In Silico Screening Using Freely Available Computational Tools to Generate Novel Peptide/Protein Pairs for Protein–Protein Ligation

Protein-protein ligation technology, such as SpyTag/SpyCatcher (ST/SC), enables the formation of functional protein complexes and has great potential for applications to biosensor development. The development of orthogonal Tag/Catcher pairs should expand the Tag/Catcher toolbox for the construction of intricately controlled complexes. Because some Tag/Catcher pairs show limited reaction rates, mutations to improve their reaction efficiency are conducted. However, they depend largely on comprehensive in vitro screening, which requires a lot of resources and laborious work. Here, in silico screening was performed using freely available computational tools including structural prediction, molecular docking, and molecular dynamics simulation to identify promising mutants. Based on the screening, four ST mutants were selected and tested in vitro. They did not react with a corresponding SC mutant, indicating that accurately predicting reactions involving covalent bond formation remains challenging under the tools and conditions employed here. Nevertheless, all selected ST mutants retained binding to the SC wild type, demonstrating that mutants of ST with partial compatibility toward SC could still be extracted. Overall, this work highlights the limitations of general-purpose computational methods in designing proteins and peptides with molecular recognition and catalysis capabilities and provides guidance for future efforts toward practical computational protein engineering.