Nature Structural & Molecular Biology
11, 1049 - 1053 (2004)
Published online: 2 November 2004; | doi:10.1038/nsmb853
SRP meets the ribosomeKlemens Wild1, 3, Mario Halic2, 3, Irmgard Sinning1
& Roland Beckmann21 Biochemie-Zentrum der Universität Heidelberg (BZH), Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany. 2 Insitute of Biochemistry, Charité, University Medical School Berlin, Monbijoustrasse 2, D-10117 Berlin, Germany. 3 These authors contributed equally to this work.
Correspondence should be addressed to Roland Beckmann roland.beckmann@charite.deCotranslational targeting directly couples synthesis of proteins to their translocation across or insertion into membranes. The signal recognition particle (SRP) and its membrane-bound receptor facilitate the targeting of the translation machinery, the ribosome, via recognition of a signal sequence in the nascent peptide chain. By combining structures of free and ribosome-bound SRP we derive a structural model describing the dynamic nature of SRP when it meets the ribosome.Cotranslational transport of secretory and membrane proteins depends on the SRP system, which consists of a cytosolic ribonucleoprotein particle (SRP) and its membrane-bound receptor (SR). Since the discovery of SRP in the early 1980s, research in this field has focused on the functional characterization of the SRP system, which is described by the SRP cycle1,
2,
3 (Fig. 1a). Newly synthesized proteins destined for secretion or membrane insertion carry a hydrophobic signal sequence at their N terminus. SRP interacts with the signal sequence as soon as it emerges from the ribosomal polypeptide exit tunnel (step I). In eukaryotes, peptide elongation is retarded upon binding of SRP to the ribosome nascent chain complex (RNC). Subsequently, the SRP−RNC complex is targeted to the endoplasmic reticulum (ER) membrane by the interaction with the SR (step II). GTP binding to SRP and SR has been shown to be a prerequisite for formation of the SRP−SR complex. The RNC is then transferred to the protein-conducting channel in the membrane (the translocon)3,
4 (step III), after which, as a result of GTP hydrolysis in SRP and SR, the SRP−SR complex dissociates (step IV), leaving the RNC bound to the translocon. The GTP dependency is thought to enable correct targeting by coordinating the presence of a signal sequence on the ribosome with the availability of a translocon in the membrane3,
4.
 | | Figure 1. Cotranslational targeting by the SRP system. | | |  | (a) Schematic overview of cotranslational targeting of proteins destined for secretion or membrane insertion (SRP cycle). SRP interacts with the signal sequence as soon as it emerges from the ribosomal polypeptide exit tunnel (step I). In eukaryotes, peptide elongation is retarded upon formation of the SRP−RNC (RNC, ribosome nascent chain complex) complex, and the complex is targeted to the ER membrane by the interaction with the SR (step II). GTP binding to SRP and SR have been shown to be a prerequisite for formation of the SRP−SR complex. The RNC is then transferred to the protein-conducting channel in the membrane (the translocon) (step III) and, triggered by GTP hydrolysis in SRP and SR, the SRP−SR complex dissociates (step IV). The coloring for the SRP−RNC complex is the same as defined in b. (b) Schematic overview of the mammalian SRP bound to the signal sequence carrying 80S ribosome (RNC) based on a cryo-EM structure17. The SRP core (SRP54 and SRP RNA helix 8) as part of the S domain is positioned near the exit of the protein tunnel of the large ribosomal subunit. The 40S and 60S ribosomal subunits are yellow and gray, respectively. The SRP RNA is red and the SRP proteins are labeled as follows: SRP54NG, turquoise; SRP54M, dark blue; signal sequence, green; SRP19 and SRP68/72, pink; SRP9, turquoise; SRP14, dark blue. Peptidyl-tRNA (p-tRNA) between the ribosomal subunits and the nascent chain in the exit tunnel are labeled.
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What is the structural basis for the activities described above? The SRP of eukaryotes, archaea and some Gram-positive bacteria consists of two distinct domains. The S domain mediates signal sequence binding and SR docking. The less well conserved Alu domain mediates retardation of peptide elongation (Fig. 1b). SRP in higher eukaryotes consists of six proteins (SRP9, SRP14, SRP19, SRP54, SRP68 and SRP72) assembled on the SRP RNA. However, the universally conserved SRP core, as part of the S domain, comprises only the protein SRP54 (Ffh for 'fifty-four-homolog' in bacteria) bound to helix 8 (domain IV in bacteria) of the SRP RNA. SRP54 is a multidomain protein with an N-terminal four-helix bundle (N domain), a central GTPase domain (G domain) and a C-terminal methionine-rich domain (M domain). It combines the two key functions of SRP: signal sequence binding (M domain)5,
6 and GTP-dependent SR interaction (NG domain)2,
3,
4. The M domain (SRP54M) attaches SRP54 to helix 8 of SRP RNA7 and is thought to accommodate hydrophobic signal sequences in a hydrophobic groove closed by a finger loop3,
4. The N-terminal domain (SRP54N) is associated with the G domain, where it may play a regulatory role8,
9. The GTPase (SRP54G) has a unique insertion box (I box, IBD) and constitutes a subclass (SRP GTPases) in the superfamily of small G proteins. Owing to their close packing, the two domains are treated as one unit (SRP54NG). In contrast to other GTPases, the nucleotide affinity of SRP54 is low10 and the protein is stable in an empty conformation as deduced from the availability of several apo SRP54 crystal structures5,
6,
8,
11,
12,
13. In the empty state, SRP54 can readily interact with a signal sequence14; however, it needs to bind GTP before interacting with its receptor. Interestingly, SR also contains an NG domain, which interacts in the GTP-bound state with SRP54NG during step II of the SRP cycle.
Despite much functional data and a growing number of SRP substructures2,
15, several fundamental questions remain unresolved: for example, how does SRP recognize and bind a signal sequence on the ribosome? How is signal sequence binding coupled to GTP binding, a prerequisite for docking of SRP on its receptor SR? And how is release of the signal sequence from SRP and transfer of the RNC to the translocon coordinated? With regard to the first questions, structure-based concepts are emerging.
Visualization of functional states of SRP
The structures of the individual domains of SRP54 from various species are known15,
16, and recently, the spatial arrangement of these domains was determined in two different functional states of SRP using X-ray13 and cryo-EM data17. The X-ray structure of SRP54 from Sulfolobus solfataricus was obtained with and without SRP RNA helix 8, both of which exhibited a very similar domain arrangement. Notably, these structures revealed the domain linkage between SRP54M and SRP54NG and the importance of this linker region for the dynamic changes between the domains13. Although the crystal packing might have influenced the arrangement of the domains, it is very likely that the overall domain arrangement indeed represents the conformation of SRP54 in the free state, as these structures were obtained in different crystal forms with different crystal packing. At the same time, the differences between these structures reflect the intrinsic flexibility of the SRP core in the free state. A cryo-EM structure17 shows the complete mammalian SRP after binding to the RNC and represents the ribosome-bound state of SRP. Here, the SRP core is part of the active targeting complex after binding both the signal sequence and the large subunit of the ribosome near the peptide exit tunnel. Although the resolution of the cryo-EM map is limited to 12 Å, docking of molecular models into the EM map allows the structural interpretation at greater detail17. Comparing the structures of SRP in the free and the ribosome-bound states allows identification of changes in the SRP core associated with the transition between these states (step I of the SRP cycle). In addition, X-ray structures of the interacting NG domains of bacterial SRP and SR provide a glimpse into the docking of the RNC−SRP complex (step II) at the target membrane18,
19.
The X-ray structure of the free SRP core13 shows SRP54 as an L-shaped molecule, with SRP54NG as the longer arm of the L, which runs parallel with helix 8 of the SRP RNA. This is referred to as the compact conformation of SRP54 (Fig. 2). SRP54NG is close but does not directly contact the SRP RNA in the SRP core structure. However, biochemical data20 and differences within the SRP54 structures13 with and without RNA indicate that such an interaction is likely to exist in the free state. SRP54NG and SRP54M are connected by a flexible linker region (Fig. 2a, orange). SRP54M itself can be divided into a flexible N-terminal and a rigid C-terminal part. The C-terminal part (MC) binds to helix 8 of SRP RNA as a rigid body and provides a stable platform for the hydrophobic groove, which is proposed to bind the signal sequence. The flexible N-terminal part (MN) includes a linker region (see below) followed by a proline-kinked helix ( M1 and M1b) and the finger loop, which in the absence of a signal sequence shields the hydrophobic groove from the aqueous solvent. The comparison with the SRP54M structure from Thermus aquaticus21 suggests that several hinge points in SRP54MN would be sufficient for anchoring to SRP54MC and for adjusting the hydrophobic groove to accommodate the signal sequence13.
| | Figure 2. Conformational changes of the SRP core. | | |  | (a,b) Schematic view of the conformational changes of the SRP core (SRP54 and SRP RNA helix 8) when switching from the free state derived from the X-ray structure13 (a) to the ribosome-bound state derived from the cryo-EM structure17 (b). The ribosome engagement causes a 50° rotation and a 50 Å shift of the SRP54NG domain as indicated. The linker region, which connects the SRP54NG with the SRP54M domain, is orange and the linker helix rotates  90°. The signal sequence is positioned in the hydrophobic groove of SRP54MN, which directly resides on the exit site and adjusts accordingly (labeled): helix M1 moves towards the groove, and helix M1b and the finger loop are shifted out of the groove and form a lid over the helical signal sequence. The positively charged N terminus of the signal sequence is in close proximity to the negatively charged SRP RNA next to the tip of helix 8, as predicted previously7. The color code is the same as in Figure 1, except that the linker region is orange (RNA, red ribbon; SRP54NG, turquoise; SRP54MN, blue; MC, dark blue; signal sequence, green).
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Compared with the structure of the free state of SRP, the cryo-EM structure of SRP bound to an active 80S ribosome reveals a strikingly different conformation of the SRP core, referred to as the open conformation (Fig. 2b). On the ribosome, SRP54NG is rotated by 50° and shifted 50 Å away from the aligned position with SRP RNA helix 8, and is found at the very tip of the SRP instead. The two distal loops of SRP54N interact with the two ribosomal proteins L23p and L29p in agreement with previous crosslink data22,
23,
24 (Fig. 3). As the remaining part of the SRP core is fixed on the ribosome, the linker region between SRP54NG and SRP54M has to accommodate the large conformational changes. Although this is not resolved at the present resolution, in the open conformation of SRP54 the signal sequence can be accommodated in the hydrophobic groove17 in an orientation that positions the positively charged N terminus of the signal sequence near the backbone of the SRP RNA helix 8, as proposed earlier7. This open conformation hardly allows for the direct participation of the SRP54NG domain in signal sequence interaction, as previously suggested25.
| | Figure 3. Model for the first steps of the SRP cycle. | | |  | A detailed scheme of the conformational events of the SRP core during the first steps of cotranslational targeting can be modeled as follows. (i) In the free state, the SRP core adopts a compact conformation. A distal loop of SRP54N contacts SRP54MN, which later on is involved in signal sequence binding. SRP54G interacts with helix 8 of the SRP RNA and the nucleotide affinity is low. (ii) In the sampling mode, SRP interacts with low affinity and transiently with translating ribosomes in order to scan for signal sequences. The contact involves at least proteins L23p and L29p of the large ribosomal region (L23p) of the large ribosomal subunit and the distal loops of SRP54N. Ribosome binding induces a change in the SRP core toward a more open conformation, which probably leads to a rotation around the linker region and renders SRP capable for signal sequence recognition and GTP binding. (iii) In the targeting mode, SRP interacts with high affinity with the RNC (including proteins L23p and L29p and ribosomal RNA helix 24, H24). Signal sequence binds to the hydrophobic groove of SRP54M and the binding is transmitted to SRP54NG by interdomain communication. As a result, the RNC-bound SRP core changes to the fully open conformation by adjusting the flexible linker region between SRP54NG and SRP54M. The SRP−RNC targeting complex induces the high-affinity GTP binding to SRP54NG, which is now primed for the interaction with the SRP receptor. (iv) The successive docking of the SRP−RNC complex to the translocon via SR is structurally unresolved. However, the interaction of the SRP−RNC complex with SR may lead to the twin-like arrangement of the SRP54 and SR NG domains, resulting in a relocalization of the SRP54 NG domain. Color code is as in Figures 1 and 2. Bold 'G', GTP.
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A flexible domain linkage between SRP54NG and SRP54M
The flexible linker region and two hydrophobic contacts within SRP54 seem to be important for its dynamic behavior and interdomain communication (Figs. 2 and 3). In the free state, a peripheral loop of SRP54N establishes a contact with M1b of SRP54MN. This is the only direct contact between SRP54N and the part of the M domain that binds the signal sequence. It contributes to the stabilization of the compact conformation in the free state of SRP and is lost in the open conformation of the ribosome-bound state of SRP54 (ref. 17). The linker region itself maintains a contact to SRP54MN in both the free and the ribosome-bound state, and undergoes the largest conformational changes between the two states. The linker region consists of three parts: a highly conserved 'LGMGD' sequence fingerprint in a loop connecting the G domain with the linker, an 18-residue long linker helix (termed ML) and a loop of variable size that ends with a conserved leucine residue13. The first loop (with two conserved glycines in the LGMGD motif permitting rotational freedom) must change conformation upon ribosome binding while the linker helix ML seems to persist as a rigid body. However, the relative orientation of the linker helix changes markedly with respect to both SRP54NG and SRP54M (Fig. 2). A rodlike density in the EM map spanning the distance from SRP54NG to SRP54M, which does not account for any other parts of the separated domains, probably represents this helix. The linker helix mainly interacts via hydrophobic interactions with helix M1 and has previously been proposed to form a greasy slide upon which the interface can be smoothly adjusted13. Similar to the LGMGD loop, the second loop, which connects the linker helix ML with SRP54MN, has to change its conformation to bring the linker helix in the correct position. This loop may also be involved in signal sequence binding as it is very close to the hydrophobic groove and often contains methionine residues. The linker region ends with a deeply buried conserved leucine (Leu329 in Sulfolobus solfataricus), which serves as an anchor point at the N terminus of helix M1 of SRP54M13.
When the interaction between the SRP54 N domain and SRP54M is lost in the ribosome-bound state, the linker region seems to provide the physical link between the signal sequence−binding domain (SRP54M) and the GTPase domain (SRP54NG). Therefore, it is easy to imagine that the change in the position of the N domain with simultaneous conformational change of the linker region communicates the signal sequence binding to the GTP-binding site (see below).
Model of the first steps of the SRP cycle
On the basis of the available structural and biochemical data, a more detailed mechanistic model can now be presented to describe the conformational changes in the SRP core during the first steps of the SRP cycle (Fig. 3).
(i) The free SRP core in solution probably adopts a compact conformation similar to that observed in the recent X-ray structure. This conformation might be stabilized by an interaction of SRP54G with the SRP RNA13. A direct contact between the distal loop of SRP54N and helix M1b of SRP54M is present and the finger loop of SRP54M covers the hydrophobic groove to protect it from aqueous solvent. In this free state, SRP is incapable of signal sequence binding26 and the GTP affinity of SRP54 is low.
(ii) In the sampling mode, SRP binds with low affinity to the ribosome and probes it for the presence of a signal sequence27. Although there are no structural data available for this mode, biochemical crosslinks indicate that the signal sequence-independent interaction is similar to that observed in the cryo-EM structure17. The ribosomal crosslink pattern of the mammalian Alu domain28 as well as that of SRP54 in mammalian23 or Escherichia coli SRP22 do not change extensively between sampling and targeting mode. Therefore, the rotation around the linker region might already take place and the hydrophobic contact between the distal loop of SRP54N and the M domain is lost owing to the interaction of SRP54N with L23p and L29p17,
22,
23,
24. This might prime SRP for binding the signal sequence on the ribosome. Notably, signal sequences of RNCs could also be crosslinked24 to the ribosomal protein L23p, making this site an ideal target for SRP54M probing for the presence of the signal sequence. The functioning of L23p as a transitory binding site for signal sequences would be in agreement with a constant affinity of SRP for signal sequences independent of chain length29. However, without a signal sequence available at the tunnel exit, the conformation of the SRP core allows only transient binding.
(iii) In the targeting mode, SRP binds with high affinity to the RNC, adopting the conformation observed in the cryo-EM structure17. The SRP core is positioned with the SRP54N and the SRP54M domains on opposite sides of the tunnel exit while the hydrophobic groove with the bound signal sequence sits directly on top of the exit (Figs. 1b and 3). It remains unclear whether the signal sequence shapes its own binding groove, or whether the conformational changes in SRP54 open the hydrophobic groove. However, a hydrophobic groove exposed to the aqueous solvent without substrate would be energetically unfavorable, suggesting an induced-fit mechanism for signal sequence accommodation. The signal sequence locks the ribosome-bound SRP54 in the open conformation, as seen in the EM structure17, and increases the affinities of SRP for both the ribosome27,
29 and GTP10. As a result, the SRP core is positioned on the ribosome in a way that allows the Alu domain to induce elongation arrest by binding in the orientation observed by cryo-EM.
(iv) Docking of the targeting complex to the SRP receptor is likely to involve the interaction of the two NG domains and additionally a direct interaction of SR with the ribosome30,
31,
32. The orientation of SRP54 on the ribosome is altered after SR binding, as shown by crosslinking studies23. The formation of the RNC−SRP−SR complex will probably move the SRP54NG domain away from the exit tunnel, which in turn would enable the transfer of the RNC to the translocon. Notably, in the X-ray structures of the interacting NG domains of bacterial SRP and SR18,
19, the N-terminal helix of both N domains is displaced out of the four-helix bundle and the C-terminal helix of the G domain (adjacent to the LGMGD linker) is shifted toward the NG interface. This might also be relevant for rearranging the SRP54NG domain on the ribosome as well as the NG domain of SR relative to the membrane.
Regulation of GTP affinity
Signal sequence binding to SRP54M must be followed by GTP binding to SRP54G in order to target the RNC to the translocation machinery at the membrane. SRP and the SRP receptor will form a complex only when the GTPases in both proteins are in the GTP-bound state. The X-ray structures of the interacting NG domains of bacterial SRP and SR in the presence of a nonhydrolyzable GTP analog18,
19 show the two nucleotides buried in the protein interface. This finding argues strongly in favor of GTP binding as a prerequisite for subsequent formation of complex.
Different models exist for the GTPase cycle during targeting31,
33,
34. One model is based on evidence that the binding of SRP to the RNC stimulates GTP binding to SRP54 (ref. 10). We suggest that the conformational change induced by the ribosome, and in particular the reorientation of the SRP54NG domain, functions as the major trigger to increase GTP affinity. GTP affinity is probably already increased in the sampling mode because even in the presence of ribosomes, without a signal sequence, the affinity of SRP54 for GTP is ten times higher than in free SRP10. As discussed previously, a tilt of SRP54N with respect to SRP54G seems to be responsible for a reorganization of GTP-binding determinants in SRP54G (for a more detailed model see refs. 1,13). The rotation of SRP54N seems to be possible only in the ribosome-bound state of SRP54, where it can be communicated via the NG interface to the nucleotide-binding pocket of SRP54G. However, in the absence of a signal sequence, SRP binding, and therefore GTP binding, remains transient. Only in the active SRP−RNC targeting complex is GTP binding to SRP54 stabilized such that targeting can proceed by docking to the SRP receptor.
The next steps
Although the structural characterization of the first step of the SRP cycle provides insight into the mechanism of signal sequence recognition, many questions remain to be answered: Does the SRP54N domain in the ribosome-bound state indeed rotate with respect to the G domain, and what is the exact conformation of the linker region? Is the signal sequence indeed bound to the hydrophobic groove of SRP54M as proposed and what is the significance of the newly defined dynamic regions of the SRP core in different functional states? Mutational and biochemical analysis as well as additional high- resolution X-ray and cryo-EM structures should provide the missing details of what happens when SRP meets the ribosome.
What happens after the SRP cycle? Additional questions arise. The comparison of the SRP−RNC structure17 with the RNC−translocon complex35 shows that both the SRP and the translocon bind near the exit of the ribosomal protein tunnel in a mutually exclusive way. It is not clear how the transfer of the signal sequence and the entire RNC from SRP to the translocon is facilitated. Visualization of an SRP−RNC−SR intermediate, possibly even in the presence of a translocon, will be required to build a more complete model of the SRP cycle.
Received 12 May 2004; Accepted 4 October 2004; Published online: 2 November 2004.
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Acknowledgments
We acknowledge support by European Union (EU) network grant QLK-CT2000-00082 and by the Deutsche Forschungsgemeinschaft (SFB 638) to I.S., by the VolkswagenStiftung, the EU and Senatsverwaltung für Wissenschaft, Forschung und Kultur Berlin, in the context of the Ultra-Structure Network and the Deutsche Forschungsgemeinschaft (SFB 449) to R.B.
Competing interests statement:
The authors declare that they have no competing financial interests. |
