The Supporting Scaffold as a Structural Module for Plant Cystatin Design

ABSTRACT Protein engineering approaches have been proposed to improve the inhibitory potency of cystatins against plant pest digestive Cys proteases, typically involving point mutations in the N‐terminal trunk or inhibitory loops known to interact with the target protease. Here, we assessed whether the loop‐supporting frame, or ‘scaffold’, could represent a valuable structural module for cystatin improvement. Hybrid cystatins were designed in silico, consisting of the N‐terminal trunk and inhibitory loops of a donor cystatin grafted onto the scaffold of an alternative, recipient cystatin. As inferred from AlphaFold models, scaffold substitutions had little impact on the cystatin fold and spatial orientation of the inhibitory loops but, in several instances, altered the spatial positioning of the N‐terminal trunk and predicted strength of the protease::cystatin complex. Synthetic versions of the hybrids produced in E. coli were assessed for their inhibitory potency against papain and the digestive proteases of a herbivorous insect model. In several cases, scaffold substitutions had an impact on the inhibitory potency of the resulting cystatins. For instance, cystatins with the N‐terminal trunk and inhibitory loops of model tomato cystatin SlCYS8 grafted onto the scaffolds of other cystatins showed K i values altered by up to 3‐fold for papain and inhibitory rates increased by up to 8‐fold against the insect proteases. Our data indicate a significant influence of the scaffold on the inhibitory activity of plant cystatins. They also suggest the potential of this structural element as a module for plant cystatin design to generate functional variability against herbivorous pest digestive proteases.