peptide cannot bind, and insertion of the conserved binding motif is sufficient to binding to TbiB1. (E and F) MALDI-TOF-MS analysis of protease action observing (E) McjA and McjAYxxP leader peptide and (F) McjA and McjAYxxP core peptide. (G and H) MALDI-TOF-MS analysis of TbiB1- and TbiB2-dependent processing of (G) McjAYxxP leader peptide and (H) McjAYxxP core peptide.

Fig. 4.

SSN of the entire PqqD protein family. SSN was constructed at a 95% representative node (Rep Node) cutoff, using the InterPro 74.0 database. Cyan nodes are colocalized near short ORFs that contain a Y/W-(X)₂-P-X-L/V/I/A/F/Y motif. Red and purple nodes are predicted to be part of lasso peptide and PQQ biosynthesis, respectively. These nodes also contain the YxxPxL motif. Remaining nodes are colored black.

Fig. 5.

(A−D) Surface models of (A) TbiB1 (copper) and TbiAα leader (blue), (B) NisB RRE (purple) and NisA leader (orange), (C) LynD RRE (green) and PatE’ (silver), and (D) CteB RRE (sky blue) and CteA (pink) indicate a conserved binding mode. (E−H) Cartoon depiction of the knob-into-hole binding of leader consensus motifs into the RREs of (E) TbiB1, (F) NisB, (G) LynD, and (H) CteB.