Structure-function of the putative I-domain within the integrin beta(2) subunit

The central region (residues 125-385) of the integrin beta (2) subunit is p ostulated to adopt an I-domain-like fold (the beta I-2-domain) and to play a critical role in ligand binding and heterodimer formation. To understand structure-function relationships of this region of beta (2), a homolog-scan ning mutagenesis approach, which entails substitution of nonconserved hydro philic sequences within the beta I-2-domain with their homologous counterpa rts of the beta I-1-domain, has been deployed. This approach is based on th e premise that beta (1) and beta (2) are highly homologous, yet recognize d ifferent ligands. Altogether, 16 segments were switched to cover the predic ted outer surface of the beta I-2-domain. When these mutant beta (2) subuni ts were transfected together with wild-type alpha (M) in human 293 cells, a ll 16 beta (2) mutants were expressed on the cell surface as heterodimers, suggesting that these 16 sequences within the beta I-2-domain are not criti cally involved in heterodimer formation between the alpha (M), and beta (2) subunits. Using these mutant alpha (M)beta (2) receptors, we have mapped t he epitopes of nine beta I-2-domain specific mAbs, and found that they all recognized at least two noncontiguous segments within this domain. The requ isite spatial proximity among these non-linear sequences to form the mAb ep itopes supports a model of an I-domain-like fold for this region. In additi on, none of the mutations that abolish the epitopes of the nine function-bl ocking mAbs, including segment Pro(192)-Glu(197), destroyed ligand binding of the alpha (M)beta (2) receptor, suggesting that these function-blocking mAbs inhibit alpha (M)beta (2) function allosterically. Given the recent re ports implicating the segment equivalent to Pro(192)-Glu(197) in ligand bin ding by beta (3) integrins, these data suggest that ligand binding by the b eta (2) integrins occurs via a different mechanism than beta (3). Finally, both the conformation of the beta I-2-domain and C3bi binding activity of a lpha (M)beta (2) were dependent on a high affinity Ca2+ binding site (K-d = 105 muM), which is most likely located within this region of beta (2).