Leukocytes target inflamed endothelium via the surface integrin LFA-1, which binds tightly to its ligand ICAM-1 when activated by “inside-out” signaling resulting from chemokine signals to the leukocyte. The ICAM-binding module of LFA-1, the I-domain, is conformationally regulated, with ligand binding upregulated by &gt;1000-fold in response to structural rearrangements involving cytoskeleton-induced forces. We have engineered chimeric proteins using the LFA-1 I-domain fused to domains from the allosteric protein calmodulin (CaM) to create ICAM binding switches in which ligand binding is regulated by exogenous peptide ligand. Switches optimized by directed evolution and rational design demonstrate upregulation of ICAM binding approaching 100-fold in response to soluble peptides that naturally bind to the CaM domains. Furthermore, directed evolution targeting the CaM domains enabled isolation of switches responding to a novel peptide ligand. Engineered ICAM-binding molecules with activity regulated by selected peptidic ligands may be useful in drug or gene targeting to sites of inflammation for therapeutic intervention.

Development of a ligand-regulated adhesive protein molecular switch

249th National Meeting (2015)

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