Understanding Conformational Regulation of the Integrin I-domain for Design of Chimeric Protein Switches

Within all complex biological processes intricate proteins are expressed to complete every niche and necessary task. Many express multiple allosterically regulated conformational states, with protein function regulated by effector molecules and other ligands. One such protein is the LFA-1 surface integrin protein and its inserted domain, the I-domain. We Isolated the I-domain for investigation of determining binding properties and understanding conformational regulations of affinity changes to its target ligand ICAM-1, for further use in chimeric protein switch design. A large change in binding affinity was found through the deletion of a sub-sequence of amino acids in I-domain known as the α7 helix. Our investigation shows that, when the α7 helix is deleted, I-domain converts into a permanent high affinity state in which binding affinity to ICAM-1 was increased, and this state can be reversed by co-expression with soluble α7 helix peptide. These results conclude that the α7 helix stabilizes the I domain in its low affinity conformation in a ligand-like manner, allowing relaxation to the high affinity conformation upon disruption of α7 helix interaction. While deletion of the α7 helix yields higher binding affinity in I-domain it cannot be applied in design of chimeric protein switches due to its permanent conformational state. Because of this, our switch design has a focus of allosterically regulating the I-domain and α7 helix through utilizing on/off switching of conformational states. I-domain is fused with EF3 and EF4 hands of calmodulin, which then regulates binding affinity to ICAM-1 through interaction with α7 helix, when the EF hands’ natural ligand peptides are present. Currently, mutant switches are being developed to alter EF hand binding specificity which, when bound to new target ligands, will cause an increase in I-domain-ICAM-1 binding affinity in switch molecules. The results of these allosteric regulations highlight the potential of chimeric protein switches for design of environmentally responsive targeting agents and suggest that, through directed evolution, regulated binding to a range of novel targets could be achieved for therapeutic intervention.