Lasso peptides are a class of knot-like polypeptides in which the C-terminal tail of the peptide threads through a ring formed by an isopeptide bond between the N-terminal amine group and a sidechain carboxylic acid. The small size (~20 amino acids) and simple topology of lasso peptides make them a good model system for studying the unthreading of entangled polypeptides, both with experiments and atomistic simulation. Here we present an in-depth study of the thermal unthreading behavior of two lasso peptides astexin-2 and astexin-3. Quantitative kinetics and energetics of the unthreading process were determined for variants of these peptides using a series of chromatography and mass spectrometry experiments and biased molecular dynamics (MD) simulations. In addition, we show that the Tyr15Phe variant of astexin-3 unthreads via an unprecedented “tail pulling” mechanism. MD simulations on a model ring-thread system coupled with machine learning approaches also led to the discovery of physicochemical descriptors most important for peptide unthreading.

Lasso peptides are a member of the superclass of ribosomally synthesized and posttranslationally modified peptides (RiPPs). Like all RiPPs, lasso peptides are derived from a gene-encoded precursor protein. The biosynthesis of lasso peptides requires two enzymatic activities: proteolytic cleavage between the leader peptide and the core peptide in the precursor protein, accomplished by the B enzymes, and ATP-dependent isopeptide bond formation, accomplished by the C enzymes. In a subset of lasso peptide biosynthetic gene clusters from Gram-positive organisms, the B enzyme is split between two proteins. One such gene cluster is found in the organism Rhodococcus jostii, which produces the antimicrobial lasso peptide lariatin. The B enzyme in R. jostii is split between two open reading frames, larB1 and larB2, both of which are required for lariatin biosynthesis. While the cysteine catalytic triad is found within the LarB2 protein, LarB1 is a PqqD homologue expected to bind to the lariatin precursor LarA based on its structural homology to other RiPP leader peptide binding domains. We show that LarB1 binds to the leader peptide of the lariatin precursor protein LarA with a sub-micromolar affinity. We used photocrosslinking with the noncanonical amino acid p-azidophenylalanine and mass spectrometry to map the interaction of LarA and LarB1. This analysis shows that the LarA leader peptide interacts with a conserved motif within LarB1 and, unexpectedly, the core peptide of LarA also binds to LarB1 in several positions. A Rosetta model built from distance restraints from the photocrosslinking experiments shows that the scissile bond between the leader peptide and core peptide in LarA is in a solvent-exposed loop.

Adeno-associated virus (AAV) is widely favored as a gene therapy vector, tested in over 200 clinical trials internationally. To improve targeted delivery a variety of genetic capsid modifications, such as insertion of targeting proteins/peptides into the capsid shell, have been explored with some success but larger insertions often have unpredictable deleterious impacts on capsid formation and gene delivery. Here, we demonstrate a modular platform for the integration of exogenous peptides and proteins onto the AAV capsid post-translationally while preserving vector functionality. We decorated the AAV capsid with leucine-zipper coiled-coil binding motifs that exhibit specific noncovalent heterodimerization. AAV capsids successfully display

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