Dissociation of I domain and global conformational changes in LFA-1: Refinement of small molecule-I domain structure-activity relationships

Richard S. Larson, Terry Davis, Cristian Bologa, Gloria Semenuk, Sreejith Vijayan, Yu Li, Tudor Oprea, Alexandre Chigaev, Tione Buranda, Carston R. Wagner, Larry A. Sklar

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29 Scopus citations

Abstract

LFA-1 (α-/32) is constitutively expressed on leukocytes, but its activity is rapidly regulated. This rapid activation has been proposed to be associated with conformation changes in the inserted ("I") domain within the headpiece of LFA-1 as well as conversion of the molecules from bent to extended forms. To study these molecular changes as they relate to affinity regulation of LFA-1, we developed and synthesized a fluorescent derivative of BIRT-377 [Kelly et al. (2001) J. Immunol.] to examine changes in LFA-1 affinity in a flow cytometer with live cells. BIRT-377 binds to the ligand-binding or "I" domain of LFA-1. Structure-activity relationships studies indicated that an aminoalkyl group could be added to the central hydantoin group without significantly affecting binding. Using this modified derivative [1-(N-fluoresceinylthioureidobutyl)-[5R]-(4-bromobenzyl)-3-(3,5-dichlorophenyl) -5-methyl-imidazolidine-2,4-dione (FBABIRT)], we analyzed the affinity of FBABIRT binding to LFA-1 on live cells. The binding affinity increases, and the dissociation rate decreases with divalent cation (Mn2+) stimulation. We then used FBABIRT with fluorescent resonance energy transfer (FRET) to show that LFA-1 changes its height relative to the cell surface when cells were treated with dithiothreitol (DTT) but not Mn2+. Competition assays among FBABIRT and BIRT derivatives defined structure-affinity relationships that refine the current model of BIRT-377 binding to the I domain. Our data supports the model in which BIRT-377 binds to the I domain and stabilizes the bent structure of LFA-1, while divalent cation activation results in a small conformational change in the I domain without significant extension of LFA-1. DTT, in contrast, induces a conversion to the extended form of LFA-1 in the presence of BIRT-377 on live cells. The structure-activity studies suggest that BIRT-377 is a fully optimized inhibitor.

Original languageEnglish (US)
Pages (from-to)4322-4331
Number of pages10
JournalBiochemistry
Volume44
Issue number11
DOIs
StatePublished - Mar 22 2005

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    Larson, R. S., Davis, T., Bologa, C., Semenuk, G., Vijayan, S., Li, Y., Oprea, T., Chigaev, A., Buranda, T., Wagner, C. R., & Sklar, L. A. (2005). Dissociation of I domain and global conformational changes in LFA-1: Refinement of small molecule-I domain structure-activity relationships. Biochemistry, 44(11), 4322-4331. https://doi.org/10.1021/bi048187k