NMR solution structure of the inserted domain of human leukocyte function associated antigen-1

The interaction between the leukocyte function-associated antigen-1 (LFA-1) and the intercellular adhesion molecule is thought to be mediated primaril y via the inserted domain (I-domain) in the alpha-subunit. The activation o f LFA-1 is an early step in triggering the adhesion of leukocytes to target cells decorated with intercellular adhesion molecules. There is some disag reement in the literature over the respective roles of conformational chang es in the I-domain and of divalent cations (Mg2+, Mn2+) in the activation o f LFA-1 for intercellular adhesion molecule binding. X-ray crystallographic structures of the I-domains of LFA-1 and Mac-1 in the presence and absence of cations show structural differences in the C-terminal a-helix; this cha nge was proposed to represent the active and inactive conformations of the I-domain. However, more recent X-ray results have called this proposal into question. The solution structure of the Mg2+ complex of the I-domain of LF A-1 has been determined by NMR methods, using a model-based approach to nuc lear Overhauser enhancement spectroscopy peak assignment. The protein adopt s the same structure in solution as that of the published I-domain X-ray st ructures, but the C-terminal region, where the X-ray structures are most di fferent from each other, is different again in the solution structures. The secondary structure of this helix is well formed, but NMR relaxation data indicate that there is considerable flexibility present, probably consistin g of breathing or segmental motion of the helix. The conformational diversi ty seen in the various X-ray structures could be explained as a result of t he inherent flexibility of this C-terminal region and as a result of crysta l contacts. Our NMR data are consistent with a model where the C-terminal h elix has the potential flexibility to take up alternative conformations, fo r example, in the presence and absence of the intercellular adhesion molecu le ligand. The role of divalent cations appears from our results not to be as a direct mediator of a conformational change that alters affinity for th e ligand. Rather, the presence of the cation appears to be involved in some other way in ligand binding, perhaps by acting as a bridge to the ligand a nd by modulation of the charge of the binding surface. (C) 2000 Academic Pr ess.