Structural Insight into a Molecular Switch in Tandem Winged-helix Motifs from Elongation Factor SelB

doi:10.1016/j.jmb.2007.05.001

J. Mol. Biol. (2007) 370, 728–741

Structural Insight into a Molecular Switch in Tandem Winged-helix Motifs from Elongation Factor SelB Nicolas Soler, Dominique Fourmy⁎ and Satoko Yoshizawa⁎ Laboratoire de Chimie et Biologie Structurales, ICSN-CNRS, 1 ave de la terrasse, 91190 Gif-sur-Yvette, France

Elongation factor SelB is responsible for co-translational incorporation of selenocysteine (Sec) into proteins. The UGA stop codon is recoded as a Sec codon in the presence of a downstream mRNA hairpin. In prokaryotes, in addition to the EF-Tu-like N-terminal domains, a C-terminal extension containing four tandem winged-helix motifs (WH1-4) recognizes the mRNA hairpin. The 2.3–Å resolution crystal structure of the Escherichia coli WH3/4 domains bound to mRNA with mutagenesis data reveal that the two WH motifs use the same structural elements to bind RNA. The structure together with the 2.6–Å resolution structure of the WH1-4 domains from Moorella thermoacetica bound to RNA revealed that a salt bridge connecting WH2 to WH3 modules is disrupted upon mRNA binding. The results provide a structural basis for the molecular switch that may allow communication between tRNA and mRNA binding sites and illustrate how RNA acts as an activator of the switch. The structures show that tandem WH motifs not only provide an excellent scaffold for RNA binding but can also have an active role in the function of protein–RNA complexes. © 2007 Elsevier Ltd. All rights reserved.

*Corresponding authors

Keywords: elongation factor; molecular switch; selenocysteine; RNA recognition; winged-helix

Introduction Selenium, an essential trace element, is found in antioxidant proteins in the form of the 21st amino acid, selenocysteine (Sec).1 Sec residues are usually located in enzyme-active sites and are essential for their activity.2–4 In all three kingdoms of life, incorporation of this unusual genetically encoded amino acid into proteins requires elongation factor SelB.5 This factor is homologous to elongation factor Tu (EF-Tu) that catalyzes the binding of aminoacyltRNAs (aa-tRNAs) to the ribosomal A site in the form of a ternary complex EF-Tu–GTP–aa-tRNA.6,7 For selenocysteine incorporation into polypeptides, a UGA stop codon is recoded as a Sec codon in the presence of a downstream selenocysteine insertion

Abbreviations used: Sec, selenocysteine; aa-tRNA, aminoacyl-tRNA; EF-Tu, elongation factor Tu; SECIS, selenocysteine insertion sequence; WH, winged-helix; RRM, RNA recognition motif. E-mail addresses of the corresponding authors: [email protected]; [email protected]

sequence (SECIS) (Figure 1(a)). Prokaryotic SelB is characterized by its unusual property of binding directly to both tRNA and the SECIS mRNA sequence. The recognition of SECIS mRNA hairpin serves as a signal for delivery of selenocysteyl-tRNA (Sec-tRNASec) at a UGA codon. SelB is composed of a N-terminal region homologous to EF-Tu that binds guanine nucleotides and Sec-tRNASec and a C-terminal extension that recognizes the SECIS hairpin8,9 (Figure 1(b)). The crystal structure of SelB from the archaeon Methanococcus maripaludis showed a structural switch in the GTPase domain that leads to exposure of residues involved in tRNA binding.8 The crystal structure of the C-terminal extension of SelB from Moorella thermoacetica revealed that it consists of four wingedhelix (WH) domains arranged in tandem.10 The WH fold was originally identified as a DNA binding motif.11,12 This motif has recently been discovered in other RNA binding proteins including the La protein,13–15 eukaryotic initiation factor 3 (eIF3k),16 the RNA 2′-phosphorotransferase17 and proteins involved in RNA metabolism.18 Genetic as well as kinetic and biochemical studies of Escherichia coli SelB suggested that changes in

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Molecular Switch in Tandem WH Domains

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Figure 1. Structure of the WH3/4–SECIS RNA complex. (a) The SECIS hairpin from the E. coli fdhF gene (left) and the RNA construct used (right). The minimal RNA fragment required for binding to SelB is boxed. The bulged U17 is essential for SelB binding. (b) Domain organization of SelB. WH3/4 represents the minimum fragment required to bind mRNA. (c) View of the WH3/4–SECIS RNA complex. The RNA is shown as a stick representation with carbon colored tan, nitrogen blue, oxygen red and phosphorus green. The protein is shown as a ribbon model and colored as a color ramp from the N to C terminus. (d) Stereo view of the 2F0–Fc electron density map contoured at 1σ showing the recognition of the bulged nucleotide U17.

the conformation of SelB, such as communication between the mRNA and tRNA-binding sites, is essential for selenocysteine incorporation.19–21 The four tandem WH motifs create an elongated L shape extension. The hinge region at the WH2-WH3 domain interface is suspected to be flexible and to contain a molecular switch that can signal the N-terminal domains I–III when SECIS mRNA is bound.10,20 The structure of the WH3/4–SECIS RNA complex from M. thermoacetica revealed a new mode of RNA recognition with a geometry that allows the complex to wrap around the small ribosomal subunit, and also suggested the possibility of SelB– ribosomal RNA interaction that may have a functional implication.22 However, this complex failed to provide insights into the proposed molecular switch. It is not yet clear how the multiple WH domains of SelB contribute to recognition of SECIS mRNA hairpin and function. Proteins can modulate their

affinity and specificity for RNA by using modular RNA binding domains.23 Most of the known RNA binding domains have been identified in tandem in many RNA binding proteins such as the RNA recognition motif (RRM),24–29 the Pumilio homology domain (PUM-HD),30,31 zinc fingers32 and KH domains.33–35 Within a protein, an RNA binding domain can also be found combined with one or several other RNA binding motifs.13,36 This modular organization of RNA binding domains in proteins permits recognition of relatively long RNA sequences.37 Here we investigate the RNA binding activity and function of the tandem WH motifs (WH1–WH4) in SelB. Since its discovery, 5 SelB has been well characterized biochemically, genetically and kinetically using E. coli SelB. We report the co-crystal structure of E. coli fragment WH3/4 and the hairpin SECIS RNA at 2.3 Å resolution. The two terminal

730 tandem WH modules synergize to establish specific interactions with SECIS hairpin. The module WH3 contacts a conserved bulged uracil (Figure 1 and Supplementary Data, Figure S1) that is crucial for high affinity binding of mRNA,38,39 while the WH4 motif binds the hairpin backbone and the loop. The importance of the observed molecular contacts with the uracil for the formation of the complex were tested using site-directed mutagenesis. Moreover, in the complex, we found that one arginine side-chain is located in the uracil binding pocket preventing the formation of a conserved salt bridge in the hinge region. This salt bridge was proposed to be a molecular switch important for communication between tRNA and the mRNA binding site.10,20 We also report the crystal structure of the complete mRNA binding fragment from M. thermoacetica bound to RNA at 2.6 Å resolution. The comparison of the WH1/4–SECIS RNA complex structure with the free form protein reveals a change of conformation at the hinge region with disruption of the conserved salt bridge. The two structures presented here provide a structural basis for the molecular switch that allows communication between tRNA and mRNA binding sites. The results also reveal that tandem WH motifs provide an excellent scaffold for RNA binding and can play an active role in the function of protein–RNA complexes.

Molecular Switch in Tandem WH Domains Table 1. Data collection and refinement statistics

A. Data collection Space group Cell dimensions a, b, c (Å) Resolution (Å)a,b Rsym or Rmergeb I/σIb Completeness (%)b Redundancyb B. Refinement Resolution (Å) No. reflections Rwork/Rfree No. atoms Protein RNA Water B-factors Protein Ligand/ion Water RMSD Bond lengths (Å) Bond angles (°) a b

WH3/4–SECIS RNA complex E. coli

WH1/4–SECIS RNA complex M. thermoacetica

C2

C2

103.50, 56.51, 48.41 18—2.30 (2.42—2.30) 0.08 (0.518) 17.4 (2.6) 96.3 (96.2) 3.6 (3.6)

158.64, 120.84, 50.87 17.56—2.6 (2.74—2.6) 0.115 (0.517) 14 (2.2) 99.7 (100) 3.7 (3.7)

18—2.30 (2.42—2.30) 11,966 0.199/0.251

17.56—2.6 (2.74—2.6) 28,977 0.222/0.283

982 491 112

3370 976 139

31. 5 30.0 35.7

29.4 30.9 22.6

0.021 2.46

0.018 2.15

One crystal was used. The highest resolution shell is shown in parenthesis.

Results Structural overview of the WH3/4 E. coli complex The 23 nt hairpin SECIS RNA and E. coli SelB WH3/4 terminal fragment (Figure 1(a) and (b)) yielded high-quality co-crystals, containing a single 1:1 protein–ligand complex in the asymmetric unit. Structure factor phases were initially obtained by molecular replacement using the 2.3–Å resolution model of the previous WH3/4–SECIS RNA complex from M. thermoacetica.22 The resulting model was then refined against 2.3 Å structure factor amplitudes by iterative rounds of molecular dynamics and manual rebuilding (Table 1). The three-dimensional structure of the E. coli WH3/4–SECIS RNA complex is illustrated in Figure 1(c). The two WH domains in WH3/4 are arranged in tandem, forming a cylindrical structure with dimensions 28 Å (diameter) × 50 Å (length). Each WH domain contains three helices (H1 to H3) flanked by a three-stranded antiparallel β-sheet (S1 to S3). Wing 1 (W1) refers to the loop between S2 and S3. The two structurally similar WH domains (root-mean-square deviation (RMSD) for 57 α−carbon pairs is 2.4 Å; calculated with DALI40) are connected by a very short two-residue linker (residues 546 and 547). The sequence identity over the compared regions (488– 503/547–562; 505–541/565–601; 543–546/602–605) is 9%. The additional nucleotide U17 that is not present in M. thermoacetica (Supplementary Data, Figure S1) but important for binding affinity in E. coli SelB is extruded from the A-form helix.

Each WH domain recognizes distinct regions of the RNA (Figure 1(c)). The WH3 contains a binding pocket for specific recognition of residue U17 (Figure 1(d)) and WH4 module recognizes the apical region of the hairpin similar to what was observed in the M. thermoacetica WH4 domain.22 Conserved amino acids in WH3 among SelB sequences that depend on the bulged uracil for high binding affinity (Figure 2(a)) form a binding pocket that discriminates a uracil from other bases. This binding site is formed by residues from the N-terminal region of helix H2, the loop W1 and strand S3 (Figure 1(c)). In WH4, the RNA sits on a basic surface that consists of conserved residues from helix H2 and the N-terminal region of helix H3. Sequence specific contacts are established with two conserved nucleotides (G23 and U24) at the tip of the RNA hairpin. The G23 binding pocket is created by the N-terminal regions of helices H2 and H3, strands S2 and S3, and loop W1 (Figure 1(c)). Overall, the two WH domains collectively recognize and bind the SECIS RNA hairpin by forming 22 hydrogen bonds and eight salt bridges. Ten water molecules are found to bridge the interactions between amino acid and nucleotide residues. Comparison with WH3/4 domain from M. thermoacetica E. coli and M. thermoacetica WH3/4 domains adopt a similar fold (RMSD for 120 α−carbon pairs is

Molecular Switch in Tandem WH Domains

731

Figure 2. Structure-based sequence alignment of bacterial SelB WH3 domains and comparison of WH3 and WH4 modules. (a) Residues are shown in red if they are identical or chemically similar in other sequences that are not displayed. The motif WVRD, important for U17 recognition, is shown in orange. RNA-interacting residues are indicated with filled green arrows. Residues with a lower degree of conservation are in blue. Conserved residues from the molecular switch are in magenta. Secondary structural elements observed in the crystal structure of the WH3/4–SECIS RNA complex are shown above the alignment. The numbering for the E. coli sequence is indicated. The sequences are: Moorella thermoacetica (M. th), Geobacter sulfurreducens (G. sul), Escherichia coli (E. coli), Shigellaflexneri (S. flex), Salmonella enterica (S. ente), Yersinia pestis (Y. pest), Haemophilus influenzae (H. inf), Pseudomonas aeruginosa (P. aer), Shewanella oneidensis (S. onei), Sinorhizobium meliloti (S. meli). (b) Superimposition of WH3 (green) and WH4 (blue) modules from E. coli. U17 and G23 nucleotides are colored accordingly.

2.1 Å). The sequence identity over the compared regions is 28%. This sequence identity value is higher than the one obtained for a comparison of individual domains WH3 and WH4 within E. coli (9%). The main difference resides in the conformation of helix H1 in WH3, which has a different orientation in the structures (Supplementary Data, Figure S2). This difference may have important functional consequences that are discussed below. The loop connecting helix H1 and strand S1 in WH3 also displays some differences. A striking feature that was observed in the WH3/ 4–RNA complex from M. thermoacetica is the small surface of interaction between the tip of the RNA hairpin and the C-terminal WH motif of SelB.22 With formation of the complex, 452 Å2 of solvent

accessible protein surface is lost in M. thermoacetica. In E. coli SelB, the surface of the protein contacting the RNA is larger (623 Å 2 (calculated with NACCESS†)) because of the coupling of the two C-terminal WH motifs. This observation may explain the higher binding affinity of E. coli SelB for mRNA. E. coli SelB binds to the SECIS hairpin with a dissociation constant of ∼1 nM,21 three orders of magnitude lower than its counterpart, M. thermoacetica.22

† Hubbard, S. J. & Thornton, J. M. (1996). NACCESS, Computer Program Version 2.1.1 (Department and Molecular Biology, University College London).

732 The same edge in E. coli WH3 and WH4 binds RNA E. coli WH3 and WH4 domains use the same edge to interact with the RNA that is related to the parallel orientation of the motifs in the cylindrical structure (Figure 1(c)). This is illustrated by the similar axis of helices H1, H2 and H3. Comparison of the RNA binding mode of WH3 and WH4 modules is shown in Figure 2(b). These domains are structurally very similar with an RMSD value of 2.4 Å for 57 Cα positions. From the view in Figure 2(b), it is clear that WH3 and WH4 use common secondary structural elements to recognize the RNA. The two nucleotides are found in similar orientations. G23 is slightly translated towards strand S2 and helix H3 in WH4. Hence, a minor difference was observed where helix H3 and strand S2 in WH4 directly contact the RNA while no direct interaction was found between these elements and U17 in WH3 (Figure 2(b)). Aromatic/aliphatic–uracil sandwiching in WH3 The bulged base U17 is inserted between aromatic and aliphatic side-chains, with interac-

Molecular Switch in Tandem WH Domains

tions between the protein and the base edge specifying uracil (Figure 3(a)). Continuous stacking interactions are observed with a motif R543W508-U17-R510. U17 is recognized by D542 (OD2–N3 3.1 Å) and sandwiched between W508 (inter-planar distance 3.9 Å) and R510 (closest approach 3.9 Å). Statistical analyses revealed that arginine guanidine groups often make cation–π interactions with uracil.41 The V509 and R510 amide groups are hydrogen bonded to U17(O4) (N–O4 3.4 Å and 2.9 Å for V509 and R510, respectively), which explains the specificity for uracil at this position. Substitution of U17 to a cytosine completely abolished selenocysteine incorporation in vivo.39 The amino group of the cytosine is expected to produce an energetically unfavorable contact with V509 and R510 backbone nitrogen. An intricate network of salt bridges contributes to orient R510 and D542 in the proper conformation to interact with U17 (K541–D542, E520–R510, R524–E520, D542–R524) (Figure 3(a)). Additionally, the U17 ribose hydroxyl group is recognized by K581 and Q585 from WH4 module via bridging water molecules (Figure 3(a) and (b)).

Figure 3. Protein RNA contacts in WH3/4–RNA complex. (a) View of the binding pocket for U17. Nitrogen is colored dark blue and oxygen red. Protein–RNA intermolecular hydrogen bonds are shown as dotted lines. WH3/4 is colored with the same color ramp as in Figure 1(c). (b) Schematic representation of the WH3/4–SECIS RNA interactions. Hydrogen bonds are represented by broken lines, salt bridges by crosses and van der Waals contacts by open circles. Amino acids are colored as in Figure 3. Six water molecules in key positions within the protein–RNA interface are depicted as red ovals. (c) and (d) Gel electrophoretic mobility shift assays of binding of mutant WH3/4 domains to the 32P-labeled SECIS RNA, W508A (c) and V509A (d). The protein concentrations (nM or μM) used are indicated above each lane.

Molecular Switch in Tandem WH Domains

733

Effect of WH3 amino acid substitutions on binding affinity

U17 is a functional link connecting WH4 to the hinge

We used gel electrophoretic mobility shift assays to monitor the binding of mutant WH3/4 domains to SECIS stem–loop (Figure 3(c) and (d)). Each residue from the conserved motif W508V509-R510-D511 as well as D542 and R543 involved in intermolecular recognition of the bulge U17 was substituted by alanine. The resulting binding affinities are listed and compared (Supplementary Data, Table S1). We observe a 12-fold and 37-fold loss in binding affinity for the V509A and D511A substitutions, respectively. Much larger reduction in binding affinity is observed for W508A and R510A substitutions, with a 1000-fold and 500-fold loss in binding affinity, respectively (Figure 3). This result is in good agreement with our structural data with W508 and R510 side-chains directly contacting U17 base. The contribution of D542 and R543 residues in RNA binding could not be evaluated, since their replacement by alanine resulted in insoluble polypeptides.

WH4 module establishes important and specific contacts with SECIS hairpin with some similarities and differences with M. thermoacetica (Supplementary Data, Figure S3). In the WH4 module, direct contacts are made between R573, R580, K581 and N601 and six consecutive helical phosphate groups (Figure 3(b) and Supplementary Data, Figure S3(b)). As described above, the conserved residue K581 and the Q585 in WH4 also contacts the hydroxyl group of U17. The bulged uracil-binding site involves an intricate network of salt bridges that span the WH3 domain (Figure 3(a)). Interestingly, D542 is establishing a contact with R524 (OD2–NE, 2.9 Å). R524 has been suspected to form a molecular switch represented by the conserved R524–E437 salt bridge (Figure 4). The salt bridge observed in the free form of the mRNA binding domain from M. thermoacetica is represented in Figure 4(a). These residues display a co-variation pattern leading to a conservation of the salt bridge. When residue 460 or 461 (M. thermoacetica numbering) is negatively charged, 551

Figure 4. Molecular switch in SelB mRNA binding domains. (a) The salt bridge of the molecular switch in free M. thermoacetica (PDB code 1lva). E552 in WH3 is in magenta and R461 in WH2 green. (b) In the M. thermoacetica bound form, the salt bridge is disrupted. (c) Superimposition of E. coli WH3/4–RNA complex (blue) and M. thermoacetica WH1/ 4-RNA (yellow) complex structures. The same color code is used for the residues of the switch (E. coli R524 in WH3 is magenta and M. thermoacetica R461 in WH2 is green). Note that the E. coli pair E437–R524 and the equivalent pair in M. thermoacetica R461–E552 are reversed. Note also the bend of the WH3 helix H1 towards helix H3 in E. coli. (d) Model of the coupling between mRNA binding and GTPase activity/tRNA binding.

734 or 552 is positively charged.10 Note that residue 524 in E. coli is shifted by one position in the C-terminal direction of helix H3 compared to the position 552 in M. thermoacetica (Figure 4(a)–(c)). However, the observed bent in WH3 helix H1 in E. coli (Figure 4(c)) would place the WH2 module closer to the WH3 helix H3 allowing the formation of the E437– R524 interaction in the free form. In the WH3/ 4–RNA complex, the guanidinium group of R524 is involved in salt bridges with D542 and E520 pre-

Molecular Switch in Tandem WH Domains

venting its interaction with E437 in the molecular switch. Upon mRNA binding, disruption of the R524–E437 pair would lead to a conformational change in the hinge region. In order to test whether SECIS RNA binding affects the hinge region conformation in solution, the E. coli WH1–4 domain was subjected to limited chymotrypsin proteolysis (Supplementary Data, Figure S4). SECIS RNA binding slightly enhances chymotrypsin susceptibility at the hinge.

Figure 5. Protein–RNA interactions in the WH1/4–SECIS RNA complex from M. thermoacetica. (a) Secondary structure of the SECIS hairpin used here and domain organization of SelB from M. thermoacetica. (b) Overall view of the complex structure. The RNA bound to WH4 is in blue and the RNA contacting the hinge region pink. (c) Comparison between the free form (red) WH1/4 domain and the complex structure (magenta). The RNA bound at the hinge region is represented as sticks. (d) Interactions of the phosphate RNA backbone with the hinge region of the protein (complex A). WH2 and WH3 are colored as in (b).

735

Molecular Switch in Tandem WH Domains

Structure of the complete mRNA binding fragment from M. thermoacetica We have generated two mutants of the complete mRNA binding fragment from M. thermoacetica in which the WH3 module has been modified to resemble the E. coli sequence. The amino acid sequences S535-F536 and S535-F536-K537-E538 from M. thermoacetica were substituted by WV (508–509 E. coli numbering) and WVRD (508–511 E. coli numbering) motifs, respectively, expecting that these mutations would favor crystallization of WH1-4–RNA complex. Combinations of WH1/4 mutants and E. coli/M. thermoacetica SECIS RNAs were assessed for co-crystallization. One mutant, the S535W-F536V in complex with SECIS RNA from M. thermoacetica (lacking the bulge uracil) (Figure 5(a)) provided crystals suitable for structure determination. The structure of the complex (Figure 5(b)) provides important new structural insights for the understanding of the function of SelB. Crystals belong to space group C2 and diffracted to 2.6 Å resolution (Table 1). A molecular replacement solution was found by using the previous WH3/4– SECIS RNA complex from M. thermoacetica.22 There are two copies of non-crystallographic related WH1/4 domains bound to two copies of SECIS RNA hairpin in the asymmetric unit. The electron density in the WH1 module is weaker compare to the other WH motifs and best visible for a region close to WH2 (for instance strands S1, S2 and S3). The observed density for the β sheet indicates that the orientation of WH1 domain relative to WH2 is slightly changed compared to the free protein. Interestingly, loop residues 475–480 in WH2 and the connecting loop W1 in WH1 that are at the WH1WH2 interface are slightly shifted compare to the free protein. Rotation of domains at the hinge region A comparison of the WH1/4–SECIS RNA complex structure with the known free form structure of the same domains reveals a change in the conformation of the protein. The two proteins adopt different conformations primarily as a result of rigid-body motions of WH1/2 and WH3/4 modules within the complete structure. The striking difference is that the WH1/2 module has rotated as a rigid body around the hinge region by 60° (Figure 5(c)). Noticeably, this rearrangement occurs at P509-S510 in the hinge region between WH2 and WH3 domains for which an expected change of conformation relevant to SelB function has been proposed10,20 (Figure 4(b)). Our result establishes that this region is indeed prone to conformational rearrangements. Unexpectedly, the hinge region that undergoes this conformational change is seen interacting with the major groove of a neighboring RNA molecule (Figure 5(b)–(d)). This interaction is limited to backbone contacts with phosphate groups and one 2′-hydroxyl group. In complex A of the asymmetric unit, the amino acid side-chains of R457, R461, R559

and H555 interact with three consecutive phosphate groups on one side of the major groove and with three other phosphate groups on the other side, such that these residues bridge the major groove (Figure 5(d)). In the second complex (B), only three phosphate groups on one strand of the major groove are contacted. In complex B, residue Glu552 is hydrogen-bonded to the 2′-hydroxyl group of C15. With binding of the RNA at the hinge, 551 Å2 of solvent accessible protein surface is lost in complex A and 415 Å2 in complex B. These values are similar to the one measured for SECIS hairpin binding to M. thermoacetica WH3/4. Side-chains of residues R457, Y458 and R461 from WH2 and H555 and R559 from WH3 form π–cation interactions and directly interact with the phosphate backbone of a SECIS RNA helix (Figure 5(d)). RNA binding residues in WH2 are located at the C-terminal part of helix H1 and at the connecting loop with strand S1 (Figure 5(d)) whereas H555, E552 and Arg559 from WH3 are located in helix H3. In the free form protein, residues R457, Y458 and R461 are already continuously stacked. By the rotational movement of WH1/2 module, they are brought above the H555 and R559 side-chains to form this long train of π-cation interactions that interact with the RNA (Figure 5(d)).

Discussion Comparison of SelB with other tandem-RNA recognition motifs Our structural analysis supplemented by a mutational study reveals that tandem WH motifs in the C-terminal sequence of elongation factor SelB can serve to modulate RNA binding affinity and specificity. The results provide an explanation for the use of multiple WH domains for high affinity RNA recognition. Some of the structural elements employed by WH3 and WH4 RNA-binding modules are common to both domains with some slight differences. These observations further support the fact that the WH motif is a highly versatile scaffold, which can be adapted for sequence-specific recognition of many different nucleic acid structures.13,14,22 Several co-crystal structures of modular RNA binding domains are available. It is of interest to compare these interactions with WH3/4 RNA recognition. These modules are usually small in size and the use of multiple copies permits recognition of extended RNAs. The RNA sequences that are recognized can be contiguous or distant. For example, in the co-crystal structure of human poly (A)-binding protein (PABP), 26 two RRMs pack together to form a continuous RNA-binding trough. On the contrary, structure of RRM1/2 of Sex-lethal protein RNA complex24 differs significantly with the RRMs forming a discontinuous RNA-binding surface. Finally, the recognition of two really distant regions of RNA by individual modules linked by flexible linkers can lead to RNA looping.29

736 In the case of WH3/4 of E. coli SelB bound to RNA, the two domains form a single elongated structure in which two separate binding surfaces contact nucleotides extruded from the helix as well as all consecutive helical phosphate groups found between U17 and G23. The solvent accessible surface area buried at the WH3–WH4 interface (528 Å2 per domain) is similar to that observed for the tightly packed RRMs in PABP. An elongated shape is also found in the crystal structures of PUM-HDs from Drosophila30 or human Pumilio1,31 where the Puf repeats align tandemly to form an extended curved molecule. These examples illustrate how the assembly of small tandem RNA binding modules that pack tightly to form elongated and rigid structures (i.e. the Puf repeats or WH domains) can serve to recognize long single-stranded RNA sequences as well as long RNA hairpins. Comparison with other WH–nucleic acid recognition complexes The present structures of E. coli WH3/4–RNA and M. thermoacetica WH1-4–RNA complexes can be compared with a recently solved La NTD-RNA complex15 as well as WH–DNA complexes. The WH4–RNA complex (Figure 6(a)) and the WH3– U17 recognition represent a new mode of recognition not seen in WH–DNA complexes22 where the structure lacks any contacts with the major or minor grooves of the RNA. Instead, binding specificity is provided by recognition of the hairpin backbone and nucleotides extruded from the helix. This mode of interaction contrasts with the RNA binding site at the hinge in M. thermoacetica WH1-4–RNA complex which spans different surfaces of WH2 (Figure 6(b)) and WH3 (Figure 6(c)) modules. WH2 and WH3 combine two different edges to bind the RNA major groove. Each contacting area contains one of the amino acid pair that forms the functionally important R461–E552 salt bridge. The N-terminal domain of La protein consists of a canonical WH motif with three additional α helices (helices H1′, H2 and H4) (Figure 6(d)). The La WH– RNA complex uses the same edge as SelB WH2 (Figure 6(b)) to bind RNA. In SelB, recognition involves the C-terminal region of helix H1 with the connecting loop to strand S1 whereas in La–NTD, this region corresponds to helices H1′ and H2. It is remarkable that one of the SelB WH–RNA interactions resembles the WH–DNA mode of recognition (Figure 6(e)). In the hinge region from M. thermoacetica WH3 in complex with RNA (Figure 6(c)), helix H3, the so called “recognition helix” in WH–DNA complexes contacts the RNA. These two complexes share similar orientation of the RNA and DNA helices; however, orientations of helices H3 differ. In SelB, the helix does not interact deeply with the narrow RNA major groove and contacts are limited to phosphate backbone. With DNA, the entire recognition helix11 and/or loop W142 penetrates deeply into the wider DNA major groove. Our structural study illustrates how the WH motif pro-

Molecular Switch in Tandem WH Domains

vides multiple binding surfaces for RNA recognition contrasting with WH–DNA interaction where, to our knowledge, the motif always uses the same edge to contact DNA. A conserved bulged uracil targeted by E. coli WH3 module All the direct hydrogen bonding and stacking contacts of the bulged U17 are with the WH3 module illustrating its importance for SECIS hairpin recognition. This nucleotide is present in most of the known SECIS RNA sequences, suggesting that the mechanism of RNA recognition used by E. coli SelB may be found in most of prokaryotes. In the previous co-crystal structure of WH3/4–SECIS from M. thermoacetica22, the complex has a V shape with a 70° angle between the RNA helix and the WH3/4 domain. Since the contacts between E. coli WH3 and the SECIS hairpin involve nucleotide U17 that is extruded from the double helix, this interaction does not affect the overall geometry of the complex (Supplementary Data, Figure S5), indicating that this feature is conserved and may be essential for SelB–tRNASec interaction with the ribosome. Compensatory mutations in genetic studies revealed the region of E. coli SelB important for mRNA binding.20,43 The E. coli WH3/4 structure described here allows us to map the mutations directly on the structure (Supplementary Data, Figure S6). Most mutations that suppress substitutions of the bulged nucleotide U17 in SECIS are clustered in a region corresponding to 28 amino acid residues in the WH4 domain. However, some mutants are double with one mutation in WH4 and an additional one in WH3. Of these mutations two out of three are changes in the binding pocket of U17 (D511V and E520G). D511 does not establish direct contacts with the RNA but is next to R510 that has stacking interaction with U17. Residue D520 belongs to this intricate network of salt bridges that form the U17 binding site and directly contacts R524. More interestingly, L525 is found mutated to proline. This amino acid is close to R524 that is involved in the molecular switch. The substitution by a proline may distort the geometry of WH3 helix H3 and alter the molecular switch. Change of conformation in the hinge region of SelB mRNA binding domain The WH1/4–SECIS RNA complex of M. thermoacetica shows that the hinge region can indeed change conformation and that, as anticipated, the hinge movement is accompanied by disruption of the R461–E552 salt bridge. The disruption of the salt bridge is accompanied by rotation of the WH1/2 domains relative to the WH3/4 domains. How the switch is triggered in M. thermoacetica in the absence of the WH3–SECIS RNA interaction is not clear. The structure of WH1-4–SECIS RNA suggests RNA binding activity at the flexible hinge

Molecular Switch in Tandem WH Domains

737

Figure 6. Comparison of protein–RNA interactions in the SelB WH–RNA complexes with other nucleic acid–WH complexes. The WH motifs are in the same orientation. (a) Protein–RNA recognition in the E. coli WH4–SECIS RNA complex. The N-terminal regions of helices H2 and H3 and β strands that interact with the RNA hairpin are in red. (b) Protein–RNA recognition in crystal contacts within the M. thermoacetica WH2–SECIS RNA complex. The connecting loop between helix H1 and strand S1 is in red. (c) Protein–RNA recognition in crystal contacts within the M. thermoacetica WH3–SECIS RNA complex. The helix H3 is colored red. (d) Protein–RNA recognition in the La NTD–RNA complex (PDB code 1ZH5). Only the WH La motif is shown. The UUUOH3′ end segment is targeted by the C-terminal region of helix H1 and insertion helices H2 and H4 (in red). (e) The recognition helix of the WH motif in the Sap-1 protein–DNA complex (PDB code 1bc8,54) is in red.

738 region involving WH2 and WH3 modules. Docking of the L-shaped mRNA binding domains to the ribosome placed the hinge region in the vicinity of ribosomal RNA helices 16 and 3322 raising the possibility of functionally important rRNA–SelB contacts. It is possible that in M. thermoacetica, ribosomal RNA binding in this region leads to structural reorganization of the hinge. Structural basis for the molecular switch Besides exhibiting a structural scaffold to recognize RNA, the tandem WH domains in SelB display functional properties. Our structures address a long-standing question associated with the molecular basis of the switch that leads to communication between the tRNA and mRNA binding sites of SelB. Addition of Sec–tRNASec increases the affinity of SelB for SECIS RNA by a factor of 10. 21 A genetic study looking for mutations in E. coli SelB that counteracted the detrimental effect of mutations in SECIS hairpin loop unexpectedly identified the E437K mutant located away from the mRNA binding site.20 Structure of the free mRNA binding domain from M. thermoacetica located the mutation in a conserved salt bridge (E. coli R524–E437) at the hinge between WH2 and WH3 modules. A functional role for the conserved salt bridge was proposed in which mRNA binding would lead to the hinge movement signaling to the N-terminal part of SelB that mRNA is bound.10 We observed that in the E. coli WH3/4–mRNA complex the guanidinium group of R524 is sequestered by salt bridges with D542 and E520, and as a consequence is not available for an interaction with E437. This explains how SECIS mRNA binding would disrupt the R524–E437 pair of the switch leading to rearrangement of the hinge (Figure 4(c) and (d)). In the E437K mutant,20 there would be no energetic cost to break the R524–E437 bridge and the R524 side-chain would be available for mRNA binding, thereby increasing affinity and restoring selenocysteine incorporation at the UGA codon. The observed structural reorganization of WH1/2 and WH3/4 modules following SECIS RNA binding may propagate to the EF-Tu like domains by a yet unknown mechanism (Figure 4(d)). The signal can reach the EF-Tu like domains through two different routes. The first one may involve a structural change within the flexible WH1 domain. A second possibility may use tRNASec. The anticodon stem– loop protrudes out of the EF-Tu–GTP–tRNA ternary complex.6 This region of tRNASec may interact with the elongated WH1/4 C-terminal extension. We docked the WH1/4–SECIS RNA complex structure of M. thermoacetica SelB into the EF-Tu– tRNA complex structure. Interestingly, the SECIS RNA helix interacting with the hinge region in a sequence independent manner can be aligned onto the anticodon stem–loop of tRNA in the ternary complex. In this conformation of the SelB—tRNA–

Molecular Switch in Tandem WH Domains

mRNA complex, WH4 is found in the vicinity of the tRNASec. We speculate that these possible contacts may lead, for example, to disruption of the salt bridge at the hinge thereby increasing SECIS affinity21 and/or affect SECIS interaction in WH4 module. The function of the mRNA SECIS hairpin is more than simply increasing the local concentration of SelB in the vicinity of the UGA stop codon. Strong evidence suggested that within SelB a conformational switch following mRNA binding is essential for recoding of the UGA stop codon.20,44,45 This mechanism prevents SelB to interfere with normal translational termination at the UGA codon. The structures presented here show how the mRNA hairpin acts as an activator of a conformational rearrangement within SelB.

Materials and Methods Sample preparation, crystallization and data collection Purification and crystallization of the E. coli WH3/4 fragment (residues 478–614) was performed as described.46 In order to obtain crystals suitable for X-ray structure determination, the length of the WH3/4 fragment as well as the RNA had to be optimized. The WH3/4 fragment was overexpressed as a GST fusion protein and was purified essentially as described.47 The M. thermoacetica full length mRNA binding domain (residues 377– 634) was expressed and purified using the same strategy from a GST fusion protein. Mutations in the WH3 domain were introduced by site-directed mutagenesis. All sequences were checked by DNA sequencing. The E. coli and M. thermoacetica SECIS RNAs were prepared as described.22,47 Both were purified by denaturing polyacrylamide gel electrophoresis (PAGE). Crystals of the WH3/4–RNA complex were grown by vapor diffusion (hanging drop) from reservoirs containing 700 μl of 0.02 M calcium chloride dihydrate, 0.1 M sodium acetate trihydrate (pH 4.6) and 30% (v/v) 2-methyl-2,4-pentanediol at 21 °C. Drops of 3 μl were prepared by mixing 1 μl of reservoir solution with 1 μl of protein/RNA solution and 1 μl of 0.5 M NaF. Prior to data collection, the crystals were transferred to a solution containing 15% (v/v) ethylene glycol and flash cooled to −180 °C. Crystals of the M. thermoacetica WH1/4–SECIS RNA complex were grown from 1.6 μl drops with the reservoir containing 25% (w/v) PEG4000, 0.1 M sodium acetate (pH 4.6), 0.2 M ammonium sulfate. Rectangular crystals reached dimensions (100 μm × 100 μm × 300–400 μm) after one week. Complete data sets were collected at 2.3 Å and 2.6 Å resolution for E. coli and M. thermoacetica, respectively, proteins under cryogenic conditions using the beamline BM30A at the European Synchrotron Radiation Facility (ESRF; Grenoble, France) (λ = 0.980 Å). Structure determination and refinement The crystals of the WH3/4–SECIS RNA complex belong to the space group C2, with unit cell dimensions of a = 103.50 Å, b = 56.51 Å, c = 48.41 Å and β = 95.60. The asymmetric unit of the crystal contains one complex with a solvent content of about 63%. The diffraction data were

739

Molecular Switch in Tandem WH Domains processed with Mosflm48 and scaled with SCALA. The structure was phased by molecular replacement using phaser in the CCP4 package 49 with the coordinates of the WH3/4–RNA complex from M. thermoacetica.22 Model building was carried out with the programs O50 or Coot.51 The resulting atomic model was refined iteratively against data to 2.3 Å using Refmac and CNS.52 We observed electron density for all nucleotides and all except the first ten and the last seven amino acid residues. The final model contains WH3/4 domains except these amino acids, the entire SECIS RNA (23 nt), and 112 water molecules. A Ramachandran plot of backbone angles gave 95.4% and 4.6% in most favored and additionally allowed regions, respectively, and none in the disallowed region. The crystals of the WH1-4–SECIS RNA complex belong to space group C2, with unit cell dimensions of a = 158.64 Å, b = 120.84 Å, c = 50.87 Å and β = 100.18. The asymmetric unit of the crystal contains two complexes. The structure was phased by molecular replacement as described for the E. coli fragment. The electron density for the WH1 domain was only visible for some segments of the chain. The final model contains segments of the WH1 domain, WH2-4 modules (residues 436–634), the entire SECIS RNA (23 nt), and 139 water molecules. Ramachandran plots were calculated by PROCHECK.53 Figures were generated using PyMOL‡. Gel mobility shift assay The WH3/4–RNA complexes were observed as a mobility shift in a non-denaturing PAGE and apparent dissociation constants were obtained as described.47 A constant concentration of (3′ -32P)-labeled RNA (3 pM to 30 pM) was incubated with various concentrations of protein in 15 μl of 10 mM sodium phosphate (pH 6.0), 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 4 μM competitor RNA, and 0.01% (v/v) Nonidet P-40. RNA oligonucleotides used for mobility shift analysis were heated to 95 °C for 5 min and cooled on ice for 10 min before adding protein. The reaction mixture was incubated at room temperature for 10 min and then on ice for 10 min. A 30% (w/v) glycerol dye was added and the complex was separated from the free RNA by electrophoresis in 10% (w/v) polyacrylamide (acrylamide/ bisacrylamide 29:1)–1X TBE gels and quantified using PhosphorImager Storm (Molecular Dynamics). Chymotrypsin digestion The 25 μg of E.coli WH1-4 (residues 364–614) complexed with or without an equimolar amount of RNA were incubated with 50 ng of chymotrypsin (Sigma) at 25 °C, in a total volume of 50 μl containing 100 mM Tris–HCl (pH7.5), 1 mM DTT and 0.5 mM EDTA. Aliquots (8 μl) were taken and the reaction was stopped by adding 20 ng of trypsin inhibitor (Sigma). Loading dye was added to samples, and the cleavage products were analyzed by SDS–acrylamide gel electrophresis. Coordinates Atomic coordinates have been deposited in the RCSB Protein Data bank under accession codes 2PJP (E. coli) and 2PLY (M. thermoacetica). ‡ http://pymol.sourceforge.net/

Acknowledgements We thank the fip group and in particular Lilian Jacquamet for assistance in data collection at ESRF Grenoble. We are also grateful to Louis Renault, Pascal Retailleau and Emmanuelle Schmitt for help with data analysis and Claudine Mayer and Stéphane Thore for helpful discussions. This work was supported by an ANR grant “young researcher” JC05-50413 (to S.Y. and D.F.).

Supplementary Data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/ j.jmb.2007.05.001

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Edited by J. Doudna (Received 12 February 2007; received in revised form 20 April 2007; accepted 1 May 2007) Available online 10 May 2007