Structural insights into how vacuolar sorting receptors recognize the sorting determinants of seed storage proteins.

Hsi-En Tsao, Shu Nga Lui, Anthony Hiu-Fung Lo, Shuai Chen, Hiu Yan Wong, Chi-Kin Wong, Liwen Jiang, Kam-Bo Wong

Proceedings of the National Academy of Sciences of the United States of America 2022 Jan 04

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In Arabidopsis, vacuolar sorting receptor isoform 1 (VSR1) sorts 12S globulins to the protein storage vacuoles during seed development. Vacuolar sorting is mediated by specific protein-protein interactions between VSR1 and the vacuolar sorting determinant located at the C terminus (ctVSD) on the cargo proteins. Here, we determined the crystal structure of the protease-associated domain of VSR1 (VSR1-PA) in complex with the C-terminal pentapeptide (468RVAAA472) of cruciferin 1, an isoform of 12S globulins. The 468RVA470 motif forms a parallel β-sheet with the switch III residues (127TMD129) of VSR1-PA, and the 471AA472 motif docks to a cradle formed by the cargo-binding loop (95RGDCYF100), making a hydrophobic interaction with Tyr99. The C-terminal carboxyl group of the ctVSD is recognized by forming salt bridges with Arg95. The C-terminal sequences of cruciferin 1 and vicilin-like storage protein 22 were sufficient to redirect the secretory red fluorescent protein (spRFP) to the vacuoles in Arabidopsis protoplasts. Adding a proline residue to the C terminus of the ctVSD and R95M substitution of VSR1 disrupted receptor-cargo interactions in vitro and led to increased secretion of spRFP in Arabidopsis protoplasts. How VSR1-PA recognizes ctVSDs of other storage proteins was modeled. The last three residues of ctVSD prefer hydrophobic residues because they form a hydrophobic cluster with Tyr99 of VSR1-PA. Due to charge-charge interactions, conserved acidic residues, Asp129 and Glu132, around the cargo-binding site should prefer basic residues over acidic ones in the ctVSD. The structural insights gained may be useful in targeting recombinant proteins to the protein storage vacuoles in seeds. Copyright © 2021 the Author(s). Published by PNAS.

Citation

Hsi-En Tsao, Shu Nga Lui, Anthony Hiu-Fung Lo, Shuai Chen, Hiu Yan Wong, Chi-Kin Wong, Liwen Jiang, Kam-Bo Wong. Structural insights into how vacuolar sorting receptors recognize the sorting determinants of seed storage proteins. Proceedings of the National Academy of Sciences of the United States of America. 2022 Jan 04;119(1)