Abstract

2'-5'-Oligoadenylate synthetases are interferon-induced enzymes that upon activation by double-stranded RNA polymerize ATP to 2'-5'-linked oligoadenylates. In our continuing effort to understand the mechanism of catalysis by these enzymes, we used photo affinity cross-linking and peptide mapping to identify the substrate-binding sites of the P69 isozyme of human 2'-5'-oligoadenylate synthetases. Radiolabeled azido 2'-5'-oligoadenylate dimers were enzymatically synthesized and used as ligands for cross-linking to the P69 protein by exposure to ultraviolet light. The radiolabeled protein was digested with trypsin, and two ligand-cross-linked peptides were purified by immobilized aluminum affinity chromatography followed by reverse phase high pressure liquid chromatography. The peptides were identified by mass spectrometry and peptide sequencing and were found to correspond to residues 420-425 and 539-547 of P69. To examine the functional importance of the cross-linking sites, specific residues in the two peptides were mutated. When residues in the two sites were mutated individually, ligand cross-linking was selectively eliminated at the mutated site, and the enzyme activity was lost almost completely. Using substrates that can serve either as a donor or as an acceptor but not both, we could identify one of the sites as the acceptor and the other as the donor site.

Highlights

  • Interferons are potent cytokines with anti-viral and cell growth modulating properties [1, 2]

  • In our continuing effort to understand the mechanism of catalysis by these enzymes, we used photo affinity cross-linking and peptide mapping to identify the substrate-binding sites of the P69 isozyme of human 2؅-5؅oligoadenylate synthetases

  • The radiolabeled protein was digested with trypsin, and two ligand-cross-linked peptides were purified by immobilized aluminum affinity chromatography followed by reverse phase high pressure liquid chromatography

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Summary

The abbreviations used are

2–5(A), 2Ј-5Ј-oligoadenylate; ds, doublestranded; ApppA, P1,P3-di(adenosine-5Ј) triphosphate; IAAC, immobilized aluminum affinity chromatography; HPLC, high pressure liquid chromatography; Wt, wild type. Because dimeric 2–5(A)s cannot activate RNase L, the biological role of P100 remains elusive Another difference between the three size classes of isozymes is their oligomeric protein compositions. We have previously identified specific residues in the carboxyl termini of the small and the medium synthetases, which are required for their oligomerization [12, 13]. We have used photo affinity cross-linking of a 2–5(A) analogue followed by peptide mapping in conjunction with point mutagenesis to identify two substrate-binding sites of the P69 isozyme. These sites are conserved in other isozymes as well

MATERIALS AND METHODS
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