Lasso peptides are bioactive compounds produced by bacteria. Their distinctive lasso-like structure gives them exceptional stability, making them resilient in harsh environments. A recent research study, featured in Nature Chemical Biology, has developed and tested theoretical models to understand the biosynthesis of these peptides and explored how this knowledge could lead to the development of lasso peptide-based medications.
Lasso peptides are bioactive compounds produced by bacteria. Their distinctive lasso-like structure gives them exceptional stability, making them resilient in harsh environments. A recent research study, featured in Nature Chemical Biology, has developed and tested theoretical models to understand the biosynthesis of these peptides and explored how this knowledge could lead to the development of lasso peptide-based medications.
“Lasso peptides are fascinating as they are linear molecules that have been intricately tied into a slip knot shape,” explained Susanna Barrett, a graduate student in the Mitchell lab (MMG). “Due to their impressive stability and the potential for modification, they are promising candidates as therapeutic agents. They’ve been shown to possess antibacterial, antiviral, and anti-cancer effects.”
Lasso peptides are synthesized by ribosomes and undergo post-translational modifications. The peptide sequence is formed by linking amino acids together, facilitated by the ribosome. After this, two enzymes—a peptidase and a cyclase—work together to transform the linear peptide into the unique knotted lasso form. Since their discovery over thirty years ago, researchers have been investigating the mechanism by which the cyclase folds these lasso peptides.
“One significant obstacle in this research has been the difficulty in working with these enzymes, as they tend to be insoluble or inactive during purification,” shared Barrett.
An exception is fusilassin cyclase, or FusC, which the Mitchell lab studied in 2019. Members of the team were able to purify the enzyme, and since then, it has been used as a model to examine the process of lasso peptide folding. However, the structure of FusC was still unknown, hindering a complete understanding of how the cyclase interacts with the peptide to create the knot.
In this new study, the research team employed the artificial intelligence tool AlphaFold to predict the structure of the FusC protein. They utilized this structure along with other AI-based resources, such as RODEO, to identify key residues in the cyclase’s active site that are crucial for interacting with the lasso peptide substrate.
“FusC consists of roughly 600 amino acids, with 120 located in the active site. These AI programs played a crucial role in our project as they enabled us to conduct ‘structural studies’ to refine which amino acids are significant in the enzyme’s active site,” Barrett noted.
Additionally, they conducted molecular dynamics simulations to analyze how the cyclase folds the lasso peptide. “With the extensive simulation data we obtained through Folding@home, we were able to visualize these interactions at an atomic level,” stated Song Yin, a graduate student from the Shukla lab. “Prior to this study, there had been no molecular dynamics simulations examining the interactions between lasso peptides and cyclases, and we anticipate that this method will be applicable to many other studies involving peptide engineering.”
Their computational analyses revealed that certain regions in the active site of various cyclases, especially the backwall area, were critical for the folding process. In the case of FusC, this corresponded to the helix 11 region. The researchers then performed cell-free biosynthesis by combining all necessary cellular components in a test tube along with enzyme variants featuring different amino acid modifications in helix 11. Ultimately, they discovered a version of FusC with a mutation in helix 11 that could successfully fold lasso peptides that the original cyclase could not produce. This finding supported the folding model developed during their computational research.
“Understanding how enzymes create a lasso knot is an intriguing question. This study offers an initial insight into the biophysical interactions behind the formation of this unique structure,” commented Diwakar Shukla, an associate professor of chemical and biomolecular engineering.
“We also demonstrated that these molecular interactions are conserved across various cyclases from different biological groups. Although we have not tested every system, we believe this model can be generalized,” Barrett stated.
In partnership with the San Diego-based company Lassogen, the researchers illustrated that their new findings could guide the engineering of cyclases to produce otherwise inaccessible lasso peptides. As a practical example, they modified a different cyclase, known as McjC, to efficiently create a powerful inhibitor of a cancer-related integrin.
“The capacity to develop a variety of lasso peptides is crucial for drug optimization,” remarked Mark Burk, CEO of Lassogen. “Natural enzymes often don’t allow us to create the desired lasso peptides, and the ability to engineer lasso cyclases significantly broadens the therapeutic applications of these remarkable molecules.”
“Our research would not have been feasible without the support of advanced computing technologies and the latest advancements in artificial intelligence and cell-free biosynthetic techniques,” said Douglas Mitchell, John and Margaret Witt Professor of Chemistry. “This work exemplifies the remarkable interdisciplinary collaborations fostered at the Carl R. Woese Institute for Genomic Biology. I am grateful to the MMG theme at IGB and our external partners at Lassogen for their contributions in tackling this complex problem.”
