Identification and reconstruction of the binding site within alpha(M)beta(2) for a specific and high affinity ligand, NIF

Engagement of the alpha(M) beta(2) (CD11b/CD18, Mac-1) integrin on neutrophils supports adhesion and induces various cellular responses. These responses can be blocked by a specific ligand of alpha M beta(2), neutrophil inhibitory factor (MF). The molecular basis of alpha(M) beta(2)-NIF interactions was studied. The single chain alpha(M) subunit, expressed on the surface of human 293 cells, bound NIF with an affinity equivalent to that of alpha(M) beta(2) heterodimer. This observation, coupled with previous data showing that the alpha(M)I domain alone supported high affinity NIF binding, indicated that the binding site for NIF is restricted to the I domain. Guided by the crystal structure of the alpha(M)I domain, 16 segments corresponding to the entire enter hydrated surface of alpha(M)I domain were switched to their counterparts sequences in alpha(L), which does not bind NIF. Surface expression and heterodimer formation were achieved for all mutants, and correct folding was confirmed. Of the 16 switches, only 5 affected NIF binding substantially, reducing affinity by 8-300-fold. These data confined the NIF-binding site to a narrow region composed of Pro(147)-Arg(152), Pro(201)-Lys(217), and Asp(248) Arg(261) of alpha(M). Verifying this localization, when these segments were introduced inter the alpha(X)I-domain, the resulting chimeric receptor was converted into a high affinity NIF-binding protein.