2-5A induces a conformational change in the ankyrin-repeat domain of RNase L.
RNase L is responsible for the 2-5A host defense system, an RNA degradation pathway present in cells of higher vertebrates that functions in both the antiviral and anticellular activities of interferon. The activity of RNase L is tightly regulated and is exerted only in the presence of 2-5A. The postulated mechanism of its regulation is as follows: the N-terminal half ankyrin-repeat domain masks the C-terminal half nuclease domain in the absence of 2-5A. On binding 2-5A at the ankyrin-repeat domain, RNase L forms a homodimer and removes the ankyrin-repeat domain from the nuclease domain to become the active form. A conformational change in the ankyrin-repeat domain is a key step in this hypothetical mechanism, but there is as yet no evidence for such a change. To clarify the events induced by 2-5A binding, we established procedures for expression and purification of the ankyrin-repeat domain of human RNase L. Fluorescence spectra of the protein showed clear difference in the presence and absence of 2-5A. The alterations in the spectra supported conformational changes of the protein. Time-resolved anisotropy measurements indicated that 2-5A binding led to a significant decrease in the rotational radius of the protein. In addition, 2-5A provided the domain with resistance to protease digestion as a result of a conformational change. These results indicated that the ankyrin-repeat domain of RNase L constricts its structure by binding of 2-5A. This observation suggests a revised model of the 2-5A-induced activation of RNase L.
Nakanishi
Masayuki
M
Laboratory of Molecular Biochemistry, Department of Biomolecular Science, Faculty of Engineering, Gifu University, Gifu, Japan.
Goto
Yoshikuni
Y
Kitade
Yukio
Y
D000227:Adenine Nucleotides / Q000737:chemistry* / Q000378:metabolism; D017089:Ankyrin Repeat / Q000235:genetics*; D000970:Antineoplastic Agents / Q000737:chemistry / Q000378:metabolism; D000998:Antiviral Agents / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D004722:Endoribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D005452:Fluoresceins / Q000737:chemistry; D005454:Fluorescence Polarization; D006801:Humans; D008954:Models, Biological; D009843:Oligoribonucleotides / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation / Q000187:drug effects; D017510:Protein Folding; D017434:Protein Structure, Tertiary / Q000187:drug effects* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000227:Adenine Nucleotides; D000970:Antineoplastic Agents; D000998:Antiviral Agents; D005452:Fluoresceins; D009843:Oligoribonucleotides; D011994:Recombinant Proteins; C023505:2',5'-oligoadenylate; C015296:5-iodoacetamidofluorescein; D004722:Endoribonucleases; C031956:2-5A-dependent ribonuclease