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Solution Structure of the GUCT Domain from Human RNA Helicase II/Gu[Beta]

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Solution Structure of the GUCT Domain from Human RNA Helicase II/Gu[Beta] proteins STRUCTURE O FUNCTION O BIOINFORMATICS Solution structure of the GUCT domain from human RNA helicase II/Gub reveals the RRM fold, but implausible RNA interactions Satoshi Ohnishi,1 Kimmo Pa¨a¨kko¨nen,1 Seizo Koshiba,1 Naoya Tochio,1 Manami Sato,1 Naohiro Kobayashi,1 Takushi Harada,1 Satoru Watanabe,1 Yutaka Muto,1 Peter Gu¨ntert,1 Akiko Tanaka,1 Takanori Kigawa,1,2 and Shigeyuki Yokoyama1,3* 1 Systems and Structural Biology Center, RIKEN, Tsurumi, Yokohama 230-0045, Japan 2 Department of Computational Intelligence and Systems Science, Interdisciplinary Graduate School of Science and Engineering, Tokyo Institute of Technology, Midori-ku, Yokohama 226-8503, Japan 3 Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan INTRODUCTION ABSTRACT a a a a Human RNA helicase II/Gu (RH-II/Gu or Deadbox Human RNA helicase II/Gu (RH-II/Gu ) and RNA helicase protein 21) is a multifunctional enzyme that unwinds dou- II/Gub (RH-II/Gub) are paralogues that share the same ble-stranded RNA in the 50 to 30 direction and folds single- domain structure, consisting of the DEAD box helicase 1–5 domain (DEAD), the helicase conserved C-terminal domain stranded RNA in an ATP-dependent manner. These (helicase_C), and the GUCT domain. The N-terminal regions RNA-unwinding and RNA-folding activities are independ- of the RH-II/Gu proteins, including the DEAD domain and ent, and they reside in distinct regions of the protein. The the helicase_C domain, unwind double-stranded RNAs. The RNA helicase activity is catalyzed by the N-terminal three- 1 C-terminal tail of RH-II/Gua, which follows the GUCT do- quarters of the molecule in the presence of Mg2 , where as main, folds a single RNA strand, while that of RH-II/Gub the RNA-foldase activity is located in the C-terminal region 1 does not, and the GUCT domain is not essential for either and functions in a Mg2 independent manner.2 As shown the RNA helicase or foldase activity. Thus, little is known in Figure 1(A), RH-II/Gua consists of three conserved about the GUCT domain. In this study, we have deter- b structural domains: the DEAD box helicase domain (DEAD mined the solution structure of the RH-II/Gu GUCT do- domain, Pfam code: PF00270), the helicase conserved C-ter- main. Structural calculations using NOE-based distance minal domain (Helicase_C domain, Pfam code: PF00271), restraints and residual dipolar coupling-based angular restraints yielded a well-defined structure with b-a-a-b-b-a- and the GUCT (NUC152) domain (Gu C-terminal domain, b topology in the region for K585-A659, while the Pfam Pfam code: PF08152) according to the Pfam HMM algo- HMM algorithm defined the GUCT domain as G571-E666. rithm (http://www.sanger.ac.uk/Software/Pfam/). The struc- This structure-based domain boundary revealed false posi- tures of the DEAD and Helicase_C domains are known tives in the sequence homologue search using the HMM (e.g., PDB codes: 1T6N,6 1VEC DOI: 10.2210/pdb1vec/pdb, definition. A structural homology search revealed that the and 3B6E DOI: 10.2210/pdb3b6e/pdb). These domains play GUCT domain has the RRM fold, which is typically found critical roles in the RNA-helicase activity of RH-II/Gua.2,5 in RNA-interacting proteins. However, it lacks the surface- In contrast, the structure of the GUCT domain has not b exposed aromatic residues and basic residues on the -sheet been solved. The GUCT domain is followed by three that are important for the RNA recognition in the canoni- FRGQR repeats and one PRGQR sequence, which are sepa- cal RRM domains. In addition, the overall surface of the rated by five amino acid residues. The 70 residue C-termi- GUCT domain is fairly acidic, and thus the GUCT domain is unlikely to interact with RNA molecules. Instead, it may interact with proteins via its hydrophobic surface around Additional Supporting Information may be found in the online version of this article. the surface-exposed tryptophan. Grant sponsor: The RIKEN Structural Genomics/Proteomics Initiative (RSGI) of the National Project on Protein Structural; Grant sponsor: Functional Analyses, Ministry Proteins 2009; 74:133–144. of Education, Culture, Sports, Science, and Technology of Japan. VC 2008 Wiley-Liss, Inc. *Correspondence to: Shigeyuki Yokoyama, Systems and Structural Biology Center, RIKEN, 1-7-22 Suehiro-Cho, Tsurumi, Yokohama 230-0045, Japan. Key words: RNA helicase; GUCT domain; NMR; residual E-mail: [email protected] Received 29 February 2008; Revised 24 April 2008; Accepted 30 April 2008 dipolar couplings; RRM domain superfamily; structural Published online 9 July 2008 in Wiley InterScience (www.interscience.wiley.com). genomics. DOI: 10.1002/prot.22138 VC 2008 WILEY-LISS, INC. PROTEINS 133 S. Ohnishi et al. Figure 1 Comparative paralogues of human RH-II/Gu. A: Comparison of the domain structures between RH-II/Gua and RH-II/Gub. The regions shown in blue, green, and red, respectively, correspond to the DEAD domain (Pfam code: PF00270), the helicase conserved C-terminal domain (Helicase_C, Pfam code: PF00270), and the GUCT domain (Pfam code: PF08152), according to the prediction by the Pfam HMM definition. B: Sequence comparison between the human RH-II/Gua and RH-II/Gub GUCT domains. The rectangular bars and arrows represent the a-helices and b-strands, conformed to the present NMR structure of the RH-II/Gub GUCT domain. C: Dendrogram of the UniProt entries found by the homologue search using the Pfam HMM profile for the GUCT domain. The human RH-II/Gua and RH-II/Gub GUCT domains are highlighted in blue. nal region, including these repeats, is necessary for the Recently, a paralogue of RH-II/Gua, referred to as RNA-foldase activity, where as the GUCT domain is not, RH-II/Gub and encoded by the human DDX50 gene, has as demonstrated by mutagenesis studies.1,2 Thus, the been identified.7,8 Both proteins localize to nucleoli, sug- functional role of the GUCT domain in this bifunctional gesting roles in ribosomal RNA production, but RH-II/ enzyme, RH-II/Gua, remains unclear. Gub also localizes to nuclear speckles containing the 134 PROTEINS Structure of the RH-II/Gub GUCT Domain splicing factor SC35, suggesting its possible involvement amino acids, with subsequent protein purification as in pre-mRNA splicing.8 These paralogues share a similar described elsewhere.10 The purified protein was concen- domain structure constituted by the DEAD domain, the trated to 0.6 mM in 20 mMd11-Tris–HCl buffer (pH Helicase_C domain, and the GUCT domain [Fig. 1(A)]. 7.0) containing 100 mM NaCl, 1 mM 1,4-DL-dithiothre- 2 The overall sequence identity is 55.6%, and those for the itol-d10 (d10-DTT), 10% H2O, and 0.02% NaN3. DEAD domain, the Helicase_C domain, and the GUCT domain are 76.1%, 87.0%, and 48.5%, respectively [Fig. NMR spectroscopy 1(B)]. Remarkably, the characteristic (F/P)RGQR repeats in the C-terminal tail of RH-II/Gua are replaced by an All spectra were recorded on Bruker Avance 600 and arginine-serine-rich sequence in the C-terminal tail of 800 spectrometers at 298 K, unless otherwise mentioned. RH-II/Gub. As predicted from the sequence comparison, As described previously, resonance assignments were in vitro assays demonstrated that RH-II/Gub has RNA- carried out using a conventional set of triple resonance 10 unwinding activity, although it is weaker than that of spectra. Interproton distance restraints were obtained 15 13 RH-II/Gua, but no RNA-folding activity.8 from N- and C-edited NOESY spectra recorded with A sequence homologue search in the uniprot database a mixing time of 80 ms. All spectra were processed using 11 12 (http://www.pir.uniprot.org/) revealed that the GUCT do- NMRPipe, and the programs KUJIRA and 13 main is unique to the eukaryotic RH-II/Gu proteins. A NMRView were used for optimal visualization and classification analysis using CLUSTAL W9 established spectral analyses. Complete resonance assignments have three primitive classes in the sequence space, with the been deposited in the BioMagResBank (accession code: RH-II/Gua GUCT domain mapped relatively distant 10213). from the RH-IIb GUCT domain in one of the classes [Fig. 1(C)]. RDC measurements Little is known about the structural and functional An aliquot of a Pf1 phage pellet, purchased from aspects of the GUCT domain, which was only defined by ASLA Biotech (Latvia), was washed with water through the characteristic sequence pattern detected by the Pfam repeated ultracentrifugation steps at 90,000 rpm HMM algorithm. In this study, we have determined the (450,000g) and 48C for 90 min, using a Hitachi himac solution structure of the human RH-II/Gub GUCT do- CS120GX ultracentrifuge with an S100AT5 angle rotor. main using NOE-based distance restraints and residual The pellet was then suspended in, approximately, twice dipolar coupling-based angular restraints, which allowed the volume of 30 mMd-Tris–HCl buffer (pH 7.0) con- redefinition of the boundary of the GUCT domain with taining 150 mM NaCl, 1.5 mMd -DTT, and 15% 2H O. the structured region. The present structure provides in- 10 2 The suspension was mixed with a freshly prepared formation about homologue classification as well as a 13C/15N-labeled protein solution in the same buffer used possible functional role. for the structural calculation with estimated Pf1 phage and protein concentrations of 10 mg/mL and 0.4 mM, respectively. This protein-liquid crystal sample MATERIALS AND METHODS 2 showed 8.5 Hz H2O quadrupole splitting at 258C, Construct design, expression, and which is indicative of 9 mg/mL Pf1 concentration.14 purification Two-dimensional 1H-15N IPAP HSQC spectra15 with 2.48 Hz/point in the indirect dimension and three- The DNA encoding the human DDX50 gene was dimensional HNCO spectra16 with 16.50 Hz/point in the subcloned by PCR from the human cDNA clone locus carbon dimension were recorded to yield a set of HN-N NM_024045 (UltimateTM ORF Clones, Invitrogen, ID: and Ca-C’ residual dipolar couplings.
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