Crystal Structure of SRP19 in Complex with the S Domain of SRP RNA and Its Implication for the Assembly of the Signal Recognition Particle

Molecular Cell, Vol. 9, 1251–1261, June, 2002, Copyright 2002 by Cell Press

Crystal Structure of SRP19 in Complex with the S Domain of SRP RNA and Its Implication for the Assembly of the Signal Recognition Particle Chris Oubridge, Andreas Kuglstatter, Luca Jovine,2 and Kiyoshi Nagai1 MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 2QH United Kingdom

Summary The signal recognition particle (SRP) is a ribonucleoprotein particle involved in GTP-dependent translocation of secretory proteins across membranes. In Archaea and Eukarya, SRP19 binds to 7SL RNA and promotes the incorporation of SRP54, which contains the binding sites for GTP, the signal peptide, and the membrane-bound SRP receptor. We have determined the crystal structure of Methanococcus jannaschii SRP19 bound to the S domain of human 7SL RNA at 2.9 A˚ resolution. SRP19 clamps the tetraloops of two branched helices (helices 6 and 8) and allows them to interact side by side. Helix 6 acts as a splint for helix 8 and partially preorganizes the binding site for SRP54 in helix 8, thereby facilitating the binding of SRP54 in assembly. Introduction The signal recognition particle (SRP) is a ubiquitous ribonucleoprotein particle essential for GTP-dependent protein translocation across the eukaryotic endoplasmic reticulum (ER) membrane or the bacterial plasma membrane (Walter and Blobel, 1983; Walter and Johnson, 1994; Lu¨tcke, 1995; Hauser et al., 1995; Keenan et al., 2001). SRP binds to the signal peptide of secretory or membrane proteins emerging from the ribosome and interacts with the ribosome. This causes the elongation of the polypeptide chain to slow down or halt, a phenomenon known as elongation arrest (Siegel and Walter, 1986; Thomas et al., 1997; Mason et al., 2000). SRP then directs the ribosome to the protein translocation channel (the translocon) in the membrane through its interaction with a membrane-bound SRP receptor such that the polypeptide chain is cotranslationally translocated across the membrane (Walter and Johnson, 1994; Fulga et al., 2001; Beckmann et al., 2001). The simplest eubacterial SRP consists of a single protein, called p48 or Ffh (fifty-four homolog, a homolog of eukaryotic SRP54 protein), and a 4.5S RNA of ⵑ120 nucleotides in length (Keenan et al., 2001). Eukaryotic SRP consists of 7SL RNA (ⵑ300 nucleotides in length) folded into a roughly Y-shaped double-stranded RNA and six proteins: SRP19, SRP54, and the SRP68/SRP72 1

Correspondence: [email protected] Present address: Department of Molecular, Cell, and Developmental Biology, Mount Sinai School of Medicine, New York, New York 10029. 2

and SRP9/SRP14 heterodimers (Figure 1A). Eukaryotic SRP can be separated into two domains with micrococcal nuclease: the Alu and S domains (Gundelfinger et al., 1983). The Alu domain, consisting of the SRP9/14 heterodimer bound to one end of the RNA, is known to play a role in elongation arrest (Weichenrieder et al., 2000). The S domain, which binds to both the signal peptide and a membrane-bound SRP receptor (SR), consists of the other four proteins bound at the opposite end of the RNA that has two branched arms called helices 6 and 8 (Siegel and Walter, 1988b). Helix 8 is similar to domain IV of 4.5S RNA, the binding site for Ffh, in sequence and secondary structure. In archaebacterial SRP, the RNA component, 7S RNA, is similar to the eukaryotic counterpart, but only two of the proteins found in eukaryotic SRPs, SRP54 and SRP19, have been identified in archaeal genomes (Bhuiyan et al., 2000). SRP54 or Ffh is the only protein component present in all SRPs from eubacteria to man and plays a key role in protein translocation (Figure 1A). SRP54 (Ffh) consists of a GTP binding domain (G domain) that has an N-terminal four-helix bundle (N domain) tightly associated with it and a C-terminal methionine-rich domain (M domain) connected to the N/G domain with a flexible linker (Freymann et al., 1997; Keenan et al., 1998). SRP54 (Ffh) binds both the RNA and signal peptides through its M domain. A bacterial SRP receptor protein, FtsY, also contains a GTP binding domain, and bacterial SRP stably associates with FtsY when both Ffh and FtsY bind GTP (Miller et al., 1993). This interaction mutually activates the GTPase activity of both Ffh and FtsY, and they dissociate upon GTP hydrolysis. The eukaryotic SRP receptor consists of two GTP binding proteins, SR␣ and SR␤. SR␣ is a direct homolog of bacterial FtsY both structurally and functionally, whereas SR␤, which can be cross-linked to a ribosomal protein, is thought to play an important role in ribosome binding (Fulga et al., 2001). The bacterial SRP54 homolog, Ffh, or its M domain binds SRP RNA (4.5S RNA) on its own, whereas human SRP54 is unable to bind to 7SL RNA without prior binding of SRP19 to the RNA (Gowda et al., 1999). Therefore, SRP19 plays an essential role in eukaryotic SRP assembly. The SRP19 binding site within 7SL RNA was first mapped by ␣-sarcin cleavage to the tips of two branched double-helical regions, helices 6 and 8 (Siegel and Walter, 1988a). This was confirmed by RNA mutagenesis and chemical protection experiments. Zwieb (1992) showed that mutation of the helix 6 tetraloop nucleotide, A149, completely abolishes the binding of SRP19 to the RNA. Recently, significant progress has been made in understanding the SRP19 structure and its interaction with RNA. The secondary structure elements (Pakhomova et al., 2001) and more recently the threedimensional structure (Pakhomova et al., 2002) of the Archeoglobus fulgidus SRP19 were determined by NMR. Furthermore, Wild et al. (2001) determined the crystal structure of the human SRP19 in complex with a 29 nucleotide RNA representing part of helix 6. This has revealed the fold of SRP19, consisting of a three-

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Figure 1. The Protein and RNA Components of the Signal Recognition Particle (A) Schematic representation of signal recognition particles from eubacteria (E. coli, left), archaebacteria (M. jannaschii, middle), and mammals (human, right). (B) Amino acid sequence alignment of M. jannaschii (Mj19) and human (Hs19) SRP19 with the secondary structure elements shown below. Green represents positions highly conserved in multiple sequence alignment, and blue represents positions where conservative changes are permitted (Zwieb and Samuelsson, 2000). Closed circles, direct contacts between SRP19 and helix 6; open circles, solvent mediated interaction between SRP19 and helix 6; red circles, contacts conserved between the Hs19-helix 6 complex (Wild et al., 2001) and the Mj19-S domain complex (present work); blue squares, direct contacts between helix 8 and Mj19.

stranded ␤ sheet with two ␣ helices, as well as molecular details of the interaction between SRP19 and helix 6. Based on this structure, Wild et al. (2001) constructed a model of SRP19 bound to both helices 6 and 8 but have not provided insights into the mechanism whereby SRP19 promotes the binding of SRP54 to 7SL RNA. We have now solved the structure of Methanococcus jannaschii SRP19 bound to a 128 nucleotide RNA that represents the entire S domain of human 7SL RNA. This RNA consists of helices 5, 6, and 8 that are joined by a three-way junction. The crystal structure has revealed that SRP19 binds to the tips of helices 6 and 8 and facilitates the tertiary interaction of the two helices. In conjunction with the structure of domain IV of 4.5S RNA in complex with the M domain of Ffh (Batey et al., 2000), our structure has allowed us to construct a model of a ternary complex between SRP19, 7SL RNA, and the M domain of SRP54. This model suggests that the M domain does not interact directly with helix 6 or extensively with SRP19. We now propose that SRP19 induces a large conformational change in 7SL RNA and partially preorganizes the otherwise flexible binding site for SRP54 in helix 8, thereby facilitating the binding of SRP54. Results and Discussion Overview of the Structure A complex of the M. jannaschii SRP19 protein (Mj19) (Figure 1B) and a 128 nucleotide fragment of human 7SL RNA was cocrystallized in space group P21212 with unit cell dimension, a ⫽ ⵑ70 A˚, b ⫽ ⵑ224 A˚, c ⫽ ⵑ43 A˚. The structure was solved by multiwavelength anomalous dispersion (MAD) using a selenomethionine-labeled protein. After solvent flattening, an electron density map (Figure 2A) was of sufficient quality to build most of the protein and RNA. The model has been refined against the single-cysteine mutant data using REFMAC and CNS to a working R factor of 26.4% and a free R factor of 29.6% (Table 1). The current model contains all protein

residues, all RNA nucleotides except A173 and G174, 30 water molecules, and 24 putative magnesium ions. The RNA in the crystal was analyzed by electrophoresis and found to be intact. SRP19 binds to the tetraloops of both helices 6 and 8, allowing the tips of these helices to interact extensively (Figure 3A). Helices 6 and 8 are clamped by SRP19 on one end and by the hinge formed by the three-way junction on the other. As a result, helix 6 lies parallel to helix 8, and the phosphate backbones of helices 6 and 8 are brought into close proximity in two additional regions (Figures 3A and 3B). Helix 5 coaxially stacks onto helix 8, and the region predicted to form helix 7 is in an extended structure with high B factors. The major groove side of the helix 6 tetraloop binds to the most basic region of SRP19 (Figure 3C), burying a wateraccessible surface of 1546 A˚2 as compared to 856 A˚2 at the SRP19-helix 8 interface. This is consistent with the stronger binding of SRP19 to helix 6 than to helix 8. The parallel arrangement of the two RNA helices in this structure is reminiscent of the group I intron P4-P6 domain (Cate et al., 1996). In both cases, there are two helices lying parallel that are brought into close contact, although this is achieved using different strategies. In the case of P4-P6, the A-rich bulge and the tetraloop receptor provide interactions to stabilize the parallel packing of the two helices, whereas in the S domain, this is accomplished through binding of SRP19. Many of the nucleotides protected from hydroxyl radical cleavage (Rose and Weeks, 2001) in the presence of SRP19 are centered around the nucleotides in close helix-helix contacts (Figure 3B): between nucleotides A139 and C140 of helix 6 and nucleotides G187 and G188 of helix 8, and between nucleotides C161 and C162 of helix 6 and nucleotides A214 and A215 of helix 8. Some magnesium ions are present in these two regions, which compensate for the phosphate charges and stabilize this RNA structure. The loss of RNA protection from ␣-sarcin (Siegel and Walter, 1988a) and hydroxyl radical

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Figure 2. Experimental Electron Density Map and the Main Chain Trace of M. jannaschii and Human SRP19 (A) Experimental electron density map contoured at 1.5 ␴ with the refined model in the region of the SRP19-helix 6 interface. The main chain amide group of Ile2 is hydrogen bonded to the phosphate oxygen of A149. The indole nitrogen of Trp4 and the phenolate oxygen of Tyr7 are hydrogen bonded to the 2⬘OH group of A149 and the phosphate oxygen of G150, respectively. (B) Overlay of the M. jannaschii SRP19 (blue) and the human SRP19 (red) in stereo. The main chain atoms of residues 1–55, 62–65, and 72–82 of Mj19 were superimposed on the corresponding residues of the human SRP19 with an rmsd of 0.98 A˚ using LSQMAN within O (Jones and Kjeldgaard, 1997).

cleavage (Rose and Weeks, 2001) in the absence of SRP19 indicates that in the free RNA, helix 6 swings away from helix 8 because of electrostatic repulsion of phosphate charges. Therefore, the binding of SRP19 to the S domain of RNA induces a large conformational change in the RNA. SRP19 and Its Interaction with Helix 6 The M. jannaschii SRP19 (Mj19) protein folds into a small domain containing a three-stranded ␤ sheet and two ␣ helices with a ␤␣␤␤␣ topology (Figures 1B and 2B). This structure has the same topology as the KH domain (Musco et al., 1996) but is distinct in that two long polypeptide loops between ␤1 and helix A (L1) and between ␤2 and ␤3 (L3) protrude from the ␣/␤ core. A sequence alignment of SRP19 proteins shows that the M. jannaschii protein is the smallest thus far identified (Zwieb

and Samuelsson, 2000; Pakhomova et al., 2002). The human protein (Hs19) has insertions in two regions around residues 60 and 70 of the M. jannaschii protein as well as extended N and C termini (Figure 1B). Superposition of the M. jannaschii and human SRP19 proteins (Wild et al., 2001) on shared regions of main chain atoms gave a root mean square deviation of 0.98 A˚, implying that the core structure of SRP19 has been well conserved during evolution (Figure 2B). Amino acid residues involved in helix 6 (circles) and helix 8 (blue squares) contacts, as well as those involved in helix 6 contacts (circles) in the human SRP19-helix 6 complex (Wild et al., 2001), are indicated in Figure 1B. The majority of these contacts are made with backbone phosphate groups (Figure 2A), and only a single base-specific contact is found between the side chain of Lys19 of Mj19 and the O6 atom of G146. The residues involved in helix

Table 1. Summary of Diffraction Data

Wavelength (A˚) Resolution (A˚) Completeness (%) Unique reflections Multiplicity Mean I/sigma (I) Rsymm (%)

SeMet Edge

SeMet Peak

SeMet Remote

“Native”a

0.9792 29.2–3.2 99.3 (99.6) 11,963 (1,707) 10.4 (11.1) 19.8 (6.3) 14.5 (48.5)

0.9788 29.2–3.2 99.3 (99.6) 11,965 (1,707) 10.8 (11.1) 23.8 (7.4) 11.4 (40.1)

0.9184 29.2–3.2 99.4 (99.6) 11,981 (1,710) 10.8 (11.1) 19.3 (6.2) 13.9 (46.4)

0.9185 38.0–2.9 91.4 (52.9) 26,760 (1,544) 6.4 (4.1) 18.7 (3.1) 8.1 (31.0)

49.1 1.81

40.4 2.39 0.824

– 1.83

Phasing statisticsb R-Cullis (%) Phasing power FOMc

The SeMet data were processed with MOSFLM and the CCP4 suite of programs, and phasing statistics are from heavy atom refinement in SHARP and density modification with Solomon. The native data were processed with DENZO and SCALEPACK. Numbers in parentheses are for the outer resolution shell: 3.37–3.20 A˚ for SeMet and 3.00–2.90 A˚ for “Native.” a The dataset that the structure was refined against was the Mj19 single cysteine mutant derivatised with methyl mercury. b Acentric reflections. c Figure of merit following density modification.

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of the SRP19-helix 6 contacts are conserved at the amino acid-nucleotide level (red circles in Figure 1B), even though these two species are distantly related. RNA-protein contacts are further strengthened in each complex by species-specific contacts (black circles in Figure 1B). The conservation of SRP19-helix 6 contacts strongly suggests that the SRP19-helix 8 contacts observed in the Mj19-human RNA complex are likely to be conserved during evolution. Our complex is heterologous between the M. jannaschii SRP19 and human RNA, but the molecular mechanism of SRP19-7SL RNA recognition observed in this complex is likely to have been conserved universally in Archaea and Eukarya. Indeed the M. jannaschii SRP19 facilitates the binding of human SRP54 to human 7SL RNA (data not shown). Mj19-Helix 8 Contacts Loop L3 of Mj19, located between the ␤2 and ␤3 strands, protrudes into the minor groove of helix 8 and makes numerous contacts (Figure 3A). The imidazole ring of His57 forms a hydrogen bond with the 2⬘OH group of U195 and N3 of A205 (Figure 4A). Together with the hydrogen bond between the 2⬘OH group of A205 and the carbonyl group of Arg55, these hydrogen bonds lock the polypeptide loop into the shallow minor groove of helix 8. The side chains of Lys51, Arg52, and Arg63 interact with the phosphate backbone on the 5⬘ strand of helix 8 (Figure 4B). The main chain amide group of Arg52 also forms a hydrogen bond with the phosphate group of G197. Another interesting feature of the Mj19RNA complex is that Mj19 inserts two additional basic side chains, Arg15 and Arg55, between helices 6 and 8. These two residues neutralize the negative charges of phosphates and stabilize close interaction of the two RNA helices. Figure 3. The Crystal Structure of the Methanococcus jannaschii SRP19 in Complex with the S Domain of Human 7SL RNA (A) Overview showing coaxially stacked helix 8 and helix 5. Mj19, shown as a blue ribbon, binds to the tetraloops of both helices 6 and 8. Helices 5, 6, and 8 are shown in light blue, light green, and yellow, respectively. (B) Secondary structure of the S domain fragment of human 7SL RNA used for crystallization. RNA is color coded as in (A). The symmetric and asymmetric loops in helix 8 are colored with dark and light orange, respectively. The consecutive AC mismatched base pairs and mismatched AG base pair in helix 6 are highlighted in darker green. The internal loop in helix 5 is highlighted in dark blue. Nucleotides in close helix 6-helix 8 contacts are boxed and joined by broken lines. Two nonnatural Watson-Crick base pairs (outlined character) are added to the ends of the RNA to permit the cleavage by hammerhead ribozyme. Nucleotides protected from hydroxyl radical cleavage in the SRP19-bound form are indicated by red circles (Rose and Weeks, 2001). (C) Surface representation of Mj19 colored according to electrostatic surface potential. Blue, positively charged; red, negatively charged. RNA is shown as a ball-and-stick model.

6 contacts are located mostly in the conserved motif of the ␤1 strand, the loop L1 that follows ␤1, loop L3, and within helix B (Figure 1B). Many of the direct RNA-protein contacts are conserved between the Mj19-S domain RNA complex and Hs19-helix 6 complex (indicated by red circles in Figure 1B). These results led us to conclude that not only the core structure of SRP19 but also many

Tetraloop-Tetraloop Interaction The tips of helix 6 and helix 8 interact not only with SRP19 but also with each other. Helix 8 contains a GNRA-type tetraloop, which is commonly found in ribosomal RNAs and the group I intron (Woese et al., 1990; Cate et al., 1996). The GNRA tetraloop is particularly stable because of a hydrogen bond between the 2-amino group of G at the first position and N7 of A at the fourth position, a hydrogen bond between the 2⬘OH group of the first G and N7 of a purine base at the third position, and stacking interaction between the second, third, and fourth bases (Jucker et al., 1996) (Figure 4C). In contrast, the GGAG tetraloop, found in helix 6, is less common. Zwieb (1992) noted that the tetraloop recognized by SRP19 and ribosomal protein S15 are both of the GNAR type. Wild et al. (2001) pointed out that a GGAG tetraloop is also used as a binding site for HIV-1 nucleocapsid protein. In the human SRP19-helix 6 complex (Wild et al., 2001), the GGAG tetraloop has a more open structure than the GNRA tetraloop. The fourth G swings away from the first G, and the 2-amino group of G147 is hydrogen bonded to the phosphate group of G150. In our Mj197SL RNA complex, the 2-amino group of G147 is hydrogen bonded to N7 of G150, and the 1-imino proton is hydrogen bonded to its phosphate group (Figures 4C and 4D). This tetraloop-tetraloop interaction requires some induced fit of the helix 6 tetraloop. The second,

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Figure 4. The Interaction of Helix 8 with SRP19 and Helix 6 (A) The loop L3 of Mj19 interacting with the minor groove of helix 8. The imidazole ring of His57 is hydrogen bonded to the 2⬘OH group of U195 and N3 of A205. (B) Interaction between the backbone phosphate groups of the helix 8 tetraloop region and the peptide loop L3 of Mj19. (C) Schematic representation of the tetraloop-tetraloop interaction. (D) Rearrangement of hydrogen bonds in the helix 6 tetraloop upon helix 8 binding. The first and fourth G of helix 6 tetraloop in the Hs19helix 6 complex (blue) (Wild et al., 2001) and in the Mj19-S domain complex (green) (present work). (E) Symmetric adenine-adenine interaction between the tetraloops of helices 6 and 8. (F) Hydrogen bonding between nucleotides of helices 6 and 8 tetraloops. Protein residues are colored in grey, and helices 6 and 8 are colored in green and yellow, respectively, except in (D).

third, and fourth bases (G148, A149, and G150) show continuous stacking interactions. These three stacked RNA bases and the RNA backbone of the 3⬘ side of the tetraloop are wedged into the minor groove side of helix 8 and make numerous contacts. Based on the structure of the human SRP19-helix 6 complex, Wild et al. (2001) proposed that, as in the hammerhead ribozyme crystal (Pley et al., 1994), the exposed A149 base at the third position of the GGAG tetraloop in helix 6 would form a base triple in the minor groove of the G197-C202 base pair immediately below

the tetraloop of helix 8. Our structure shows that this is clearly not the case. A149 instead forms a symmetric A-A base pair (hydrogen bonding between the 6-amino group of adenine with the N1 of the partner) with A201 at the fourth position of the GAAA tetraloop in helix 8 (Figures 4C and 4E). The N3 atom of A149 also forms a hydrogen bond with the 2⬘OH of G197. The O6 atom of the second tetraloop nucleotide (G148) of helix 6 is hydrogen bonded to the 6-amino group of A200 at the third position of the GAAA tetraloop in helix 8. The 2-amino group of G150 also forms a hydrogen bond

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of SRP19. However, in the Mj19-7SL RNA crystal, these AC base pairs form mismatched base pairs distinct from A⫹C wobble base pairs (Figure 5A). The 6-amino group of A139 forms a bifurcated hydrogen bond with the 2-keto and N3 atom of C158, whereas the 4-amino group of C140 forms a hydrogen bond with the N1 atom of A157. Our Mj19-7SL RNA complex crystal was grown at a more physiological pH (pH 7.4), and the hydrogen bonding pattern of these base pairs is consistent with unprotonated adenine. Another mismatched base pair is found in helix 6. The 2-amino group of G164 forms a hydrogen bond with N7 of A133, and the 6-amino group of A133 is hydrogen bonded to the 2⬘OH group of G164 (data not shown). Helix 6 is expected to be rigid because of the continuous stacking of predominantly WatsonCrick and GU wobble base pairs (Figures 3A and 3B).

Figure 5. Close Interactions between Helices 6 (Green) and 8 (Yellow) (A) Two consecutive AC mismatched base pairs found in helix 6 at the close helix-helix contact. (B) Close helix 6-helix 8 contact near the asymmetric loop of helix 8. Four adenines in helix 8 stack continuously, and A215 is in syn conformation. (C) Close contacts between helices 6 and 8. G188 is in syn conformation and shows unusual base pairing with U211.

with the 2-keto oxygen and the 2⬘OH group of C202 (Figure 4F). An RNA mutagenesis experiment by Zwieb (1992) showed that mutation of A149 to any other nucleotide abolishes the binding of SRP19 to 7SL RNA. Mutation of G150 also severely affects the binding of SRP19. Our structure readily accounts for the effect of these mutations. Helix 6 Helix 6 of human 7SL RNA is base-paired throughout with Watson-Crick and GU wobble base pairs with the exception of three mismatched base pairs: A133-G164, A139-C158, and C140-A157 (Figures 3A and 3B). In the crystal structure of the Hs19-helix 6 complex (Wild et al., 2001), the two consecutive AC base pairs have perfect wobble base-pair geometry. These crystals were grown at low pH (pH 5.6), and hence this geometry is attributable to protonation of N1 of both A139 and A157. Wild et al. (2001) proposed that these consecutive A⫹C base pairs open up the major groove and facilitate the binding

Helix 8 Helix 8 in human 7SL RNA is closely related to domain IV of the E. coli SRP RNA (4.5S RNA), the binding site for Ffh (SRP54 homolog). Both contain a symmetric loop with four consecutive non-Watson-Crick base pairs and an asymmetric internal loop. The symmetric internal loop of helix 8 of human 7SL RNA has exactly the same mismatched base pairs, C191-A208, A192-C207, G193G206, and G194-A205, as in the E. coli 4.5S RNA to which the M domain of Ffh binds in the minor groove (Figure 3B). As expected from the sequence identity, the four mismatched base pairs show the same hydrogen bonding pattern in both E. coli and human RNAs, except for the G193-G206 base pair (Batey et al., 2000; Jovine et al., 2000). An NMR study by Schmitz et al. (1999) showed that the asymmetric loop of free 4.5S RNA with one nucleotide on the 3⬘ strand and four nucleotides on the 5⬘ strand is flexible and undergoes a hinge motion (Figure 6C). The asymmetric loop in human RNA with five nucleotides on the 5⬘ strand and three A’s on the 3⬘ strand is larger than that of 4.5S RNA and hence likely to be even more flexible in the absence of SRP19. In our crystal structure, bases in the shorter 3⬘ strand of the asymmetric loop stack continuously with helical geometry across the asymmetric loop. The phosphate backbone of the 3⬘ strand around A215 comes into close proximity with the phosphate backbone of C161 and C162 in helix 6 (Figure 5B). The two strands interact in such a way that the phosphate groups are staggered to minimize the electrostatic repulsion between the phosphate charges. The close packing of RNA helices observed here is similar but distinct to the “ribose zipper” in the P4-P6 domain of the group I intron (Cate et al., 1996). In the P4-P6 domain, pairs of riboses interact by hydrogen binding between the 2⬘OH and pyrimidine O2 (purine N3) of one strand and the 2⬘OH group of its partner, but in the Mj19-S domain complex, we observe no hydrogen bonding between the two strands. The two strands are rather forced to be in close proximity by SRP19 binding. Nevertheless, the two strands are locked with each other by the interdigitating backbone arrangement. This interaction must be important for the stabilization of the shorter 3⬘ strand into the helical conformation. The four consecutive adenines in the 3⬘ strand show continuous stacking interactions, and the base of A215 has an unusual syn confor-

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Figure 6. SRP19 Promotes the Binding of SRP54 by Stabilizing the Structure of the Asymmetric Loop in Helix 8 (A) Conformation of the longer 5⬘ strand of the asymmetric loop in helix 8. (B) A model of the SRP19/SRP54/RNA complex. The E. coli SRP54 M domain (EcFfh-M) was placed into the SRP19-S domain complex structure by overlaying the symmetric loop of the E. coli M domain-RNA complex (Batey et al., 2000) onto the symmetric loop of the Mj19RNA complex. Red, domain IV of the E. coli 4.5S RNA (Batey et al., 2000); yellow, helix 8 of 7SL RNA in the Mj19-S domain RNA complex; green, helix 6; blue, M. jannaschii SRP19 (Mj19); orange, the M domain of the E. coli Ffh. The structure of the longer 5⬘ strand of the asymmetric loops is shown as a ball-and-stick model. (C) The asymmetric loop of 4.5S RNA has been shown to undergo a hinge motion by NMR (Schmitz et al., 1999). In the absence of SRP19, helix 6 swings away from helix 8, as indicated by a loss of protection from hydroxyl radical cleavage (Rose and Weeks, 2001). (D) A model of the mammalian SRP. The S domain RNA with SRP19 and the SRP54 M domain are as in (B), and the Alu domain RNA with SRP9/14 heterodimer is based on a model by Weichenrieder et al. (2000). The A form RNA linker between the S and Alu domains (gray) is built as an A form helix with 38 base pairs. Although the structure of the N/G domain of Ffh is known, its position with respect to the M domain is uncertain (Keenan et al., 1998). There is no structural information for the SRP68/72 heterodimer. Approximate length of the model is in good agreement with Andrews et al. (1987).

mation (Figure 5B). Similarly in the M domain-bound 4.5S RNA (Batey et al., 2000) but not in free 4.5S RNA (Schmitz et al., 1999), the bases in the 3⬘ strand continuously stack.

Stacking of bases in the longer 5⬘ strand of the asymmetric loop is discontinuous between A183 and A184, and the backbone undergoes a twist at the junction (Figure 6A). The nucleotides in this region are poorly

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ordered. The Watson-Crick face of G182 is pointing towards the Hoogsteen face of A215, and O6 of G182 forms a hydrogen bond with N6 of A214 (Figure 6A). Electron density for the base of A183 is poor but suggests that it is in syn conformation and stacks onto the base of G182. The base of A184 is on the same plane as the bases of G182 and A215, but they do not form a base triple. The base of C185 stacks onto the base of A184. The bases of A213 and C186 are too far apart to form a base pair, but they both stack onto the adjacent Watson-Crick G187-C212 base pair (Figure 6A). Helices 6 and 8 come into close contact again between A139 and C140 of helix 6 and G187 and G188 of helix 8 (Figure 5C). The distance between the O2P atom of G188 and the O5⬘ atom of A139 is only 3.4 A˚. The phosphate groups are again staggered to minimize electrostatic repulsion. The two consecutive AC base pairs (A139-C158 and C140-A157) in helix 6 may be important in allowing the close helical contact. The base of G188 takes a syn conformation, and its 6-keto oxygen is hydrogen bonded to the 3-imino group of U211. The G188U211 base pair is flanked by normal Watson-Crick GC base pairs, and it is not clear why G188 takes a syn conformation instead of forming a GU wobble base pair. It is noteworthy that both A215 and G188 are adjacent to the nucleotides at close helix-helix contacts, and a nucleotide with a syn conformation may be important to accommodate this backbone conformation. It would be interesting to investigate the structure of human helix 8 in solution to see if G188 and U211 form a wobble base pair in the absence of such a tertiary contact. Interaction between Helix 8 and SRP54 The amino acid sequence of the M domain is highly conserved from bacteria to man, and the crystal structures of the M domain from Thermus aquaticus, E. coli, and human show that its three dimensional structure has remained virtually unchanged throughout evolution (Keenan et al., 1998; Clemons et al., 1999; Batey et al., 2000). The E. coli M domain recognizes predominantly the minor groove of the symmetric loop by forming a dense network of direct and solvent-mediated hydrogen bonds. The noncanonical base pairs of the symmetric loop and the key amino acid residues of the M domain involved in the recognition of the symmetric loop are largely conserved during evolution. This strongly suggests that the contacts between the M domain and the symmetric loop are also conserved. Hence, the position of the M domain with respect to the symmetric loop must be maintained in the human and E. coli complexes. A comparison between the crystal structures of the free and the M domain-bound 4.5S RNA (Batey et al., 2000; Jovine et al., 2000) showed that the symmetric loop undergoes virtually no structural change upon binding the M domain. Based on these observations, we superimposed the four mismatched base pairs of the symmetric loop of helix 8 of our complex (yellow helix in Figure 6B) and the M domain-bound E. coli 4.5S RNA (red helix in Figure 6B) to model the M domain into our SRP19-S domain RNA complex structure. The asymmetric loop of free RNA is flexible and undergoes a hinge motion (Schmitz et al., 1999), whereas the longer 5⬘ strand of the asymmetric loop becomes

ordered and forms a platform when it interacts with the M domain of E. coli Ffh (Batey et al., 2000). The binding of the M domain to 4.5S RNA is therefore accompanied by a considerable ordering of the asymmetric loop. Our crystal structure of the Mj19-7SL RNA shows that the helix 6-helix 8 interaction stabilizes the 3⬘ strand of the asymmetric loop of helix 8 into a helical conformation with continuous base stacking as in the M domainbound 4.5S RNA. In contrast, the longer 5⬘ strand retains substantial flexibility, as indicated by high temperature factors. A comparison of the asymmetric loop structure between the M domain-bound 4.5S RNA (red) and the Mj19-bound 7SL RNA (yellow) is shown in Figure 6B. The striking difference between these two structures is that the 5⬘ strand of the asymmetric loop of human helix 8 is much further away from the symmetric loop and would have to move up closer to the M domain in order to interact with the M domain as observed in the E. coli complex. The 5⬘ strand of the asymmetric loop is poorly ordered in the Mj19-7SL RNA complex and hence retains the ability to undergo a conformational change upon binding of the M domain to form a platform as in the E. coli complex (Batey et al., 2000). A Large Conformational Change of the S Domain RNA Induced by SRP19 Facilitates SRP54 Binding Human SRP54 does not readily bind to 7SL RNA in the absence of SRP19, so how does SRP19 facilitate the binding of SRP54 or its M domain to 7SL RNA? The major binding site for the M domain, the minor groove of the symmetric loop, is facing away from helix 6, and hence, the M domain is most unlikely to interact with helix 6. Although human SRP19 has N- and C-terminal extensions and two loop insertions, our model of the ternary complex suggests that SRP19 is unlikely to interact extensively with the M domain (Figure 6B). Therefore, direct interaction between the M domain and helix 6 or SRP19 is unlikely to account for the facilitated SRP54 binding to 7SL RNA in the presence of SRP19. Our structure supports the idea that SRP19 enables SRP54 binding by inducing a conformational change in 7SL RNA. In the absence of SRP19, neither helix 6 nor 8 is protected from hydroxyl radical or ␣-sarcin cleavage (Siegel and Walter, 1988a; Diener and Wilson, 2000; Rose and Weeks, 2001). This shows that helices 6 and 8 swing away from each other in the absence of SRP19 (Figure 6C). Considering the arrangement of three helices in hammerhead and VS ribozymes as studied by electrophoretic and fluorescent energy resonance transfer (FRET) methods (Bassi et al., 1995; Lafontaine et al., 2001), it is likely that helix 6 swings away substantially from helix 8 (Figure 6C) in the absence of SRP19. The three helices may undergo a dynamic hinge motion around the three-way junction. Furthermore, Schmitz et al. (1999) showed that domain IV of E. coli 4.5S RNA undergoes a hinge motion around the asymmetric loop (Figure 6C). Hence, the closely related helix 8 would also undergo a hinge motion around the asymmetric loop. The S domain RNA contains at least two flexible joints: one at the three-way junction and the other at the asymmetric loop in helix 8. We have shown that SRP19 binds to the tetraloops of both helices 6 and 8 and allows these tetraloops to

Crystal Structure of the SRP19-S Domain RNA Complex 1259

interact extensively. As a result, helices 6 and 8 interact side by side because they are clamped by SRP19 on one end and by the three-way junction on the other. The hydroxyl radical cleavage protection (Rose and Weeks, 2001; Diener and Wilson, 2000) is entirely consistent with our crystal structure. Helix 6, base paired throughout and likely to be a rigid structure, acts as a splint for helix 8 to reduce the conformational freedom of the asymmetric loop and prevent the hinge motion of the asymmetric loop as observed in free E. coli 4.5S RNA (Schmitz et al., 1999). As the shorter 3⬘ strand of the helix 8 asymmetric loop is stabilized into a helical conformation as a consequence of the helix 6 and helix 8 interaction, the binding of the M domain to helix 8 would primarily involve a conformational change of the longer 5⬘ strand of the asymmetric loop. Our crystal structure shows that SRP19 would facilitate the binding of SRP54 by partially preorganizing the binding site for SRP54 and reducing the entropic cost of ordering the asymmetric loop. Therefore SRP19 plays a crucial role in SRP assembly by stabilizing the tertiary structure of 7SL RNA and enabling SRP54 to bind to helix 8. Overall Structure of the Eukaryotic SRP Our structure has provided important insights into the tertiary interaction within 7SL RNA as well as the overall structure of the eukaryotic SRP. The binding site for the SRP68/SRP72 heterodimer has been mapped in the region near the three-way junction by ␣-sarcin protection (Siegel and Walter, 1988a). The four protein subunits, SRP19, SRP54, SRP68, and SRP72, assemble around helices 5, 6, and 8, which are arranged in a rod shaped structure. Based on the low-resolution structure of the SRP14/SRP9 heterodimer in complex with a variant Alu domain RNA, it was proposed that the Alu domain RNA folds back and wraps around the SRP14/ SRP9 heterodimer (Weichenrieder et al., 2000). The S and Alu domains are connected by a long RNA duplex interspersed with internal loops. Based on this available structural information, the overall length of the human SRP is estimated to be around 260 A˚ (Figure 6D), which is in good agreement with the dimension inferred by electron microscopy (Andrews et al., 1987). In our structure, helix 5 is kinked around the asymmetric loop whose high B factor suggests substantial flexibility. When the S domain binds to a signal peptide emerging from the ribosome exit site, the long connector RNA helix could therefore curve around the surface of the large ribosomal subunit such that the Alu domain could reach an effector binding site at the interface between the 60S and 40S ribosomal subunits. This could provide a mechanism for a temporary arrest of peptide elongation, although the binding site for the Alu domain and the mechanism of the elongation arrest have yet to be elucidated (Weichenrieder et al., 2000). Experimental Procedures Production of the Wild-Type and Mutant SRP19 A DNA sequence encoding SRP19 was PCR amplified from M. jannaschii genomic DNA and cloned into pRET3a (a generous gift of Dr S. Tan, Pennsylvania State University). A single-cysteine mutant (Cys61→Ala, Cys63→Arg, Cys80→Ala, and Gln74→Cys) gene was constructed by cassette mutagenesis. A coding sequence for a

mutant with five methionines (Ile2→Met, Leu41→Met, Leu43→Met, Leu75→Met, and Leu76→Met) was constructed by PCR-based sitedirected mutagenesis (Sarkar and Somer, 1990). A culture of C41(DE3) cells (Miroux and Walker, 1996) harboring the SRP19 expression vector was grown at 37⬚C until OD600 ⫽ 0.7, induced with 1 mM IPTG, and harvested 7 hr after induction. A selenomethioninelabeled protein of the methionine mutant was produced in C41(DE3) cells as described by van den Ent et al. (1999). Cells were suspended in 50 mM sodium citrate, 5 mM dithiothreitol (DTT), and 100 mM Tris-HCl (pH 7.4), and 1.5 tablets of complete EDTA-free protease inhibitor cocktail (Roche Diagonostics, Mannheim, Germany) were added. Cells were frozen, thawed, and then lysed by ultrasonication. 1.6 M sodium citrate (pH 7.0) was added to the cleared lysate to a final concentration of 250 mM. The protein solution was heated to 70⬚C in a hot waterbath and then immediately cooled on ice. After denatured E. coli proteins were removed by centrifugation, the supernatant was diluted 3.5-fold with 100 mM Tris-HCl (pH 7.4) and then passed through a 300 ml bed volume DEAE Sepharose (Amersham Biosciences UK, Little Chalfont, UK) column equilibrated with the same buffer. The protein solution was then loaded onto a 45 ml bed volume heparin Sepharose (Pharmacia) column preequilibrated with 50 mM Tris-HCl (pH 7.4), 75 mM NaCl, and 5 mM DTT. The Mj19 protein was eluted by a linear gradient of 75→650 mM NaCl. The peak fractions were pooled and dialysed against 50 mM TrisHCl (pH 7.4), 100 mM NaCl, and 5 mM DTT prior to loading onto a 45 ml bed volume CM Sepharose (Pharmacia) column. The Mj19 protein was eluted with a linear gradient of 100→500 mM NaCl. The pooled peak fractions were dialysed against 20 mM Tris-HCl (pH 7.4), 100 mM sodium citrate (pH 7.0), and 5 mM DTT and then stored at liquid nitrogen temperature in small aliquots of 1 mg/ml solution. Synthesis of a 7SL RNA Fragment A synthetic gene containing T7 RNA polymerase promoter and a 128 nucleotide fragment (Figure 3B) of human 7SL RNA gene flanked by 5⬘ and 3⬘ hammerhead ribozymes was constructed by assembling six overlapping oligonucleotides and was cloned into pUC19 vector (Price et al., 1995). This plasmid was linearised with HindIII at the end of the 3⬘ ribozyme sequence and used as a template for in vitro transcription. The 7SL RNA fragment was purified by polyacrylamide gel electrophoresis, eluted from gel pieces by a crush and soak method, precipitated with ethanol, dissolved in water, and stored in small aliquots at ⫺20⬚C. Crystallization Crystallization was carried out by hanging drop vapor diffusion. 1.5–3 ␮l of complex at ⵑ5 mg/ml in 100 mM ammonium acetate, 10 mM Tris-HCl (pH 7.4), 10% glycerol, 5 mM MgCl2, and 5 mM DTT were mixed with an equal volume of 12% (w/v) PEG4000, 0.1 M Tris-HCl (pH 7.4), and 50 mM magnesium acetate. Drops were placed over 0.5 ml of the well buffer and incubated at 27.5⬚C. Crystals grew to a size suitable for data collection in 3 days (native protein and cysteine mutant) to 2 weeks (SeMet protein). Crystals were cryoprotected by gradually increasing the glycerol concentration in the crystallization drop to 20% (v/v) and subsequently flashcooling to 100 K for data collection. Data Collection and Structure Determination X-ray diffraction data were collected on a MAR Research CCD detector at the BM14U CRG beamline at ESRF, Grenoble, France. The data were processed and scaled with MOSFLM (Leslie, 1992) and SCALA (CCP4, 1994) or the HKL suite (Otwinowski and Minor, 1997) (Table 1). The selenomethionine derivative of the Mj19 protein with five Met substitutions was used for multiwavelength anomalous dispersion (MAD) phasing. X-ray diffraction from these crystals was highly anisotropic with, crystals typically diffracting to 3.0 A˚ in the b* and c* directions, but only to 4.5 A˚ in the a* direction. Four initial selenium sites were determined using SOLVE (Terwilliger and Berendzen, 1999), two additional sites were found, and their heavy atom parameters were refined with SHARP (de La Fortelle and Bricogne, 1997). Initial phases were solvent flattened using SOLOMON (Abrahams and Leslie, 1996), and an experimental electron density map of the selenomethionine derivative calculated at 3.2 A˚ was used for model building with O version 8 (Jones and Kjeldgaard,

Molecular Cell 1260

1997). Initial refinement was carried out in REFMAC5 (Murshudov et al., 1999) until a free R factor of 35.1% and a working R factor of 32.2% were achieved at a resolution of 3.2–29.5 A˚. The model at this stage included all protein residues except the five C-terminal residues and all RNA nucleotides except the helix 7 region and the internal loop in helix 5. However, we were unable to refine the structure further because of poor data quality due to high anisotropy. Crystals of the methylmercury derivative of the single-cysteine mutant (see above), prepared for possible mercury MAD phasing, diffracted more isotropically to 2.9 A˚ and hence were used for further analysis. The structure of the Hg derivative was solved by molecular replacement in CNS (Bru¨nger et al., 1998) using the selenomethionine derivative structure as a search model. The structure has been refined to a free R factor of 29.6% and a working R factor of 26.4% at 2.9 A˚ resolution with good geometry (rmsd bond lengths of 0.007 A˚ and rmsd angles of 1.1⬚) using TLS refinement within REFMAC 5.1, CNS 1.1, and maps calculated by EDEN 3.3 (Szo¨ke et al., 1997) as a guide (Table 1). Stereochemistry of Mj19 investigated by Procheck (Laskowski et al., 1993) showed that 81% residues are in the most favored region, and 19% are in the additional allowed region of a Ramachandran plot. Illustrations were generated using RIBBONS (Carson, 1997), GRASP (Nicholls et al., 1991), and MOLSCRIPT (Kraulis, 1991). Acknowledgments We thank Tobias Hainzl for his valuable contribution at the early stage of the project and Phil Evans, Venki Ramakrishnan, Jade Li, Andrew Leslie, Ditlev Broderson, Ben Luisi, and Adelaine Leung for their help and critical reading of the manuscript. We thank Andy Thompson and Martin Walsh (ESRF), Massimo Degano and Edoardo Busetto (Elettra), and Sam Yong Park (Spring8) for their assistance with data collection. Assistance of Pietro Roversi, Gwyndaf Evans, and Gerald Bricogne with SHARP, of Garib Murshudov with REFMAC, and of Hanna Szo¨kes with EDEN was essential and is greatly appreciated. The work was supported by the Medical Research Council. A.K. was also supported by a Boehringer Ingelheim predoctoral fellowship. L.J. was supported by a Human Frontier Science Program long-term fellowship. This paper is dedicated to the late Max Perutz for his encouragement. Received: February 7, 2002 Revised: March 26, 2002 References

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