Abstract
In αI integrins, including leukocyte function-associated antigen 1 (LFA-1), ligand-binding function is delegated to the αI domain, requiring extra steps in the relay of signals that activate ligand binding and coordinate it with cytoplasmic signals. Crystal structures reveal great variation in orientation between the αI domain and the remainder of the integrin head. Here, we investigated the mechanisms involved in signal relay to the αI domain, including whether binding of the ligand intercellular adhesion molecule-1 (ICAM-1) to the αI domain is linked to headpiece opening and engenders a preferred αI domain orientation. Using small-angle X-ray scattering and negative-stain EM, we define structures of ICAM-1, LFA-1, and their complex, and the effect of activation by Mn2+ Headpiece opening was substantially stabilized by substitution of Mg2+ with Mn2+ and became complete upon ICAM-1 addition. These agents stabilized αI-headpiece orientation, resulting in a well-defined orientation of ICAM-1 such that its tandem Ig-like domains pointed in the opposite direction from the β-subunit leg of LFA-1. Mutations in the integrin βI domain α1/α1' helix stabilizing either the open or the closed βI-domain conformation indicated that α1/α1' helix movements are linked to ICAM-1 binding by the αI domain and to the extended-open conformation of the ectodomain. The LFA-1-ICAM-1 orientation described here with ICAM-1 pointing anti-parallel to the LFA-1 β-subunit leg is the same orientation that would be stabilized by tensile force transmitted between the ligand and the actin cytoskeleton and is consistent with the cytoskeletal force model of integrin activation.
Highlights
In ␣I integrins, including leukocyte function–associated antigen 1 (LFA-1), ligand-binding function is delegated to the ␣I domain, requiring extra steps in the relay of signals that activate ligand binding and coordinate it with cytoplasmic signals
Hi3-intercellular adhesion molecule-1 (ICAM-1) with a C-terminal His tag was made in HEK293S GnTIϪ/Ϫ cells and purified using nickel-nitrilotriacetic acid–Sepharose, anion exchange, and gel filtration
We have visualized how the headpiece of integrin LFA-1 binds to its physiological ligand, intercellular adhesion molecules (ICAMs)-1, and demonstrated a remarkably well-defined orientation in EM as well as in solution that we discuss in relation to the pathway for force transmission through the integrin between ICAM-1 and the actin cytoskeleton
Summary
In ␣I integrins, including leukocyte function–associated antigen 1 (LFA-1), ligand-binding function is delegated to the ␣I domain, requiring extra steps in the relay of signals that activate ligand binding and coordinate it with cytoplasmic signals. In a crystal structure of the ␣L2 headpiece, the ␣I domain was in the closed conformation, and crystal lattice contacts stabilized an extreme tilt toward the ␣-subunit [10] (Fig. 1D). These findings raise the question of whether the ␣I domain has a well-defined orientation, either in the closed conformation when not bound to the platform through the internal ligand or in the open conformation when bound through its internal ligand to the platform, and illustrate the importance of the use of techniques orthogonal to crystallography, such as EM and small angle X-ray scattering in solution (SAXS), as in the current study.
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