Phytocystatin 6 is a context-dependent, tight-binding inhibitor of <i>Arabidopsis thaliana</i> legumain isoform β

Plant legumains are crucial for processing seed storage proteins and are critical regulators of plant programmed cell death. Although research on legumains boosted recently, little is known about their activity regulation. In our study, we used pull-down experiments to identify AtCYT6 as a natural inhibitor of legumain isoform β (AtLEGβ) in Arabidopsis thaliana. Biochemical analysis revealed that AtCYT6 inhibits both AtLEGβ and papain-like cysteine proteases through two separate cystatin domains. The N-terminal domain inhibits papain-like proteases, while the C-terminal domain inhibits AtLEGβ. Furthermore, we showed that AtCYT6 interacts with legumain in a substrate-like manner, facilitated by a conserved asparagine residue in its reactive center loop. Complex formation was additionally stabilized by charged exosite interactions, contributing to pH-dependent inhibition. Processing of AtCYT6 by AtLEGβ suggests a context-specific regulatory mechanism with implications for plant physiology, development, and programmed cell death. These findings enhance our understanding of AtLEGβ regulation and its broader physiological significance.