Abstract
RNase L is activated by the binding of unusual 2',5'-linked oligoadenylates (2-5A) and acts as the effector enzyme of the 2-5A system, an interferon-induced anti-virus mechanism. Efforts have been made to understand the 2-5A binding mechanism, not only for scientific interests but also for the prospects that the understanding of such mechanisms lead to new remedies for viral diseases. We have recently elucidated the crystal structure of the 2-5A binding ankyrin repeat domain of human RNase L complexed with 2-5A. To determine the contributions of amino acid residues surrounding the 2-5A binding site, point mutants and a deletion mutant were designed based on the crystal structure. These mutant proteins were analyzed for their interaction with 2-5A using a steady-state fluorescence technique. In addition, full-length RNase L mutants were tested for their activation by 2-5A. The results reveal that pi-pi stacking interactions of Trp60 and Phe126, electrostatic interactions of Lys89 and Arg155, and hydrogen bonding by Glu131 make crucial contributions to 2-5A binding. It was also found that the crystal structure of the ankyrin repeat domain L.2-5A complex accurately portrays the 2-5A binding mode in full-length RNase L.
Highlights
Interferons are immunomodulatory cytokines that trigger anti-pathogenic and anti-proliferative mechanisms in the cells [1]
RNase L activity is regulated by its N-terminal ankyrin repeat domain, at which 2–5A is recognized and bound [9]
In human RNase L, the ankyrin repeat domain is composed of eight complete and one partial ankyrin motifs, which suppresses the activity of the C terminus ribonuclease domain of RNase L [9, 11, 12]
Summary
Interferons are immunomodulatory cytokines that trigger anti-pathogenic and anti-proliferative mechanisms in the cells [1]. We have recently reported the crystal structure of the ankyrin repeat domain of human RNase L complexed with 2–5A [12, 14] This structure shows a typical ankyrin repeat configuration; i.e. each repeat consists of pairs of anti-parallel ␣-helices stacked side by side that are connected by a series of intervening -hairpin motifs [10]. To determine the contribution of these residues and the extra helix to the binding of 2–5A, we have designed nine point mutants and a deletion mutant of the ankyrin repeat domain protein and full-length RNase L. Biochemical characterization of these mutants allowed us to determine the 2–5A binding determinants of human RNase L
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