Insight into Weak Interactions between Carrier Proteins and their Substrates by Chemo-Enzymatic and Improved NMR Methods

Non-ribosomal peptides are a class of microbial secondary metabolites synthesized by non-ribosomal peptide synthetases (NRPSs) often with pharmaceutical and medicinal properties. There is an emerging demand to engineer NRPSs to develop novel pharmaceuticals but gaps in understanding molecular mechanisms hamper progress. During synthesis, substrates are attached to 20 Å phosphopantetheinyl moieties of carrier protein (CP) domains via thioester bonds. CPs then visit partner domains in a series of sequential transient interactions. Substrates were seen to dock with CP protein cores in a manner that may affect domain interactions. Further, such docking events are yet to be observed for the canonical NRPS substrates, amino acids. However, substrates often rapidly fall off CPs due to hydrolysis of the thioester bond, thus challenging structural studies. Here, we employ a chemo-enzymatic route to load a cysteine onto a carrier protein through an inert amide bond. This approach overcomes disulfide bond formation, transthioesterification and disulfide-thiol exchange inherent to the thiol functional group of cysteines. Using NMR spectroscopy, we identified weak but well-defined chemical shift perturbations indicating a transient interaction between the substrate and the protein core. To describe this interaction at the molecular level, we developed a time-shared filtered NOESY NMR experiment to measure distances between substrates and CP cores. Our results will help understand whether and how different substrates interact with CPs as they are presented to partner domains, thus guiding NRPS engineering to produce novel metabolites.