The structure of the chloroplast signal recognition particle (SRP) receptor reveals mechanistic details of SRP GTPase activation and a conserved membrane targeting site

FEBS Letters 581 (2007) 5671–5676

The structure of the chloroplast signal recognition particle (SRP) receptor reveals mechanistic details of SRP GTPase activation and a conserved membrane targeting site Katharina F. Stengel, Iris Holdermann, Klemens Wild, Irmgard Sinning* Biochemie-Zentrum der Universita¨t Heidelberg, INF 328, D-69120 Heidelberg, Germany Received 19 October 2007; revised 7 November 2007; accepted 8 November 2007 Available online 20 November 2007 Edited by Richard Cogdell

Abstract Two GTPases in the signal recognition particle and its receptor (FtsY) regulate protein targeting to the membrane by formation of a heterodimeric complex. The activation of both GTPases in the complex is essential for protein translocation. We present the crystal structure of chloroplast FtsY (cpFtsY) ˚ resolution. The comparison with FtsY structures in difat 1.75 A ferent nucleotide bound states shows structural changes relevant for GTPase activation and provides insights in how cpFtsY is pre-organized for complex formation with cpSRP54. The structure contains an amino-terminal amphipathic helix similar to the membrane targeting sequence of Escherichia coli FtsY. In cpFtsY this motif is extended, which might be responsible for the enhanced attachment of the protein to the thylakoid membrane.  2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Chloroplast SRP system; Protein targeting; cpFtsY; SRP GTPase; Membrane targeting sequence

1. Introduction The signal recognition particle (SRP) mediates the co-translational targeting of secretory or membrane proteins to the endoplasmic reticulum in eukaryotes or the plasma membrane in prokaryotes [1–3]. SRP binds to the signal sequence as it emerges from the ribosome. Subsequent interaction with the SRP receptor leads to the transfer of the ribosome nascent chain complex (RNC) to the SecYEG translocation pore. Cytosolic SRP is a ribonucleoprotein particle and the SRP RNA was shown to play more than a structural role as it participates actively in catalysis and accelerates complex formation of SRP and the SRP receptor [4–6]. The composition of SRP varies in the different organisms, but the SRP core (SRP54/helix 8 of the SRP RNA) and the principle of nucleotide dependent complex formation with the membrane bound

* Corresponding author. Fax: +49 6221 544790. E-mail address: [email protected] (I. Sinning).

Abbreviations: SRP, signal recognition particle; FtsY, SRP receptor in prokaryotes; cp, chloroplast; SIMIBI, for signal recognition particle, MinD, and BioD; MTS, membrane targeting sequence

SRP receptor (FtsY in prokaryotes) are universally conserved [2,3]. The chloroplast SRP (cpSRP) which targets membrane proteins to the thylakoid membrane presents a remarkable exception [7–9]. It consists of cpSRP54, a homologue of SRP54, but does not contain an SRP RNA [10]. Chloroplast encoded substrate proteins are recognized by cpSRP associated with translating ribosomes [11] while nuclear encoded proteins interact with cpSRP after import into the chloroplast. Therefore, cpSRP is able to recognize signal sequences in two fundamentally different contexts. In addition, for post-translational targeting of nuclear encoded proteins ribosomes are not involved. A novel protein component, cpSRP43, forms a complex with cpSRP54 and participates in substrate recognition [12–15]. The light-harvesting chlorophyll binding proteins (LHCPs) are the major nuclear encoded substrates of cpSRP. In the stroma the LHCP substrate, cpSRP43 and cpSRP54 form the so-called transit complex which is targeted to the thylakoid membrane via the membrane associated SRP receptor chloroplast FtsY (cpFtsY) [16–18]. Together with Alb3, a functional homologue of the YidC and Oxa1 proteins [19], cpFtsY assists in the insertion of integral membrane proteins into the thylakoid membranes [13,18,20,21]. Protein targeting by SRP and its receptor is regulated by small G proteins present in SRP54 and FtsY [22,23]. The SRP GTPases belong to the SIMIBI (for signal recognition particle, MinD, BioD) class of NTP binding proteins which are characterized by the formation of nucleotide dependent dimers [24] as described for the heterodimeric complex of the two NG domains of SRP54/FtsY [25,26] (Bange and Sinning, unpublished data). In the SRP54/FtsY complex (targeting complex) the GTPases activate each other [27] and GTP hydrolysis induces dissociation of the complex [28]. The activation of GTP hydrolysis in the targeting complex is crucial for efficient protein translocation [29,30]. For Escherichia coli FtsY we have recently shown that only FtsY mutants which allow to activate GTP hydrolysis in the SRP/FtsY complex at the membrane are functional in vivo and FtsY mutants that cannot be activated due to a compromised membrane targeting sequence (MTS) lead to an accumulation of ribosomes at the membrane [31]. FtsY of E. coli interacts with membrane phospholipids [32,33] and the Sec translocon [34]. It contains a conserved MTS at the N-terminus of the N domain [33]. While in E. coli only a fraction of FtsY is located at the plasma membrane [35], in chloroplasts cpFtsY is predominantly located

0014-5793/$32.00  2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2007.11.024

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at the thylakoid membrane [16,18]. However the mechanism of membrane association of cpFtsY is not known. We have solved the crystal structure of cpFtsY from Arabid˚ resolution with bound malonate. The opsis thaliana at 1.75 A malonate induces structural changes which are similar to the activated conformation in the targeting complex.

2. Materials and methods 2.1. Cloning, expression, purification, crystallization and structure determination cpFtsY has been cloned and expressed in E. coli and crystallized following standard protocols [33,36] with modifications as described in Supplementary data. The crystals belong to space group I222 with cell ˚ , b = 84.15 A ˚ , c = 99.3 A ˚ and contain one moldimensions a = 87.89 A ecule per asymmetric unit with a solvent content of 60%. The cpFtsY structure was solved by molecular replacement. For details of structure determination and refinement see Supplementary data. Refinement statistics are given in Supplementary Table 1. 2.2. Lipid binding experiments Lipid binding experiments were performed as described for E. coli FtsY [33]. Briefly, thylakoid lipids (Lipid Products/England) in the appropriate concentrations [37] were mixed, dried and resuspended in reaction buffer. After incubation of the protein with the liposomes the sample was applied to an Optiprep reaction buffer gradient and ultra centrifuged (with 154 000 · g). Four fractions were taken from top to bottom. Detection of the proteins was performed via SDS– PAGE and Western Blot with an anti-His antibody. The soluble protein thioredoxin served as a negative control and the NG + 1 FtsY construct from E. coli as a positive control [33].

3. Results and discussion 3.1. cpFtsY shares the domain arrangement of SRP GTPases cpFtsY is a member of the SRP GTPases which form a subfamily within the SIMIBI class of NTP binding proteins [24] with only three members: the SRP54 protein involved in signal sequence binding, the SRP receptor protein FtsY and FlhF involved in the assembly of polar flagella [38] (Fig. 1A). In contrast to E. coli FtsY, cpFtsY does not contain an A domain. The mature form of cpFtsY from A. thaliana (lacking the 41 amino acid long transit sequence for import into the chloroplast) is purified as a monomer without bound nucleotide (not shown). The crystal structure of cpFtsY was determined ˚ resolution. The atomic coordinates and the structure at 1.75 A factors have been deposited in the Protein Data Bank under the accession code 3b9q. For data and refinement statistics see Supplementary Table 1. The structure of cpFtsY contains residues 65–366 (disordered are only the first 23 residues of the mature protein, residues 151–152 and residues 226–228), and shows the typical domain structure of SRP GTPases with two segments: the N domain comprising four a-helices (aN1 to aN4; residues 65–150), and the C-terminal G domain (residues 153–366) with the typical I-box insertion (residues 205–247) (Fig. 1B). Residues 42–64 are absent in the crystal structure and are predicted to form an extension of the N-terminal helix aN1a. The overall structure of cpFtsY is very similar to FtsY from E. coli which is the only apo structure of an SRP GTPase ˚ over to date (root mean square deviation (r.m.s.d.) of 1.7 A 275 aa residues) [23,33]. Sequence alignments with FtsY homologs show the preservation of five highly conserved sequence motifs involved in nucleotide binding and hydrolysis (G1–G5

Fig. 1. Structure of cpFtsY. (A) Scheme of the domain arrangement of SRP GTPases. (B) Overall structure of cpFtsY with bound malonate in the nucleotide binding site. The N domain is shown in blue and the G domain in green with the I-box in cyan. Conserved sequence motifs in the NG interface (ALLEADV, DARGG, GQ) are highlighted in yellow and the N-terminal amphipathic helix aN1a is drawn in red. The N- and C-termini and the conserved G motifs are indicated (G1– G5). (C) Sequence alignments of conserved motifs in the NG interface. Abbreviations are as follows: A.t.: Arabidopsis thaliana; T.m.: Thermotoga maritima; E.c.: Escherichia coli; T.a.: Thermus aquaticus; M.m.: Mycoplasma mycoides. (D) Sequence alignments of the conserved G motifs.

motifs, Fig. 1D). Notably, only cpFtsY contains three subsequent glycines in the P-loop (G1). In addition, three conserved sequence motifs specific for SRP GTPases are present in the NG interface, the ‘ALLEADV’, ‘GQ’ and ‘DARGG’ motifs which are involved in adjusting the NG interface in response to the nucleotide load and/or the presence of a signal sequence (reviewed in [2]) (Fig. 1C). The observed conservation suggests that the regulation of the GTPase activity of cpFtsY should follow the same general principles as described for SRP54 and FtsY GTPases. 3.2. The nucleotide binding site is changed by the presence of malonate The nucleotide binding affinity of cpFtsY (and cpSRP54) was recently reported to be significantly higher [39] than in FtsY and SRP54 from E. coli [31,40,41]. Together with an enhanced complex formation with cpSRP54 [39] this might explain why SRP RNA is not needed in chloroplasts. However, cpFtsY is purified and crystallized without bound nucleotide and malonate from the crystallization solution is present in the nucleotide binding site (Figs. 1B and 2A). It induces a number of conformational changes compared to

K.F. Stengel et al. / FEBS Letters 581 (2007) 5671–5676

Fig. 2. Comparison of the nucleotide binding site of cpFtsY with different FtsY structures. (A) cpFtsY with bound malonate. The G1 (P-loop) to G3 motifs are shown together with relevant side chains as mentioned in the text. Final 2mFo-DFc electron densities (contoured at 2.0r) are given for malonate, coordinated water molecules (shown as red spheres) and Arg204 from the G2 motif. The hydrogen bonding network around the malonate is indicated by dashed lines. The same view was chosen for panels (B)–(F). (B) The apo form of E. coli FtsY (2qy9 used here; 1fts) is the only empty FtsY structure to date. (C) GDP bound T. aquaticus FtsY (2iyl) with an unstructured G2 motif. (D) Sulfate bound FtsY from M. mycoides (1zu4) resembles the apo form (B), the GDP (C), and citrate (F) bound structures. (E) GMPPCP bound FtsY from the targeting complex of T. aquaticus (1okk). The three G motifs are placed for GTP hydrolysis. The P-loop is opened up, the G2 motif is ordered by the presence of the Mg2+ ion, and the G3 motif is moved toward the nucleotide correlated with a peptide flip of the conserved glycine within the G3 motif. (F) Citrate bound T. maritima FtsY (1vma) resembles the structures shown in (B)–(D).

the nucleotide-free protein (Fig. 2B), which are highlighted by the comparison of FtsY structures with different ligands (Fig. 2B–F). Malonate interacts with the backbone nitrogens of three subsequent glycine residues (Gly174–176) and the side chain hydroxyl groups of the two threonine residues (Thr178–179) in the P-loop, as well as with Arg204 of the G2 motif. Arg204 is strictly conserved and has been suggested as the catalytic arginine for GTP hydrolysis in SRP GTPases [25,26]. Malonate occupies the position taken by the a- and b-phosphate groups in the nucleotide bound structures [25,26,42] (Fig. 2C and E) and is arranged with its long axis perpendicu-

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lar to the connection between the two phosphate groups. Due to malonate, the P-loop is opened and shifted upwards similar to the targeting complex (Fig. 2E).This is not observed in the apo form [33] (Fig. 2B), and the GDP [42] (Fig. 2C), sulfate [43] (Fig. 2D), and citrate (PDB entry code: 1vma) (Fig. 2F) bound FtsY structures. In addition, the P-loop is shifted towards the G4 motif and away from the G3 motif. Binding of malonate might be facilitated in cpFtsY by the unique presence of three subsequent glycine residues in the P-loop. The P-loop contains a strictly conserved asparagine residue (Asn173 in cpFtsY) which in the targeting complex interacts with the 3 0 OH group of the ribose moiety of GMPPCP in trans [25,26]. It has been implicated to participate in GTPase activation and/or formation of the heterodimer [44] and to specify the order of nucleotide hydrolysis [25,26]. In the apo form, and the GDP, sulfate and the citrate bound structures of FtsY this asparagine is locked in between the P-loop and the G3 motif and forms a hydrogen bond with the nitrogen of the conserved glycine residue within the G3 motif (Gly257 in cpFtsY) (Fig. 2). In the malonate bound structure of cpFtsY, as well as in all structures of the targeting complex, the G3 motif adopts a similar conformation and the P-loop is moved (see above) which leads to a loss of the interaction between Asn173 and Gly257. As a major consequence, Asn173 is withdrawn from the active site and the peptide flip of the glycine in the G3 motif, previously observed only in the targeting complex is already induced in cpFtsY. The G2 motif is involved in Mg2+ ion coordination and contributes a conserved arginine (Arg204 in cpFtsY) to the nucleotide binding site which has been suggested as the catalytic arginine in SRP GTPases [25,26]. In cpFtsY the G2 motif has moved into the binding site compared with the apo form, the GDP, sulfate, and citrate bound structures. The interaction of Arg204 with malonate induces a closed conformation of the G2 motif. The guanidinium group of Arg204 occupies a similar position as the c-phosphate in the targeting complex and the conformation of the G2 motif resembles the one observed in FtsY structures without the Mg2+ ion (apo form, sulfate and citrate bound structures). In cpFtsY the conformation of the G4 motif is similar to the apo form (not shown). The conserved lysine (packing against the guanine base, Lys319 in cpFtsY) interacts directly with the P-loop and indirectly through a water molecule with the bound malonate. The sidechain of Asp321, the strictly conserved aspartate important for nucleotide specificity, is poorly ordered and compared with the overall structure the G4 and G5 motifs reveal higher B values probably due to the absence of bound nucleotide. The comparison of the G motifs shows that although malonate occupies the position of the a- and b-phosphates it induces changes in the P-loop and the G3 motif as observed in the targeting complex. Thereby cpFtsY is already pre-organized in a conformation that would allow complex formation with cpSRP54 with only minor conformational changes. The G4 and G5 motifs, however, remain unaffected.

3.3. The interface between the N and G domains is similar to the apo structure SRP GTPases are multidomain proteins and in order to ensure productive protein translocation, the two GTPases of SRP and FtsY are synchronized by the formation of the

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the shift of the DARGG helix does not occur (Fig. 3B). In cpFtsY the orientation of the four helix bundle with respect to the G domain is similar to FtsY from E. coli in its apo form [23,33] (Fig. 3A). However, the ‘ALLEADV’ and ‘GQ’ motifs ˚ away from the and the DARGG helix are shifted by about 2 A G domain, resulting in a 10 tilt of the helices aN2 and aN3 between the two structures. The shift of the DARGG helix as seen in the targeting complex has not occurred (Fig. 3B). Taken together, the presence of malonate in the active site does not induce the rearrangements in the NG interface as seen in the targeting complex.

Fig. 3. NG domain interface of cpFtsY. (A) Superposition of FtsY structures in the apo form (red, 2qy9), with malonate (green) and from the targeting complex (blue, 1okk). The superposition is based on the G domain core, which is represented by cpFtsY (grey) for clarity. The N to G domain orientation of the malonate bound form is close to the apo form (cpFtsY is tilted 10 to the back resulting in the visible translation of helices aN2 and aN3). The missing aN1 helix within the targeting complex allows for the adjustments in the NG interface and the large rotation (about 30) of N relative to G (within the plane of the figure to the left). (B) Close-up view of the NG interface in the same colour code. The DARGG motif and the adjacent helix are given for all structures. The superposition shows the conformational changes upon formation of the targeting complex (blue).

Fig. 4. The N-terminus of cpFtsY is conserved in FtsY homologs. (A) Sequence alignment of the MTS containing region of FtsY from various origins (based on aN1a) and the MTS of MinD from Listeria monocytogenes (L.m.). For abbreviations see Fig. 1C. O.s.: Oriza sativa; Z.m.: Zea mays. The motifs M-I and M-II of cpFtsY are indicated. Conserved residues are given in blue. (B) Helical wheel representation of the M-I and M-II motifs from cpFtsY.

3.4. The N-terminus of cpFtsY contains a membrane targeting sequence FtsY from E. coli interacts with anionic phospholipids and this interaction activates the GTPase [32]. A conserved MTS was identified in the N-terminal extension of the NG domain which forms an amphipathic helix (aN1a) [33] (Fig. 4A). It resembles the MTS of MinD [45] which is also a member of the SIMIBI class of NTP binding proteins [24]. The MTS is crucial for the correct cellular distribution of FtsY and for the stimulation of GTP hydrolysis in the targeting complex [31]. cpFtsY contains two conserved sequence motifs at its N-terminus (M-I and M-II, Fig. 4A) which are both very similar to the MTS in E. coli FtsY. The region of F48 to R75 is predicted to form an a-helix, but M-I (F48-E58) has a stronger amphipathic character than M-II (D63-R72) (Fig. 4B). The sequence of M-I is more closely related to the MTS of E. coli FtsY than M-II. The differences in amino acid sequence between FtsY homologs might reflect an adaptation to the lipid composition of its respective membrane as also suggested for MinD [46]. In the structure of cpFtsY only a short helical segment corresponding to M-II is ordered at the N-terminus (Fig. 1B), similar to aN1a in the FtsY structures of E. coli, Mycoplasma mycoides [43] and Thermotoga maritima (pdb 1vma). In order to test the membrane interaction of cpFtsY we performed flotation assays [33]. The composition of the liposomes was adapted to resemble the thylakoid membrane [37]. Indeed, cpFtsY interacts with membrane lipids (data not shown). The duplication of the membrane interaction motif might contribute to the observed higher membrane binding affinity of cpFtsY [16,18]. Clearly, the membrane interaction of cpFtsY needs to be analysed in more detail. Likewise, whether the extended N-terminus of cpFtsY is important for an activation of the GTPase upon membrane interaction as seen in E. coli [31] or for the stabilization of the transit complex at the membrane is not known at present. In the absence of ribosomes, the interaction between the transit complex and Alb3 might need to be stabilized for efficient protein translocation.

4. Conclusions SRP54/FtsY heterodimer in the targeting complex. A maximum rotation of up to 30 of the N vs. the G domain is observed in the targeting complex compared to the domain arrangement in the individual proteins [25,26] (Fig. 3A). In addition, the DARGG helix (following the ‘DARGG’ motif) is shifted along (and rotated around) its helical axis towards G4. In the individual SRP GTPases only minor changes in the NG interface are observed upon nucleotide binding and

The effects of malonate on the nucleotide binding site and the NG interface described here, show that the ligand induces a conformation which can be regarded as a chimera between the apo form and nucleotide bound FtsY structures. The presence of three subsequent glycine residues in the P-loop is unique for cpFtsY and seems to allow for a conformation of the P-loop and the G3 motif, which in other FtsY structures is only

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induced by the formation of the SRP54/FtsY heterodimer in the targeting complex. Therefore, the higher intrinsic flexibility of the P-loop in cpFtsY may contribute to the higher nucleotide binding affinity and thereby enhance the rate of complex formation with cpSRP54 as observed [39]. The absence of the SRP RNA in the chloroplast might be compensated in part by the observed modification of the P-loop in cpFtsY. However, in order to understand the complete story of how efficient protein translocation is achieved in the absence of the SRP RNA, more biochemical and structural data on complexes of cpSRP with cpFtsY and with substrate proteins are needed. Acknowledgements: We thank M. Groves and U. Du¨rrwang for cloning of cpFtsY, G. Stjepanovic for support with the lipid binding assay, and I. Tews for assistance during data collection at the ESRF. We acknowledge access to the beamlines ID14 and ID23 at the ESRF (Grenoble, France) and thank the beamline scientists for excellent support. This work was supported by the DFG (SFB638).

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.febslet.2007. 11.024.

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