Abstract
The integrin LFA-1 (αLβ2) is an αβ heterodimeric adhesion molecule critical in the effective trafficking of leukocytes and in facilitating subsequent antigen-specific inter-action. Participation of αLβ2 in multiple steps critical for T-cell-mediated immunity in vivo makes αLβ2 a valid therapeutic target for anti-inflammation therapy. Many small-molecule antagonists to αLβ2 have been developed as anti-inflammatory agents, out of which polysubstituted (S)-2-benzoylamino propionic acids, represented by XVA143 (XVA), have emerged as the most potent antagonists. αLβ2 is a large glycoprotein with a complex multi-domain organization, where a conserved von Willebrand factor-type A domain is contained in each subunit, the inserted (I) domain in the α-subunit and the I-like domain in the β subunit. The α-subunit I domain directly binds ligand, whereas the β-subunit I-like domain is thought to play a regulatory role by interacting with a part of the I domain. Thus far, it remains to be elucidated which domain the antagonists bind to and how they inhibit αLβ2 function. Here we investigate a mechanistic basis of XVA activity. XVA blocked the αLβ2-ICAM-1 interaction with EC50 of < 1 nM and suppressed mixed lymphocyte reaction as potently as cyclosporin A. XVA did not block ligand binding by αLβ2 directly, as it did not block αLβ2 containing a mutant I domain that is stabilized in the high-affinity conformation. Rather, XVA interfered with conformational changes that convert the I domain to the high-affinity state. Surface plasmon resonance analysis using an isolated I domain showed that XVA did not target the I domain. Interestingly, XVA stabilized non-covalent αβ association sufficiently to make it resistant to denaturation with SDS. Stabilization of mutant αβ complexes was utilized to test compound binding to αLβ2 mutants and locate the inhibitor-binding site. As binding of XVA was found to be metal-dependent, alanine-scanning of the metal binding sites indicated that this compound binds to the metal ion-dependent adhesion site in the I-like domain, where it disrupts the interaction of the I-like domain with the I domain. XVA inhibits αLβ2 allosterically by perturbing the inter-domain communication that is critical to relay conformational signals which induce the active I domain conformation. Furthermore, XVA stabilized a global conformation of αLβ2 in the active extended form, whereas the ligand binding I domain was left in the inactive conformation, as demonstrated by exposure of activation-dependent epitopes in αLβ2 on the cell surface and electron microscopic images of the soluble recombinant αLβ2. The results strongly suggest that XVA would serve as a mimetic for the intrinsic ligand that is involved in receptor-ligand like interaction between the I domain and I-like domain. This inhibitor revealed a crucial intersection for relaying conformational signals within the integrin αLβ2. While blocking signals in one direction (to the I domain), the antagonists induce the active conformation of the I-like domain as well as the rest of domains, and thus transmit conformational signals in the opposite direction toward the transmembrane domains.
The Advantages of EPR Spectroscopy in Exploring Diamagnetic Metal Ion Binding and Transfer Mechanisms in Biological Systems
