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The RNA Binding Domain of Ribosomal Protein L11: Three-Dimensional Structure of the RNA-Bound Form of the Protein and Its Interaction with 23 S Rrna

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The RNA Binding Domain of Ribosomal Protein L11: Three-Dimensional Structure of the RNA-Bound Form of the Protein and Its Interaction with 23 S Rrna J. Mol. Biol. (1997) 274, 101±113 The RNA Binding Domain of Ribosomal Protein L11: Three-dimensional Structure of the RNA-bound Form of the Protein and its Interaction with 23 S rRNA AndrewP.Hinck1,MichelleA.Markus1,ShengrongHuang1 StephanGrzesiek2,IrinaKustonovich3,DavidE.Draper4 andDennisA.Torchia1* 1Molecular Structural The three-dimensional solution structure has been determined by NMR Biology Core, National Institute spectroscopy of the 75 residue C-terminal domain of ribosomal protein of Dental Research, National L11 (L11-C76) in its RNA-bound state. L11-C76 recognizes and binds Institutes of Health, Bethesda tightly to a highly conserved 58 nucleotide domain of 23 S ribosomal MD, 20892-4326, USA RNA, whose secondary structure consists of three helical stems and a central junction loop. The NMR data reveal that the conserved structural 2Forschungszentrum Juelich core of the protein, which consists of a bundle of three a-helices and a Juelich, Germany two-stranded parallel b-sheet four residues in length, is nearly the same 3Hebrew University, Jerusalem as the solution structure determined for the non-liganded form of the Israel protein. There are however, substantial chemical shift perturbations 4 which accompany RNA binding, the largest of which map onto an Department of Chemistry extended loop which bridges the C-terminal end of a-helix 1 and the ®rst The Johns Hopkins University strand of parallel b-sheet. Substantial shift perturbations are also Baltimore, MD, USA observed in the N-terminal end of a-helix 1, the intervening loop that bridges helices 2 and 3, and a-helix 3. The four contact regions identi®ed by the shift perturbation data also displayed protein-RNA NOEs, as identi®ed by isotope-®ltered three-dimensional NOE spectroscopy. The shift perturbation and NOE data not only implicate helix 3 as playing an important role in RNA binding, but also indicate that regions ¯anking helix 3 are involved as well. Loop 1 is of particular interest as it was found to be ¯exible and disordered for L11-C76 free in solution, but not in the RNA-bound form of the protein, where it appears rigid and adopts a speci®c conformation as a result of its direct contact to RNA. # 1997 Academic Press Limited Keywords: L11, heteronuclear NMR; protein-RNA; 23 S ribosomal RNA; *Corresponding author ribosome Introduction The C-terminal domain of ribosomal protein L11 interacts speci®cally with a 58 nucleotide domain Abbreviations used: L11-C76, recombinant protein oflargesubunit23SribosomalRNA(Xing& produced in E. coli, whose amino acid sequence Draper,1996).Boththeproteinanditscognate corresponds to the C-terminal 75 residues of Bacillus RNA are highly conserved: each is present among stearothermophilus ribosomal protein L11, with an archaebacterial, prokaryotic, and eukaryotic organ- additional N-terminal methionine residue; fL11-C76, isms and both are known to be components of the designates the form of L11-C76 free in solution; large subunit GTPase center. Reconstituted ribo- bL11-C76, designates RNA-bound form L11-C76 (1:1 somes lacking native L11 synthesize protein two- complex with its target RNA of 58 nucleotides); nt, nucleotide; RMSD, root mean square deviation; ppm, fold more slowly than normal ribosomes, and are parts per million; NOE, nuclear Overhauser defective in elongation factor-G (EF-G) dependent enhancement; EF, elongation factor; 2D, 3D, two and GTPhydrolysis(Stark&Cundliffe,1979)and three-dimensional, respectively; HSQC, Heteronuclear releasefactor-1dependenttermination(Tateetal., single quantum correlation; NOESY, NOE spectroscopy. 1984).NativeL11alsoformspartofthebinding 0022±2836/97/460101±13 $25.00/0/mb971379 # 1997 Academic Press Limited 102 NMR Studies of the Interaction of L11 with 23 S rRNA site for the thiazole family of antibiotics. Such anti- Limitedproteolysisexperiments(Xing&Draper, biotics bind cooperatively with L11 and inhibit 1996) have shown that native L11 consists of two ribosome function by interfering with the inter- functional domains: the N-terminal domain is action of EF-G GTP and EF-Tu aminoacyl responsible for the cooperative binding of L11 and tRNA GTPcomplÁexeswiththelargÁesubunit thiostrepton to RNA, whereas the C-terminal (ThompsoÁ netal.,1979). domain is the RNA binding domain. The disasso- The speci®city of L11 binding for a limited 58 ciation constant measured for the C-terminal nucleotide domain of 23 S rRNA (nt 1051 to 1108, domain of the protein complexed to nt 1029 to Escherichia coli numbering) was ®rst identi®ed by 1126 of 23 S rRNA, as determined by quantitative ribonuclease T1 digests of L11 bound to naked 23 S ®lterbindingassays,is 0.1mM(Xing&Draper, rRNA(Schmidtetal.,1981).Subsequentchemical 1996).TworecentindependentNMRstudies protection studies of the protein-RNA complex (Markusetal.,1997;Xingetal.,1997)haveshown (Egebjergetal.,1990),site-speci®cmutagenesisstu- that the C-terminal 75 residues of recombinant diesoftheRNA(Ryan&Draper,1991;Ryanetal., Bacillus stearothermophilus L11 (which, along with 1991),andanalysesofthethermodynamicsof an N-terminal methionine, is designated L11-C76) unfoldingofboththeRNAbyitself(Laing& folds into a compact structure consisting of a bun- Draper,1994)andtheL11-RNAcomplex(Xing& dle of three a-helices and a short two-stranded par- Draper,1996)stronglysupporttheideathatthe allel b-sheet. Although L11 lacks any detectable RNA adopts a speci®c tertiary structure. The ter- sequence homology with other known nucleic acid tiary structure of the RNA has been shown to be binding proteins, it was noted that the arrange- stabilizedbymono-(Draperetal.,1995;Wangetal., ment of the three helices of L11-C76 free in sol- 1993)anddivalentcations(Laingetal.,1994;Lu& ution (fL11-C76) is strikingly similar to that found Draper,1994),ribosomalproteinL11(Xing& in the homeodomain family of eukaryotic tran- Draper,1996),andtheantibioticthiostrepton scriptionfactors(Xingetal.,1997).Moreover,sev- (Draperetal.,1995;Ryanetal.,1991;Xing& eral conserved residues in helix 3 which appear to Draper,1996). be required for rRNA recognition, align with con- Figure 1. Ribosomal RNA and L11 sequences used for NMR studies and a comparison of L11-RNA and homeodo- main-DNA contact sites. (a) A 58 nucleotide fragment of E. coli 23 S rRNA, modi®ed at position 1061 (E. coli number- ing) by a U to A substitution. Bases which are protected by native L11 in hydroxyl radical footprinting experiments areindicatedbygrayshading(Rosendahl&Douthwaite,1993).(b)AprimarysequencealignmentoftheOct-1 (Klemmetal.,1994)andMAT-a2(Lietal.,1995)homeodomains.Homeodomainresiduesarenumberedaccording theconventionpreviouslyestablished(Lietal.,1995).Thehelicalboundariesandaminoacidresidueswhichcontact theDNA,arethosereportedfortheOct-1(Klemmetal.,1994)andMAT-a2(Lietal.,1995)homeodomain-DNAcom- plexes, respectively. The three helical regions are indicated symbolically above the amino acid sequences, whereas the protein-DNA contact sites are identi®ed by residue shading. Residues shaded black correspond to those which engage in base-speci®c contacts, whereas those shaded gray correspond to those which exhibit either phosphate or ribose contacts. (c) Primary sequence, deduced secondary structure,and sites of protein-RNA contacts for the C-term- inal fragment (75 residues plus N-terminal initiator methionine) of Bacillus stearothermophilus L11. The secondary structure is indicated schematically above the amino acid sequence, whereas the protein-RNA contact sites are indi- cated by residue shading. The latter were identi®ed on the basis of ®ltered NOE experiments, as described in Materials and Methods. NMR Studies of the Interaction of L11 with 23 S rRNA 103 served residues in homeodomain helix 3 which one of the four internal methionine residues. The engageinbase-speci®cDNAcontacts(Xingetal., latter conclusion follows from the observation that 1997).Inordertoprovidemoredetailedstructural we were able to assign a number of backbone and information as to the extent of protein-RNA inter- side-chain 1H and 13C side-chain resonances for actions, and to investigate the effects of RNA bind- residues T2 and F3. These reside in the highly ¯ex- ing on protein structure and dynamics, we have ible N-terminal sequence, as assessed by backbone used NMR spectroscopy to study the 27 kDa com- 15Nrelaxationmeasurements(seeFigure5(c), plex of L11-C76 with its 58 nucleotide binding site below), and this accounts for their rapid amide in23SrRNA(Figure1).Herein,wereportnearly exchange. Side-chain 1H, 13C, and 15N assignments complete backbone and side-chain sequential were generally complete, although there were a assignments, the three-dimensional structure for few exceptions involving Lys Ce/He, Ile Cg1/Hg1, the protein component of the L11-RNA complex, and Leu Cg/Hg resonances. and the sites on L11-C76 that interact with the The secondary structure of the protein was 58 nt RNA target. deduced from the pattern of short and medium 3 range NOE data, JHNHa coupling constants, and hydrogen-exchange rate constants, as summarized Results and Discussion inFigure3(a).Aconsensusofallindicesindicates the presence of three -helical regions and two Assignments and secondary structure a b-strands. The former was supported by small (<6 Hz) 3J couplings, strong d (i,i 1), Triple-resonance methodology was used to HNHa N-N obtain nearly complete sequential backbone and weak da-N(i,i 1), observable da-N(i,i 3) NOE side-chain resonance assignments for bL11-C76 connectivities, and contiguous stretches of slowly using samples in which the protein
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