Abstract
Binding of lymphocyte function-associated antigen-1 (LFA-1) to intercellular adhesion molecule-1 (ICAM-1) mediates leukocyte adhesion under force. Using a biomembrane force probe capable of measuring single bond interactions, we showed ICAM-1 binding to LFA-1 at different conformations, including the bent conformation with the lowest affinity. We quantify how force and conformations of LFA-1 regulate its kinetics with ICAM-1. At zero-force, on-rates were substantially changed by conditions that differentially favor a bent or extended LFA-1 with a closed or open headpiece; but off-rates were identical. With increasing force, LFA-1/ICAM-1 bond lifetimes (reciprocal off-rates) first increased (catch bonds) and then decreased (slip bonds). Three states with distinct off-rates were identified from lifetime distributions. Force shifted the associated fractions from the short- to intermediate- and long-lived states, producing catch bonds at low forces, but increased their off-rates exponentially, converting catch to slip bonds at high forces. An internal ligand antagonist that blocks pulling of the α(7)-helix suppressed the intermediate-/long-lived states and eliminated catch bonds, revealing an internal catch bond between the αA and βA domains. These results elucidate an allosteric mechanism for the mechanochemistry of LFA-1/ICAM-1 binding.
Highlights
Using a biomembrane force probe capable of measuring single bond interactions, we showed intercellular adhesion molecule-1 (ICAM-1) binding to lymphocyte function-associated antigen-1 (LFA-1) at different conformations, including the bent conformation with the lowest affinity
Using force clamp [25] and thermal fluctuation [26] experiments to measure single bond interactions by a biomembrane force probe (BFP), here we show that lymphocyte functionassociated antigen-1 (LFA-1), an ␣A domain-containing integrin ␣L2, forms catch-slip bonds with intercellular adhesion molecule-1 (ICAM-1) in three cation conditions and in the presence of a chemokine that triggers inside-out signaling, 2 The abbreviations used are: MIDAS, metal ion-dependent adhesion site; FN, fibronectin; BFP, biomembrane force probe; RBC, red blood cell
Using a BFP (Fig. 1A and supplemental video S1), we measured frequencies of adhesions and lifetimes of bonds between LFA-1 on a Jurkat cell (Fig. 1, A and B, right) and recombinant ICAM-1 or anti-LFA-1 monoclonal antibodies coated on a glass bead attached to the apex of a RBC (Fig. 1, A and B, left)
Summary
Proteins, and Chemicals—Jurkat cells purchased from ATCC (Manassas, VA) were cultured in RPMI 1640 medium with 10% fetal calf serum plus L-glutamine (4 mM) and penicillin/streptomycin (0.1 mg/ml). To determine the Mac-1 expression, Jurkat cells or PMNs washed as described in the preceding paragraph were incubated with 10 g/ml PE-conjugated anti-␣M mAb at 37 °C for 30 min in binding butter containing Ca2ϩ/Mg2ϩ or Ca2ϩ/Mg2ϩ/ CXCL12, washed and re-suspended in the same buffer at 4 °C for immediate flow cytometric analysis. To determine the CXC chemokine receptor 4 (CXCR4) expression, Jurkat cells washed as previously described in the preceding paragraph were incubated in binding butter containing Ca2ϩ/Mg2ϩ or Ca2ϩ/Mg2ϩ/CXCL12 at room temperature for 1 or 2 h, incubated with 10 g/ml PE-conjugated antiCXCR4 mAb at room temperature for 30 min, washed and resuspended in the same buffer for immediate flow cytometric analysis. The zero-force offrates were obtained from the negative slopes of the linear fits to the ln(survival frequency) versus lifetime data measured by the thermal fluctuation assay (see Fig. 2E). Based on the degrees of the freedom and the F statistic, a p value can be calculated. p Ͻ 0.01 means that the three-states model is significantly better than the two-state model
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