Domain Interactions in E. coli SRP: Stabilization of M Domain by RNA Is Required for Effective Signal Sequence Modulation of NG Domain

Molecular Cell, Vol. 1, 79–87, December, 1997, Copyright 1997 by Cell Press

Domain Interactions in E. coli SRP: Stabilization of M Domain by RNA Is Required for Effective Signal Sequence Modulation of NG Domain Ning Zheng*† and Lila M. Gierasch†‡ * Molecular Biophysics Program University of Texas Southwestern Medical Center at Dallas Dallas, Texas 75235 † Department of Chemistry University of Massachusetts at Amherst Amherst, Massachusetts 01003

Summary The E. coli protein, Ffh, binds to 4.5S RNA through its M domain to form the signal recognition particle (SRP). The other domain of Ffh (NG) is a GTPase, which binds and is coordinately regulated by its receptor, FtsY. We find that the helical M domain is inherently flexible. Binding of 4.5S RNA to Ffh stabilizes the M domain yet has little apparent effect on the binding of signal peptides. However, in the absence of the RNA, signal peptide binding results in a global destabilization of Ffh, which is prevented by binding of 4.5S RNA. Signal peptide binding to isolated NG domain also causes a pronounced destabilization, implicating the NG domain in direct recognition of signal peptide. Introduction Significant progress has been made in the elucidation of the functions of the signal recognition particle (SRP) in protein targeting and translocation processes of both eukaryotes and prokaryotes (Walter and Johnson, 1994; Lu¨tcke, 1995), yet the structural features enabling SRP to play its biological roles are only beginning to emerge. In mammalian cells, SRP has long been believed to be the major cellular component that mediates the targeting of presecretory proteins to the endoplasmic reticulum membrane. In E. coli, extensive genetic studies failed to identify SRP in protein translocation pathways, yet recent studies provide increasing evidence suggesting that SRP is involved in the E. coli protein export process (Luirink et al., 1992; Phillips and Silhavy, 1992; Powers and Walter, 1997; Ulbrandt et al., 1997). Identification of SRP in many other organisms has further confirmed its universal importance (compiled by Larsen and Zwieb, 1996). Mammalian SRP has six protein subunits and one RNA backbone, 7S RNA. The most crucial function of SRP, recognizing signal sequences on nascent polypeptides, has been assigned to the methionine-rich M domain of the 54 kDa protein subunit (SRP54) (Zopf et al., 1990; Lu¨tcke et al, 1992). This same domain also contains the binding site for 7S RNA, which is proposed to act as a backbone to hold together all the protein subunits (Romisch et al., 1990; Zopf et al., 1990). Structurally, the 7S RNA can be divided into four domains, labeled domain I to domain IV. Domain IV has a unique ‡ To whom correspondence should be addressed.

predicted secondary/tertiary structure (Zwieb et al., 1996), containing one tetra loop and two bulges, and is believed to bind specifically to the SRP54 M domain. The E. coli SRP, on the other hand, consists of only two components, an SRP54 homolog (Ffh) and 4.5S RNA. Ffh has the same domain structure as mammalian SRP54 (Bernstein et al., 1989; Romisch et al., 1989); the 4.5S RNA is shorter than 7S RNA but has a structural motif similar to the 7S RNA domain IV region (Poritz et al., 1990). Interactions between Ffh and 4.5S RNA are apparently analogous to those between mammalian SRP54 and 7S RNA, since the components of these complexes are interchangeable (Poritz et al., 1990; Bernstein et al., 1993). Thus, E. coli SRP provides an ideal model system for structural characterization where large quantities of materials are needed. A central puzzle is how SRP54 and its homologs specifically bind signal sequences, among which no sequence homology exists. Based on secondary structure prediction through sequence analysis, Bernstein et al. (1989) proposed that the M domains of mammalian SRP54 and Ffh bind signal sequences with so-called “methionine bristles.” Confirmation of this model awaits structural analysis of the M domain. Indeed, several results have raised the possibility that signal sequence binding is not restricted to the M domain (Lu¨tcke et al., 1992; Zopf et al., 1993; Newitt and Bernstein, 1997). After the binding of signal sequence, the next critical function of SRP is to interact with its receptor on the membrane. This step is accomplished through the N-terminal NG domain found in both SRP54 (Ffh) and the SRP receptor (named FtsY in bacteria) (Bernstein et al., 1989; Romisch et al., 1989). The NG domain is a GTPase. Binding and mutually regulated GTP hydrolysis of SRP54 (Ffh) and SRP receptor (FtsY) provide a mechanism to release the nascent chain from SRP after it reaches the membrane and subsequently to release SRP from its receptor (Walter and Johnson, 1994; Bacher et al., 1996). The crystal structures of the NG domains of both Ffh (Freymann et al., 1997) and FtsY (Montoya et al., 1997) have recently been solved, revealing a Ras-like GTPase fold packed against a four-helix bundle (the N domain). The structural relationship between the M domain and the NG domain of either SRP54 or Ffh remains unclear. Cross-talk between these domains is suggested by previous studies showing that synthetic signal peptides can inhibit the GTPase activity of both Ffh and mammalian SRP54 in complexes with their respective RNAs (Miller et al., 1993, 1994). The fact that one can see this modulation with a simple system (i.e., no ribosomes, no nascent chains, stripped-down SRP) invites structural studies to explore how the binding of signal sequence modulates the GTPase activity of the NG domain. What is the role of the SRP RNA? The multisubunit composition of the mammalian SRP leads to a logical need for a scaffold to hold the components of the particle together. By contrast, the E. coli complex consists of only the SRP54 homolog and a segment of RNA bearing the domain IV region. The fact that the domain IV

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Results

Figure 1. The Ffh M Domain Has a Significantly Higher Protease Susceptibility than the NG Domain (A) Time course of V8 digestion of Ffh. The digestion reaction (Ffh:V8 5 100:1 w/w), as described in the Experimental Procedures, was stopped at the indicated time point and analyzed on SDSPAGE. Proteolytically generated M domain was digested at a much greater rate than the NG domain. (B) Overexpressed His-tagged Ffh M domain was treated with V8 protease (Ffh:V8 5 100:1 w/w) at room temperature under the same conditions as (A).

part of the SRP RNA is evolutionarily conserved suggests that it is indispensable. Its role may be more than that of a structural linker: not only is 4.5S RNA required for the mutual stimulation of GTP hydrolysis by Ffh and its receptor (FtsY) (Miller et al., 1994), but it is also essential for the in vivo stability of Ffh (Jensen and Pedersen, 1994). In order to understand the structure–function relationship of E. coli SRP, we have initiated structural characterization of the Ffh M domain and biochemical studies of the interaction between Ffh, signal peptides, and 4.5S RNA. We find that, distinct from the NG domain, the Ffh M domain has a highly helical yet flexible “moltenglobule”-like structure. Specific binding of signal peptides causes a dramatic and global destabilization of the Ffh protein. Intriguingly, and counter to expectations from past work, this effect is also seen when signal peptides bind to isolated NG domains. Binding of 4.5S RNA inhibits this catastrophic effect of signal peptide binding to Ffh by locally stabilizing the M domain. These results provide insight into the interdomain functional coupling of SRP and the importance of 4.5S RNA beyond a molecular scaffold in the cellular apparatus. Moreover, the fact that the functional signal peptide-induced destabilizing effect can be observed for the isolated NG domain suggests that the NG domain is directly involved in signal peptide binding.

The Ffh M Domain Is Proteolytically Labile and Has Features of an a-Helical “Molten Globule” State In order to study the structural characteristics and ligand binding of the Ffh M domain, we prepared the M domain by both limited V8 protease digestion of the Ffh protein and bacterial overexpression of a His-tagged polypeptide corresponding to the predicted M domain. Monitoring the time course of the Ffh digestion by SDS-PAGE, we found that the M domain is significantly more protease susceptible than the NG domain. As Figure 1A shows, the protease first rapidly cut the protein into two transiently stable domains, as observed in mammalian SRP54 (Romisch et al., 1990; Zopf et al., 1990). Yet, as digestion proceeded, the proteolytically generated M domain was completely digested whereas the bulk of the NG domain remained intact for more than 2.5 hr. Since both domains have multiple glutamate residues dispersed throughout their sequences, their susceptibility to V8 should be comparable in the absence of stable structure. The contrasting behavior of the M domain and NG domain in V8 protease digestion suggests that the M domain has a much more unstable nature than does the NG domain. This is further confirmed by the treatment of the overexpressed M domain alone with V8 protease, which was also rapidly digested (Figure 1B). Despite its high protease susceptibility, the expressed M domain is functional. We determined its 4.5S RNAbinding activity with both a filter binding assay (Figure 2A) and DEAE chromatography (data not shown). The basic M domain exhibits the same RNA-binding affinity as that of native Ffh protein (Walter and Johnson, 1994), indicating that it is in a biologically active form and that the RNA-binding capability of the intact Ffh can be attributed to the M domain. Previous secondary structure analysis has predicted that the Ffh M domain consists of three helices connected by loops. A secondary structure measurement by circular dichroism (CD) verified that the protein is indeed rich in a helix (Figure 2B). The high secondary structure content and protease susceptibility paint a picture of a poorly packed, “molten globule”-like structure for the Ffh M domain. This conclusion is supported by 1H and 15N NMR analysis of the M domain, which yielded spectra of low resolution and poor proton dispersion (data not shown). Moreover, thermal denaturation of the expressed M domain, monitored by CD at 222 nm, where the ellipticity of a helical

Figure 2. Functional Ffh M Domain Has “Molten-Globule”–like Features (A) Overexpressed Ffh and the Ffh M domain have the same 4.5S RNA binding affinity (z5 nM) measured by a filter binding assay. (B) The CD spectrum of the isolated Ffh M domain shows a predominantly a-helical structure. The ellipticity minima at 208 nm and 222 nm are characteristic of a helix. (C) Thermal denaturation of the Ffh M domain was monitored by CD at 222 nm. The unfolding of the Ffh M domain represents a noncooperative process in contrast to the transition observed in intact Ffh (see Figure 5F).

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polypeptide reaches a minimum (Figure 2B), revealed a noncooperative melting transition (Figure 2C). These results are in contrast to what is expected for a compactly folded protein with a well-defined tertiary structure (Pace et al., 1989). The nearly linear ellipticity decrease with increasing temperature indicates that the helices of the structural domain are not well packed. Binding of the Ffh M Domain to 4.5S RNA Leads to a Stable, Cooperatively Folded Domain Given the affinity of the interaction between Ffh and 4.5S RNA and the relative amount of both molecules in bacterial cells (Jensen and Pedersen, 1994), Ffh should always be in a complex with 4.5S RNA in vivo. Thus, the flexibility of the M domain that we observed could be a result of the absence of 4.5S RNA. Using purified 4.5S RNA, we repeated the protease digestion assay to test the behavior of the M domain in the presence of its physiological partner. Whereas 4.5S RNA did not prevent the initial cleavage of the Ffh protein into two domains by V8 protease, it completely prevents the protease from further digesting the M domain (Figure 3A). In the presence of the 4.5S RNA, the M domain is as stable as the NG domain during the whole course of protease digestion (2 hr). This result strongly suggests that, upon binding to 4.5S RNA, the flexibility of the M domain is greatly reduced. The presence of 4.5S RNA also decreased the rate of cleavage of Ffh into two domains. This is probably due to the bulky size of the RNA, which can cause some steric hindrance to the protease access. However, this potential steric effect cannot account for the protease resistance of the M domain bound to 4.5S RNA since the digestion of M domain was not simply retarded, but was completely abrogated, even when incubation time reached 6 hr (data not shown). Thermal denaturation monitored by CD provides further evidence that the binding of RNA leads to a cooperative folding of the M domain. In order to carry out these spectroscopic studies, we replaced the complete 4.5S RNA molecule with a 41-base oligonucleotide encompassing the domain IV region (4.5S RNA dIV). As expected, 4.5S RNA dIV binds to the Ffh protein and has the same stabilizing effect as 4.5S RNA on the M domain in the protease digestion assay (see Figure 3B). To compare the unfolding of the free M domain with M domain in complex with 4.5S RNA dIV, we took advantage of the fact that the contribution of the RNA to the CD spectrum is negligible at 222nm and does not change during thermal denaturation (Figure 3D). The ellipticity at this wavelength reflects only the contribution of the highly helical protein. As shown in Figure 3C, the unfolding curve of the M domain becomes significantly more cooperative upon forming a complex with 4.5S RNA dIV, which is consistent with what we have observed in the protease digestion assay. Signal Peptide Binding to Ffh Causes Significant Structural Changes in the NG Domain The observed effect of the 4.5S RNA on the proposed peptide binding domain of Ffh raises a question about its potential effect on signal sequence recognition. To

Figure 3. 4.5S RNA and Its Domain IV Fragment Stabilize the Ffh M Domain (A) Ffh was incubated with equimolar amount of 4.5S RNA at room temperature for 10 min and then treated with V8 protease as described in methods. Proteolytically generated M domain stayed intact as did the NG domain. (B) V8 protease digestion of Ffh, with or without 4.5S RNA dIV, indicates that the domain IV region of 4.5S RNA is sufficient for M domain stabilization. (C) Comparison of the thermal denaturation curves of the Ffh M domain alone and the Ffh M domain in complex with 4.5S RNA dIV (bottom) shows a clear transition in the presence of the RNA. The thermal denaturation profile was obtained by monitoring the CD signal of the samples at 222 nm. (D) Thermal denaturation of 4.5S RNA dIV alone monitored at 222 nm (top). The near zero ellipticity of RNA at 222 nm did not change as temperature increased. CD spectra of 4.5S RNA dIV alone and Ffh M domain/4.5S RNA dIV complex. As 4.5S RNA shows near zero ellipticity at 222 nm, the helical Ffh M domain contributes a characteristic negative peak.

explore this, we used an approach previously reported by Miller et al. (1994) and monitored the Ffh GTPase inhibition by the synthetic signal peptides. We used a LamB signal peptide analog (Table 1), KRRnoW, which has enhanced aqueous solubility (for the rationale behind the introduction of KRR sequence into LamB signal peptide analogs, (see Wang et al., 1993, and Table 1). As Figure 4A shows, the LamB signal peptide causes half inhibition of the GTP hydrolysis at z1 mM, whereas the nonfunctional analog KRR13W14D shows little inhibition. Adding the 4.5S RNA does not alter the inhibitory effect of signal peptides on the Ffh GTPase activity (Figure 4B). Our data in the presence of RNA are entirely consistent with those reported earlier by Miller et al. (1994), who have seen a similar effect of signal peptides on the stimulated GTPase of Ffh/FtsY/4.5S RNA. Note the small increase in basal GTPase activity of Ffh upon RNA binding to the M domain (also observed by Miller

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Table 1. Synthetic Signal Peptide Analogs Peptide

Sequence

LamB KRRnoW KRR13W14D OmpA K 5W(AL)10

MMITLRKLPLAVAVAAGVMSAQAMA MMITLRKRRKLPLAVAVAAGVMSAQAMA MMITLRKRRKLPLAVWDAAGVMSAQAMA MKKTAIAIAVALAGFATVAQA/APKD KKKKKWALALALALALALALALALAL

Three positively charged amino acids have been introduced into the N-terminal part of LamB signal peptide to increase solubility. This modification is justified, as a comparable mutation in which two charges (RK) introduced following the Leu in position 5 of the wild-type sequence led to a fully functional signal sequence (Carlson, 1993). In that case, two additional positive charges were inserted at the same site. Since introduction of a charged residue in the hydrophobic core leads to loss of signal sequence function (Stader et al., 1986; Gierasch, 1989), we use KRR13W14D as a nonfunctional control.

et al., 1994), which indicates that RNA mediates communication between the two domains even though it is not essential for signal sequence recognition. GTPase modulation by signal sequences requires interdomain communication if signal sequence binding is confined to the M domain. This modulation may be facilitated by a stabilization of the M domain upon signal sequence binding, as observed upon binding of its RNA ligand. We included a functional signal peptide analog in the protease digestion assay to monitor its influence on the stability of Ffh and its M domain. Surprisingly, as free Ffh (1 mM) became gradually cleaved into two domains, the presence of 100 mM KRRnoW caused a profound increase in proteolytic susceptibility of Ffh— the protein was almost completely digested by the first time point we checked (12 min; Figure 5A) (without generating two transiently stable domains). The concentration range required for the enhanced proteolytic lability (Figure 5B) is thus comparable to that for half maximal GTPase inhibition (see above). As a control experiment, we replaced KRRnoW-LamB with a nonfunctional LamB signal peptide analog, KRR13W14D. In contrast to KRRnoW, KRR13W14D showed little effect on the proteolytic susceptibility of Ffh (Figure 5C). The destabilization caused by the LamB signal peptide is not specific to this sequence, as another signal peptide, that of OmpA, has the same effect (Figure 5D), as does an idealized longer hydrophobic peptide, K5W(AL)10 (data not shown, but see Figure 5H). The latter peptide is an appropriate transmembrane sequence model for the inner membrane proteins whose biogenesis seems most dependent on Ffh (Ulbrandt et al.,1997). Monitoring thermal melting of Ffh in the presence and absence of signal peptide shows that the enhanced proteolytic susceptibility arises from global destabilization of structure and not solely from exposure of selected new proteolytic sites. As shown in Figure 5E, the CD spectrum of Ffh in the presence of signal peptide shows significant change relative to the sum of the spectra of protein and peptide. It is not possible to attribute the observed change to a specific conformational change in the protein, as the peptide might be also undergoing conformational rearrangement and both of

Figure 4. Inhibition of Ffh GTPase Activity by Synthetic Signal Peptides (A) GTP hydrolysis by Ffh was monitored following the previously published methods (Miller et al., 1994). The Ffh concentration in the 100 ml reaction was 100 nM. No 4.5S RNA was added. KRRnoW shows similar inhibitory activity as wild-type LamB peptide whereas nonfunctional KRR13W14D peptide has negligible effect on the GTP hydrolysis. (B) Comparison of the peptide concentration dependence of the Ffh GTPase inhibition in the presence and absence of 4.5S RNA. Whereas the basal GTPase activity of Ffh is slightly increased upon binding to 4.5S RNA, KRRnoW half inhibits the GTP hydrolysis at a similar concentration both with and without 4.5S RNA.

these effects will contribute to the CD. However, the change in the CD of the mixture upon heating is very informative. The separate components show, respectively, a linear shift to more negative ellipticity for the essentially random coil signal peptide and a cooperative melt, with a component of linearly increasing ellipticity, for the Ffh protein (Figure 5F, upper panel). Summing these two leads to a highly cooperative melt with very little linear shift to either higher or lower ellipticity (Figure 5F, lower panel). Strikingly, this transition is completely abolished in the melting profile of the mixture (Figure 5F, lower panel). Even if there were a pronounced change in the peptide conformation upon binding, it is extremely unlikely that it could account for a transition of the magnitude necessary to negate the transition from Ffh. Therefore, the observed result must reflect a global loss of cooperative folding in the protein. These results indicate that although Ffh is capable of specifically binding functional signal peptides, the loading of the peptide substrates causes the whole protein to reach a globally unstable state. Cross-linking experiments have suggested that signal sequence binding is mediated by the M domain of SRP, yet our results point to a profound effect of signal peptide on the whole of Ffh, including the NG domain. Is the effect observed on the NG domain dependent on interaction of the signal peptide with the M domain? Strikingly, we find that addition of the KRRnoW signal peptide or the idealized transmembrane peptide, K5W(AL)10 , to isolated NG domain leads to the same rapid and complete proteolytic degradation upon V8 protease treatment as observed for intact Ffh (Figures 5G and 5H), strongly suggesting a direct interaction of signal peptide with the NG domain.

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Figure 5. Effects on the Stability of Ffh and the NG Domain by Binding of Signal Peptides (A) Ffh (1 mM) was mixed with 100 mM KRRnoW and then treated with V8 protease (right half) as described in the Experimental Procedures. On the left, no KRRnoW was included. (B) Titration of KRRnoW in the V8 protease digestion assay of Ffh. 5 mM KRRnoW is enough for half maximum effect of enhanced proteolytic lability of Ffh (1 mM). (C) Nonfunctional signal peptide KRR13W14D was titrated in the digestion assay with Ffh. At all the concentrations examined, no effect on the protease accessibility of Ffh was observed. (D) Ffh (1 mM) was mixed with 20 mM OmpA signal peptide and then treated with V8 protease as described in the Experimental Procedures. Similar dramatically increased protease susceptibility of Ffh was observed. (E) The CD spectra of 4 mM Ffh (1) and 40 mM KRRnoW (2), both in 10 mM NaHEPES (pH 7.6) at 258C, were taken individually and then summed. The resulted spectrum (4, dotted) is compared with the spectrum of the mixture of 4 mM Ffh and 40 mM KRRnoW (3). (F) The thermal denaturation profiles of 4 mM Ffh and 40 mM KRRnoW (upper panel), both in 10 mM NaHEPES (pH 7.6), were obtained by monitoring the ellipticity changes at 222 nm. The sum of these two profiles results in a highly cooperative curve (lower panel, dotted), in contrast to the flat melting curve of the mixture of 4 mM Ffh and 40 mM KRRnoW (lower panel). (G–H) Ffh (4 mg/ml) was treated with V8 protease (Ffh:V8 5 50:1) as in Figure 1A. The V8 protease was less active in this specific batch yet provided a convenient “marker” for its existence. After 1 hr digestion, the sample is diluted from 4 mg/ml to 0.05 mg/ml (i.e., 1 mM) (total Ffh) by the same digestion buffer system and transferred to ice. The digested sample was then passed through cation exchange CM resin equilibrated with the same buffer and the supernatant was collected. This step removes any undigested Ffh and the Ffh M domain. Subsequently, the sample is shifted to 258C and the digestion is continued from 1 hr. Aliquots of the sample were taken at indicated time point for SDSPAGE analysis. (H) Same as (G), except KRRnoW (left panel) and K5W(AL) 10 (right panel) were added to the sample after it was passed through the CM resin. The final concentrations of KRRnoW and K5W(AL) 10 were 20 mM and 10 mM, respectively.

Binding of 4.5S RNA to the M Domain Stabilizes the NG Domain in the Presence of Signal Sequence The effect of signal peptide on the NG domain likely reflects a physiologically critical conformational change during targeting and release of the nascent chain. In vivo, the Ffh protein would only bind signal sequence with the 4.5S RNA bound to the M domain. To assess the effect of RNA on the conformational change revealed by signal peptide binding, we examined the proteolytic lability of Ffh/4.5S RNA upon binding to KRRnoW. As shown in Figure 6 (compare lanes 4 and 5), 4.5S RNA totally prevents the signal peptide-induced global destabilization. Again, without KRRnoW, 4.5S RNA slows

down the cleavage of Ffh by V8 into two domains (Figure 6, lanes 2 and 3). As expected, 4.5S RNA dIV caused the same effect on Ffh in the presence of KRRnoW (data not shown), indicating that it is the local interaction between M domain and its RNA partner that changes the whole protein’s structural behavior upon peptide binding. Note that upon KRRnoW binding, the linker region between the M and NG domains becomes protected against protease (Figure 6, compare lanes 3 and 5), suggesting that the arrangement of the two domains is significantly altered by signal peptide binding. An order-of-addition experiment reveals that the effect of signal peptide on the Ffh protein in the absence of RNA is completely reversible. We added either RNA

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Figure 6. 4.5S RNA Inhibits the Globally Destabilizing Effect of Signal Peptides on Ffh

Figure 7. A Model for the Essential Structural and Functional Roles Played by 4.5S RNA in E. coli SRP

In lane 2, Ffh alone was treated with V8 protease. In lane 3, Ffh was incubated with 4.5S RNA first and then treated with V8 protease. KRRnoW (20 mM) was added to Ffh solution in lane 4 to 6. No 4.5S RNA was added in lane 4. 4.5S RNA was added first in lane 5 whereas it was added 30 min after the addition of KRRnoW in lane 6.

In the absence of 4.5S RNA, the Ffh M domain is in a molten globule– like state (disorganized helices and broken outline) in contrast to the well-folded NG domain. Upon binding to a signal peptide, although peptide-binding induces domain contact, both NG and M domains (fuzzy filling and no outline) become highly unstable because of an improper interface. RNA binding to the M domain, which stabilizes the polypeptide and results in a more specific structure (organized helices), establishes the correct communication between the two domains. This domain–domain interaction stabilizes the NG domain under the influence of signal sequence binding. See text for more discussion. The final complex of Ffh/4.5S RNA/signal peptide is functionally competent to interact with FtsY on the membrane.

or KRRnoW to the protein solution first, leaving 30 min incubation time, then the other ligand, and followed by addition of the protease. As shown in Figure 6, lanes 5 and 6, the order of adding the RNA and the peptide has no effect. This result shows that the global destabilization, while reflecting a major conformational change, does not lead to irretrievable loss of structure or aggregation. As a control, we tested whether RNA addition had any effect when signal peptide was mixed with isolated NG domain. As expected, there was no diminution in the destabilization caused by signal peptide binding to the separated domain (data not shown), showing that domain–domain interaction mediated by the M domain was responsible for the RNA effect on Ffh.

Discussion Using biochemical and biophysical approaches, we have carried out a series of experiments to probe the direct interactions among Ffh, 4.5S RNA, and signal peptides. The results of our studies have revealed unusual characteristics of the Ffh protein and its interaction with signal peptides, as well as a crucial role of 4.5S RNA in E. coli SRP. Figure 7 shows a model that emerges from our studies. It depicts the signal peptideinduced domain–domain interaction and the essential role of 4.5S RNA. In the absence of 4.5S RNA, the M domain is distinct from the NG domain based on its inherent flexibility (Figure 7, upper left). The loosely packed tertiary structure of the M domain can be partially due to the fact that it contains a large number of positively charged residues (its pI is around 10). These residues will likely compromise the stability of the protein by electrostatic repulsion. To keep the polypeptide in a well-folded state, other structural components are needed for a delicate balance among all forces. The structural behavior of the M domain is reminiscent of de novo–designed helical bundles, which require specific interactions to become

cooperatively folded (Bryson et al., 1995). It is very likely that 4.5S RNA plays such a role (Figure 7, lower left), both in neutralizing the concentrated positive charges and in providing specific “linking” interactions for tertiary packing of the helical M domain. Intriguingly, whereas 4.5S RNA stabilizes the M domain, the binding of the polypeptide also induces a conformational change in the RNA as detected by a fluorescence assay (Lentzen et al., 1994), indicating a specific molecular engagement. Given its structural behavior, the Ffh M domain is similar to some ribosomal proteins that have a helical, yet flexible, RNA-binding motif (Yonath and Franceschi, 1997), suggesting that SRP might have originated from a part of ribosome. The Ffh protein appears to be able to recognize signal sequences specifically, either in the presence or absence of the RNA (Figure 7, left to right). A central question related to this signal recognition event is where the signal sequence binding site is on the Ffh protein. Our results provide direct evidence that the NG domain recognizes signal peptide, either alone or in combination with the M domain. Previous conclusions assigning signal peptide binding exclusively to the M domain of SRP were based on crosslinking studies (Zopf et al., 1990; Lu¨tcke et al., 1992) and may thus have missed a contribution from the NG domain. Moreover, cross-linking efficiency is affected by proximity, among other factors, and as such does not necessarily correlate with direct binding. In the recently reported crystal structure of the Ffh NG domain from T. aquaticus (Freymann et al., 1997), a four-helix subdomain (N) packs against the Ras-like GTPase domain. It is tempting to speculate that the N domain, whose function is not known, plays a role in signal peptide binding. Interestingly, this potential role is consistent with the recent mutagenesis studies by

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Newitt and Bernstein (1997). In their studies, SRPs containing SRP54 N domain mutations showed defects in elongation arrest activity, suggesting that the N domain might be involved in signal sequence recognition. Present evidence does not exclude the possibility that the M domain also participates in signal peptide binding. During the characterization of the Ffh M domain, its plasticity initially attracted our attention. Although our studies showed that the unusual flexibility of the Ffh M domain is greatly reduced upon interaction with domain IV of the 4.5S RNA, the “resolution” of these assays is relatively low. We can not rule out the possibility that residual tertiary structural plasticity, besides the proposed methionine sidechains, still exists in the M domain complexed with the RNA molecule and might help the peptide binding domain to accommodate various substrates. Overall, we propose that the two helical domains, N and M, are both involved in the signal sequence binding, presenting yet another new peptide binding mechanism (Stanfield and Wilson, 1995). For proper physiological function, signal sequence binding by SRP must be coupled to membrane targeting and release. These latter functions are dependent on the conformational states of the NG domain. Our studies show that the 4.5S RNA plays a pivotal role in mediating the effective interdomain communication of Ffh by enabling signal sequence modulation of the NG domain without disruption of its structural integrity. In the absence of RNA, binding to signal peptides triggers conformational changes of the protein, but a proper interface between the two domains cannot be established (Figure 7, upper right). The interdomain interaction results in a global structural perturbation under these conditions as shown by high protease susceptibility. Binding of the M domain to 4.5S RNA, which specifically tightens the fold of the polypeptide and creates a specific structure, leads to a functional interface (Figure 7, lower right). Elucidation of the effect of RNA on Ffh clarifies the observed effects of signal sequence on the Ffh GTPase activity. Although signal sequence recognition in both the presence and absence of RNA results in GTPase inhibition of the protein, we believe that the peptide binding–induced structural change of the NG domain is physiologically relevant only when 4.5S RNA is present. In the absence of the RNA, recognition of the signal sequence by Ffh induces conformational changes within the NG domain that lead to structural destabilization, owing to the lack of correct contact with the M domain. In vivo, Ffh will always be in complex with 4.5S RNA, and thus the biological relevance of the destabilization of Ffh structure by signal peptide is indirect. Ironically, only by study of the effects of both RNA and signal peptide ligands individually have we been able to dissect successfully the domain relationships and the ligand interactions of Ffh. The unique structural and functional relationships among the Ffh M domain, NG domain, 4.5S RNA and signal sequences present a challenging case for a complete understanding of the signal recognition event. Our results have revealed the peptide binding function of the NG domain and the crucial role of 4.5S RNA in organizing the domains of Ffh for proper and functional communications upon signal sequence binding. Further

structural analysis of the SRP will be required to provide a detailed picture of its functions. In particular, questions such as where exactly the signal sequence binding sites are, how 4.5S RNA stabilizes the M domain, and how signal sequence binding and the M domain modulate the GTPase activity of the NG domain remain to be answered.

Experimental Procedures Preparation of Proteins and Peptides The genes encoding Ffh and its predicted M domain (Met-297–Arg253) (from pAraffh, a gift from Dr. Gregory J. Phillips, College of William and Mary) were subcloned into the pET16b vector (Novagene) using PCR-generated DNA fragments with NdeI and XhoI restriction sites introduced in oligonucleotide primers. The proteins were overexpressed in BL21(DE3)pLysS host cells in LB medium. Purification of Ffh was first carried out on BioCAD Sprint system with a POROS CM column. The buffer system followed the one published before (Samuelsson, 1992). Fractions containing Ffh proteins were pooled and applied to a Ni2 1-NTA column. Imidazole (100 mM) was included in the buffer to elute the protein. The Ffh M domain was purified from inclusion bodies of the overexpressing cells. The purification procedure using Ni 21-NTA column was the same as described above except 8 M urea was included in every buffer to generate denaturing conditions. Purified Ffh M domain was then dialyzed against water with no urea to refold the polypeptide. Inclusion of salt would induce aggregation of the polypeptide. To purify Ffh M domain/4.5S RNA dIV complex, purified Ffh was first incubated with equal molar of 4.5S RNA dIV with V8 protease as described below. After 1 hr of digestion at room temperature, the sample was passed through a Ni 21-NTA column to remove any undigested His-tagged Ffh and NG domain with the His tag. The sample in the flow-through was then applied to a POROS CM column on a BioCAD Sprint system and eluted by a gradient of NaCl from 0 M to 1 M. Fractions containing both the Ffh M domain and 4.5S RNA were pooled and concentrated for further studies. Preparation of the Ffh M domain/4.5S RNA dIV complex by mixing directly the His-tagged M domain with the RNA at high micromolar concentration resulted in irreversible precipitation. KRRnoW and KRR13W14D LamB signal peptide analogs, OmpA signal peptide, and K5 W(AL)10 transmembrane peptide were synthesized and purified according to the methods published before (Wang et al., 1993; Jones and Gierasch, 1994) for KRR peptide.

Preparation of 4.5S RNA and 4.5S RNA Domain IV Overproduction and purification of 4.5S RNA followed the procedures described previously (Bourgaize et al., 1984) with the following modifications. The initial chromatography steps were omitted. After two rounds of electrophoresis purification with 10% polyacrylamide gel, the RNA was extracted from the gel slices by 2 hr of electroelution at 200 V using the Elutrap system (Schleicher and Schuell). The purity of the final product was checked by 10% polyacrylamide gels under both denaturing and nondenaturing conditions. 4.5S RNA dIV was prepared by standard in vitro transcription methods (Milligan and Uhlenbeck, 1989) with a pair of synthetic DNA templates encoding the T7 RNA polymerase promoter region and the domain IV region of 4.5S RNA. Purification of the transcription products was carried out by electrophoresis with a 10% polyacrylamide gel under denaturing condition, followed by electroelution described above.

Filter Binding Assay The 39 terminus of 4.5S RNA was labeled with [59-32P]pCp. 32Plabeled RNA (0.1 nM) was incubated with protein in a 50 ml solution containing 50 mM Tris-Acetate, 150 mM KCl, 20 mM Mg-Acetate, 1 mM DTT, and 40 mg/ml BSA. The reaction was carried out at room temperature for 10 min and then filtered through nitrocellulose filters. Following a quick wash by 500 ml reaction buffer without the RNA and the protein, radioactivity of the filters were determined.

Molecular Cell 86

Protease Digestion Analysis For protease digestion studies without signal peptides, reactions were carried out at room temperature in a final volume of 50–100 ml with a buffer system of 10 mM NaHEPES (pH 7.5), 150 mM NaCl, 1 mM DTT, 10 mM MgCl2, and 10% glycerol. The ratio of Ffh to V8 is usually 100:1 unless specifically labeled. The final concentration of Ffh was 4 mg/ml. Equal molar amount of 4.5S RNA was added when necessary. To monitor the time course of digestion at each time point 5 ml sample was taken out and mixed with SDS-PAGE loading buffer immediately and boiled for 5 min and then stored on ice. For studies to test the effects of addition of signal peptides, the final volume of each sample is 200 ml and Ffh final concentration is 1 mM (z0.05 mg/ml). The reactions were stopped by adding TCA solution to reach a final concentration of 10% (v/v). After TCA precipitation, the samples were directly analyzed on SDS-PAGE. CD Analysis CD spectra were acquired on either Aviv Model 62DS CD spectrometer or Jasco J-715 spectropolarimeter. Quartz cells (1 mm path length) were used for the measurements. The spectra were average of three consecutive scans. For thermal denaturation, temperature is increased at 18C/step with 3 min equilibrating time. The concentration of the protein or protein/RNA complex samples is 1–5 mM. Acknowledgments We thank Gregory Phillips for plasmid pAraffh and Lillian Hsu for the 4.5S RNA overexpression plasmid. We are grateful to Uma Kuchibhotla for helpful advice on RNA characterization and comments on the manuscript, Linda Rotondi for peptide synthesis, and Joanna Feltham and Martin Wiedmann for critical reading of the manuscript. This work was supported by National Institute of Health grant GM34962 to L. M. G. Received May 5, 1997; revised August 25, 1997. References Bacher, G., Lu¨tcke, H., Jungnickel, B., Rapoport, T.A., and Dobberstein, B. (1996). Regulation by the ribosome of the GTPase of the signal-recognition particle during protein targeting. Nature 381, 248–251. Bernstein, H.D., Poritz, M.A., Strub, K., Hobben, P.J., Brenner, S., and Walter, P. (1989). Model for signal sequence recognition from amino-acid sequence of 54K subunit of signal recognition particle. Nature 340, 482–486. Bernstein, H.D., Zopf, D., Freymann, D.M., and Walter, P. (1993). Functional substitution of the signal recognition particle 54-kda subunit by its Escherichia coli homolog. Proc. Natl. Acad. Sci. USA 90, 5229–5233. Bourgaize, D.B., Farrell, C., Langley, K.H., and Fournier, M.J. (1984). Physical properties of the E. coli 4.5S RNA: first results suggest a hairpin helix of unusual thermal stability. Nucleic Acids Res. 12, 2019–2034. Bryson, J.W., Betz, S.F., Lu, H.S., Suich, D.J., Zhou, H.X., O’Neil, K.T., and DeGrado, W.F. (1995). Protein design: a hierarchic approach. Science 270, 935–941.

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