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2‐5A induces a conformational change in the ankyrin‐repeat domain of RNase L
Author(s) -
Nakanishi Masayuki,
Goto Yoshikuni,
Kitade Yukio
Publication year - 2005
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.20474
Subject(s) - ankyrin repeat , conformational change , rnase p , nuclease , ankyrin , biophysics , binding domain , rnase h , chemistry , microbiology and biotechnology , biochemistry , biology , binding site , rna , dna , gene
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. Proteins 2005. © 2005 Wiley‐Liss, Inc.