The antiviral and antiproliferative activities of human type I interferons (IFNs) are mediated by two transmembrane receptor subunits, IFNAR1 and IFNAR2. To elucidate the role of IFNAR1 in IFN binding and the establishment of biological activity, specific residues of IFNAR1 were mutated. Residues (62)FSSLKLNVY(70) of the S5-S6 loop of the N-terminal subdomain of IFNAR1 and tryptophan-129 of the second subdomain of IFNAR1 were shown to be crucial for IFN-alpha binding and signaling and establishment of biological activity. Mutagenesis of peptide (278)LRV in the third subdomain shows that these residues are critical for IFN-alpha-induced biological activity but not for ligand binding. These data, together with the sequence homology of IFNAR1 with cytokine receptors of known structure and the recently resolved NMR structure of IFNAR2, led to the establishment of a three-dimensional model of the human IFN-alpha/IFNAR1/IFNAR2 complex. This model predicts that following binding of IFN to IFNAR1 and IFNAR2 the receptor complex assumes a "closed form", in which the N-terminal domain of IFNAR1 acts as a lid, resulting in the activation of intracellular kinases. Differences in the primary sequence of individual IFN-alpha subtypes and resulting differences in binding affinity, duration of ligand/receptor association, or both would explain differences in intracellular signal intensities and biological activity observed for individual IFN-alpha subtypes.
Site-directed mutagenesis and chemical modification of the two cysteine residues of the MurC L-alanine-adding enzyme from Escherichia coli were undertaken to study their possible role in activity and stability. Their replacement by alanine was not critical for activity. However, C230 played a role in enzyme stability and substrate binding. N-Ethylmaleimide alkylation led to monoalkylated and dialkylated proteins. The monoalkylated protein had mostly unmodified C230 residues. The extent of alkylation of C230 paralleled the loss of activity, whereas that of C426 did not. Protection against inactivation by L L,Q Q-imidoadenosine 5P-triphosphate implied the involvement of C230 in the ATP binding site.z 1998 Federation of European Biochemical Societies.
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