Hidden potential of the supporting scaffold as a structural module for plant cystatin design

Protein engineering approaches have been proposed to improve the inhibitory properties of plant cystatins towards herbivorous pest digestive Cys proteases, typically involving sequence alterations in the inhibitory loops and/or N-terminal trunk of the protein interacting with specific amino acid residues of the target protease. In this study, we assessed whether the loops-supporting frame, or scaffold, would represent a valuable structural module for cystatin function improvement. Twenty hybrid cystatins were designed in silico, consisting of the N-terminal trunk and two inhibitory loops of a given donor cystatin grafted onto the scaffold of an alternative, recipient cystatin. Synthetic genes for the hybrids were expressed in E. coli, and the resulting proteins assessed for their potency to inhibit model Cys protease papain and the digestive Cys proteases of Colorado potato beetle (Leptinotarsa decemlineata) used as an insect pest model. In line with the occurrence of positively selected amino acids presumably influencing inhibitory activity in the scaffold region of plant cystatins, grafting the N-terminal trunk and inhibitory loops of a given cystatin onto the scaffold of an alternative cystatin generally had an effect on the inhibitory potency of these function-related elements against Cys proteases. For instance, hybrid cystatins including the three structural elements of model tomato cystatin SlCYS8 grafted on the scaffold of cystatins from other plant families showed Ki values altered by up to 3-fold for papain, and inhibitory efficiencies increased by up to 8-fold against L. decemlineata cathepsin L-like proteases, compared to wild-type SlCYS8 bearing the original scaffold. Our data point to a significant influence of the cystatin scaffold on the inhibitory activity of the N-terminal trunk and protease inhibitory loops. They also suggest the potential of this structural element as a module for plant cystatin design to generate functional variability against Cys proteases, including the digestive proteases of herbivorous pests.