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 component of exchanging amide protons. The latter was sup- 15 3 the complex was labeled with either N or ported by large (>8 Hz) JHNHa couplings, and 15 13 N/ C, and the RNA component was unlabeled. weak or medium dN-N(i,i 1) and strong Upon completion of the assignments, all amide d -N(i,i 1) NOE connectivities. Evidence support- a 1H-15N correlations, with the exception of the ing pairing of the two strands in a parallel fashion, N-terminal residues M1, T2, and F3, were ident- asshowninFigure3(b),includesseveralHN-Ha i®edinthe2D1H/15NHSQCspectrum(Figure2). and HN-HN long range NOEs (A33 HN ±I72 Ha, The N-terminal methionine residue is missing due V73 HN ±T34 Ha, E75 HN ±K36 Ha, and V73 HN ± to its post-translational cleavage from the recombi- I35 HN) and slowly exchanging amide protons nant protein product, whereas the amides of T2 (I35, V73, and E75). The regions of secondary struc- and F3 are missing owing to rapid exchange of ture, as identi®ed by the chemical shift index their amide protons with solvent. The former con- (Wishart&Sykes,1994),wereincompleteagree- clusion was based on the observation that (a) only ment with the secondary structure identi®ed using four of the ®ve methionine He/Ce resonances were the criteria described above. There were, however, identi®ed in a 2D constant time HSQC spectrum three additional short b-strands predicted by the (Vuister&Bax,1992),and(b)thateachofthese index, I4-T7, I20-G23, and A53-S55, which are not four methyl peaks was sequentially assigned to generally supported by the J-coupling, hydrogen
Figure2.Two-dimensional 1H/15N HSQC spectrum of bL11-C76. The peaks are labeled by their assign- ments, with the one-letter amino acid code and their position in the amino acid sequence. The arginine He/Ne and Hz/Nz resonances, are singly and doubly folded into the spectrum, respectively. Their true chemical shifts are their apparent chemical shifts minus 30.2 ppm and 60.4 ppm, respectively. 104 NMR Studies of the Interaction of L11 with 23 S rRNA
Figure 3. Secondary structure of bL11-C76. (a) Second- ary structure as deduced from sequential and short 3 range NOE connectivities, hydrogen exchange, JHNHa couplings,andthechemicalshiftindex(Wishart& Sykes,1994).Hydrogenexchange:openboxesindicate residues whose backbone amides could be observed at 0.27 hour, half-®lled boxes correspond to those which could be detected at 1.53 hours, and ®lled boxes corre- spond to those which could be observed longer than 1.53 hours. Three-bond HN-Ha couplings: open boxes 3 correspond to JHNHa < 6.0 Hz, half-®lled boxes corre- 3 spond to 6.0 Hz < JHNHa < 8.0 Hz, and ®lled boxes 3 correspond to JHNHa > 8.0 Hz. Sequential NOE connec- tivities (dN-N(i,i 1) , da-N(i,i 1) , db-N(i,i 1)) are classi®ed according to relative intensities, the bar height corre- sponding to increasing NOE intensity. Short range NOE connectivities are indicated by horizontal bars which link the ®rst residue involved in the crosspeak to the second. Chemical shift index (CSI): Ca,Cb,CO, and Ha consen- sus values are shown, upward boxes corresponding to b-conformation, downward boxes corresponding to a-confor- mation, and other regions corresponding to coil. The secondary structure of bL11-C76, as deduced by the program PROCHECK(Laskowskietal.,1993),isdiagrammedonthelowerpartoftheFigure.(b)Adiagramaticillustrationof the parallel b-sheet of bL11-C76. Sequential and long range NOE connectivities that have been identi®ed are indicated by double-ended arrows; the single broken arrow corresponds to an expected NOE connectivity which could not be veri®ed experimentally owing to resonance overlap. The horizontally broken lines correspond to hydrogen bonds that have been included in the calculation of the bL11-C76 structures.
exchange, and NOE data. Although the origin of the experimental restraints (26/50) is shown in this discrepancy is not fully understood, one poss- Figure4(a).Thesetofcalculatedstructuresshowed ible source is the fact that these regions adopt gen- good covalent geometry and low overall energies; erally extended, but non-regular secondary a summary of the statistics describing these struc- structures. turesisprovidedinTable1.Thestructureshave been deposited with the Protein Data Bank (Brook- Solution structure of bL11-C76 haven Natiotional Laboratory, Broookhaven, NY, energy minimized average PDB code 1FOY, The solution structure for bL11-C76 was calcu- lated using the distance geometry/simulated ensemble of accepted structures PDB code 2FOW). annealing protocol as implemented in X-PLOR 3.8. A ribbon diagram depicting the energy minimized The input data for the calculation, summarized in averagestructureisshowninFigure4(b)and Table1,consistedofatotalof684interresidue reveals that the overall structure consists of a bun- NOE-distance and dihedral angle restraints. The dle of three a-helices and a two-stranded parallel ensemble of calculated structures consistent with b-sheet four residues in length. NMR Studies of the Interaction of L11 with 23 S rRNA 105
Table 1. Distance and dihedral restraint statistics for the the length of the protein backbone. In contrast, the structure of bL11-C76 calculated structures reveal elevated RMSD values A. Restraint summary intheN-terminalsegment(M1toK6)andinloop Total 684 1(I20toN30)(Figure5(a)).ResiduesM1toK6of Total NOE distance restraints 545 bL11-C76 provide a clear example of disorder cor- Intraresidue (i j 0) 0 related with ¯exibility: the ill-de®ned conformation Sequential (|i j| 1) 241 of this segment is a direct consequence of its Short range (1 < |i j| 4 5) 149 Longrange(|i j| >5)155 internal ¯exibility and a lack of NOE restraints Totalhydrogenbon drestraintsa46 whichde®neitsconformation(Figure5(b)).Resi- Total dihedral restraints 60 dues I20 to N30 provide an example of disorder f 47 uncorrelated with ¯exibility: the precision of this w1 13 Acceptedstructuresb26/50 part of the structure is limited by the fact that only short-range and intraresidue intraprotein NOEs are B. RMSD from idealized covalent geometry and experimental restraints observed between residues 26 and 30. However, as c c will be seen, several loop 1 residues have NOEs to bL11-C76 r {bL11-C76} Bonds (AÊ )h 0.002i 0.002 0.0001 the RNA. Angles ( ) 0.57 0.60 Æ 0.01 Æ Impropers () 0.44 0.40 0.01 Distance restraints (AÊ ) 0.019 0.025 Æ 0.002 Comparison to L11-C76 free in solution Æ Angle restraints () 0.000 0.05 0.08 3 Æ JHNHarestraints(Hz)0.440.51 0.03 The secondary structure of bL11-C76, as d Æ FNOE(kcal/mol) 11.018.2 2.9 deducedbyPROCHECK(Laskowskietal.,1993), e Æ FDihedral(kcal/mol) 0.000.02 0.08 is in good agreement with that found for L11- F (kcal/mol)f6.38.5Æ1.1 HNHa Æ C76freeinsolution(fL11-C76)(Markusetal., a Includes two restraints per hydrogen bond. 1997),althoughthereareminordifferencesinvol- b Acceptance criteria: NOE distance violations < 0.3 AÊ , dihe- 3 ving the end points of the helix 1, helix 3, and dral angle violations < 3, and JHNHa coupling viola- tions < 1.5 Hz. the two b-strands. Helix 1 extends from A10 to c {bL11-C76} is the ensemble of 26 accepted structures;
Figure 4. NMR solution structures of bL11-C76. (a) A stereoview of the ensemble of 26 accepted structures, superim- posed on the basis of the deduced secondary structure (residues 10 to 17, 33 to 46, 56 to 67, 72 to 75). Only backbone heavyatomsareshown.(b)AMOLSCRIPT(Kraulis,1991)ribbondiagramoftheenergy-minimizedaveragebL11- C76 structure in which the regular elements of secondary structure have been identi®ed. distance restraints involving residues 24, 25, 31, etal.,1997).Loop1wasshowntobe¯exiblefor and 32, which are present in the bL11-C76 fL11-C76 with internal motions occurring on two restraint data set. timescales: slow (milliseconds to seconds) motions Interestingly, fL11-C76 differs from bL11-C76 in corresponding to cis:trans isomerization of the that the disorder among the calculated structures E26±P27 peptide bond, superimposed upon clearly correlates with internal ¯exibility for both much faster segmental motions (ns to ps) of the the N-terminal segment from M1 to T7, as well loopbackbone(Markusetal.,1997).Another astheloop1segmentfromG19toV32(Markus major difference between the free and RNA- bound forms of L11-C76 is in the apparent lack of conformational heterogeneity involving cis:trans isomerization of the E26±P27 prolyl peptide bond. Evidence supporting this contention fol- lows from the observation that there were no unassigned peaks of signi®cant intensity corre- sponding to the second isomer present in the 2D 1H/15N HSQC spectrum of bL11-C76. Attempts to identify such peaks by collecting very high sig- nal-to-noise 2D 1H/15N HSQC of bL11-C76 revealed several weak peaks in the random coil region of the spectrum, the strongest of which was less than 6% of the average HN/NH peak intensity of bL11-C76. Additionally, P8, P9, and P27 have been identi®ed as having a trans pep- tide bond on the basis of strong Xaa Ha ±Pro Hd NOEs and the lack of Xaa Ha ±Pro Ha NOEs. The former NOE is characteristic of the a trans prolyl peptide bond, whereas, the latter is characteristic ofacisprolylpeptidebond(Hincketal.,1993). (For P49, it was not possible to de®nitively ident- Figure 5. Disorder and ¯exibility of bL11-C76. (a) Resi- ify either Xaa Ha ±Pro Hd or Xaa Ha ±Pro Ha due RMSD values for the ensemble of 26 accepted struc- NOEs). Taken together, these data indicate that tures versus the mean structure. (b) The number of NOE loop 1 becomes ordered upon binding to its cog- distance restraints on a per-residue basis. (c) Backbone 15 nate RNA. There are two limiting models that N transverse relaxation times for bL11-C76 at 42C in 2 could explain the ordering: indirect effects buffer consisting of 95% H2O/5% H2O with 10 mM Na2HPO4, 25 mM KCl (pH 6.1). The data were recorded induced by RNA binding, or direct effects that at a ®eld strength of 11.7 T. are the result of loop 1 contacts to the RNA. NMR Studies of the Interaction of L11 with 23 S rRNA 107
Table 2. Coordinate RMSD of bL11-C76 All Ordered Secondary Helices StructuresabbbHeavybbHeavybbHeavybbHeavy {bL11-C76} versus bL11-C76 2.26 2.84 0.64 1.24 0.50 1.15 0.48 1.20 h i bL11-C76 versus bL11-C76 r 1.14 1.49 0.56 0.79 0.46 0.70 0.44 0.72 hbL11-C76i versush fL11-C76i 3.06 4.00 1.74 2.58 1.26 1.97 1.13 1.88 h ir h ir In units of AÊ . a {bL11-C76} is the ensemble of 26 accepted structures; bL11-C76 is the average of {bL11-C76} bL11-C76 is obtained by h i h ir restrainedminimizationoftheaverageof{bL11-C76}; fL11-C76 r corresponds to the energy minimized average structure for fL11-C76(Markus,etal,1997;PDB1FOW). h i b bb includes the backbone atoms, NH Ca, and CO; heavy includes all non-hydrogen atoms; All includes residues 1 to 76, ordered includes 10 to 20, 31 to 50, 57 to 76, secondary structure includes 10 to 18, 33 to 36, 37 to 48, 57 to 68, 72 to 75, and helices includes 10 to 18, 37 to 48, and 57 to 68.
NMR evidence supporting the latter model is pre- regions of the structure: the N-terminal portion of sented below. a-helix 1 (T7 to A11), loop 1 (I20 to A33), the C-terminal end of a-helix 2 and loop 2 (L46 to Chemical shift perturbations and A54), and throughout a-helix 3 and into the turn protein:RNA NOEs linking a-helix 3 and the second b-strand (A58 to G71). The chemical shift is sensitive to small pertur- Such perturbations can be broadly classi®ed as bations in local geometry and conformation, the being caused by a direct interaction with the RNA, anisotropy of nearby magnetic ®elds, and local by structural changes that occur within the protein electrostatics(Old®eld,1995),andhastherefore upon RNA binding, or both. Because of the latter proven to be a useful tool for monitoring the effect, chemical shift perturbations must be inter- effects of ligand binding and conformational changes that occur within biological macromol- ecules. A comparison of the 2D 1H/15N HSQC spectrum of bL11-C76 with that of fL11-C76 showed that the binding to RNA has a major in¯u- enceonthechemicalshiftsoftheprotein(Markus etal.,1997).Herein,wehaveextendedsuchacom- parison to include Ha,HN,Ca,Cb,Co, and NH shift perturbations, several examples of which are pre- sentedinFigure7,andusedthesetocalculatea composite shift perturbation index (bottom panel). The largest shift perturbations fall within four
Figure 7. Chemical shift differences between the RNA- bound form of the protein, described here, with those Figure 6. A comparison of the backbone Ca traces for previously reported for L11-C76 free in solution fL11-C76(Markusetal.,1997)tobL11-C76.Thestruc- (Markusetal.,1997).Panels,fromtoptobottom,include tures have been superimposed on the basis of the backbone 15N, Ca,Cb, and Ha shift perturbations, deduced secondary structure of bL11-C76 (residues 10 to respectively, where the chemical shift difference (Ád) is 17, 33 to 46, 56 to 67, and 72 to 75). bL11-C76 is indi- calculated by subtracting shift values for the free form cated by a continuous line, whereas, fL11-C76 is indi- of the protein from those of the RNA-bound form of the cated by a broken line. Residues are numbered protein. The bottom panel includes the composite back- according to the starting and ending points of the regu- bone 15N, CO,Ca,Cb,HN, and Ha shift perturbations, as lar secondary structure of bL11-C76. described in Materials and Methods. 108 NMR Studies of the Interaction of L11 with 23 S rRNA
Table 3. A summary of bL11-C76±RNA NOEs identi®ed in 13C-®ltered NOE spectra Chemical shift Protein group RNA protons (ppm)
A10 Hb 4.56, 5.88 A11 Hb 3.84, 4.47 S24 HN 6.16 E26 Ha 6.30 d d P27 H ,H 0 6.30 L51 Hd 4.32, 4.42, 6.17 d2 d2 N52 H ,H 0 5.50, 5.76 A53 Ha 4.09, 4.75 A53 Hb 4.32 A54 HN 4.08, 4.74 b Figure 8. Correspondence of chemical shift pertur- A54 H 4.09, 4.71 bations that occur upon RNA binding with protein- A58 Hb 4.15, 4.33, 4.65 a RNA intermolecular NOEs that have been identi®ed in M62 H 5.90 13 M62 He 4.34, 5.72, 6.17, 7.30 C-®ltered NOE experiments. (a) Composite chemical T66 HN 5.91 shift perturbations, calculated as described in Materials T66 Hg2 5.88 and Methods, mapped onto a ribbon diagram of the d d R68 H ,H 0 4.52, 5.84 bL11-C76 structure. The magnitude of the shift pertur- S69 HN 6.37 bation is indicated by the differential shading of the pro- M70 HN 6.35 tein backbone; the smallest shift perturbations are in M70 Ha 4.38, 4.58, 6.03 b b blue, intermediate perturbations are in white, and the M70 H ,H 0 4.37 largest perturbations are in red. (b) A schematic depic- M70 He 3.84, 4.37 tion of those parts of the bL11-C76 structure which were The Hd protons of P27 are non-degenerate and are designated shown to display NOE contacts to the its cognate RNA. d d as P27 H and P27 H 0; each displays an NOE to RNA at Those residues which displayed protein-RNA NOEs are 6.3 ppm. 13 13 indicated in a pink color, whereas those which did not Two 3D C-®ltered NOE experiments were acquired: C- are indicated in blue. (a) and (b) were both generated selected in F1 with 13C ®ltering in F3 and 15N-selected in F1 with 13C-®ltering in F3. withtheprogram,GRASP(Nichollsetal.,1991). preted carefully because RNA-induced structural changes are not necessarily relegated to sites of The other protein residues that have NOEs to direct protein-RNA contact. The structural com- the RNA are S24, E26, and P27 in loop 1. Thus, parison of bL11-C76 and fL11-C76 presented herein residues located near the center of loop 1 associate shows that there are minimal structural differences intimately with target RNA. Moreover, the dra- in the ordered regions of the sequence, P8 to G19 matic loop 1 structural and dynamic changes and K31 to D76. Hence, the shift perturbations detailed earlier, appear to be the direct result of the observed for T7 to A11, L46 to A54, and A58 to interaction of L11-C76 with its target RNA, not G71 likely arise from direct contacts with the RNA. merely an indirect structural/dynamic change that This conclusion was con®rmed by 13C-®ltered/13C- accompanies RNA binding. In regards to the large or 15N-selected NOESY experiments which shift perturbations which occur within loop 1, two strongly select for protein-RNA NOEs on samples effects appear to predominate: one related to struc- containing uniformly 15N/13C labeled protein and tural perturbations which occur within the loop, unlabeledRNA(Folkersetal.,1993).Atotalof40 and another related to electrostatic and ring-cur- unique protein-RNA NOEs have been assigned rent effects of RNA binding. usingthisapproach;alistingisprovidedinTable3. Protein residues having NOEs to RNA include A10 The role of helix 3 in RNA binding and A11 in the N-terminal end of -helix 1, L51, a Previous structural work on fL11-C76 revealed N52, A53, and A54 in loop 2, A58, M62, and T66 in the interesting structural similarity between the a-helix 3, and R68, S69, and M70 in loop 3. The homeodomain family of eukaryotic helix-turn-helix datapresentedinFigure8showthatthereisgood transcription factors and the three-helix bundle of agreement between the location of protein-RNA fL11-C76(Markusetal.,1997;Xingetal.,1997).The NOEs and the three regions of the protein whose structure of bL11-C76 is similarly related to the chemical shifts are most strongly affected by RNA homeodomain motif. A structural based search of binding (T7 to A11, L46 to A54 and A58 to G71). It the Protein Data Bank using the DALI algorithm is interesting to note that the shift perturbations (Holm&Sander,1993)revealsthatthethreeheli- and protein-RNA NOEs all map onto the face of cal domains of bL11-C76 superimpose with pair- themoleculewhichincludesa-helix3(Figure8). wise RMSD values of 1.6 and 1.3 AÊ with the Oct-1 This observation is intriguing in light of the fact Pou and MAT-a2 homeodomains, respectively that it has been suggested that helix 3 plays an (Table4).Homeodomainsrecognizetheirtarget important role in mediating protein-RNA contacts DNA sequences speci®cally by positioning helix 3 (Markusetal.,1997;Xingetal.,1997). into the major groove. This arrangement is stabil- NMR Studies of the Interaction of L11 with 23 S rRNA 109
Table 4. Structural alignments of bL11-C76 to homeodomains DALIaThreehelicesbHelix2/helix3c bL11-C76 versus Oct-1 2.68 1.62 0.90 hbL11-C76i versus MAT-a2 2.46 1.34 0.78 h i a Alignment of L11-C76 and homeodomain amino acid sequences on the basis of structural homol- ogy of bL11-C76 with the Oct-1 and MAT-a2 homeodomains, respectively. For the L11-C76±Oct-1 comparison, residues K6 to P9, V12 to K16, A18 to E21, V32 to M48, and A59 to G71 of bL11-C76 align with E9 to I12, V14 to K18, S19 to E22, Q24 to N39, and E41 to R53 of Oct-1, respectively. For the L11-C76-MAT-a2 comparison, residues K6 to P9, V12 to K16, A18 to E21, V32 to M48, and A59 to G71 of bL11-C76 align with residues T9 to N12, R14 to S18, W19 to K22, N23 to S39, and S41 to R53 of MAT-a2, respectively. Numbers reported are pairwise RMSD values in AÊ between backbone atoms of the two structures. b Sequences are aligned as in column 1, but include only residues of the helical domains (resi- dues V12 to K16, R37 to L46, and A59 to A67 of L11-C76). c Sequences are aligned as in column 1, but include only residues of helix 2 and helix 3 (residues R37 to L46 and A59 to A67 of L11-C76). ized by an array of basic residues that interact shiftof7.30ppm(Table3).Onthebasisofchemi- with the phosphate backbone, along with base- cal shifts alone, these are likely to be adenine or speci®c contacts involving a universally conserved guanine H8, adenine H2, or uridine or cytosine H6 Asnatposition51(Figure1(b)).Additionalmajor protons, which would be consistent with the inter- groove base-speci®c contacts that typify most action of a-helix 3 with a distorted major or minor homeodomain-DNA complexes involve residues groove. It is tempting to speculate that the internal found at positions 47, 50, and 54. Additional home- loop of stem 2 leads to distortions in the helical odomain-DNA interactions occur by wrapping the framework, thereby exposing a suitable interaction N-terminal tail into the adjacent minor groove and surface for helix 3 of L11-C76. Several lines of evi- by interacting with the DNA via conserved basic dence, including universal conservation of A1077 residues. and mutagensis studies of U1060 and the U1060- The current NMR data strongly support the U1078mismatch(Lu&Draper,1994;Ryanetal., hypothesis that helix 3 of L11-C76 plays an import- 1991)implicatetheinternalloopasbeingimport- ant role in RNA binding: G65, R68 and S69, all ant for RNA tertiary structure formation. Structural undergo signi®cant shift perturbations in response studies of the RNA component of the complex, to RNA binding, whereas the latter two display which are clearly needed, should provide infor- protein-RNANOEsaswell(Figure1(c)).Moreover, mation regarding the extent to which stem 2 forms the chemical shift perturbations and protein-RNA a helical structure and the details by which it inter- NOEs generally correlate with mutagenesis studies acts with helix 3. oftheprotein(Xingetal.,1997)whichhaveshown that substitution of helix 3 residues G65 and T66 Interactions outside helix 3 are important in by alanine and valine, respectively, lowers the RNA binding binding af®nity for the target RNA by greater than 35-fold. Alanine substitutions for other residues in On the basis of the shift perturbation data and helix 3 and loop 3 have smaller effects on binding, the protein-RNA NOE data, three additional R61A weakens binding 5.0-fold, R68A 2.7-fold, and regions of L11-C76 outside of helix 3 contact the S69A 9.5 fold. These observations highlight the RNA. These include the N-terminal end of helix 1, structural and thermodynamic importance of helix loop 1, and the C-terminal end of helix 2 including 3 residues in RNA binding. loop 2. It is of interest that the ¯exible N-terminal The RNA fragment to which L11-C76 binds is tail of bL11-C76 is not among these. The N termi- composed of three helical stems, one of which has nus is a conserved region of homeodomain a number of sites protected in hydroxyl radical sequences, and is ¯exible in the free homeodomain, footprinting studies of native L11 bound its target but upon binding DNA, it inserts into the minor RNA(Rosendahl&Douthwaite,1993;Figure1(a)). grooveinanorderedconformation(Ottingetal., Although an A-form RNA does not form a suitable 1990). interaction surface for an a-helix, irregular RNA We note that unlike L11-C76, simple three helix helices that result from internal loops, bulge loops, homeodomains generally do not have contact sites and mismatches can accommodate an a-helix, as that fall outside of helix 3, although protein-DNA has been recently suggested by the solution NMR backbone contact sites are sometimes found within structure of the HIV rev peptide bound to its target loop1andhelix2(Figure1(b)).Morerecentstruc- site,therevresponseelement(Battisteetal.,1996). turalworkonthePU.1ETS(Kodandapanietal., The present work does not provide any direct 1996)andHNF-3/forkhead(Clarketal.,1993) information regarding the L11 contact sites on the DNA binding proteins complexed with their target RNA, although it is apparent that helix 3 must be DNA sequences has shown that sequence-speci®c in close proximity to the bases of the RNA as helix DNA recognition can often be achieved by the use 3 residue M62 displays an NOE with a chemical of additional loops and b-strands ¯anking a-helix 3 110 NMR Studies of the Interaction of L11 with 23 S rRNA of a core homeodomain fold. These additional exact nature of the RNA interaction surface is not regions are referred to as wings. The fold that known. The chemical shifts of the RNA protons these proteins adopt, which is classi®ed as a b contacted by loop 1 residues are in the 6.2 to helix-turn-helix, consists of a core homeodomain 6.3 ppm range and are probably either anomeric plus additional b-sheet regions. Loop extensions ribose protons or cytosine H6 protons, whereas and b-strand insertions are common among the loop 2 residues have NOE contacts to RNA pro- loops connecting the three homedomain helices. tons primarily in the 4.0 to 5.0 ppm, and are prob- Critical DNA contact sites often include residues ably ribose H20, H30, H40, H50, or H500 protons. The located within the loops connecting the homeodo- exception to this are L51 and N52 which display main helices, as in the case of PU.1 ETS, or by the NOEs to protons at 6.2 and 5.5/5.8 ppm, respect- loops which serve to connect the b-strands, as in ively, which again are probably either anomeric the case of HNF-3/forkhead. ribose protons or cytosine H6 protons. Further Although the topology and three-dimensional NMR studies are clearly needed to assign the RNA structure of L11-C76 is not identical to any of the resonances in the L11-C76±RNA complex, and to winged homedomains whose structure has been use these along with NOE data to elucidate a thus determined, it appears that L11-C76 employs three-dimensional structure for the complex. a similar strategy to interact with its target RNA. Loop 1 is considerably longer than corresponding Conclusion loops in minimalist homeodomains such as Oct-1, engrailed, and Mat a2. Additionally, the loop con- The C-terminal domain of ribosomal protein L11 tains a single b-strand insertion, and is ¯exible in is the newest member of the family of ribosomal fL11-C76(Markusetal.,1997),butisrigidandresi- proteins whose structure has been studied at near- dues S24, E26, and P27 display RNA contacts upon atomicresolution(Ramakrishnanetal.,1995).Sig- complex formation. Although none of the L11-C76 ni®cantly, this is the ®rst ribosomal protein whose loop 1 residues is fully conserved across the three interaction with ribosomal RNA has been studied major phylogenetic domains, several, such as G23, using a structure-based approach. These studies S24, and P27 of eubacteria, are highly conserved have shown that the RNA binding domain of L11 withinasingledomain(Xingetal.,1997). interacts with its target RNA by employing a-helix Additionally,mutagenesisstudies(Xingetal., 3 of a homeodomain DNA-binding motif, and by 1997)haveshownthatthesethreeresiduesinpar- the use of loop regions that serve to connect the ticular contribute signi®cantly to L11-C76±RNA three helices of the homeodomain. The loop binding af®nities: the G23P, S24A, and P27G sub- regions of L11-C76 are lengthened relative to those stitutions diminish protein-RNA binding af®nities of minimalist homeodomains and probably have 13, 2.4, and 13-fold, respectively. Within loop 2 of adapted through evolution to optimize binding to L11-C76, there are four residues, L51, N52, A53, 23 S rRNA. The RNA-binding strategy employed and A54, which display both substantial RNA shift by L11-C76 appears to be similar in principle to perturbations and protein-RNA NOEs. Although that used by winged homeodomain DNA binding these residues are not well conserved among the proteins, such as HNF-3/forkhead and PU.1 ETS, threemajorphylogeneticdomains(Xingetal., although the placement of b-strands and use of 1997),theirRNAcontactsmayberelevant,asloop loop regions differ in detail. In the absence of any 2 is considerably longer than that found in minim- direct structural information about the RNA itself, alisthomeodomains(Figure1(b)and(c))andsev- it is premature to speculate further about the inter- eral protein-DNA contacts were found among action. One potential practical outcome of the type PU.1 ETS loop 2 residues and the DNA oligomer of structural information reported here would be with which it was co-crystallized. The L11-C76± to aid in the design of new classes of antibiotics RNA contacts identi®ed in the N-terminal segment that speci®cally disrupt the interaction between of a-helix 1 may also play a role in RNA recog- key ribosomal proteins and their cognate RNA. nition, in a manner similar to residues located in The design of such drugs are of increasing import- the N-terminal end of a helix-1 of PU.1 ETS which ance as many of the drugs that selectively target contact the phosphate backbone. prokaryotic ribosomes are no longer effective. In summary, the major RNA contact sites in bL11-C76 include a-helix 3 and residues located Materials and Methods within loop regions ¯anking its two ends. The extensive interaction surface that is formed from Sample preparation these contacts, as delineated by the shift pertur- bationandprotein-RNANOEdata(Figure8), L11-C76,whosesequenceisshowninFigure1(c),was suggests a model for RNA recognition in which produced in E. coli using a pET11a expression vector the primary interaction surface, -helix 3, is (Novagen, Madison, WI) and was labeled uniformly a with either 15N or 15N/13C and puri®ed as described anchored on both ends by additional protein-RNA (Markusetal.,1997;Xing&Draper,1996).TheRNA, interactions involving loops 1 and 2. On the basis whosenucleotidesequenceisshowninFigure1(a),was 1 of the H chemical shifts of the protein-RNA produced using T7-based in vitro runoff transcription NOEs, it appears that helix 3 may contact the RNA with a plasmid template and was puri®ed by prepara- in the context of a groove, although at present the tive scale polyacrylamide gel electrophoresis (8% (w/v) NMR Studies of the Interaction of L11 with 23 S rRNA 111 gel)andelectroelutionasdescribed(Laing&Draper, Structure calculations 1994).SamplesoftheL11-C76±RNAcomplexforNMR spectroscopy were prepared by ®rst exchanging each of Structure calculations were carried out with the dis- 2 tance geometry/simulated annealing protocol with the components into buffer containing 5 % (v/v) H2O, XPLOR 3.8. The input data are described below. Dis- 10 mM K2HPO4, 25 mM KCl, 2 mM MgCl2 at pH 6.1 (Centricon-10; Amicon, Beverly, MA) followed by step- tance restraints for structure calculations were derived wise addition of small aliquots of the RNA solution. The by interactively analyzing the three NOE data sets using addition of RNA was continued until peaks arising from theprogramPIPP(Garrettetal.,1991).Theinitialsetof the non-liganded form of the protein were no longer pre- NOE crosspeaks were limited to those de®ning the sec- sent in 2D 1H/15N HSQC spectra. The samples were ondary structure of the protein, along with several 2 unambiguous NOEs involving residues that pack in the then lyophilized, redissolved in either 5% H2O/95% 2 hydrophobic core. In successive iterations, NOEs were H2O or 99.99% H2O in a volume of approximately 220 ml, and transferred to a 5 mm NMR microcell added to the restraint tables provided that either the 13 (Shigemi, Allison Park, PA). The ®nal concentration symmetry-related crosspeak was present in the C-edi- of the L11-C76-RNA complex, as judged on the basis ted NOE data set, as in the case of aliphatic-aliphatic of the extinction coef®cient of the RNA (1.00 A NOEs, or the observed NOE crosspeak was present in 260 15 15 13 37.2 mg/ml, S.H., unpublished observation), was 0.5 mM. both the N-edited and N-edited/ C-edited NOE data sets, as in the case of amide-aliphatic NOEs. NOE cross- peak intensities were converted into distances by (a) ®rst normalizing each to the intensity of the corresponding NMR Spectroscopy diagonal peak, and (b) by using a modi®ed form of the isolatedtwo-spinapproximation(Suri&Levy,1993, NMR spectra were acquired at ®eld strengths of 1995) to calculate distances. The calculated distances, either 11.7 or 14.2 Tesla using either a Bruker AMX or along with a 1.8 AÊ lower distance bound and appropri- DMX spectrometer equipped with a pulsed-®eld gradi- ate pseudo-atom corrections for methylene, methyl, and ent unit and a triple-resonance 5 mm probe. The majority non-stereospeci®cally assigned methyl groups of leucine of data were collected at a temperature of 47 C, although and valine residues, were then used as input for struc- the 3D CBCA(CO)NH data set was collected at 42 C. ture calculations Distance restraints for hydrogen bonds, Spectral quality improved at 47 C owing to the more N to O and HN to O, were added for those residues favorable relaxation properties of the complex. Sequen- which were found in regular elements of secondary tial backbone assignments of the protein were made structure and whose amide proton was protected from usingthe3DCT-HNCA(Grzesiek&Bax,1992b)and3D exchangewithsolvent(Figure3(a)). CBCA(CO)NH(Grzesiek&Bax,1992a)pairofexper- Backbone torsion angle restraints for , derived from iments. Side-chain assignments were made by extending f HN-Ha coupling constants, were applied to only those the sequentially assigned HN resonances using the 3D residues found in regular elements of secondary struc- C(CO)NH(Grzesieketal.,1993)and3DHBHA(CO)NH ture, and were set to 65( 25) for 3J < 6 Hz and (Grzesiek&Bax,1993)pairofexperiments,andbyusing HNHa 120( 40) for 3J > 8.0Æ Hz. Phi torsion angles the3DHCCH-TOCSY(Baxetal.,1990)experiment, HNHa amongÆ the regular elements of secondary structure, were although the 3D 13C-edited NOESY was used in a few additionally restrained by re®ning the measured 3J instances. Asparagine Hd protons were assigned from HNHa couplings against a reparametrized form of the Karplus their NOESY crosspeaks to the assigned Hb resonances, equation(Wang&Bax,1995).Side-chaintorsionangles methionine He resonances were assigned from a 2D for 6/7 Ile, 1/4 Thr, and 5/5 Val residues, derived from HMBCexperiment(Baxetal.,1994),andarginineHeres- 2D N-Cg SED-ctHSQC, 2D CO-Cg SED-ctHSQC, and 3D onances were assigned by their NOESY crosspeaks to HaHb data sets, were restrained to one of the staggered the assigned Hd resonances. rotamers, 60, 60, or 180, with an error range of Distance restraints were derived from three 3D 30. NOESY data sets: 13C-edited, 15N-edited, and an 15N-edi- Æ An additional calculation was carried out by placing ted experiment with 13C-editing and 13C chemical shift all of the calculated distances into either strong (d 2.5 evolution in the F2 dimension. Protein-RNA NOE con- to 3.0 AÊ ), medium (d 3.0 to 4.0 AÊ ), weak (d 4.0 to tacts were delineated by a pair of 15N and 13C-edited 5.0 AÊ ), or very weak (d> 5.0 AÊ ) categories. The structures NOESY experiments, modi®ed with a ®lter that selects calculated from such restraints compared favorably with for 12C-bound protons following the NOE transfer step. those calculated using NOE distance restraints derived The 3J coupling constant was derived by averaging HN-Ha from the isolated spin pair approximation. Thus, all cal- the calculated couplings from two independently culations reported here were carried out using the for- acquired3DHNHA(Vuister&Bax,1993)datasets. mer approach. Side-chain torsion angle restraints for Ile, Thr, and Val 3 3 residues were made by measuring JN-Cg, JCO-Cg, and 3 13 JHa-Hb using the 2D CO spin echo difference constant Chemical shift perturbations timeHSQC(SED-ctHSQC;Grzesieketal.,1993),2D15N SED-ctHSQC(Vuisteretal.,1993),and3DHAHBexper- Chemical shift perturbations of L11-C76 that occur iments(Grzesieketal.,1995),respectively.Hydrogen upon RNA binding were calculated by subtracting the exchange rates were measured by dissolving a lyophi- valuesforfL11-C76previouslyreported(Markusetal., lized, protiated sample of 15N L11-C76, in standard 1997)fromthevaluesreportedhereforbL11-C76.Since 2 NMR buffer containing 99.99% H2O and by following the temperature at which the fL11-C76 shifts were 1 15 amide proton intensities with a series of 2D H/ N recorded (25C) differed from that of bL11-C76 (47 C), HSQC spectra at 37C. Spectra were acquired at 0.27, the bL11-C76 shifts were ®rst temperature-corrected to a 0.60, 0.93, 1.53, 3.18, 6.15, 10.1, 14.1, 18.1, 22.0 and 155.5 common reference temperature of 25 C. A composite 2 15 hours after H2O addition. Backbone N transverse absolute shift perturbation index was calculated by ®rst relaxation times were measured by the 1H-detected pulse calculating the absolute shift for all residues of a given schemeasdescribed(Kayetal.,1992). atom type and by normalizing the maximum to a value 112 NMR Studies of the Interaction of L11 with 23 S rRNA of 1.0. These absolute shifts were then summed over all Grzesiek, S. & Bax, A. (1993). Amino acid type determi- of the atom types compared (HN, Ha, Ca, Cb, CO, and nation in the sequential assignment procedure of NH) to give the composite absolute shift perturbation. uniformly 13C/15N-enriched proteins. J. Biomol. NMR, 3, 185±204. Grzesiek, S., Anglister, J. & Bax, A. (1993). Correlation Acknowledgments of backbone amide and aliphatic side chain reson- ances in 13C/15N enriched proteins by isotropic We gratefully acknowledge Frank Delaglio for devel- mixing of 13C magnetization. J. Magn. Res. 101, oping and supporting the NMR processing package 114±119. nmrPipe/nmrDraw, and Dan Garrett for the data anal- Grzesiek, S., Kuboniwa, H., Hinck, A. P. & Bax, A. ysis package, PIPP. We also acknowledge Dr Larry (1995). Multiple-quantum line narrowing for Fischer for providing assistance in purifying the 15N- measurement of Ha-Hb J couplings in isotopically labeled protein. This work was supported by the AIDS enriched proteins. J. Am. Chem. Soc. 117, 5312±5315. Targeted Antiviral Program of the Of®ce of the Director Hinck, A. P., Eberhardt, E. S. & Markley, J. L. (1993). on the National Institutes of Health. 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