Structural and biochemical analyses of concanavalin A circular permutation by jack bean asparaginyl endopeptidase

Plant Cell. 2021 Aug 31;33(8):2794-2811. doi: 10.1093/plcell/koab130.


Over 30 years ago, an intriguing posttranslational modification was found responsible for creating concanavalin A (conA), a carbohydrate-binding protein from jack bean (Canavalia ensiformis) seeds and a common carbohydrate chromatography reagent. ConA biosynthesis involves what was then an unprecedented rearrangement in amino-acid sequence, whereby the N-terminal half of the gene-encoded conA precursor (pro-conA) is swapped to become the C-terminal half of conA. Asparaginyl endopeptidase (AEP) was shown to be involved, but its mechanism was not fully elucidated. To understand the structural basis and consequences of circular permutation, we generated recombinant jack bean pro-conA plus jack bean AEP (CeAEP1) and solved crystal structures for each to 2.1 and 2.7 Å, respectively. By reconstituting conA biosynthesis in vitro, we prove CeAEP1 alone can perform both cleavage and cleavage-coupled transpeptidation to form conA. CeAEP1 structural analysis reveals how it is capable of carrying out both reactions. Biophysical assays illustrated that pro-conA is less stable than conA. This observation was explained by fewer intermolecular interactions between subunits in the pro-conA crystal structure and consistent with a difference in the prevalence for tetramerization in solution. These findings elucidate the consequences of circular permutation in the only posttranslation example known to occur in nature.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • Canavalia / enzymology
  • Catalytic Domain
  • Circular Dichroism
  • Concanavalin A / chemistry*
  • Concanavalin A / genetics
  • Concanavalin A / metabolism*
  • Crystallography, X-Ray
  • Cysteine Endopeptidases / chemistry*
  • Cysteine Endopeptidases / genetics
  • Cysteine Endopeptidases / metabolism*
  • Hydrogen-Ion Concentration
  • Methylmannosides / metabolism
  • Models, Molecular
  • Protein Conformation
  • Protein Precursors / chemistry
  • Protein Precursors / genetics
  • Protein Precursors / metabolism*
  • Protein Stability
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Solutions


  • Methylmannosides
  • Protein Precursors
  • Recombinant Proteins
  • Solutions
  • pro-concanavalin A
  • Concanavalin A
  • methylmannoside
  • Cysteine Endopeptidases
  • asparaginylendopeptidase