Characterization of Specificity Determinants in the Hydrolysis of Lasso Peptide Astexin-2 by its Isopeptidase

Abstract

Lasso peptides are a class of small, bioactive, ribosomally derived natural products whose unique shape confers upon them exceptional physical and chemical stability. Previous inquiries into a lasso peptide gene cluster found in Asticcacaulis excentricus have reported on the heterologous expression of two lasso peptides (astexin-2 and astexin-3) as well as a divergently transcribed gene (atxE2 ) on chromosome 2 annotated as a protease that cleaves their isopeptide bonds. Astexin-1, a product of a separate gene cluster on chromosome 1 of A. excentricus has also previously been characterized. It has been found that AtxE2 hydrolyzes full-length astexin-2 and -3 but not astexin-1 peptides and that this isopeptidase must recognize a knotted structure in order to function. This work attempts to characterize the specificity of AtxE2 to astexin-2 through mutagenesis approaches. As hypothesized by the fact that AtxE2 does not hydrolyze unthreaded astexin-2, this work intends to confirm that recognition elements do not include specific residues of the peptide but instead are related to its topology. First, we conducted alanine-scan mutagenesis of the macrolactam ring (aside from residue Ala7) and the loop to determine whether these residues were important for maturation and more specifically whether changing these residues would prevent hydrolysis from occurring. Just as previously unpublished work has shown that the astexin-2 tail is not integral to hydrolysis by AtxE2, obtained results revealed that Gln14 was critical for maturation and that all variants produced were capable of being hydrolyzed by AtxE2. Secondly, due to the observed decrease at lower enzyme concentrations in AtxE2 activity from mutating Leu8, we further explored the seventh and eighth residues near the isopeptide bond. It was established that while not preventing hydrolysis, mutations in these residues severely decrease the rate of hydrolysis of the produced variants. Additionally, it was noted that though AtxE2 is non-specific towards particular astexin-2 residues, it maintains specificity to its cluster, indicating no discernible effect on other Class II lassos or lassos with highly similar sequences, like astexin-1. Finally, because reaction trajections showed preferential cutting of astexin-2 truncated variants and NMR structures revealed spatial differences in the tail region of astexin-1 and astexin-2 and -3, we tested AtxE2 on truncated variants of astexin-1. While the tail region affects the rate of enzymatic activity, it is by no means the only part of the peptide involved in specific recognition by AtxE2. These results provide insight into specificity determinants that are important for understanding the interactions between astexin-2 and AtxE2 before physical structures can be solved.