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bioRxiv · 10.1101/2021.12.09.471967

Structural basis for substrate recognition, ligation and activation by a hyperactive Asn peptide ligase from Viola yedoensis

Lescar, J.·Hu, S.·El Sahili, A.·Kishore, S.·Hemu, X.·Liu, C. F.·Tam, J. P.·Goh, B. C.·Wong, Y. H.·Wang, Z.
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Abstract

Peptide asparaginyl ligases (PALs) belong to a limited class of enzymes from cyclotide-producing plants, that perform site-specific ligation reactions after a target peptide Asx (Asn/Asp) binds to the ligase active site. How PALs specifically recognize their polypeptide substrates has remained elusive especially at the prime binding side of the enzyme. Here we captured VyPAL2, a catalytically efficient PAL from Viola yedoensis, in an activated state, with and without a bound substrate. The bound structure shows one ligase with the N-terminal polypeptide tail from another ligase molecule trapped at its active site, revealing how Asx inserts in the enzymes S1 pocket and why a hydrophobic residue is required at the substrate P2 position. Beside illustrating the role played by P1 and P2 residues as primary anchors for the enzyme reaction, these results provide a mechanistic explanation for the role of the "Gatekeeper" residue at the surface of the S2 pocket, in shifting the non-prime portion of the substrate and, as a result, the activity towards either ligation or hydrolysis. These results detail the molecular events that occur during proenzyme maturation in the plant vacuolar compartment, suggest a mechanism for ligation, and will inform the design of peptide ligases with tailored specificities. One sentence summaryWe captured VyPAL2, a catalytically efficient plant peptide ligase with a bound substrate, providing the molecular basis for substrate recognition and ligation.

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Lescar, J., Hu, S., El Sahili, A., Kishore, S., Hemu, X., Liu, C. F., Tam, J. P., Goh, B. C., Wong, Y. H., Wang, Z.. 2021-12-09. Structural basis for substrate recognition, ligation and activation by a hyperactive Asn peptide ligase from Viola yedoensis. https://doi.org/10.1101/2021.12.09.471967

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