title: Structures of the first and second double-stranded RNA-binding domains of human TAR RNA-binding protein.
abstract: The TAR RNA-binding Protein (TRBP) is a double-stranded RNA (dsRNA)-binding protein, which binds to Dicer and is required for the RNA interference pathway. TRBP consists of three dsRNA-binding domains (dsRBDs). The first and second dsRBDs (dsRBD1 and dsRBD2, respectively) have affinities for dsRNA, whereas the third dsRBD (dsRBD3) binds to Dicer. In this study, we prepared the single domain fragments of human TRBP corresponding to dsRBD1 and dsRBD2 and solved the crystal structure of dsRBD1 and the solution structure of dsRBD2. The two structures contain an α-β-β-β-α fold, which is common to the dsRBDs. The overall structures of dsRBD1 and dsRBD2 are similar to each other, except for a slight shift of the first α helix. The residues involved in dsRNA binding are conserved. We examined the small interfering RNA (siRNA)-binding properties of these dsRBDs by isothermal titration colorimetry measurements. The dsRBD1 and dsRBD2 fragments both bound to siRNA, with dissociation constants of 220 and 113 nM, respectively. In contrast, the full-length TRBP and its fragment with dsRBD1 and dsRBD2 exhibited much smaller dissociation constants (0.24 and 0.25 nM, respectively), indicating that the tandem dsRBDs bind simultaneously to one siRNA molecule. On the other hand, the loop between the first α helix and the first β strand of dsRBD2, but not dsRBD1, has a Trp residue, which forms hydrophobic and cation-π interactions with the surrounding residues. A circular dichroism analysis revealed that the thermal stability of dsRBD2 is higher than that of dsRBD1 and depends on the Trp residue.
authors.affiliation: RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.
authors.forename: Seisuke
authors.initials: S
authors.lastname: Yamashita
authors.forename: Takashi
authors.initials: T
authors.lastname: Nagata
authors.forename: Masahito
authors.initials: M
authors.lastname: Kawazoe
authors.forename: Chie
authors.initials: C
authors.lastname: Takemoto
authors.forename: Takanori
authors.initials: T
authors.lastname: Kigawa
authors.forename: Peter
authors.initials: P
authors.lastname: Güntert
authors.forename: Naohiro
authors.initials: N
authors.lastname: Kobayashi
authors.forename: Takaho
authors.initials: T
authors.lastname: Terada
authors.forename: Mikako
authors.initials: M
authors.lastname: Shirouzu
authors.forename: Motoaki
authors.initials: M
authors.lastname: Wakiyama
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Muto
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Yokoyama
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D018360:Crystallography, X-Ray; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D055503:Protein Multimerization; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012330:RNA, Double-Stranded / Q000737:chemistry*; D034741:RNA, Small Interfering / Q000737:chemistry; D016601:RNA-Binding Proteins / Q000737:chemistry*; D016415:Sequence Alignment; D000099253:Trans-Activation Responsive RNA-Binding Protein
chemical_list: D012330:RNA, Double-Stranded; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D000099253:Trans-Activation Responsive RNA-Binding Protein