Abstract
Many ribosomally synthesized and posttranslationally modified peptide classes (RiPPs) are reliant on a domain called the RiPP recognition element (RRE). The RRE binds specifically to a precursor peptide and directs the posttranslational modification enzymes to their substrates. Given its prevalence across various types of RiPP biosynthetic gene clusters (BGCs), the RRE could theoretically be used as a bioinformatic handle to identify novel classes of RiPPs. In addition, due to the high affinity and specificity of most RRE-precursor peptide complexes, a thorough understanding of the RRE domain could be exploited for biotechnological applications. However, sequence divergence of RREs across RiPP classes has precluded automated identification based solely on sequence similarity. Here, we introduce RRE-Finder, a new tool for identifying RRE domains with high sensitivity. RRE-Finder can be used in precision mode to confidently identify RREs in a class-specific manner or in exploratory mode to assist in the discovery of novel RiPP classes. RRE-Finder operating in precision mode on the UniProtKB protein database retrieved ∼25,000 high-confidence RREs spanning all characterized RRE-dependent RiPP classes, as well as several yet-uncharacterized RiPP classes that require future experimental confirmation. Finally, RRE-Finder was used in precision mode to explore a possible evolutionary origin of the RRE domain. The results suggest RREs originated from a co-opted DNA-binding transcriptional regulator domain. Altogether, RRE-Finder provides a powerful new method to probe RiPP biosynthetic diversity and delivers a rich data set of RRE sequences that will provide a foundation for deeper biochemical studies into this intriguing and versatile protein domain.IMPORTANCE Bioinformatics-powered discovery of novel ribosomal natural products (RiPPs) has historically been hindered by the lack of a common genetic feature across RiPP classes. Herein, we introduce RRE-Finder, a method for identifying RRE domains, which are present in a majority of prokaryotic RiPP biosynthetic gene clusters (BGCs). RRE-Finder identifies RRE domains 3,000 times faster than current methods, which rely on time-consuming secondary structure prediction. Depending on user goals, RRE-Finder can operate in precision mode to accurately identify RREs present in known RiPP classes or in exploratory mode to assist with novel RiPP discovery. Employing RRE-Finder on the UniProtKB database revealed several high-confidence RREs in novel RiPP-like clusters, suggesting that many new RiPP classes remain to be discovered.
Highlights
Many ribosomally synthesized and posttranslationally modified peptide classes (RiPPs) are reliant on a domain called the RiPP recognition element (RRE)
The precision-mode profile hidden Markov models (pHMMs) are primarily based on known RiPP classes—in most cases, representative RRE-containing proteins from these classes have been verified to bind their cognate precursor peptide through biophysical experiments, such as X-ray crystallography or fluorescence polarization binding assays
Depending on the end user’s objective, RRE-Finder can be used in precision mode to accurately predict the presence of an RRE domain as well as the likely RiPP class in which the precursor peptide belongs
Summary
Many ribosomally synthesized and posttranslationally modified peptide classes (RiPPs) are reliant on a domain called the RiPP recognition element (RRE). Given its prevalence across various types of RiPP biosynthetic gene clusters (BGCs), the RRE could theoretically be used as a bioinformatic handle to identify novel classes of RiPPs. In addition, due to the high affinity and specificity of most RRE-precursor peptide complexes, a thorough understanding of the RRE domain could be exploited for biotechnological applications. We introduce RRE-Finder, a method for identifying RRE domains, which are present in a majority of prokaryotic RiPP biosynthetic gene clusters (BGCs). With a few notable exceptions, the precursor peptide is genetically encoded adjacent to one or more genes encoding proteins that bind with high specificity and affinity to the leader region of the precursor This interaction facilitates subsequent posttranslational modification of the core residues [3]. Considering that the RRE domain appears to be the most conserved class-independent feature in RiPP BGCs, it theoretically could be used as an imperfect but useful bioinformatic handle to expand known RiPP sequence-function space by identifying new RRE-dependent RiPP classes
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