@article {Nonis2020.10.28.360099, author = {Samuel G. Nonis and Joel Haywood and Jason W. Schmidberger and Charles S. Bond and Joshua S. Mylne}, title = {Structural basis for a natural circular permutation in proteins}, elocation-id = {2020.10.28.360099}, year = {2020}, doi = {10.1101/2020.10.28.360099}, publisher = {Cold Spring Harbor Laboratory}, abstract = {Over 30 years ago, an intriguing post-translational modification was discovered to be responsible for creating concanavalin A (conA), a carbohydrate-binding protein found in the seeds of jack bean (Canavalia ensiformis) and commercially used for carbohydrate chromatography. Biosynthesis of conA involves what was then an unprecedented rearrangement in amino acid sequence, whereby the N-terminal half of the gene-encoded conA precursor is swapped to become the C-terminal half of conA. The cysteine protease, asparaginyl endopeptidase (AEP), was shown to be involved, but its mechanism was not fully elucidated. To understand the structural basis and consequences of conA circular permutation, we generated a recombinant jack bean conA precursor (pro-conA) plus jack bean AEP (CeAEP1) and solved crystal structures for each to 2.1 {\r A} and 2.7 {\r A} respectively. By reconstituting the biosynthesis of conA in vitro, we prove CeAEP1 alone can perform both the cleavage and cleavage-coupled transpeptidation to form conA. CeAEP1 structural analysis reveals how it is capable of carrying out both these reactions. Biophysical assays illustrated that conA is more thermally and pH stable than pro-conA, consistent with fewer intermolecular interactions between subunits in the pro-conA crystal structure. These findings elucidate the consequences of circular permutation in the only post-translation example known to occur in nature.Competing Interest StatementThe authors have declared no competing interest.}, URL = {https://www.biorxiv.org/content/early/2020/10/29/2020.10.28.360099}, eprint = {https://www.biorxiv.org/content/early/2020/10/29/2020.10.28.360099.full.pdf}, journal = {bioRxiv} }