Computational design of a leucine-rich repeat protein with a predefined geometry

Significance Repeat proteins are used in nature to bind to proteins and peptides. The shape of their binding surfaces can vary substantially, even for proteins within the same family. This variability likely arose because they evolved to match the proteins they interact with geometrically. Repeat proteins are often engineered to develop binders specific to new target proteins. It would be highly beneficial to design repeat proteins with predefined geometrical shapes because such a method would enable development of engineered repeat proteins that are shape-optimized to their targets. Here, we demonstrate that repeat proteins with a predefined shape can be designed using a computational design method. The approach is exemplified by the design of a protein that forms a ring structure not seen in nature. Structure-based protein design offers a possibility of optimizing the overall shape of engineered binding scaffolds to match their targets better. We developed a computational approach for the structure-based design of repeat proteins that allows for adjustment of geometrical features like length, curvature, and helical twist. By combining sequence optimization of existing repeats and de novo design of capping structures, we designed leucine-rich repeats (LRRs) from the ribonuclease inhibitor (RI) family that assemble into structures with a predefined geometry. The repeat proteins were built from self-compatible LRRs that are designed to interact to form highly curved and planar assemblies. We validated the geometrical design approach by engineering a ring structure constructed from 10 self-compatible repeats. Protein design can also be used to increase our structural understanding of repeat proteins. We use our design constructs to demonstrate that buried Cys play a central role for stability and folding cooperativity in RI-type LRR proteins. The computational procedure presented here may be used to develop repeat proteins with various geometrical shapes for applications where greater control of the interface geometry is desired.

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