(188cv) Engineering Ligand-Regulated Adhesion Proteins Targeting ICAM-1

Many natural proteins involved in complex biological processes such as ligand binding and protein folding demonstrate multiple, allosterically-regulated conformational states, with protein activity regulated by effector molecules. The alpha L integrin and its inserted domain (I domain) is one example of such a protein. Binding of the effector molecule (talin or filamin) to the cytoplasmic domain of the integrin will switch the binding affinity between the I domain and its ligand intercellular adhesion molecule-1 (ICAM-1) from low to high affinity. We have engineered new chimeric proteins based on this conformationally regulated adhesion molecule. Alpha L I-domain fused with EF3 and EF4 hands of calmodulin demonstrate autonomously-regulated interactions with ICAM-1 when calmodulin-binding peptides are present. Furthermore, we have constructed I domains truncated to remove an alpha helix near the C-terminus (the alpha7 helix) and mutants altered at key positions defining the interface of the alpha7 helix with the rest of the structure, and we compared the affinity of these altered I domains to ICAM-1. The results suggest a model wherein the alpha7 helix acts similarly to an induced-fit ligand, stabilizing the I domain in its low affinity conformation and allowing relaxation to the high affinity conformation upon disruption of alpha7 helix interaction. These results highlight the potential of this novel allosteric system, which could be leveraged for the design of environmentally-responsive targeting agents or therapeutics.