Structure-guided design of protein attachment points for functional augmentation of complex molecular machines

Protein-protein conjugation systems are a powerful way of creating fusion proteins and enable the dynamic combination of protein domains with diverse functionalities. However, the insertion of these systems into enzymes is often performed with little consideration of the structural impact they might have. This is particularly relevant when modifying complex molecular machines that transition between numerous conformational states. Here, we address this issue by developing SIMPLIFE, a computational workflow that supports the design of optimal insertion sites for conjugation tags based on the structure of the proteins involved and performs localised residue redesign where needed. We demonstrate how SIMPLIFE can be used to effectively augment the function of T7 RNA polymerase using the DogCatcher-DogTag system, enabling diverse and dynamically varying mutations within a targeted region of DNA. This work demonstrates the power of combining biophysical and machine learning based approaches for protein structure prediction to efficiently augment the function of molecular machines, accelerating our ability to combine complex biochemical functionalities in new ways.