local folding coupled to rna binding in the yeast ribosomal protein l30

Article No. jmbi.1999.3044 available online at http://www.idealibrary.com on J. Mol. Biol. (1999) 292, 345±359

Local Folding Coupled to RNA Binding in the YeastRibosomal Protein L30

Hongyuan Mao1,2 and James R. Williamson2*

1Department of ChemistryMassachusetts Institute ofTechnology, Cambridge, MA02139, USA2Department of MolecularBiology, The Scripps ResearchInstitute, La Jolla, CA92037, USA

E-mail address of the [email protected]

Abbreviations used: L30, yeast Saribosomal protein L30, formerly knodesignates the L30 protein free in sodesignates the L30 protein in the RNmRNA, precursor messenger RNA;magnetic resonance; NOE, nuclear OMW, molecular weight; MBP-L30, rmaltose-binding protein with L30; Kconstant; CD, circular dichroism; HSsingle quantum correlation; 2D, 3D,dimensional, respectively; RMSD, rodeviation; EDTA, ethylenediaminetedithiothreitol; SA, simulated annealserum albumin.

0022-2836/99/370345±15 $30.00/0

The ribosomal protein L30 from yeast Saccharomyces cerevisiae auto-regu-lates its own synthesis by binding to a structural element in both its pre-mRNA and its mRNA. The three-dimensional structures of L30 in thefree (fL30) and the pre-mRNA bound (bL30) forms have been solved bynuclear magnetic resonance spectroscopy. Both protein structures containfour alternating a-helices and four b-strands segments and adopt an over-all topology that is an aba three-layer sandwich, representing a uniquefold. Three loops on one end of the aba sandwich have been mapped asthe RNA binding site on the basis of structural comparison, chemicalshift perturbation and the inter-molecular nuclear Overhauser effects tothe RNA. The structural and dynamic comparison of fL30 and bL30reveals that local dynamics may play an important role in the RNA bind-ing. The fourth helix in bL30 is longer than in fL30, and is stabilized byRNA binding. The exposed hydrophobic surface that is buried uponRNA binding may provide the energy necessary to drive secondarystructure formation, and may account for the increased stability of bL30.

# 1999 Academic Press

Keywords: ribosomal protein L30; NMR; three-dimensional structure;protein-RNA complex; free and bound comparison
*Corresponding author
Introduction

The yeast Saccharomyces cerevisiae ribosomal pro-tein L30 (MW � 11 kD) (Mager et al., 1997), for-merly known as L32 (Dabeva & Warner, 1987), isan essential protein for yeast growth (Dabeva &Warner, 1987). The L30 protein has no homolog inprokarya, but apparently is ubiquitous in eukaryaand archaea. The sequence conservation of L30protein across species in eukarya (Figure 1) impliesan important role for the L30 in the ribosome func-tion. However, the rRNA binding site within ribo-

ng author:

ccharomyces cerevisiaewn as L32; fL30,lution; bL30,A complex; pre-

NMR, nuclearverhauser effect;

ecombinant fusion of

d, dissociationQC, heteronucleartwo and three-ot-mean-squaretraacetic acid; DTT,

ing; BSA, bovine

some is currently not known. The best understoodaspect of yeast L30 protein function is the negativefeedback regulation of pre-mRNA splicing andmRNA translation upon over-production of L30(Eng & Warner, 1991; Li et al., 1996). The presenceof L30 bound to its pre-mRNA near the 50 splicesite prevents the completion of spliceosome assem-bly in vitro (Vilardell & Warner, 1994). Binding ofL30 close to the translational initiation codon in itsmRNA provides another level of auto-regulation(Li et al., 1996). Both splicing and translationalregulation involve binding of L30 protein to aconsensus sequence in the L30 mRNA that formsan internal loop structure (Eng & Warner, 1991; Liet al., 1996).

Ribosomes are the essential machinery for pro-tein synthesis in all organisms. A typical eukary-otic ribosome contains about 78 different proteins(32 in the 40 S subunit and 46 in the 60 S subunit)(Mager et al., 1997), as compared to some 52 pro-teins in a prokaryotic ribosome. A surprisingly lim-ited amount of structural information is availablefor eukaryotic ribosomal proteins, while severalstructures of prokaryotic ribosomal proteins havebeen solved recently (Liljas & al-Karadaghi, 1997).As an initial step toward studying the structureand function of the eukaryotic ribosome, it isadvantageous to examine individual components.

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Figure 1. Sequence alignment of 60 S ribosomal protein L30 from various species, including yeast S. cerevisiae,Kluyveromyces lactis, Schizosaccharomyces pombe, chicken, human, Trypanosoma brucei brucei, and Leishmania major. Thenumbering of residues refers to the yeast S. cerevisiae sequence. Absolutely conserved residues are highlighted in red,relatively conserved hydrophobic residues in blue, and charged residues in green. The alignment was performedusing the BLAST program (Altschul & Lipman, 1990) on protein sequences in the NCBI database.

346 Structures of the Free and Bound L30 Protein

We have successfully prepared pure and activeyeast L30 protein by over-expression in Escherichiacoli, and solved the high-resolution structure of theprotein in free form (fL30) using heteronuclearmagnetic resonance spectroscopy. In addition, wehave recently completed the structure of the L30-mRNA complex (unpublished results), and forcomparison, we present here the bound form ofL30 extracted from the complex as bL30. The three-dimensional structures obtained from simulatedannealing reveal an aba three-layer sandwich top-ology in both fL30 and bL30. Detailed comparisonsbetween the free and bound forms reveal insightsto the structure and function of L30 protein.Speci®cally, side-by side comparisons of chemicalshifts, resonance linewidths, and relaxation timesfor the free and RNA-bound proteins indicatechanges in the protein local environment, structure,and dynamics that accompany RNA binding. TheL30 structure provides insights into the role of con-served residues in folding of the protein and inRNA binding.

Results

Protein binding and CD analysis

Although yeast L30 is a relatively small protein(MW � 11 kD), previous biochemical studies of theinteractions between the L30 and its autoregulatoryRNAs were primarily carried out with a 53 kDmaltose-binding protein-L30 fusion (MBP-L30)(Vilardell & Warner, 1994; Li et al., 1995). Prior tostructural studies using NMR, it is essential toobtain the active L30 protein with a suitable size.Combining the advantages of the MBP-L30 fusionprotein for af®nity puri®cation and the chargedifferences between L30 and MBP proteins uponFactor Xa protease cleavage for separation, a newpuri®cation scheme has been developed whichprovides milligrams of native and full-length L30protein. Indeed, the puri®ed full-length L30 protein

maintains an RNA binding af®nity similar to thatof MBP-L30 fusion (Kd � 4(�1) nM and 10(�4) nM,respectively) using an RNA construct designed forNMR studies (Figure 2). Furthermore, a stoichio-metric-binding assay indicates that the L30 proteinbinds to the RNA construct with a 1:1 ratio, con-sistent with the results for the MBP-L30/RNAcomplex (Li et al., 1995).

Gel-shift assays have shown that the puri®edL30 protein is competent to bind RNA, but it wasnot known if the folding of L30 is induced by thepresence of its cognate RNA. In many cases, a pro-tein may undergo signi®cant conformationalchanges upon ligand binding and, in some extremecases, binding is accompanied by global folding(Spolar & Record, 1994). Because the refolding ofL30 protein in vitro is very dif®cult (data notshown), we initially suspected that L30 protein freein solution may not adopt a folded structure.Hence, circular dichroism (CD) measurementswere conducted to estimate the secondary structur-al content of the free L30 protein. Unexpectedly,CD spectra of the free (fL30) and RNA-bound(bL30) proteins are very similar (Figure 3), andboth suggest the presence of a-helix and b-sheetsecondary conformations. The CD signals of fL30suggest a secondary structure consisting ofapproximately 50 % a-helix, 25 % b-sheet and 25 %coil. The increase in the CD signal at 208 nm forbL30 could be the result of an increase in regularsecondary structure upon RNA binding, but it isdif®cult to rule out a potential contribution fromthe RNA at 208 nm.

Despite comparable secondary structural con-tents, the two forms of L30 appear to have verydifferent thermal stability, judging from the CDmonitored thermal-melt pro®les (see the insets inFigure 3). The fL30 has a shallow and elongatedmelting curve, and becomes turbid around 45 �Cwith irreversible denaturation. The bL30 protein inthe pre-mRNA complex, on the other hand,remains folded at temperatures up to 60 �C, and

Figure 2. (a) The secondarystructure of the L30 autoregulationpre-mRNA site designed for NMRstudies. The wild-type nucleotidesare in upper case and are num-bered according to the pre-mRNAsequence, and the non-wild typenucleotides are represented inlower case. (b) Gel-shift bindingassay of the RNA to MBP-L30fusion (lanes 1 to 15) and L30(lanes 16 to 30) performed onnative 10 % (w/v) polyacrylamideat 4 �C. From left to right, the MPB-L30 concentration is 2000.0, 1000.0,

500.0, 250.0, 125.0, 62.5, 31.7, 16.0 8.0, 4.0, 2.0, 1.0, 0.5, 0.3 and 0.1 nM; and the L30 protein concentration is 8000.0,4000.0, 2000.0, 1000.0, 500.0, 250.0, 125.0, 62.5, 31.7, 16.0 8.0, 4.0, 2.0, 1.0 and 0.5 nM.

Structures of the Free and Bound L30 Protein 347

undergoes a cooperative thermal denaturationtransition that is presumably accompanied bymelting of the RNA. A survey of conditions usingCD reveal that the folded fL30 protein can only bemaintained at low temperature and high salt con-centration, and the protein structure is highlystabilized by RNA binding.

Figure 3. CD spectra of (a) fL30 and (b) bL30 recordedwith 25 mM samples in 10 mM potassium phosphatebuffer (pH 6.5) at 4 �C. The insets in (a) and (b) showthe thermal denaturation spectra (from 4 �C to 88 �C) ofthe respective fL30 and bL30 proteins monitored at218 nM. The arrow in the inset in (a) indicates thetemperature when the fL30 becomes denatured andturbid.

Chemical shift assignments and secondarystructure mapping

The structures of fL30 and bL30 have beencharacterized using multidimensional NMR spec-troscopy at 10 �C and 30 �C, respectively. The opti-mal temperature for NMR data acquisition wasdifferent for the two forms, and it was not possibleto record high quality spectra of the free proteinand the protein-RNA complex under the same con-ditions. In fact, fL30 is only stable under high salt(300 mM NaCl) and low temperature conditions(below 15 �C), but the high molecular mass of theL30-RNA complex (MW � 22 kDa) resulted inbroad lines under these conditions. Fortunately,the bL30 protein is stabilized in the complex, allow-ing NMR data to be recorded at temperatures upto 40 �C. Under their respective optimal conditions,both free and bound proteins exhibit high qualityNMR spectra, as demonstrated by the 2D 15N-edi-ted heteronuclear single quantum coherence(HSQC) spectra acquired at 10 �C and 30 �C(Figure 4(a) and (b), respectively). Nearly all of theamide cross-peaks (except for Ala2 and Val81)were observed in the fL30 HSQC spectrum, and allthe expected peaks were observed for bL30.Assignments of the backbone 1HN, 15N, 13C0 and13Ca resonances were based on standard triple res-onance experiments, including 3D HNCO, HNCA,and HN(CO)CA spectra collected on 13C/15N-labeled fL30 and 13C/15N-labeled bL30 complexedwith unlabeled RNA. Analyses of 3D CBCA(CO)NH (for fL30) or HNCACB (for bL30), incombination with 3D (1H, 13C, 1H) HCCH-TOCSY,(1H, 1H, 15N) TOCSY, (1H, 1H, 13C) and (1H, 1H,15N) NOESY spectra, made it possible to assignmost side-chain resonances. Overall, the resonances(1H, 13C, and 15N) for 95 % of the backbone and82 % of the side-chains were assigned in fL30.Exchange broadening of the backbone amide res-onances between residues 74 to 88 (Figure 4(a))made assignments for some of these residuesambiguous or incomplete. In contrast to fL30, res-onances for residues 74 to 88 have been unambigu-

Figure 4. The (1H,15N) HSQCspectra of (a) fL30 recorded at10 �C, pH 6.5 and (b) bL30 at 30 �C,pH 5.5. The peaks are labeled withtheir assignments. The side-chainamide protons for Asn and Gln areconnected with horizontal lines.The Arg e side-chain peaks arefolded in both spectra. In (a), peakswith signi®cant broader linewidthare highlighted in green; in(b), resonances experiencing largechemical shift changes (�0.5 ppmin 1H dimension) upon RNA bind-ing are highlighted in cyan.


ously identi®ed in bL30, since the amide reson-ances in this region sharpened upon RNA binding.Near complete backbone assignments (1H, 13C, and15N) were made to the bL30 protein, except forAla2, Thr48, and Ala105. The side-chain assign-ments of bL30 were somewhat incomplete due tothe much shorter transverse relaxation time in theRNA-protein complex. The 1H, 13C, and 15N assign-

ments for the fL30 protein at 10 �C and pH 6.5 andbL30 at 30 �C and pH 5.5 are available asSupplementary Material, Tables 1 and 2, respect-ively.

The obvious spectral differences between fL30and bL30 are in several speci®c regions in the L30sequence. For instance, one missing and ten out ofthe 12 weak amide cross-peaks in the (1H, 15N)


HSQC spectrum of fL30 were from the regionbetween Gly72 and Gly88 (Figure 4(a)). Thesemissing or weak peaks are the direct result of alocal conformational exchange process in fL30.When L30 protein is bound to RNA, resonances inthis region appear with much sharper linewidths,indicating stabilization of the protein upon bind-ing. The dispersion of backbone amide chemicalshifts also increases in L30 upon RNA binding. Theamide proton resonances range from 6.5 to 9.3ppm in fL30, as compared to 5.9 to 9.9 ppm inbL30. Most of the large changes (up to 0.5 ppm)are localized to three segments in the L30sequence: between Leu25 and Thr30, betweenAsp47 and Arg52, and between Thr79 and Gly88(Figure 4(b)). Moreover, three distinct pairs of argi-nine guanidinium side-chain resonances are alsoobserved in bL30 (ranging from 6.6 to 7.9 ppm for1H and from 62 to 75 ppm for 15N), suggesting theformation of hydrogen bonds.

The secondary structural elements of the freeand RNA-bound proteins were derived from ana-lyzing their patterns of short and medium-rangeNOEs, 3JHNHa coupling constants, hydrogen-exchange rate constants, and chemical shift indexvalues. A schematic description of the diagnosticNMR parameters for bL30 is illustrated Figure 5.For example, four helices (e.g. Ser9 to Ile18, Tyr27to Glu36, Val50 to Leu62, and Asn74 to Val81)were identi®ed from their slowly exchangingamide protons (except for the ®rst four amide pro-tons at each N terminus), 3JHNHa coupling con-stants less than 6 Hz, medium-strong dNN(i, i � 1),daN(i, i � 3) and dab(i, i � 3) NOEs, and down®eld13Ca chemical shifts (Figure 5(a)). Four b-strands(e.g. Lys22 to Gly26, Leu41 to Ala45, Lys66 toPhe70, and Val89 to Leu93) were supported bytheir larger than 8 Hz 3JHNHa coupling constants,weak or missing dNN(i, i � 1), and strongdaN(i, i � 1) NOEs, and up®eld 13Ca chemical shifts(Figure 5(a)). The arrangement of these b-strandswas derived from the inter-strand, long-range1H-1H NOEs and slowly exchanging amide protonsinvolving hydrogen bonds (Figure 5(b)). Most ofthese NMR parameters were also seen in fL30, butseveral differences were also observed. One of thedistinctive differences is the much faster 2H2Oexchange rate in fL30; almost all amide protonshave exchanged away in an hour after exchanginginto 2H2O. This fast solvent penetration rate indi-cates that the free protein may undergo moltenglobule-like breathing. Another major difference infL30 is the lack of most sequential NOEs, andincomplete assignments of 13Ca shifts and 3JHNHacoupling constants in the region of residues 74 to88 in fL30, thus it is dif®cult to derive the second-ary structure in this region.

Three-dimensional structure calculation

The three-dimensional structures of fL30 andbL30, have been obtained using simulated anneal-ing protocols in X-PLOR (BruÈ nger, 1992) and

AMBER (Cornell et al., 1995) programs. Initial cal-culations were performed in X-PLOR, but the ®nalre®nement was performed in AMBER due to themore complete parameter set for nucleic acid calcu-lations. For both structure calculations, NMRexperimental restraints consisted of NOE-derivedinterproton distances, backbone f and side-chainw1 torsion angles, and backbone hydrogen bonds(Tables 1A). Aside from intraresidue distancerestraints, the input data for fL30 contain 698 inter-residue distance restraints and 139 dihedral anglerestraints. Signi®cantly more inter-residue NOEshave been identi®ed for bL30, with a total of 1175inter-residue distance restraints and 135 dihedralangle restraints. A total of 56 hydrogen bondrestraints used in bL30 were identi®ed throughslowly exchanging amide protons. Hydrogen bondrestraints used in fL30 (a total of 62) were basedprimarily on regular secondary structures observedin the initial calculations, because most amide pro-tons in fL30 exchanged rapidly with solvent. The®nal bL30 structure calculation was conducted inthe presence of the RNA, including 73 inter-mol-ecular protein-RNA restraints (data to be presentedelsewhere).

Ensembles of 21 converged structures for thefree and bound forms are shown in Figure 6(a) and(c), respectively. The ensembles of both the freeand bound protein showed good covalent geome-try and low overall energies, and a summary ofthe statistics describing these structures is providedin Table 1B. The deviations from ideal covalentgeometry and from experimental constraints arereasonably small, and the ®nal AMBER energyterms are fairly low. The qualities of both struc-tures have been checked using the PROCHECKprogram (Laskowski et al., 1996). A total of 85 % ofthe residues in bL30 and 74 % of the residues in thefL30 protein have been located within the mostfavorable region in the Ramachandran plot. Exceptfor the N terminus (residues 2 to 8) and C terminus(residues 101 to 105), most bL30 protein coordi-nates are very well de®ned over the ensemble ofstructures. The fL30 protein coordinates are wellde®ned for the most part, but an additional disor-dered region has been found in fL30 from residue74 to 88. The average root-mean-squared devi-ations (RMSD) of the free and bound structures arelisted in Table 1C. When only the residuesassigned to elements of regular secondary structureare considered, the backbone (N, Ca, C0 and O)RMSD from the mean structure for the free protein(residues 9 to 70 and 89 to 100) is 0.56 AÊ , and thebound form (residues 9 to 100) is 0.36 AÊ . When theentire molecule is considered, the N terminus andC terminus (Ala2 to Glu8 and Leu101 to Ala105,respectively) consistently appear as relativelypoorly de®ned regions in both fL30 and bL30. Inthe fL30, residues between Gly72 and Gly88 alsostand out as a poorly de®ned region in the ensem-ble of structures.

Figure 5. (a) Summary of NMRdata used to identify the secondarystructure elements in bL30. Theseinclude the amide proton 2H2Oexchange rates, 3JHNHa couplingconstants, sequential and mediumNOEs and 13Ca secondary chemicalshifts. A helical conformation issupported by a stretch of slowlyexchanging amide protons, small3JHNHa couplings (<6 Hz), medium-strong dNN(i, i � 1), daN(i, i � 3)and dab(i, i � 3) NOEs, and positive13Ca secondary chemical shifts.The b-strand conformation wassupported by large 3JHNHa coup-lings (>8 Hz), weak or missingdNN(i, i � 1), and strong daN(i, i � 1)NOEs, and negative 13Ca secondarychemical shifts. The broken linearound the helix a4 region high-lights the major difference betweenthe free and bound L30 protein.(b) NMR evidence for the arrange-ment of the four-stranded b-sheetin bL30 protein. Inter-strand, long-range 1H-1H NOEs are indicated bydouble-headed arrows, and slowlyexchanging amide protons areshown in bold.


Description of the free and bound L30protein structures

Backbone ribbon diagrams depicting the energyminimized average free and bound L30 structuresare shown in Figure 6(b) and (d), respectively.They reveal an overall three-layer aba sandwichtopology in both forms. The three-dimensionalstructure of fL30 consists of four a-helices (residues8 to 18, 27 to 35, 50 to 62 and 76 to 79) and fourb-strands (residues 22 to 26, 41 to 45, 66 to 70 and89 to 93). The four-stranded mixed b-sheet is sand-wiched between two layers of a-helices. The struc-ture of bL30 has a similar topology, except that a4is extended from Gly73 to Val81. The arrangementof the b-strands and the resulting helical packingare unique structure features in both forms. Start-ing with b1 at one edge of the b-sheet, strands b1,b4 and b2 run antiparallel to each other. Strand b3,however, runs parallel with b2. Helices a2 and a3run almost parallel with each other and are packed

on one side of the b-sheet platform, while a1 anda4 are on the other side of the b-sheet. Based onanalysis with program SCOP (Murzin et al., 1995),the topology of yeast L30 appears to be unique.

The structures of L30 appear to be stabilized pre-dominantly by hydrophobic interactions. The sec-ondary structure is stabilized by hydrogenbonding, and salt bridges provide intrahelical(Lys53 and Glu57), interhelical (Arg35 and Glu55),and intra-strand (Lys22 and Glu94) stabilization.Nonetheless, much of the folding energy is likelydue to burying a large number of hydrophobicresidues within the protein core. A number of phy-logenetically conserved hydrophobic and aromaticresidues are important for de®ning the tertiary foldof the protein, and the slight curved b-sheet pro-vides hydrophobic packing surfaces on both sides(Figure 7). For instance, on one side of the b-sheet,there are conserved non-polar residues such asVal67 and Tyr69 in b3, Ile42 and Ile44 in b2, andVal 89 in b4. These residues make further contacts

Table 1. Summary of structural statistics of the free and bound L30 proteins based on simulated annealing (SA) cal-culations in the AMBER program

A. NMR constraints fL30 bL30

Distance and dihedral angle constraints 1619 2264Total distance constraints 1480 2129

Intraresidue 720 898Interresidue 698 1175Sequential (ji ÿ jj � 1) 291 512Short-range (1 < ji ÿ jj4 4) 149 328Long-range (ji ÿ jj > 4) 258 335Protein-RNA NOEs 73Hydrogen bond 62 56

Total dihedral angle constraints 139 135Backbone f 80 76Side-chain w1 59 59

B. Structure statistics fL30 bL30Constraint violations

Number of distance >0.2 AÊ 7.2 � 3.8 0.5 � 0.4Largest distance (AÊ ) 0.36 0.23Number of dihedral >2 � 1.3 � 0.7 2.0 � 0.5Largest dihedral (deg.) 3.1 4.9

Constraint energy (kcal/mol) 12.3 � 3.7 4.2 � 1.3a

Deviation from idealized covalent geometryBond (AÊ ) 0.0108 � 0.0003 0.0105 � 0.0002a

Angles (deg.) 2.25 � 0.04 2.13 � 0.04a

AMBER energy (kcal/mol) ÿ1345 � 41 ÿ1355 � 22a

PROCHECK evaluation (most favorable region) (%) 74 85

C. RMSD among the 21 refined structures Pairwise RMSD RMSD to averageHeavy atoms (C*, N* and O*)

fL30 residues 9-70, and 89-100 (AÊ ) 1.26 0.87bL30 residues 9-100 (AÊ ) 0.72 0.50

Protein backbone (N, Ca, C0 and O)fL30 residues 9-70, and 89-100 (AÊ ) 0.80 0.56bL30 residues 9-100 (AÊ ) 0.52 0.36

a These statistics in bL30 were calculated for the protein prior to docking against RNA.


with residues Val31 and Leu34 in a2, and Leu56and Tyr59 in a3, thus orienting the two helices. Onthe other side of b-sheet, Tyr68 and Phe70 of b3,Ile43 of b2, and Val90 and Ile92 of b4 interact withIle10, Leu14, and Ile18 on a1, and Leu77 and Val81on a4, and thus orient helices a1 and a4. Ile43 fromb2 is particularly important in bridging three of theb-strands (b2, b3, and b4) and two of the a-helices(a1 and a4). The one known functional L30mutation is �I43, which results in a slow growthrate and a crippling of the 60 S subunit of S. cerevi-siae (Vilardell & Warner, 1997), consistent with animportant role of this residue in folding. Con-served hydrophobic residues are also seen withinthe four helices, which further orient the positionsof the helices with respect to each other. Most ofthese hydrophobic residues are highly conservedin phylogeny (Figure 1), which is likely due tohydrophobic interactions that are critical for L30folding.

Discussion

Structural comparison between the free andRNA-bound protein

NMR and CD data both indicate that the fL30adopts a folded tertiary structure in the absence of

its cognate RNA. The NOEs, J-couplings, and sec-ondary chemical shift alignments are similar formost of the free and bound L30. Indeed, the ter-tiary structures obtained from molecular modelingexhibit a common folding topology, namely, anaba three-layer sandwich. Based on the overlay ofthe backbone of fL30 and bL30 average structure(Figure 8(a)), most of the signi®cant structural fea-tures are preserved in both forms. Disregardingthe unordered regions at N and C termini, theoverall backbone RMSD between two averagestructures (residues 9 to 100) is about 1.67 AÊ . Themajority of the four-stranded b-sheet (residues 22to 25, 41 to 44, 66 to 70, and 89 to 92), which formsthe core of the protein structures shows the closestresemblance, with a backbone RMSD less than0.75 AÊ . Additionally, helices a1, a2, and a3 arecomparable in both structures, with helix a1 (resi-dues 8 to 18) showing the best-®tted backboneRMSD (0.95 AÊ ), followed by helix a3 (residues 51to 62) with RMSD 1.34 AÊ . These similaritiessuggest that the protein has adopted a pre-foldedconformation prior to RNA binding, and no dra-matic structural rearrangement occurs upon RNAbinding.

However, signi®cant differences have also beenobserved in several regions upon RNA binding.First, the N and C-terminal regions (residues 2 to 8

Figure 6. Schematic representations of the structures of fL30 (upper panel) and bL30 (lower panel). (a) and(c) Show the stereoviews of a best-®t backbone superposition of the 21 simulated annealing structures of fL30 andbL30, respectively. Only backbone atoms (N, Ca, C') are shown. The coloring schemes are as follows: Ala2 to Ser20,Gly72 to Gly88, and Glu94 to Ala55 in green, Gly21 to Gly26, Leu41 to Ala46, Lys66 to Gln71, and Val89 to Leu93are in magenta, and Tyr27 to Lys40 and Asn47 to Thr65 are in cyan. (b) and (d) Show the ribbon diagrams of therespective fL30 and bL30 average structures, depicting the secondary structural elements with the correspondinglabeled. The regular secondary structures are colored in green (a1 and a4), magenta (b-sheet), and cyan (a2 and a3),and the loop regions are colored in white. The ribbon diagrams were generated with RIBBONS (Carson, 1987).


and 101 to 105, respectively) have the largestRMSD (>3 AÊ ) between the two average structures.Second, there is a minor adjustment in the orien-tation of helix a2 with respect to a3, and there aremajor differences in the loop connecting a2 and b2,the loop between b2 and a3, and the regionbetween Gly72 and Gly88. Most importantly, theregion between Gly72 and Gly88 appears largelyas a random coil in fL30, but as an a-helix fromresidues 73 to 81 in the bL30 ensemble. The differ-ences observed in the average free and boundstructures may be partly due to the number of

Figure 7. Stereoview of the conserved hydrophobicresidues within the core of bL30 protein. The backboneof the bL30 average structure is traced with white rib-bon. The conserved hydrophobic residues are shownwith their side-chain heavy atoms colored in red andare labeled with their one-letter names and sequencenumbers.

long-range distance restraints in the two data sets.On average, bL30 clearly exhibits more inter-resi-due distance restraints than fL30 (22.6 per residuesversus 13.4 per residue, respectively). In particular,the region between residues 72 and 88 has 19.4inter-residue distance restraints per residue inbL30, but only 6.6 per residue in fL30. The numberof long-range restraints available is in¯uenced bydisorder in the structure, as well as the presence ofexchange broadening. The lower density ofrestraints in residues 72 to 88 results in a largerRMSD for this segment, but it is dif®cult to deter-mine the extent of the effects of dynamics on thestructure in this region.

Dynamic comparison between the free andbound L30

Dynamics within a protein are of particularinterest because ¯exible regions in a protein with-out its bound ligand usually exhibit motions onthe micro- to millisecond time-scales (Gitschieret al., 1998; Eliezer et al., 1998). It is clear from thestructure ensembles of both free and bound L30proteins that several regions of ¯exibility exist,including the N and C terminus (residues 2 to 8and 101 to 105, respectively) in both fL30 andbL30, and residues 72 to 88 in fL30. However, it isunclear whether the ¯exibility observed from theensembles coincides with local dynamics. Flexibleregions characterized by large RMSD in the ®nalstructure ensemble can also be attributed to a lackof long-range distance restraints in the structure

Figure 8. (a) Superposition of the fL30 and bL30 average structures ®tted from residues 9 to 99. The backboneN, Ca, C0 atoms are traced with blue (for fL30) and red (for bL30) ribbons. (b) Mapping of the chemical shift pertur-bations upon RNA binding in bL30. The yellow segments highlighted in the ribbon diagram are residues experiencinglarge perturbations in 1Ha, 1HN, 13Ca, 13C0 and 15N chemical shifts upon RNA binding. In both (a) and (b), the N andC termini and segments of the regular secondary structures are labeled according to their position in bL30.(c) GRASP (Nichols et al., 1991) representation of the bL30 surface potential. Regions of the surface with electrostaticpotentials greater than �10 kT, equal to 0 kT, and less than ÿ10 kT are colored blue, white, and red, respectively. Thepositively charged residues surrounding RNA binding site and hydrophobic residues contacting RNA are labeled inyellow and black. The orientations of bL30 are the same in all three Figures.


calculation. Motion on the picosecond (ps) time-scale and conformational exchange broadening(micro- to millisecond time-scales) can lead to anabsence of observable NOEs. However, other fac-tors such as spectral overlap and chemical shiftdegeneracy may have contributed to the absence oflong-range restraints. In order to distinguish the

¯exibility caused by real motion from that due toartifacts from an incomplete restraint set duringthe structure calculation, it is necessary to carryout dynamic studies. NMR is an excellent tool forthese studies because it provides direct informationregarding motions on the pico- to millisecondtime-scales that can be accessed by various

Figure 9. Plots of the backbone relaxation parametersof T1, T1r, and NOE of (a) fL30 and (b) bL30. The datafor fL30 and bL30 were recorded on a 600 MHz spec-trometer at 10 �C and 30 �C, respectively. The measuredT1 and T1r data with error bars are shown in (i) and (ii),respectively, and 15N-1H NOE data in (iii). The missingdata points in fL30 are due to either broad or missingcorrelation peaks, or resonance overlaps in the 2D (1H,15N) HSQC spectra, whereas those missing peaks inbL30 are all due to resonance overlaps.


relaxation parameters (e.g. T1, T1r, and heteronuc-lear NOE).

The local motions of both free and bound L30proteins have been qualitatively investigated using15N backbone dynamic studies. The highly ¯exiblenatures of the N and C-terminal residues 2 to 8and 101 to 105, and residue G72 in both proteinstructures are con®rmed by backbone 15N T1, T1r,and heteronuclear NOE measurements. As can beseen in Figure 9(a) depicting the dynamics of fL30,both the regions of the N and C terminus and G72exhibit T1r greater than the average of 89 ms andNOE values less than 0.7, both of which are charac-teristic of motions on the pico- to nanosecond time-scales. Consequently, these regions appear as dis-ordered in the modeled structural ensembles. Simi-lar dynamic trends for the N and C termini andG72 have also been observed in bL30 (Figure 9(b)).The motions of these ¯exible regions are very fast(pico- to nanoseconds), and are unlikely to beassociated directly with RNA binding. A majordifference in dynamics between the free andbound protein has been identi®ed in the regionbetween residues 74 to 88. Broad backbone amidepeaks were observed in this region for fL30, soaccurate relaxation parameters could not beobtained. The broadening in fL30 implies thatintermediate (micro- to milliseconds) time-scalechemical exchange is present. bL30, on the otherhand, residues in this region have dynamic par-ameters similar to other ordered regions. Thus thedisordered region between residues 74 to 88 in thefL30 structure ensemble probably correlates withincreased ¯exibility in the absence of cognateRNA, and is most likely associated with the RNA-binding behavior of the L30 protein. The rest of theresidues in bL30 have relatively uniform relaxationparameters. For example, the average T1r value forfL30 was 89 ms at 10 �C, and for bL30 was about59 ms at 30 �C; the heteronuclear NOE had anaverage value about 0.75 for both fL30 and bL30.These T1r and heteronuclear NOE relaxation par-ameters suggest that most parts of the fL30 andbL30 do not undergo signi®cant internal motion.

Mapping the RNA binding surface onL30 protein

The structural and dynamic comparison betweenthe free and bound proteins, the chemical shift per-turbation data, and the intermolecular protein-RNA NOEs have been used to map the RNA bind-ing site on the L30 protein. For instance, the loopbetween b2 and a3 and the region from residues74 to 88 show large differences between the twoaverage structures (Figure 8(a)). In particular, theregion between 74 and 88 appears to be a largelydisordered coil in fL30 but a more de®ned andordered segment in the bL30. Dynamic studies alsosuggest that residues 74 to 88 in fL30 experiencemotion on the micro- to millisecond time-scale thatis characteristic of a binding site in fL30. Thesedifferences between the free and bound forms are

most likely affected by the presence of RNA bind-ing.

Chemical shift is very sensitive to the localenvironment and geometry (Old®eld, 1995), andRNA binding should be directly re¯ected throughchemical shift changes. The effect of RNA bindingis seen by comparing 2D (1H, 15N) HSQC spectraof the free and bound L30 protein (Figure 4). Atstoichiometric concentrations of L30 protein andRNA, changes are observed in the backbone amide1H resonances of the protein up to 0.5 ppm. Theshift perturbation effects are also extended to otherresonances, such as 1Ha, 1Hb, 13Ca, 13Cb, 13C0 and15N chemical shifts. The most signi®cant chemicalshift changes induced by binding of RNA fallwithin three regions of the protein: the C-terminalportion of b1 and the adjacent N-terminal portionof a2 (Leu25 to Thr30), the loop connecting b2 anda3 (Ala46 to Arg52), and the region between b3and b4 (Gly72 to Val89) (Figure 8(b)). Almost allchemical shift changes are located on one side ofthe aba sandwich, but the opposite face is essen-tially unaffected.

Structural alteration and chemical shift changesmay give a good indication of inter-molecular


interaction, but a simple conformational changealone is suf®cient to induce chemical shift changeswithout direct RNA contact. The most direct evi-dence for RNA binding comes from intermolecularNOEs obtained from samples containing 13C/15N-labeled protein complexed with unlabeled RNA.Intermolecular NOEs have been observed in threeregions (Leu25 to Thr30, Asn47 to Arg52 andLys83 to Gly88), which correspond directly to thethree regions where shift perturbations wereobserved. In contrast, residues between Gly72 andGly83 show no intermolecular NOEs to RNA,suggesting that the observed chemical shift pertur-bations might be associated with the structuralchanges (i.e. from coil to a-helix).

Based on the structural comparison, the chemicalshift perturbation, and the inter-molecular NOEsdata, the L30 protein seems to employ a loop-bind-ing strategy to interact with its target RNA.Detailed examination of the structure of bL30shows a concavity (Figure 8(c)) formed by threeloops between b1 and a2, b2 and a3, and b3 andb4 on one end of the aba sandwich. Several posi-tively charged residues, such as Lys28, Lys32,Arg52, Lys83 and Arg86 are found within andadjacent to the binding site. Many residues withinthe binding site (e.g. Leu25, Gly26, Asp47, Pro49,Arg52, Lys83, Phe85 and Arg86) display absoluteor a high degree of sequence conservation acrossthe phylogeny (Figure 1). Both the charged andhydrophobic residues within the binding site couldpotentially play functional roles for protein-RNArecognition. Further biochemical studies on theprotein will address these issues.

The observation of similar folding topology butdifferent local structures between the free andbound proteins suggests that the L30 protein-RNAcomplex may form according to a local ``induced-®t'' mechanism (Creighton, 1984) by coupling localfolding to site-speci®c binding. Such a recognitionmechanism has been observed in many protein-DNA interactions (Spolar & Record, 1994), andhad been predicted in ribosomal protein L11 (frag-ment 11-76)-RNA interaction (Hinck et al., 1997). Inthe case of L30, the transition from an unorderedloop in free protein to a stable helix in bound formis a characteristic of an induced-®t recognition.Furthermore, induced-®t interaction also accompa-nies a large negative free energy change. Detailedexamination of the structures shows an exposedhydrophobic surface in both protein forms, com-prising residues Leu25, Leu84, Phe85 and Val87. InfL30 the solvent-exposed hydrophobic residuesmay cause the protein to unfold by intra-molecularnon-speci®c hydrophobic interactions, or to aggre-gate through inter-molecule non-speci®c hydro-phobic interactions. In contrast, the structure of theprotein-RNA complex reveals that these residuesare sequestered from solvent upon RNA binding(unpublished results). Burial of these hydrophobicresidues may help to drive RNA binding, andresults in the observed thermal stability of theprotein-RNA complex.

Relationship to other RNA-bindingprotein structures

The topology of bL30 (a four-stranded mixedb-sheet sandwiched between two sets of helices)appears to be unique. An automated search of theBrookhaven protein structure database with theprogram DALI (Holm & Sander, 1993) was con-ducted, and the closest three-layer aba sandwichfold found was the glutaredoxin protein family thatcarry thioredoxin folds (Martin, 1995). However,the thioredoxin fold, a bab and a bba motif with aconnecting helix, is somewhat different from bL30in the order of the secondary structure elements.The arrangement of the four-stranded mixedb-sheet in L30 has also been observed in RNase Pprotein (Stams et al., 1998), the 70 residue COOH-terminal domain of prokaryotic ribosomal proteinS5 (Ramakrishnan & White, 1992) and the 117 resi-due domain IV of elongation factor G (EF-G)(Czworkowski et al., 1994). However, close inspec-tion reveals that there are signi®cant differences inthe b-strand connections and organization and,most important of all, these proteins differ from L30with an unusual left-handed bab crossovertopology.

Although the L30 fold is technically unique, thesplit b-a-b motif in L30 protein is reminiscent ofthe abc/ad-unit in many RNA-binding proteins(E®move, 1995). The abc/ad-unit (Figure 10) hascharacteristic right-handed b-a-b superhelix formedby the b, c and d regions, while strand a is locatedbetween strands b and d in the direction antiparal-lel with them (Rao & Rossmann, 1973). ManyRNA-binding proteins, including the U1A, aminoa-cyl-tRNA synthetases, many prokaryotic ribosomalproteins (e.g. S5, S6, L1, L6, L7/L12, L9, L22 andL30) and proteins from RNA viruses (e.g. tobaccomosaic virus and bacteriophage MS2 protein) carrythis motif (Arnez & Caverelli, 1997). In theeukaryotic L30 protein, b4, b2, a2 and b1 can beconsidered as the respective a, b, c and d regionswithin the abc/ad-unit, while a3, b3 and a4 can beregarded as a large insertion between the a and bregions (Figure 10). The long loop connecting the aand b regions has been observed in the structure ofchorismate mutase (Chook et al., 1993). If only thesegment of the abc/ad region is considered, thensegments of at least four other proteins are foundto share a close similarity with L30 protein, includ-ing the N terminus of S8 (Davies et al., 1996),DNase I (Weston et al., 1992), DNA methyltransfer-ase (Reinisch et al., 1995) and EPSP synthase(Stalling & Kishore, 1991).

Conclusion

The yeast L30 is the newest member in thefamily of eukaryotic ribosomal proteins whosestructures have been studied at near-atomic resol-ution. Signi®cantly, this is the ®rst ribosomal pro-tein whose interaction with its pre-mRNA bindingsite RNA has been studied using an NMR-based

Figure 10. Schematic representation of the topologiesof two basic abc/ad-units (A) and (B), and the abc/ad-unit in L30. Segments a, b, c, and d are highlighted inred, green, yellow and cyan, respectively. The topologyof L30 is a variant of (B), with a long loop (in pink)inserted between the a (in red) and b (in green) seg-ments. For clari®cation, the L30 is labeled with its sec-ondary segments (i.e. a2, b1).


approach. These studies have shown that the L30protein interacts with its target RNA using threeloops at one end of an a/b sandwich. The structureof the free and bound forms of L30 reveal thatRNA binding is accompanied by a local foldingevent and burial of hydrophobic surface. It will beinteresting to compare the RNA binding propertiesof L30 with its rRNA site to learn how the uniquestructure of the L30 protein may have speci®callyevolved towards a specialized function in theeukaryotic ribosome.

Materials and Methods

Protein expression and purification

The yeast S. cerevisiae ribosomal protein L30 (withoutthe ®rst methionine residue) was over-expressed as a sol-uble maltose-binding protein fusion (MBP-L30) in E. colistrain JM109, hosting plasmid pMalc-L30 (Vilardell &Warner, 1994). Unlabeled protein was prepared fromcells grown in a medium containing 25 g/l of LuriaBroth (Gibco) and 100 mg/l ampicillin. Uniformly 15N or13C/15N-labeled cells were grown in M9-based minimalmedium containing 0.7 g/l (15NH4)2SO4 alone or 1.0 g/l(15NH4)2SO4 and 1.0 g/l D-[13C6]glucose (Cambridge Iso-tope Laboratories, Cambridge, MA) with supplements oftrace metals (750 mg CaCl2 �2H2O, 30 mg Na2EDTA,25 mg FeCl3 �6H2O, 240 mg CuSO4 �5H2O, 180 mgMnSO4 �5H2O, 27 mg ZnSO4 �7H2O, and 270 mg CoCl2 perliter), and vitamins (2 ng biotin, 2 ng folic acid, 5 ngthiamine, 5 ng calcium pantothenate, 0.1 ng vitamin B12,5 ng nicotinamide, and 5 ng ribo¯avin per liter). Whenthe cell density reached 108 cells/ml (A600 5 0.6), the cul-ture was shifted to 25 �C, and protein expression wasinduced with 0.5 mM isopropyl-b-D-thiogalactopyrano-side (IPTG), and an additional 100 mg of ampicillin wasadded. After four hours of induction, the cells were har-vested by centrifugation, and lysed by sonication at 0 �Cin ``lysis'' buffer (20 mM Tris �HCl (pH 7.0) 200 mMNaCl, 1 mM EDTA, 5 mM DTT) containing 20 mg/mlphenylmethylsulfonyl ¯uoride (PMSF) and 0.1 unit/mlDNase. Nucleic acids and negatively charged proteins

were precipitated with the addition of polyethyleneimine (pH 8.0) to a ®nal concentration of 0.15 % andmoderate stirring on ice for 30 minutes. The supernatantcontaining the target MBP-L30 fusion protein was loadedonto a carboxyl methyl cellulose (CM) column (TOYO-PEARL) equilibrated with the same lysis buffer. MBP-L30 bound to the column under these conditions, andwas eluted with a salt gradient of 200 mM to 500 mMNaCl. Fractions containing MBP-L30 eluting at � 0.3 MNaCl were pooled and further puri®ed on an amyloseaf®nity column (New England Biolabs) that selectivelybinds to the MBP portion of the protein in order toremove any RNase contaminants. The MBP-L30 proteinwas eluted using the lysis buffer containing 10 mM mal-tose. The free L30 protein was released from the MBP bythe protease Factor Xa cleavage reaction at the engin-eered recognition site (Ile-Glu-Gly-Arg) (Nagai &Thogersen, 1984) in 18 to 36 hours at 4 �C. The separ-ation of L30 protein from the proteolytic cleavage mix-ture was achieved through CM column chromatographywith a salt gradient from 200 mM to 1 M NaCl concen-tration prepared in 10 mM sodium phosphate buffer(pH 7.0). This puri®cation procedure yields an averageof approximately 5 mg of homogeneous L30, as quanti-®ed by either a BioRad protein assay or UV absorbanceat 280 nm (e280 � 8976 Mÿ1cmÿ1). The identity and integ-rity of the protein was con®rmed by electro-spray massspectrometry. The puri®ed L30 shows a single molecularmass of 11,295.1, which is comparable to the calculatedmass of 11,294.4 at pH 4.0.

In vitro RNA binding assay andstoichiometry determination

Binding assays of both MBP-L30 fusion and L30protein are similar to published procedures (Li et al.,1995). The buffer used in this study contained 30 mMTris-HCl (pH 8.0), 75 mM KCl, 2 mM MgCl2, 1 mMDTT, 50 ng/ml bovine serum albumin (BSA) (NewEngland Biolab), 40 ng/ml tRNA (Sigma), and 0.2unit/ml RNase inhibitor (Promega). Dissociation con-stants were determined by titration of 0.0 to 1.9 mMof MBP-L30 fusion protein or 0.0 to 8.0 mM of L30protein into less than 0.01 nM (50-32P)-end-labeledRNA (5Kd) to achieve pseudo-®rst order binding.Protein-RNA stoichiometry was determined fromtitration of 10 nM to 10 mM MBP-L30 fusion proteininto 300 nM unlabeled RNA and trace amount of(50-32P)-labeled RNA. After incubation, binding mixturewas loaded onto 10 % (w/v) native gels (29 to 1 ratioof acrylamide to bisacrylamide) with 0.5 � TBE run-ning buffer at 200 V/20 cm in a 4 �C cold room. Theresulting bandshift data were quanti®ed using a phos-phoimager (Molecular Dynamics, Inc.). All bindingdata were analyzed with the Igor Pro3.0 program(Wavemetrics).

Circular dichroism

Circular dichroism spectra were recorded on eitheran Aviv 60 DS or Aviv 292 SF spectrophotometers.Scans from 200-300 nm (0.5 nm steps, 15 second aver-aging time) were acquired (0.1 cm path-length) on400 ml of 25 mM samples in 10 mM potassium phos-phate buffer (pH 6.5) at 4 �C. While potassium phos-phate buffer was used for background correction ofthe free form protein signal, free RNA in the samebuffer was used for the complex. The signal output,


millidegree (m�), was converted to molar ellipticity [y]via equation ([y] � 100 �m� � (l � c � Nres), where l isthe path-length of the cell (in centimeters), c is theconcentration of the sample (in millimolar), and Nres isthe number of residues in the protein). The percentageof helix and sheet contents were estimated from molarellipiticity [y] according to the literature (Chen et al.,1974). Thermal-melts were carried out by monitoringsignal at 218 nm and increasing temperature from 4 �Cto 88 �C (2�C temperature step, one minute equili-bration, and 30 seconds average time). The molar elli-piticity ([y]) was ®tted to a hyperbolic curve in IgorPro3.0 program (Wavemetrics) to derive the meltingtemperature.

NMR spectroscopy

NMR spectra of fL30 protein were recorded on Var-ian Inova 600 (MIT), FBML 600 (MIT) and BrukerAMX 500 (TSRI) spectrometers equipped with a pulse-®eld gradient unit and triple-resonance 5 mm probe at10 �C. Protein samples ranging 0.5 to 0.7 mM are dis-solved in buffer containing 300 mM NaCl, 0.02 % (v/v)NaN3, 0.1 mM EDTA, and 2.0 mM potassium phos-phate buffer (pH 6.5). Sequential backbone assign-ments of the free L30 protein were accomplishedusing triple resonance experiments HNCA (Ikura et al.,1990), HN(CO)CA (Yamazaki et al., 1994), HNCO(Muhandiram & Kay, 1994; Kay et al., 1992), CBCA(CO)NH (Grzesiek & Bax, 1992), and side-chainassignments with 3D HCCH-TOCSY (Kay et al., 1993)with sensitivity enhancement water suppression.Three-dimensional 13C or 15N-edited NOESY wereused in a few instances. Glutamine He and asparagineHd protons were assigned from their NOESY cross-peaks to the assigned Hg and Hb resonances, respect-ively; methonion He resonances were assigned from2D 13C-edited CT-HSQC, and arginine He resonanceswere assigned by their NOESY cross-peak and15N-edited TOCSY to the assigned Hd resonances.Two-dimensional 15N relaxation (T1, T1r, NOE)measurements of the fL30 were recorded on VarianInova 600 instrument using published pulse sequences(Dayie & Wagner, 1994). The T1 of each backboneamide proton was derived from ®tting six delaypoints: 10.02, 20.04, 40.08, 120.24, 300.6, and 601.2 msto a single exponential decay curve, and the T1r wasderived from six delay points: 2.74, 5.48, 21.93, 41.11,65.78, and 104.15 ms. The 1H-15N NOE was obtainedby measuring the ration of the peak intensitiesbetween with-NOE/without-NOE spectra.

NMR spectra of bL30 were recorded on Varian Inova600 (MIT) and 750 (Harvard) spectrometers at 30 �C. Pro-tein-RNA complex samples ranging from 0.8 to 1.7 mMwere dissolved in buffer containing 0.02 % NaN3,0.1 mM EDTA, 10.0 mM potassium phosphate buffer(pH 5.5). Sequential backbone assignment of the free L30protein were accomplished using triple resonance exper-iments HNCA (Ikura et al., 1990), HN(CO)CA (Yamazakiet al., 1994), HNCO (Muhandiram & Kay, 1994; Kay et al.,1992), HNCACB (Muhandiram & Kay, 1994), and 3DHCCH-TOCSY (Clore et al., 1990) with WATERGATEsolvent suppression. The 15N relaxation parameters(T1, T1r, NOE) of the bL30 were measured using similarapproaches to those of the fL30 except that eight T1

delays points: 10.02, 20.04, 40.08, 150.3, 300.6, 601.2,801.6, and 1202.0 ms, and seven T1r delays points: 2.55,5.10, 10.21, 20.42, 38.28, 61.25, and 96.98 ms were col-lected. All data were processed with NMRPIPE (Delaglio

et al., 1995) and analyzed with CAPP and PIPP (Garrettet al., 1991) or NMRView (Johnson & Blevins, 1994)programs.

Structure calculation

Distance and dihedral restraints were used for thestructure calculation. Distance restraints were derivedfrom three 3D NOESY data sets: 3D 13C-editing, 15N-edit-ing NOESY-HSQC and 13C, 15N simultaneous detectingNC-NOESY-HSQC (Pascal et al., 1994). Protein-RNA con-tacts were derived from 15N and 13C-edited NOESYexperiments. Backbone 3JHNHa coupling constants for thebound form were derived from two independent exper-iments: HNHA (Vuister & Bax, 1993) and HMQC-J (Kayet al., 1989) experiments. Side-chain w1 torsion angleswere derived from 11 ms TOCSY in combination with3D 13C-edited or 15N-edited NOESY (50 ms) experiment.Hydrogen exchange rates were measured by dissolvinga lyophilized, protonated sample of 15N-labeled L30 pro-tein samples in its respective NMR buffer containing99.98 % 2H2O and by following amide proton intensitieswith a series of 2D 1H/15N HSQC spectra at 10 �C and30 �C, respectively. Backbone amide relaxation par-ameters T1, T1r, and NOE rates were measured by the1H-detected pulse scheme as described (Dayie &Wagner, 1994).

Distance restraints for structure calculations werederived by analyzing the three NOE data sets using thePIPP program (Garrett et al., 1991). NOE cross-peakintensities were converted into distances by normalizingeach to the intensity of the corresponding diagonal peakand calculated distances were based on those of the sca-lar-coupled protons. The lower bound for distancerestraints was set to 1.8 AÊ , and the upper bound was setto 3.0 AÊ (strong), 4.0 AÊ (medium), 5.0 AÊ (weak) or 6.0 AÊ

(very weak), with appropriate pseudo-atom correctionsfor methylene and methyl groups. Distance restraints forhydrogen bonds, N to O, and HN to O, were added forthose residues which were found in regular elements ofsecondary structure, and whose amide proton was pro-tected from solvent exchange. Backbone torsion anglerestraints for f, derived from HN-Ha coupling constants,were set to ÿ60(�25) for 3JHNHa < 6 Hz and ÿ120(�40)�for 3JHNHa > 8.0 Hz. Side-chain w1 angles were addition-ally restrained to one of the staggered rotamers, �60 �,ÿ60 � or 180 �, with an error range of �30 �.

The structure of the free protein was calculated usingboth X-PLOR (version 3.1) (BruÈ nger, 1992) and AMBER(Cornell et al., 1995). Initial structure calculations wereconducted in X-PLOR, using on distance and dihedralrestraints described above. Starting from random coordi-nates, 50 initial structures were generated using the stan-dard simulated annealing (SA) protocols optimized forprotein structure calculation (e.g. random.inp, dgsa.inpand re®ne.inp) (BruÈ nger, 1992). The 50 X-PLOR struc-tures were subsequently subjected to 20 ns SA re®ne-ment in AMBER (Sander 5.1), with constant SA at1000 K for the ®rst 10 ns and slow cooling to 0.5 K forthe second 10 ns. Half of the 50 structures converged tolow energy and restraint violation (EAMBER cutoff ofÿ1266 kcal molÿ1), 21 fL30 structures were chosen tocompare with the bound form. The bound protein wascalculated using similar strategy prior to docking withthe RNA in the presence of protein-RNA intermolecularNOEs and subsequent re®nement (unpublished results).A total of 21 of L30-mRNA complex structures were


obtained. The structures for bL30 presented here weretaken from the coordinates of the protein-RNA complex.

Protein Data Bank accession numbers

The coordinates of both the free L30 protein and theL30-mRNA complex have been deposited into theBrookhaven Protein Data Bank (accession codes 1CN6and 1CN8 for the free protein and the complex averagestructures, respectively; 1CN7 and 1CN9 for the free pro-tein and the complex structural ensembles, respectively).The NMR constraints of the free L30 protein and thecomplex have also been deposited into the BrookhavenProtein Data Bank (accession codes R1CN6MR andR1CN8MR, respectively). The 1H, 13C, and 15N chemicalshifts of both the free and bound proteins are availablefrom the BioMagResBank (accession codes 4339 and 4345respectively).

Acknowledgements

We thank Dr Susan White at Bryn Mawr College,Dr Jonathan Warner and Dr Joseph Vilardell at AlbertEinstein College for providing the pMalc-L30 plasmid.We thank Dr Kwaku Dayie and Dr John Chung at TSRIand Dr Christopher Turner at MIT for their assistancewith NMR spectrometers, and Dr Radha Plachikkat forhelpful discussions about X-PLOR. We thank JasonSchnell for assistance with X-PLOR to AMBER restraintconversion, and we thank both Jason Schnell andDr Lena Maler for valuable discussions about AMBER.This project is supported by a grant from the NationalInstitute of Health (GM-53320) to J.R.W.

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Edited by D. E. Draper

(Received 24 May 1999; received in revised form 15July 1999; accepted 16 July 1999)

http://www.academicpress.com/jmb

Supplementary material comprising two Tables isavailable from JMB Online.

http://www.academicpress.com/jmb

local folding coupled to rna binding in the yeast ribosomal protein l30

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