The inserted (I) domain of αLβ2 integrin (LFA-1) contains the entire binding site of the molecule. It mediates both rolling and firm adhesion of leukocytes at sites of inflammation depending on the activation state of the integrin molecule. The affinity change of the entire integrin can be mimicked by the I domain alone through mutations that affect the conformation of the molecule. High affinity mutants of the I domain have been discovered previously using both rational design and directed evolution.

We have developed a novel sorting method to separate firmly adherent I domain molecules out of a library of mutants under shear flow. Using conditions analogous to those experienced by cells along the endothelium, we have identified mutations that support firm adhesion not found using typical directed evolution techniques that select for binding to soluble ligands. Characterization of these mutants is underway to support a greater understanding of the structure-function relationship of the αL I domain.

We have also engineered the I domain as an allosteric switch regulated by ligand binding to secondary domains that flank the I domain. Yeast displaying this I domain fusion on the surface exhibit rolling adhesion along ICAM-1 surfaces under shear flow, and the rolling velocity of these yeast decreases upon addition of the soluble secondary ligand. Work is ongoing to refine this switchable adhesive interaction by directed evolution to support rolling adhesion in the off state, and firm adhesion in the on state.