title: Functional characterization of 2',5'-linked oligoadenylate binding determinant of human RNase L.
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.
authors.affiliation: Department of Biomolecular Science, Faculty of Engineering, Gifu University, Yanagido 1-1, Gifu 501-1193, Japan.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Nakanishi
authors.forename: Nobutada
authors.initials: N
authors.lastname: Tanaka
authors.forename: Yasuaki
authors.initials: Y
authors.lastname: Mizutani
authors.forename: Masahiko
authors.initials: M
authors.lastname: Mochizuki
authors.forename: Yoshihito
authors.initials: Y
authors.lastname: Ueno
authors.forename: Kazuo T
authors.initials: KT
authors.lastname: Nakamura
authors.forename: Yukio
authors.initials: Y
authors.lastname: Kitade
mesh_terms: D000227:Adenine Nucleotides / Q000737:chemistry*; D001120:Arginine / Q000737:chemistry; D018360:Crystallography, X-Ray; D004305:Dose-Response Relationship, Drug; D004722:Endoribonucleases / Q000737:chemistry / Q000235:genetics*; D004789:Enzyme Activation; D017353:Gene Deletion; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D006860:Hydrogen Bonding; D008239:Lysine / Q000737:chemistry; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry; D009843:Oligoribonucleotides / Q000737:chemistry*; D010649:Phenylalanine / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D014364:Tryptophan / Q000737:chemistry
chemical_list: D000227:Adenine Nucleotides; D009841:Oligonucleotides; D009843:Oligoribonucleotides; D010649:Phenylalanine; C023505:2',5'-oligoadenylate; D014364:Tryptophan; D001120:Arginine; D005982:Glutathione Transferase; D004722:Endoribonucleases; C031956:2-5A-dependent ribonuclease; D008239:Lysine