Characteristics of cation binding to the I domains of LFA-1 and MAC-1. The LFA-1 I domain contains a Ca2+-binding site.
The crystal structures of the I domains of integrins MAC-1 (alphaM beta2; CD11b/CD18) and LFA-1 (alphaL beta2; CD11a/CD18) show that a single conserved cation-binding site is present in each protein. Purified recombinant I domains have intrinsic ligand binding activity, and in several systems this interaction has been demonstrated to be cation-dependent. It has been proposed that the I domain cation-binding site represents a general metal ion-dependent adhesion motif utilized for binding protein ligands. Here we show that the purified recombinant I domain of LFA-1 (alphaLI) binds cations, but with significantly different characteristics compared with the I domain of MAC-1 (alphaMI). Both alphaLI and alphaMI bind 54Mn2+ in a conformation-dependent manner, and in general, cations with charge and size characteristics similar to Mn2+ most effectively inhibit 54Mn2+ binding. Surprisingly, however, physiological levels of Ca2+ (1-2 mM) inhibited 54Mn2+ binding to purified alphaLI, but not to alphaMI. Using 45Ca2+ and 54Mn2+ in direct binding studies, the dissociation constants (KD) for the interactions between these cations and alphaLI were estimated to be 5-6 x 10(-5) and 1-2 x 10(-5) M, respectively. Together with the available structural information, the data suggest differential affinities for Mn2+ and Ca2+ binding to the single conserved site within alphaLI. Antagonism of LFA-1, but not MAC-1, -mediated cell adhesion by Ca2+ may be related to the Ca2+ binding activity of the LFA-1 I domain.
Discovery Pharmacology, Searle Research and Development, Monsanto Company, St. Louis, Missouri 63198, USA.
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Griggs
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Schmidt
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Carron
D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002412:Cations / Q000378:metabolism*; D002448:Cell Adhesion; D018360:Crystallography, X-Ray; D004926:Escherichia coli; D008024:Ligands; D016169:Lymphocyte Function-Associated Antigen-1 / Q000737:chemistry / Q000378:metabolism*; D016177:Macrophage-1 Antigen / Q000737:chemistry / Q000378:metabolism*; D008345:Manganese / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D002412:Cations; D008024:Ligands; D016169:Lymphocyte Function-Associated Antigen-1; D016177:Macrophage-1 Antigen; D011994:Recombinant Proteins; D008345:Manganese; D002118:Calcium