L11 domain rearrangement upon binding to RNA and thiostrepton studied by NMR spectroscopy

Published online 14 December 2006 Nucleic Acids Research, 2007, Vol. 35, No. 2 441–454 doi:10.1093/nar/gkl1066 L11 domain rearrangement upon binding to RNA and thiostrepton studied by NMR spectroscopy Hendrik R. A. Jonker1, Serge Ilin1, S. Kaspar Grimm1,2, Jens Wöhnert1,2,* and Harald Schwalbe1,* 1 Johann Wolfgang Goethe-University, Institute for Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic Resonance, Max-von-Laue-Strasse 7, 60438 Frankfurt am Main, Germany and 2University of Texas Health Science Center SA, Department of Biochemistry, 7703 Floyd Curl Drive, San Antonio, TX 78229, USA Received October 11, 2006; Revised and Accepted November 20, 2006 ABSTRACT INTRODUCTION The ribosome is a large ribonucleoprotein complex that translates the genetic code from mRNA into a polypeptide *To whom correspondence should be addressed. Tel: +69 7982 9737; Fax: +69 7982 9515; Email: *Correspondence may also be addressed to Jens Wöhnert. Tel: +1 210 567 3743; Fax: +1 210 567 6595; Email: Present address: Serge Ilin, Sloan-Kettering Institute, Structural Biology Program, 1275 York Avenue, New York, NY 10021, USA  2006 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Ribosomal proteins are assumed to stabilize specific RNA structures and promote compact folding of the large rRNA. The conformational dynamics of the protein between the bound and unbound state play an important role in the binding process. We have studied those dynamical changes in detail for the highly conserved complex between the ribosomal protein L11 and the GTPase region of 23S rRNA. The RNA domain is compactly folded into a well defined tertiary structure, which is further stabilized by the association with the C-terminal domain of the L11 protein (L11ctd). In addition, the N-terminal domain of L11 (L11ntd) is implicated in the binding of the natural thiazole antibiotic thiostrepton, which disrupts the elongation factor function. We have studied the conformation of the ribosomal protein and its dynamics by NMR in the unbound state, the RNA bound state and in the ternary complex with the RNA and thiostrepton. Our data reveal a rearrangement of the L11ntd, placing it closer to the RNA after binding of thiostrepton, which may prevent binding of elongation factors. We propose a model for the ternary L11–RNA– thiostrepton complex that is additionally based on interaction data and conformational information of the L11 protein. The model is consistent with earlier findings and provides an explanation for the role of L11ntd in elongation factor binding. chain during protein synthesis. Detailed structural information is presently known that reveals the two subunit complex structure of the RNA and the associated proteins, resulting in a wealth of information about protein–RNA interactions (1–6). Various Cryo-EM and X-ray structures are currently available of the ribosome trapped in different states of the translation process, including mRNAs, tRNAs, translation factors, release factors and antibiotics (7–21). Some regions in the molecular structure turned out to be rather flexible and at the same time are possible targets for antibiotics and are involved in regulation. Studies of these individual regions by high-resolution structural techniques largely contribute to understanding the dynamical properties and mechanisms of the proteins involved in ribosomal protein synthesis. The complex between the ribosomal protein L11 and the 23S rRNA domain is an essential part (22,23) of the ribosomal GTPase-associated region (GAR). L11 is a highly conserved two-domain protein and has a specific role both in EF-G dependent GTP hydrolysis and in release factor 1 (RF-1) dependent termination (24,25). The C-terminal domain of L11 (L11ctd) is primarily involved in binding to a well-conserved 58 nt sequence in the GAR region. This ribosomal RNA region shows a compact fold, which is stabilized by extensive tertiary contacts (26,27). An essential monovalent ion-binding-site must be occupied for the RNA to fold (28) and Mg2+, which is essential under most conditions, can be replaced by high concentrations of monovalent ions (29,30). Biochemical experiments have shown that the RNA structure is further stabilized by the presence of L11ctd (31–33). The structures of full-length L11 and L11ctd in its free form have been solved by NMR (34–36). Moreover, the structure of L11 in complex with its cognate RNA has been characterized by NMR for L11ctd and solved by X-ray crystallography for L11ctd as well as full-length L11 (26,27,37). Cryo-EM and X-ray experiments show that the location of the N-terminal domain of L11 (L11ntd) differs upon binding of EF-G (38), the release factors 1 and 442 Nucleic Acids Research, 2007, Vol. 35, No. 2 MATERIALS AND METHODS Sample preparation The L11 protein (1–141 from Thermotoga maritima) was prepared essentially as described before (36). Labeled (15N) protein was obtained by over-expression in Escherichia coli strain BL21(DE3) using 0.5 g/l 15NH4Cl (CIL) and 4.0 g/l 12 C-glucose in minimal media. Triple labeled (2D,15N,13C) L11 was obtained using stable isotope labeled OD2 CDN media (Silantes). The RNA fragment corresponds to 1050–1109 nt of E.coli 23S rRNA (57). The 60 nt RNA sequence (50 -GGGCAGGAUGUAGGCUUAGAAGCAGCCAUCAUUUAAAGAA AGCGUAAUAGCUCACUGCCC-30 ) differs slightly from E.coli in the last four 30 and 50 nt and by a single base substitution, U1061A, to stabilize the tertiary structure (58). Unlabeled RNA was prepared by in vitro transcription with T7 RNA polymerase from linearized plasmid DNA templates (59). The unlabeled rNTPs were purchased from Sigma. The DNA template consisted of a T7 promoter region followed by the RNA coding sequence and a SmaI restriction site overlapping with the 30 end of the coding sequence. The pUC19-plasmid containing the appropriate insert was amplified in E.coli strain DH5a and purified using a QiagenMega purification kit. The plasmid was linearized with SmaI and subsequently purified. The in vitro transcription for production of the RNA was performed for 4 h at 37 C in 30 ml [200 mM Tris–Glutamic acid (pH 8.1), 20 mM 1,4-DTT, 2 mM spermidine, 40 mM Mg(OAc)2, 5 mM of rNTP mixture, 50 mg/ml DNA template and 50 mg/ml T7 RNA polymerase]. The RNA was purified on a diethylaminoethyl (DEAE) sepharose fast flow column (APB) developed with a sodium acetate buffer step gradient (0–3 M, pH 5.5). The RNA in the selected fractions (denaturing PAA gels) was precipitated with 4· vol of ethanol overnight at 20 C. The air dried pellet (centrifugation in a Heraeus #7588 rotar for 30 min at 9000 g, 4 C) was dissolved in water to a concentration of 150 OD260/ml and purified by h (...truncated)