Pandonodin: A Proteobacterial Lasso Peptide with an Exceptionally Long C-Terminal Tail

Abstract

Lasso peptides are a family of ribosomally synthesized and post-translationally modified peptides (RiPPs) defined by their threaded-ring topology. The N-terminus of the peptide forms an isopeptide bond with an aspartate or glutamate side chain to create a 7−9 amino acid (aa) macrocyclic ring through which the rest of the peptide is threaded. The result is a highly constrained three-dimensional structure. Even though they share a threaded-ring feature, characterized lasso peptides vary greatly in sequence and size, ranging from 14 to 26 aa. Using genome mining, we identified a new lasso peptide gene cluster with a predicted lasso peptide that is 33 aa long. Here we report the heterologous expression of this new peptide, pandonodin, its NMR structure, and its unusual biophysical properties. Pandonodin has a long, proteolytically resistant 18residue tail of low sequence complexity, which limits its water solubility. Within this tail is a 6 aa disulfide-bonded macrocycle that serves as a steric lock to maintain the lasso structure. This disulfide bond is unusually stable, requiring both heat and high concentrations of reductants for cleavage. Finally, we also show that segments of the C-terminal tail of pandonodin can be replaced with arbitrary sequences, allowing for the construction of pandonodin−protein fusions.

Cellulonodin-2 and Lihuanodin: Lasso Peptides with an Aspartimide Post-translational Modification

“…1,2 Structurally, lasso peptides are [1]rotaxanes, in which an isopeptide bond joins the N-terminus of the core peptide to the side chain of a D or E residue, forming a 7-9 amino acids (aa) macrocyclic ring. [3][4][5] The C-terminal tail portion of the peptide, ranging from 6 to 25 aa, 6,7 is threaded through the ring, resulting in a right-handed, lassoed three-dimensional conformation. The size of the lasso peptide loop, which extends from the isopeptide bond and threads through the ring, varies in size from as few as 4 aa up to 18 aa.…”

“…6,8 Likewise, the C-terminal tail of the peptide can vary from as few as 2 aa up to 18 aa. 7,9 Lasso peptide biosynthesis follows the general biosynthetic steps of other RiPPs. [10][11][12][13][14][15][16] The precursor peptide, encoded by the A gene, is first processed by a cysteine peptidase, encoded by the B gene, which hydrolyzes the peptide bond between the leader and the core peptide.…”

Lasso Peptides: Heterologous Production and Potential Medical Application

“…Firstly, the gene cluster panABCD was under an inducible tet promoter. Secondly, the panA gene was engineered under an inducible T5 promoter, while the intact putative operon panBCD was engineered under the control of a constitutive promoter ( Cheung-Lee et al, 2019a ). Thirdly, the incorporation of an E. coli optimized RBS also significantly increased the yields of other lasso peptides, like caulosegnins I–III ( Hegemann et al, 2013a ), astexin-1 ( Zimmermann et al, 2013 ), astexin-2, astexin-3 ( Maksimov and Link, 2013 ).…”

“…Pandonodin that was identified from Pandoraea norimbergensis has the longest (18 residues) proteolytically resistant tail (Cheung-Lee et al, 2019a). Lasso peptides, burhizin-23, mycetohabin-16, and mycetohabin-15 were firstly isolated from endosymbiotic bacteria (Bratovanov et al, 2020).…”

Class IV Lasso Peptides Synergistically Induce Proliferation of Cancer Cells and Sensitize Them to Doxorubicin

“…In class III, the disulfide bridge links the tail to the macrolactam ring, whereas in class IV the disulfide bridge is located at the tail itself. So far only two class IV peptides have been characterized, LP2006 from the actinomycete bacterium Nocardiopsis alba (PDB accession number 5JPL ; Tietz et al., 2017 ), and pandonodin from Pandoraea norimbergensis (PDB accession number 6Q1X ; Cheung-Lee et al., 2019 ).…”