Title : Structure-based rational design of a
Toll-like receptor 4 (
TLR4 ) decoy
receptor with high binding affinity for a target
protein
Abstract :
- Repeat proteins are increasingly attracting much attention as alternative scaffolds to immunoglobulin antibodies due to their unique structural features
- Nonetheless, engineering interaction interface and understanding molecular basis for affinity maturation of repeat proteins still remain a challenge
- Here, we present a structure-based rational design of a repeat protein with high binding affinity for a target protein
- As a model repeat protein , a Toll-like receptor4 ( TLR4 ) decoy receptor composed of leucine leucine-rich repeat (LRR) modules was used, and its interaction interface was rationally engineered to increase the binding affinity for myeloid differentiation protein 2 ( MD2 )
- Based on the complex crystal structure of the decoy receptor with MD2 , we first designed single amino acid substitutions in the decoy receptor , and obtained three variants showing a binding affinity (K(D)) one-order of magnitude higher than the wild-type decoy receptor
- The interacting modes and contributions of individual residues were elucidated by analyzing the crystal structures of the single variants
- To further increase the binding affinity, single positive mutations were combined, and two double mutants were shown to have about 3000- and 565-fold higher binding affinities than the wild-type decoy receptor
- Molecular dynamics simulations and energetic analysis indicate that an additive effect by two mutations occurring at nearby modules was the major contributor to the remarkable increase in the binding affinities