The ability of leukocytes to adhere and extravasate at sites of inflammation is crucial for immune responses. While the initial transition from moving at the free stream velocity to rolling along the endothelium can be achieved through selectin interactions, αLβ2 integrin (LFA-1) can mediate both rolling adhesion and firm adhesion of leukocytes depending on the activation state of the molecule through interaction with intercellular adhesion molecule-1 (ICAM-1). The global conformational change that LFA-1 undergoes during activation also causes a conformational change in the inserted (I) domain of the α subunit, which contains the entire ligand-binding site, and increases affinity for ICAM-1 approximately 10,000 fold. We have displayed wild-type I domain on the surface of yeast and validated expression using I domain specific antibodies and flow cytometry. Surface display of I domain supports yeast rolling on ICAM-1-coated surfaces under shear flow. I domain mutants were also expressed, and soluble ICAM-1 binding studies validated that these mutants have a range of affinities for the ligand. Expression of the locked open, high-affinity I domain mutant supports firm adhesion of yeast under shear flow, while yeast displaying intermediate-affinity I domain mutants exhibit a range of rolling phenotypes. These results indicate that unstressed binding affinity is not the only molecular property that determines adhesive behavior under shear flow, as rolling behavior for these mutants fails to correlate with ligand binding affinity. Work to validate this observation on a larger scale is underway utilizing I domain mutant libraries to characterize the molecular structure-function relationship via directed evolution.

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