Structural insights into how vacuolar sorting receptor recognizes the C‐terminal sorting determinant of a vicilin‐like seed storage protein

During seed development, vacuolar sorting receptors (VSRs) recognize a sequence‐specific vacuolar sorting determinant located at the C terminus (ctVSD) of storage proteins, thereby sorting them into protein storage vacuoles. The protease‐associated (PA) domain of VSRs is responsible for interacting with the ctVSD of cargo proteins. Here, we report the crystal structure of the PA domain of Arabidopsis vacuolar‐sorting receptor 1 (VSR1) in complex with the C‐terminal pentapeptide ( 507 SDRFV 511 ) of vicilin‐like seed storage protein 22 (VL22). Structural comparison with the apo form of VSR1 reveals conformational changes in four switch regions in the PA domain. VL22 binds to a cradle of VSR1 formed by residues in the cargo‐binding loop, the switch I and III regions. The C‐terminal carboxyl group of VL22 is recognized by forming salt bridges with the invariant Arg95 of VSR1. Compared with the structure of VSR1‐PA in complex with the ctVSD of cruciferin 1, VL22 makes extra hydrophobic interactions with the cargo‐binding loop and hydrogen bonds with switch I residues in VSR1. Tagging the C‐terminal sequence of VL22, but not VL22‐R509P, VL22‐V511P, VL22‐R509P‐V511P nor vicilin‐like seed storage protein 43 (VL43), redirected secretory red fluorescent protein (spRFP) to the vacuoles in Arabidopsis protoplasts. Scanning mutagenesis identified an E519S substitution converting the C‐terminal sequence of VL43 to a sorting determinant that can redirect spRFP to the vacuoles, suggesting that charge–charge repulsion prevents the receptor–cargo interactions between VL43 and VSR1. The recognition of ctVSD by VSRs is likely promiscuous, resulting from the additive effect of individual preference of residues in the ctVSD.