A R T I C L E S

The La protein is a ubiquitous, abundant, monomeric phosphoproteinfound in the nucleus of eukaryotic cells. It associates predominantlywith a short polyuridylate sequence (UUUOH) at the 3′ end of almostall nascent pol III transcripts1,2. In yeast, the La protein (Lh1p1) asso-ciates with RNAs transcribed by pol II that also contain 3′ poly(U)sequences1,2. The specific binding of La to precursor RNA moleculesprotects them from exonuclease digestion3,4 and thereby regulatesdownstream processing. For example, La helps to ensure the correctendonucleolytic digestion of the 5′ and the 3′ extensions of pre-tRNA3,4 and acts as a chaperone to promote the correct folding ofthese molecules5. La also serves to retain precursor RNA molecules inthe nucleus6–8. Moreover, La assists the assembly of functional ribo-nucleoprotein particles9, an activity that may be promoted by its asso-ciation with RNA helicases10.

In addition to its rather complex functional profile, La also has arole in translation1,2. For example, La can bind to the internal ribo-some entry sites (IRES) of hepatitis C virus (HCV)11 and the X-linkedinhibitor of apoptosis protein12, in both cases stimulating translationinitiation. In the case of the HCV IRES, which lacks a 3′-UUUOH, Laseems to bind specifically to an internal sequence near the initiatorAUG codon13.

Human La (hLa) is a multidomain protein of 408 amino acids(Fig. 1). Recognition of UUUOH sequences is a function of the N-terminal domain (NTD), the most conserved portion of the protein. This region contains two subdomains, the La motif and the central RRM (Fig. 1). The structure of the La motif, an ∼ 80-residue domain, has been debated: although some predict that it

folds as a canonical RRM, others contend that it adopts a predomi-nantly helical configuration1,2,14. Both the La motif and the adjacentRRM seem to be required for high-affinity RNA binding15–17, and ithas been suggested that the role of the La motif is to provide specificrecognition for UUUOH sequences1,2,16. Notably, the coupling of a Lamotif to an RRM is observed in a subset of La motif–containing proteins that are otherwise unrelated to La1.

The C-terminal domain of hLa exhibits considerable variationamong eukaryotes1,2. Recent structural work has revealed the presencein this domain of an unusual RRM, encompassing residues 229–326,followed by a long, flexible polypeptide that contains a short basicmotif (SBM), a regulatory phosphorylation site on Ser366 and anuclear localization signal (NLS)18 (Fig. 1). The C-terminal RRM hasa β-sheet comprising five rather than four strands and contains a longC-terminal helix that binds across the putative RNA-binding site,potentially impeding interactions with nucleic acid18. This helix alsoincorporates a functional nuclear retention element (NRE), whichhelps to ensure appropriate localization and processing of pre-tRNAmolecules7,18. The C-terminal RRM is not required for specific, high-affinity interactions with poly(U) RNA, although it remains possiblethat this RRM, in conjunction with the SBM downstream, contributesto La interactions with non-poly(U) RNA targets such as viral RNAs,TOP mRNAs and 5′-triphosphate groups1–3,11,13,19–22.

To shed light on the molecular mechanism of the NTD of hLa,which contains the predominant RNA-binding activity, we have usedNMR to determine the solution structures of its composite domainsand mapped its interactions with poly(U) RNA. We find that the La

1Biophysics Laboratories, Institute of Biomedical and Biomolecular Sciences, University of Portsmouth, St. Michael’s Building, Portsmouth PO1 2DT, UK.2Department of Molecular Structure, Walter and Eliza Hall Institute, Parkville, Victoria 3052, Australia. 3Biomedical NMR Centre, National Institute for MedicalResearch, The Ridgeway, Mill Hill, London NW7 1AA, UK. 4Biophysics Section, Department of Biological Sciences, Blackett Laboratory, Imperial College London,London SW7 2AZ, UK. 5Present address: Department of Biochemistry and Biophysics, Texas A&M University, MS 2128, College Station, Texas 77843-2128, USA.6These authors contributed equally to this work. Correspondence should be addressed to M.R.C. (sasi.conte@port.ac.uk).

Published online 7 March 2004; doi:10.1038/nsmb747

Structural analysis of cooperative RNA binding by the Lamotif and central RRM domain of human La proteinCaterina Alfano1,6, Domenico Sanfelice1,6, Jeff Babon2, Geoff Kelly3, Amanda Jacks1,5, Stephen Curry4 & Maria R Conte1

The La protein is a conserved component of eukaryotic ribonucleoprotein complexes that binds the 3′ poly(U)-rich elements of nascent RNA polymerase III (pol III) transcripts to assist folding and maturation. This specific recognition is mediated by the N-terminal domain (NTD) of La, which comprises a La motif and an RNA recognition motif (RRM). We have determined the solution structures of both domains and show that the La motif adopts an �/� fold that comprises a winged-helix motifelaborated by the insertion of three helices. Chemical shift mapping experiments show that these insertions are involved in RNA interactions. They further delineate a distinct surface patch on each domain—containing both basic and aromaticresidues—that interacts with RNA and accounts for the cooperative binding of short oligonucleotides exhibited by the La NTD.

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motif is not an RRM but folds into a largely helical domain that seemsto be an elaborated winged-helix module. The central RRM has a classical structure consisting of a four-strand β-sheet backed by two α-helices but has an unusual C-terminal α-helix that projects up fromthe β-sheet RNA-binding surface. NMR analysis also indicates that theLa motif and the central RRM are independently folded and maintainthe same three-dimensional structure in the context of an NTD frag-ment (La(1–194)) that contains both modules. We confirm that, whenpresent in isolation, neither the La motif nor the central RRM has sub-stantial affinity for oligo(U) RNA; instead, the domains seem to actsynergistically, as the minimal fragment of hLa that has specific RNA-binding activity comprises the La motif and the central RRM. Thisbehavior is explained by chemical shift mapping of the interactions ofshort oligo(U) RNAs with the intact La NTD, which identify distinctsurface patches on each domain that are involved in RNA binding.

RESULTSStructure determinationTo characterize the structure of the NTD ofhuman La, three constructs were expressedand purified: La(1–103) (the La motif),La(105–202) (the central RRM) andLa(1–194), a fragment that incorporates bothdomains. The three-dimensional structures

of the La motif and central RRM were determined using standard heteronuclear multidimensional NMR techniques (see Methods).Analysis of the backbone 1HN and 15N chemical shifts of the isolatedLa(1–103) and La(105–202) show that these are not substantiallyaltered in La(1–194) (data not shown); this indicates that the struc-tures of the two component domains are largely conserved in the tan-dem construct and suggests that the modules do not form stableinterdomain interactions in the absence of RNA. We therefore chose tosolve the structures of the isolated domains, as their reduced sizes andsuperior solubilities simplified the analysis of NMR spectra obtainedwith double-labeled 13C-15N samples and allowed a higher-resolutionstructure determination. A final family of 20 superposed structures forthe La motif and the central RRM is shown in Figure 2. The overallr.m.s. deviation between the family and the mean coordinate positionsis 0.53 and 0.55 Å for backbone atoms in the structured regions of thetwo domains, respectively.

Tertiary structure of the La motifThe structure of the La motif has been controversial during the pastdecade. The tertiary structure presented here is consistent with theNMR-based secondary structure assignment23 and reveals that the Lamotif does not adopt an RRM-like fold. In fact, its compact structure comprises six α-helices and a three-stranded antiparallel β-sheet (Fig. 2).

The structure of the La motif is dominated by a very long amphi-pathic α-helix (α1; residues 8–25), which, after a small kink at Gly26,is followed by a shorter helix identified ambiguously in our initial sec-ondary structure assignment23 (and labeled α1′ for consistency). The

Figure 1 Domain organization of human La and deletion mutants used inthis study. Conventionally1,2 human La is divided in two halves, N-terminaldomain, comprising the La motif (domain 1) and the adjacent RRM (domain2), and C-terminal domain, encompassing the RRM (domain 3), the SBMand the NLS (see text).

Figure 2 Structural analysis of the La motif andthe adjacent RRM. (a,b) Superposition of thebackbone traces of 20 refined structures for (a) the La motif (residues 6–100) and (b) thecentral RRM of hLa (residues 106–195). The N and C termini and the helices are indicated; the β-strands are numbered 1–4. (c) Diagram ofthe La motif showing protein secondary structure.Helices that are insertions into the canonicalwinged-helix motif are blue. Selected basicresidues are color-coded by atom type (carbon,gray; nitrogen, blue; oxygen, red). (d) Structure ofthe central RRM of hLa. Blue helices areadditional features to the core RRM structure.Selected basic residues are shown. (e) Structure ofthe C-terminal domain of human La18. (f) Close-upview of polar and hydrophobic residues involved instabilizing helix α3 relative to the surface of the β-sheet. Atoms are color-coded as in c. Panels c–fand Figure 3 were prepared using MolScript52,PyMOL (http://www.pymol.org) and MolMol50.

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polar flank of helix α1 is largely solvent-exposed but the opposite apolar face makeshydrophobic contacts with every other sec-ondary structure element in the La motif,forming the hydrophobic core of the domain.The twisted three-strand β-sheet contributesapolar residues to the domain core but alsopresents a solvent-exposed surface on theopposite side of the domain to helix α1. Helixα1 seems to provide a scaffold around whichthe rest of the domain can fold.

Apart from the N- and C-terminal portionsof the polypeptide chain, the loop connectinghelix α5 and strand β2 is the most poorlydefined part of the molecule; {1H}-15N NOEanalysis indicates that this region undergoesfast internal motion on the pico- to nanosec-ond timescale. The C-terminal extension ofstrand β3 contains a well-defined loop fol-lowed by an extended arm that folds back ontothe molecule, contacting helix α1′ and theloops connecting it to α1 and α2 (Fig. 2a,c).

According to a search of the ProteinStructure Matching server at EMBL-EBI(http://www.ebi.ac.uk/msd-srv/ssm/), the Lamotif is an elaborated version of a winged-helix protein, a small module found in manyDNA-binding proteins that folds withαβααββ topology to give a three-strand β-sheet packed against three helices24. The‘wings’ of the domain are formed by the loopbetween β2 and β3 and the polypeptide extension following β3. Structural alignmentshows that the winged-helix motif within the La motif is formed by the elementsα1–β1–α3–α5–β2–β3; thus, α1′, α2 and α4may be considered insertions within the con-served module. Two of the most similar struc-tures to the La motif (Q ≥ 0.2; r.m.s. deviation∼ 3.3 Å) are the Zα domain of ADAR1 (ref. 25)and the central core domain of TFIIEβ26.

Notably, members of this family exhibit variable modes of inter-action with DNA. Many winged-helix proteins bind in the majorgroove of B-DNA, largely via contacts with the so-called ‘recognition’helix α3 (corresponding to α5 in the La motif), with wings 1 and 2making variable contributions to DNA recognition24,27. The versatil-ity of this domain is underscored by the finding that the Zα domainof ADAR1 binds left-handed Z-DNA, again via the recognitionhelix25. Moreover, in other cases, the recognition helix has only aminor role in DNA binding, whereas the wings or other surface features dominate the interactions with nucleic acid26,28. The Lamotif, a winged-helix protein that binds RNA, is an interesting newstructural variant on this theme (see below).

Tertiary structure of the central RRMAccording to the three-dimensional structure of La(105–202), thisdomain adopts a classical RRM-type fold (Fig. 2d). In our preliminarysecondary structure assignment29 we described an additional β-strand (β4′) inserted between α2 and β4; however, the three-dimensional structure shows that although this section of extendedpolypeptide (residues 173–176) makes antiparallel contacts with β4, it

is not itself a β-strand because it lacks appropriate interstrand hydrogenbonds. The C-terminal helix (α3; residues 185–194) is connected to β4by an extremely short, single-residue linker (Phe184) and is located onthe β4 side of the RNA-binding surface of the β-sheet, mainly stabilizedby hydrophobic contacts between side chains from the helix (Tyr188and the aliphatic portions of Lys185, Asp186 and Asp187) and Tyr114,Leu183 and Phe184 on the surface of the β-sheet (Fig. 2f). There mayalso be a salt bridge interaction between Asp186 and Lys109. The C-terminal helix also abuts a short 310-helical turn at the N terminus ofthe domain (α0; residues 108–111), reminiscent of one observed inhnRNP A1 (refs. 30,31). Helix α3 is predominantly hydrophilic andprotrudes away from the body of the domain and toward the solvent;this unusual spatial arrangement with respect to the rest of the mole-cule has not previously been observed in RRM domains bearing a C-terminal α-helix32,33. This configuration is also in marked contrastto the long, amphipathic helix α3 of the C-terminal RRM of hLa, whichforms an extensive network of hydrophobic interactions with an apolarpatch on the β-sheet, thereby obscuring the putative RNA-binding surface18 (Fig. 2e). Thus the β-sheet surface of the central RRM seemsto be largely available for RNA interactions.

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Figure 3 Analysis of La-oligo(U) interaction. (a,b) Structure mapping of the chemical shift variationsobtained on RNA binding for the La motif (a) and the central RRM (b) and comparison with theelectrostatic surface potential. The positions of residues that show substantial shifts are indicated onthe protein secondary structures (top) and surfaces (middle) whereas the electrostatic surface potentialin each case is shown below. The orientations in the left-hand column for each protein are similar tothose in Figure 2. Magnitudes of chemical shift changes are color-coded as follows: yellow, 0.05 ≤∆δAV ≤ 0.07; orange, 0.07 ≤ ∆δAV ≤ 0.1; red ∆δAV ≥ 0.1 (Supplementary Table 1 online). Selectedresidues showing large chemical shift changes on addition of RNA are labeled. Electrostatic surfacepotentials are red and blue for acidic and basic regions, respectively.

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The conserved RNP motifs predicted for the central RRM of hLa(113-VYIKGF-118 and 151-KGSIFVVF-158), located on the centralpair of β-strands, contain two of the three residues (Tyr114 andPhe155) that are generally conserved in RRM domains as aromaticside chains that can make base-stacking interactions with RNA32; thethird aromatic residue has been substituted by Ser153. Notably, Tyr114participates in hydrophobic contacts with the base of the C-terminalhelix; if it is involved in RNA binding, this interaction will require dis-placement of helix α3.

Protein structure matching via the EMBL-EBI server showed thatthe central RRM of hLa is most similar to RRM1 of the sex-lethal pro-tein34 and RRM1 of hnRNP D0 (ref. 35) (Q > 0.4; r.m.s. deviation∼ 2.3 Å). The C-terminal RRM of hLa was only the 97th best structuralmatch, and structural alignment of the central and C-terminal RRMsof the protein indicate substantial differences in addition to those dis-cussed above. In particular, the position of helices α1 and α2 on the‘rear’ of the domains relative to the β-sheet differs considerablybetween the two domains (Fig. 2). Of more relevance to RNA interac-tions, loop β2-β3 in the central RRM, a feature often implicated inRNA interactions, contains five basic residues, which may form elec-trostatic interactions with the sugar-phosphate backbone of RNA

(compared with two basic residues in thesame loop of the C-terminal RRM).Moreover, in contrast to the C-terminalRRM, the β-sheet surface of the central RRMis entirely devoid of acidic residues18.Together, these make the β-sheet surface ofthe central RRM much more basic and proba-bly account for its vastly superior RNA-binding activity (see below).

The α3 helix of the C-terminal RRM of hLa(residues 316–332) contains an NRE thathelps to retain pre-tRNAs in the nucleus forpost-transcriptional processing7,18. AlthoughLa homologs in the yeasts Saccharomyces cere-visiae and Schizosaccharomyces pombe lack adomain corresponding to the C-terminalRRM found in higher eukaryotes, a similarNRE was identified in S. pombe La (spLa;residues 233–255)7, which, on the basis ofsequence alignment with hLa, maps onto theC-terminal helix of the central RRM. Thus, aconserved structural and functional elementis associated with different RRM domains inthe La proteins of yeasts and vertebrates.

RNA-binding analysis of the La NTDTo examine the RNA-binding properties ofthe La motif and central RRM constructs usedin our structural analysis, we carried out gelmobility shift assays with a ten-nucleotideoligo(U) RNA. We confirmed that the Lamotif (La(1–103)) does not bind this RNAand that the truncation mutant La(104–408)reduces the binding activity to a weak, non-specific interaction16,17 (see SupplementaryFig. 1 online). In contrast to its tight interac-tion with HCV IRES13, we found that, on itsown, the central RRM (La(105–202)) doesnot bind ten-nucleotide oligo(U) RNA.Addition of the C-terminal RRM to this

domain (construct La(105–334)) conferred no improvement in RNAbinding (Supplementary Fig. 1 online). As we have previously shown,addition of the N-terminal La motif restores binding affinity observedwith oligo(U) to a level similar to that observed for full-length La18.This is consistent with data from other laboratories obtained with avariety of RNA ligands that also terminate in UUUOH

16,17,36.

Chemical shift analysis of RNA recognitionThe high-affinity RNA-binding activity of the hLa NTD was furtherevaluated using 1H-15N HSQC NMR experiments to monitor thebackbone amide chemical shift changes in La(1–194) upon titration often-nucleotide oligo(U) RNA37. This sensitive method allows detec-tion of amide chemical shift perturbation due to direct RNA bindingor conformational changes induced by ligand interaction, and cantherefore delineate the regions directly affected by complex formation.However, the method cannot probe regions of severe spectral overlapand may not report the full extent of the interaction surface. In thepresent case, in addition to scattered surface residues (SupplementaryTable 1 online) the resonances of the interdomain linker (residues99–105) could not be unambiguously assigned in the spectra of thetandem construct and are not included in the analysis.

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�4 �3 �1 �2 �1 �1´ 10 20 30 40 50 60 l s ls mll lll ll l mms ll ms H. sap. MAENGDNEKMAALEAKICHQIEYYFGDFNLPRDKFLK-EQIKLDEGWVPLEIMIKFNRLNRL M. mus. MAENGDNEKMTALEAKICHQIEYYFGDFNLPRDKFLK-EQIKLDEGWVPLETMIKFNRLNRL X. laev. MAENGDKEQKLDSDTKICEQIEYYFGDHNLPRDKFLK-QQILLDDGWVPLETMIKFNRLSKL D. mel. -AKNGDAKKDPAQERAIIRQVEYYFGDANLNRDKFLR-EQIGNEDGWVPLSVLVTFKRLASL C. eleg. MGET-AAAVHDDADQKIIKQLEYYFGNINLPRDKFLQ-EKLKEDDGWVPITTMLNFNRLASI T. bruc. MPLSSENK------QKLQKQVEFYFSDVNVQRDIFLKGKMAENAEGFVSLETLLTFKRVNSV S. pombe --EDDGKKDLSFDEAEVLKQVEFYFSDTNLPHDKFLW-TTSQKNDGWVPIQTIANFKRMRRF S. cerev. ---------TPEVLDRCLKQVEFYFSEFNFPYDRFLR-TTAEKNDGWVPISTIATFNRMKKY 70 80 90 100 110 120

�5 �0 �2 �1 �3

l l s s s mllls l H. sap. TTDFNVIVEALSKSKAELMEISEDKTKIRRSPSKPLPEVTDEYKNDVKNRSVYIKGFPTD--- M. mus. TTDFNVIVQALSKSKAKLMEVSADKTKIRRSPSRPLPEVTDEYKNDVKNRSVYIKGFPTD-- X. laev. TTDFNTILQALKKSKTELLEINEEKCKIRRSPAKPLPELNDEYKNSLKHKSVYIKGFPTS-- D. mel. STDLSEIVAALNKSEEGLVEISEDKLSLRRHPERPIPEHNEERRKEIQERTAYAKGFPLD-- C. eleg. SKDTEKIANAVKNSGSGIISVSEDNQKIRRNEENPVPENSLEYWQKIKHRTVYMKGFSTD-- T. bruc. TTDVKEVVEAIRPS--EKLVLSEDGLMVRR--RDPLPES-----IQTDHQTVYVKPVPPT-- S. pombe -QPLEAIVNALRKS-PELLEVDEAGEKVRRMIPLVRVD-----NKSVMERSVYCKGFGDE-- S. cerev. RP-VDKVIEALRSS--EILEVSADGENVKRRVPLDLTAARNARIEQNQ-RTLAVMNFPHEDV

�1 �2 �3 �2 130 140 150 160 m l s ls lll l ssl l ls sl m H. sap. ------ATLDDIKEWLEDKGQVLNIQMRR-TLHK-------AFKGSIFVVFDSIESAKKFVE M. mus. ------ATLDDIKEWLDDKGQILNIQMRR-TLHK-------TFKGSIFAVFDSIQSAKKFVE X. laev. ------AILDDVKEWLKDKGPIENIQMRR-TLQR-------EFKGSIFIIFNTDDDAKKFLE D. mel. ------SQISELLDFAANYDKVVNLTMRK-HYDK-------PFKGSIFLTFETKDQAKAFLE C. eleg. ------TQLDDIIQWANQFGETENVLMRR-LKPGDR-----TFKGSVFITYKTREEA-EAAQ T. bruc. ------ATLEQLTEFFSKHGTVQAVWRRY-FAGKKDAPPESRTKPSVFVVFNSSEEAEAFQK S. pombe ------KDDTQIKFFEENAGPISAVRMRR-DDDK-------KFKGSVFVEFKEPDVANKFLE S. cerev. EASQIPELQENLEAFFKKLGEINQVRLRRDHRNK-------KFNGTVLVEFKTIPECEAFLK 170 180 190 200 mll H. sap. TPGQK--------YKETDLLILFKDDYFAKKNEERKQNKVE M. mus. IPGQK--------YKDTNLLILFKEDYFAKKNEERKQSKVE X. laev. NRNLK--------YKDNDMTVLSREEYHAKKNEERKLNKSE D. mel. QEKIV--------YKERELLRKWQVDYLKEKQEEYAQKNEK C. eleg. KAEVK--------FGETELTKMMQDEYWTLKNKETKEARAA T. bruc. AP-PM--------YDDVQLTAEMKTTYLERKAEEIAAKKSS S. pombe KVKLK--------WGEDELTIMSKKEYVDMKAELHKNDPPK S. cerev. SYSNDDESNEILSYEGKKLSVLTKKQFDLQREASKSKNFSG

�4 �3

Figure 4 Alignment of the La motif and central RRM sequences for La homologs. Invariant residues arehighlighted in blue, and conserved residues in red. The secondary structure elements are are shownabove the amino acid sequence and color-coded as in Figure 2. Letters s, m and l indicate residuesaffected in the RNA chemical shift experiment: s, small, 0.05 ≤ ∆δAV ≤ 0.07; m, medium, 0.07 ≤∆δAV ≤ 0.1; l, large, red ∆δAV ≥ 0.1.©

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The results of the 1H-15N HSQC chemical shift mapping experi-ments with La(1–194) indicate that a substantial subset of the assignedresonances in both the La motif and the central RRM are shifted uponaddition of ten-nucleotide oligo(U) (Supplementary Fig. 2 online).The location of residues with a weighted average of the backbone 15Nand 1HN shift variation upon binding >0.05 (δ∆AV, see Methods andSupplementary Table 1 online) is shown in a surface representation ofthe two domains in Figure 3. For the central RRM, as expected for thistype of module, the residues affected by addition of RNA are mainlyclustered in a large area spread across the face of the domain thatencompasses the central β-sheet, the β2-β3 loop, the C-terminal α-helix and the N-terminal 310-helix (Fig. 3). A few generally isolatedresonance shifts are located on loops α1-β2 and β3-α2 and on theinner surface of helices α1 and α2. These results are compatible withthe assumption that the conformation of the domain is largely preserved upon RNA binding (with the possible exception of the C-terminal helix), and that RNA binding occurs across the β-sheetsurface, in agreement with previously studied RRM–RNA com-plexes32,34,38. Consistent with this interpretation, the binding regionidentified by chemical shift mapping is the most basic feature on thesurface of the domain (Fig. 3).

On the La motif, the largest chemical shift changes associated withRNA binding map to helices α1, α1′, α2, α3 and α4 and are clusteredinto two surface patches on one edge of the molecule. The oppositeface of the protein almost completely lacks perturbed residues,although three small shifts occur in residues of the β-sheet (Fig. 3).Notably, resonances belonging to helix α5, which corresponds to thecanonical recognition helix α3 of winged-helix proteins (see above),are unaffected in the titration experiment, as is also observed forTFIIEβ26. Notably, the structural elements (α1′, α2 and α4) that areinsertions in the La motif relative to the archetypal winged-helix motif

are all implicated in RNA binding. This suggests that the interaction ofthe La motif with RNA may represent a new mode of binding forwinged-helix proteins. The notion that the principal RNA-bindingregion is on the helical face of the La motif centered at the base of α1(on the opposite side of the domain to the β-sheet) is supported by theobservation that the area most affected by RNA coincides with thelargest basic region on the surface of the domain. Notably, this regionis very close to the linker peptide extending from β3 that connects theLa motif to the central RRM; this peptide, which corresponds to wing2, may also be involved in RNA interactions.

Although the La motif and the central RRM use radically differentstructural elements to interact with RNA, the identified binding sur-faces both contain clusters of conserved solvent-exposed aromatic sidechains that may form base-stacking interactions with RNA32.Although Phe28 in the La motif and Phe155 and Tyr188 from the cen-tral RRM are associated with large shifts upon addition of RNA(Figs. 3 and 4), the lack of observed shifts for neighboring surface-exposed aromatics is in part due to peak overlap in the HSQC spec-trum, which hampers the identification of affected residues(Supplementary Table 1 online).

DISCUSSIONOur structural analysis reveals that the N-terminal domain of humanLa protein, which contains the primary RNA-binding activity of theprotein, is composed of a La motif, a previously undetected variationof the winged-helix module that has three helical insertions, attachedby a short linker to an RRM domain terminated by a helix that projectsup from the β-sheet RNA-binding surface. The unusual features ofboth domains have been shown to participate in RNA binding.

In fact, chemical shift analysis shows large areas of perturbation onboth domains and indicates that the domains work together as a func-tional unit to bind RNA. Synergy may arise from the RNA lying lin-early across a contiguous binding surface formed by the two modules,somewhat akin to the interaction observed between the poly(A)-binding protein and its cognate RNA38. Alternatively, RNA bindingcould promote a closer association of the La motif and the RRM toform a binding pocket in which the binding surfaces from eachdomain contact opposite sides of the RNA. In favor of the latter model,a shorter five-nucleotide oligo(U) molecule, which probably cannotspan the full length of an open binding site, induces a very similar pattern of chemical shift perturbation to that of the decamer oligo(U)(data not shown). However, further structural work is required to testthis hypothesis.

The residues that undergo chemical shift changes upon RNA bind-ing in hLa NTD are highly conserved in the La proteins of differenteukaryotes, including yeasts (Fig. 4), suggesting that these orthologsadopt a similar mode of RNA binding. Indeed, this possibility mayextend to members of the larger family of La motif-containing pro-teins that also have an adjoining RRM1.

Almost 20 years after the cloning of human La39, the initial struc-tural survey of the component modules of La is now complete. Ourfindings provide a structural framework for the design of new experi-mental approaches to arrive at deeper insights into the function of thisversatile protein and its many orthologs. The challenge now is to putthe pieces together to determine the structure of a La–RNA complex.

METHODSPlasmid construction and protein expression. Plasmids encoding the full-length human La (pET-La(1–408)) and the N-terminal domain (pET-La(1–194))16 were provided by J. Keene and D. Kenan. La(1–408) andLa(1–194) and were expressed and purified as described18.

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Table 1 Structural statistics for the La motif and the central RRMof hLa

NMR restraints La motif RRM

Total distance restraints (inter-residue)

Short to medium range (residue i to i + j, j = 1–4) 436 292

Long range (residue i to i + j, j > 4) 195 140

Hydrogen bond 40 56

Total dihedral angle restraints 146 144

φ 73 73

ψ 73 71

NH residual dipolar coupling restraints 29 21

Restraints violations

Distance restraint violation > 0.3 Å 0 0

Dihedral restraint violation > 5° 0 0

NH residual dipolar coupling restraint violation > 2 Hz 0 0

Average r.m.s. deviation (Å) among the 20 refined structures

Backbone of structured regionsa 0.53 0.55

Heavy atoms of structured regions 1.06 1.09

Backbone of all residuesb 0.61 0.69

Heavy atoms of all residues 1.15 1.21

Ramachandran statistics of 20 structures

Most favored regions (%) 78.2 82.0

Additional allowed regions (%) 18.0 13.9

Generously allowed regions (%) 3.5 2.5

Disallowed regions (%) 0.2 1.6

aResidues selected on the basis of 15N backbone dynamics. La motif: residues 11–75, 79–97;RRM: residues 108–116, 121–134, 138–142, 154–170, 174–192. bLa motif: residues9–97; RRM: residues 108–192.

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A plasmid encoding La(104–408) with an N-terminal His6-tag17 was a giftof R. Maraia. La(105–202) (the central RRM) was subcloned into a modifiedpET-28a vector that adds a short N-terminal His6-tag as described29.La(1–103) (the La motif) and La(105–334) were cloned in a pET-30 vectorusing the LIC methodology (Novagen)23. All His-tagged proteins wereexpressed in Escherichia coli strain BL21(DE3) pLysS grown on either richmedia or minimal media supplemented with 0.8 g l–1 [15N]ammonium chlo-ride and 2 g l–1 [13C]glucose. Cell pellets were lysed in 20 mM Tris-HCl,300 mM NaCl, 10 mM imidazole, pH 8, and purified by affinity chroma-tography on a Ni-NTA resin (Qiagen) using the manufacturer’s protocol. Theeluted proteins were dialyzed in 20 mM Tris-HCl, 100 mM KCl, 1 mM DTT,pH 7.25, loaded on a 5 ml Hi-Trap Heparin column (Amersham PharmaciaBiotech) and eluted with a linear 0–2.0 M KCl gradient in buffer A (50 mMTris-HCl, 0.2 mM EDTA, 1.5 mM MgCl2, 10% (v/v) glycerol, pH 7.25). ForNMR studies, La(1–103) was dialyzed in 50 mM Tris-HCl, 100 mM KCl,5 mM Ca2Cl, pH 8, and cleaved with factor Xa (Novagen) to remove the tag asdescribed23. The cleaved tags were removed by reapplying the sample ontothe Ni-NTA column; the purified protein was dialyzed against 20 mM Tris-HCl, 100 mM KCl, 1 mM DTT, pH 7.

NMR spectroscopy. NMR sample preparation and the 1H, 15N and 13C reso-nance assignments for the La motif and the central RRM have beenreported23,29. NMR samples of unlabeled, 15N-labeled and/or 15N-13C labeledLa(1–194) were prepared by dialyzing the purified proteins against buffers con-taining 20 mM Tris-HCl, 100 mM KCl, 1 mM DTT, pH 7. NMR spectra wererecorded at 293 K on Varian Inova spectrometers operating at 14.1 and 18.8 T,processed using NMRPipe and NMRDraw40 and analyzed using XEASY41. Forstructure calculation, distance restraints were obtained from 1H-15N and 1H-13C edited NOESY-HSQC experiments42 and dihedral φ and ψ angles wereobtained as described23,29. Hydrogen bonded amide protons were identified byacquiring a series of 1H-15N HSQC spectra up to 12 h after buffer-exchangingthe proteins in D2O. T1, T2 and{1H}-15N NOE experiments were done usingthe pulse sequences adapted from standard schemes43. 1DNH residual dipolarcouplings for La(1–103) and La(105–202), were measured at 293 K in a ternarycomplex composed of ~5% (v/v) alkyl-poly(ethylene glycol) C8E5, ~0.8%(v/v) n-octanol and 10 mM Tris-HCl, 50 mM KCl, 0.5 mM DTT, pH 7, and20 mM sodium phosphate,100 mM KCl, pH 6, respectively44. The liquid crys-talline media gave a stable quadrupolar splitting of the D2O signal of 29.9 and33.6 Hz, respectively. The final concentration of the proteins in this media was∼ 0.15 mM. Precise measurements of 1JNH splittings were obtained from 1JNH-modulated 2D spectra45.

RNA titration experiments were done by adding increasing amounts of unla-beled synthetic oligonucleotides ten-nucleotide oligo(U) (5′-UUUUUUU-UUU-3′) and five-nucleotide oligo(U) (5′-UUUUU-3′) (Dharmacon) to15N-labeled protein (La(1–194)). 1H-15N HSQC spectra were recorded atRNA/protein mole ratios of 0.2:1, 0.5:1, 0.8:1, 1:1, 1.2:1 and 1.5:1 to follow theresonances perturbed by RNA binding to the protein. The titration buffer usedwas 20 mM Tris-HCl, 100 mM KCl, 1 mM DTT, pH 7. The backbone amideassignments of La(1–194) were transferred in the complex to the resonancesthat would have the smallest δ∆AV. The weighted average of 15N and 1HN chem-ical shift variation was calculated as follows: ∆δAV = {0.5[∆δ(1HN)2 + (0.2 ∆δ(15N))2]}1/2. Chemical shift variation due to RNA binding ∆δAV wasdivided in three major categories: 0.05 ≤ ∆δAV ≤ 0.07; 0.07 ≤ ∆δAV ≤ 0.1; ∆δAV ≥0.1 (see Supplementary Table 1 online).

Structure calculation. The solution structures of the La motif and the centralRRM of hLa were calculated using a combined torsion angle and Cartesiancoordinates dynamics protocol executed in X-PLOR46 modified to includedipolar coupling restraints47. The structures of the La motif were calculatedfrom random starting coordinates on the basis of 631 NOE distance restraints,composed of 436 short-range (residue i to residue i + j, where 1 < j ≤ 4) and 195 long-range (residue i to residue i + j, where j > 4) connectivities, 146 dihedral angle restraints, composed of 73 φand 73 ψ angles, 40 hydrogen bonddistance restrains and 29 NH residual dipolar coupling restraints. The struc-tures of the central RRM were calculated as described above using 432 NOE dis-tance connectivities (292 short-range and 140 long-range restraints), 144dihedral angle restraints (73 φ and 71 ψ angles), 56 hydrogen bond distance

restraints and 21 NH residual dipolar coupling restrains. Dihedral anglerestraints were obtained using the backbone torsion angle prediction packageTALOS48. The NH residual dipolar coupling restraints were incorporated dur-ing the Cartesian coordinate dynamics phase of the simulated annealing proto-col as harmonic restraints. Initial estimates of the magnitude and rhombicity ofthe alignment tensor, obtained using the maximum-likelihood method49 andnot improved on by iteration during structure calculation were the following:La(1–103), magnitude –19.4 Hz, rhombicity 0.30; La(105–202), magnitude–17.25 Hz, rhombicity 0.25.

The structures were displayed and analyzed using MolMol50 andPROCHECK-NMR51. The final family for both modules comprised the 20structures of lowest total energy; structure statistics are summarized in Table 1.

Gel mobility shift assays. The affinities of several deletion mutants of La forradiolabeled RNA targets were assessed using gel shift binding assays. Thedecameric synthetic RNA oligonucleotide, ten-nucleotide oligo(U) (5′-UUU-UUUUUUU-3′), was prepared and gel-purified by Dhamarcon Research. Thegel shift experiments were conducted as described18.

Coordinates. Structure coordinates of the La motif and central RRM havebeen deposited in the Protein Data Bank (accession codes 1S7A and 1S79,respectively).

Note: Supplementary information is available on the Nature Structural & MolecularBiology website.

ACKNOWLEDGMENTSWe are grateful to D. Kenan, J. Keene, N. Sonenberg and R. Maraia for the gift ofplasmids containing constructs of human La. We thank S. Wolin and K. Reinischfor communicating results before publication. We are indebted to The WellcomeTrust for financial support. C.A. and D.S. are Institute of Biomedical andBiomolecular Sciences research students. A list of NMR assignments and restraintsis available from M.R.C. (sasi.conte@port.ac.uk).

COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.

Received 26 January; accepted 19 February 2004Published online at http://www.nature.com/natstructmolbiol/

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