Structural Basis of Leader Peptide Recognition in Lasso Peptide Biosynthesis Pathway

Abstract

Lasso peptides are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) with a unique 3Dinterlocked structure, in which an N-terminal macrolactam ring is threaded by a linear C-terminal part. The unique structure of lasso peptides is introduced into ribosomally translated precursor peptides by lasso peptide synthetase encompassing proteins B and C or B1, B2, and C when the B enzyme is split into two distinct proteins. The B1 protein recognizes the leader sequence of the precursor peptide, and then the B2 protein cleaves it. The C protein catalyzes the formation of the macrolactam ring. However, the detailed mechanism of lasso peptide maturation has remained elusive, due to the lack of structural information about the responsible proteins. Here we report the crystal structure of the B1 protein from the thermophilic actinobacteria, Thermobifida f usca (TfuB1), complexed with the leader peptide (TfuA-Leader), which revealed the detailed mechanism of leader peptide recognition. The structure of TfuB1 consists of an N-terminal β-sheet and three C-terminal helices. The leader peptide is docked on one edge of the N-terminal β-sheet of TfuB1, as an additional β strand. Three conserved amino acid residues of the leader peptide fit well on the hydrophobic cleft between the β-sheet and adjacent helices. Biochemical analysis demonstrated that these conserved residues are essential for affinity between TfuB1 and the TfuA-Leader. Furthermore, we found that TfuB1 and the leader peptide jointly form a hydrophobic patch on the β-sheet, which includes the highly conserved TfuA Phe-6 and TfuB1 Tyr33. Homology modeling and mutational analysis of the B1 protein from a firmicute, Bacillus pseudomycoides (PsmB1), revealed that the hydrophobic patch is conserved in a wide range of species and involved in the cleavage activity of the B2 protein, indicating it forms the interaction surface for the B2 protein or the core part of the precursor peptide.

Factors Governing the Thermal Stability of Lasso Peptides

“…Lasso peptides are biosynthesized in three steps. First, the precursor peptide (A) is recognized and bound by a so‐called RiPP‐recognition element (RRE) protein . RREs are defined by a topology comprising three N‐terminal β‐strands and three C‐terminal α‐helices and can be found in many different RiPP families .…”

“… A selection of representative RRE domain and protein structures from various RiPP systems. A) the CteB RRE domain with bound leader peptide (PDB ID: 5WHY), B) the LynD RRE domain with bound leader peptide (PDB ID: 4V1T), C) the NisB RRE domain with bound leader peptide (PDB ID: 4WD9), D) the PqqD RRE protein (PDB ID: 5SXY), and E) the lasso peptide RRE protein TfuB1 with bound leader peptide (PDB ID: 6JX3) . TfuB1 stems from Thermobifida fusca and is involved in the biosynthesis of the lasso peptide fusilassin/fuscanodin …”

Efficient in vivo synthesis of lasso peptide pseudomycoidin proceeds in the absence of both the leader and the leader peptidase

“…As can be seen from Fig. S10, † digestion with carboxypeptidase led to accumulation of products ending at His, 23 Ala, 20 Gly, 19 Gln, 18 and Gln. 10 Peaks corresponding to each of these intermediates were split, with a characteristic 18 Da difference indicating that they were produced from both intact and the Y-shaped pseudomycoidin.…”

“…Although both PsmB1 and PsmB2 are functional in vivo, 18 the unexpected result suggested that in the absence of these two proteins their role is carried out by some host proteins. To conrm further functionality of PsmB1 and PsmB2, an in vitro experiment was performed using puried recombinant proteins and synthetic PsmA precursor peptides.…”

Identification and Heterologous Expression of the Biosynthetic Gene Cluster Encoding the Lasso Peptide Humidimycin, a Caspofungin Activity Potentiator

“…Then, the B2 protein cleaves the leader peptide. The structural basis of leader peptide recognition by these so-called "split-B" proteins have been characterized in depth in Actinobacteria and Firmicutes [20].…”