Identification and reconstruction of the binding site within alphaMbeta2 for a specific and high affinity ligand, NIF.
Engagement of the alphaMbeta2 (CD11b/CD18, Mac-1) integrin on neutrophils supports adhesion and induces various cellular responses. These responses can be blocked by a specific ligand of alphaMbeta2, neutrophil inhibitory factor (NIF). The molecular basis of alphaMbeta2-NIF interactions was studied. The single chain alphaM subunit, expressed on the surface of human 293 cells, bound NIF with an affinity equivalent to that of alphaMbeta2 heterodimer. This observation, coupled with previous data showing that the alphaMI 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 alphaMI domain, 16 segments corresponding to the entire outer hydrated surface of alphaMI domain were switched to their counterparts sequences in alphaL, 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 Pro147-Arg152, Pro201-Lys217, and Asp248-Arg261 of alphaM. Verifying this localization, when these segments were introduced into the alphaXI-domain, the resulting chimeric receptor was converted into a high affinity NIF-binding protein.
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Joseph J. Jacobs Center for Thrombosis and Vascular Biology, Department of Molecular Cardiology, The Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
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D001665:Binding Sites; D018360:Crystallography, X-Ray; D005434:Flow Cytometry; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D015801:Helminth Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D016177:Macrophage-1 Antigen / Q000378:metabolism*; D008565:Membrane Proteins; D008958:Models, Molecular; D008970:Molecular Weight; D016296:Mutagenesis; D018696:Neuroprotective Agents / Q000378:metabolism*; D011487:Protein Conformation
D006023:Glycoproteins; D015801:Helminth Proteins; D016177:Macrophage-1 Antigen; D008565:Membrane Proteins; D018696:Neuroprotective Agents; C086581:NIF protein, Ancylostoma caninum