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Proc Natl Acad Sci U S A. Feb 10, 2009; 106(6): 1754–1759.
Published online Jan 27, 2009. doi:  10.1073/pnas.0808573106
PMCID: PMC2644110

Multiple conformational switches in a GTPase complex control co-translational protein targeting


The “GTPase switch” paradigm, in which a GTPase switches between an active, GTP-bound state and an inactive, GDP-bound state through the recruitment of nucleotide exchange factors (GEFs) or GTPase activating proteins (GAPs), has been used to interpret the regulatory mechanism of many GTPases. A notable exception to this paradigm is provided by two GTPases in the signal recognition particle (SRP) and the SRP receptor (SR) that control the co-translational targeting of proteins to cellular membranes. Instead of the classical “GTPase switch,” both the SRP and SR undergo a series of discrete conformational rearrangements during their interaction with one another, culminating in their reciprocal GTPase activation. Here, we show that this series of rearrangements during SRP-SR binding and activation provide important control points to drive and regulate protein targeting. Using real-time fluorescence, we showed that the cargo for SRP—ribosomes translating nascent polypeptides with signal sequences—accelerates SRP·SR complex assembly over 100-fold, thereby driving rapid delivery of cargo to the membrane. A series of subsequent rearrangements in the SRP·SR GTPase complex provide important driving forces to unload the cargo during late stages of protein targeting. Further, the cargo delays GTPase activation in the SRP·SR complex by 8–12 fold, creating an important time window that could further improve the efficiency and fidelity of protein targeting. Thus, the SRP and SR GTPases, without recruiting external regulatory factors, constitute a self-sufficient system that provides exquisite spatial and temporal control of a complex cellular process.

Keywords: conformational change, fluorescence spectroscopy, protein targeting and translocation, signal recognition particle

SRP-mediated co-translational protein targeting delivers roughly one-third of proteins to their correct subcellular destinations, including the eukaryotic endoplasmic reticulum and the bacterial plasma membrane. This pathway involves a sequential series of molecular steps (13), including (i) recognition and loading of cargo (ribosomes translating nascent polypeptides with signal sequences) on the SRP; (ii) delivery of cargo to the target membrane via complex formation between SRP and SR; (iii) unloading and transfer of cargo from the SRP to the protein conducting channel (PCC); and (iv) disassembly of the SRP·SR complex and recycling of free SRP and SR for subsequent rounds of protein targeting. Like many cellular processes, this complex series of molecular interactions are spatially and temporally regulated by members of the GTPase superfamily, in this case, two highly homologous and directly interacting GTPases in both the SRP and SR.

SRP and SR provide a notable exception to the “GTPase switch” paradigm established for classical signaling GTPases (4). These GTPases do not exhibit substantial conformational changes depending on whether GTP or GDP is bound (57), and further, their intrinsic nucleotide exchange rates are 102–104-fold faster than those of classical GTPases (8, 9). Thus, no external GEFs are required to switch these GTPases from the GDP- to the GTP-bound state, and the facilitation of nucleotide exchange by an external GEF cannot be the mechanism to turn these GTPases to the “on” state. Moreover, both the SRP and SR reciprocally stimulate each other's GTP hydrolysis activity when they form a complex with one another (8, 10). Thus, no external GAPs are required either to switch these GTPases from the GTP- to the GDP-bound state, and the stimulation of GTP hydrolysis by an external GAP cannot be the mechanism to turn these GTPases to the “off” state. In contrast, these GTPases undergo a series of discrete conformational changes driven by heterodimeric interactions between the two GTPases (Fig. 1; ref. 1013). Both proteins, starting in an inactive, “open” conformation, quickly bind one another to form a transient “early” intermediate independently of GTP (Fig. 1 step 1; ref. 14, 15). The presence of GTP bound at both GTPase active sites induces a conformational rearrangement in both proteins to form a stable “closed” complex (Fig. 1 step 2; ref. 11, 13, 16). A subsequent rearrangement involving the activation loops in both proteins activates GTP hydrolysis (Fig. 1 step 3; ref. 11, 12), which drives disassembly of the complex (Fig. 1 step 4; ref. 17).

Fig. 1.
Multiple conformational changes during SRP·SR complex formation and activation (11, 14), as described in the text, and the positions of fluorescence probes that detect the different conformational stages, as described in the text.

If these conformational rearrangements during SRP·SR binding and activation are integral to the regulatory role of these GTPases in protein targeting, then they should be responsive to the biological events they are monitoring. To test this hypothesis, we examined the effects of cargo loading on the kinetic and thermodynamic features of the SRP and SR GTPase cycle. Our results demonstrate that the SRP and SR GTPases can use each of the conformational changes during their binding and activation cycle to sense temporal cues, such as cargo loading, and, in response, substantially change the free energy landscape of the different conformational states in the SRP·SR GTPase complex. These cargo-induced responses allow these GTPases to drive the efficient delivery and unloading of cargo to the target membrane, and to potentially improve the fidelity of protein targeting via kinetic proofreading mechanisms.


General Experimental Approach.

To monitor the different conformational stages of the SRP·SR complex, we used fluorescence resonance energy transfer (FRET) between donor and acceptor probes incorporated on both the SRP and SR. FRET provides a highly sensitive assay that allows us to detect the transient early intermediate (Fig. 1; ref. 14). Further, this intermediate can be distinguished from the subsequent conformations because it has a lower FRET value than the closed and activated complexes (Fig. 1; ref. 14). In addition, an environmentally sensitive probe, acrylodan, labeled at residue 235 of the SRP, detects formation of the closed and activated complexes but not the early intermediate (Fig. 1 and Fig. S1), thereby simplifying kinetic and thermodynamic analyses of these later conformations. Finally, acrylodan labeled at residue 356 of the SR near its catalytic loop specifically detects the activated complex (Fig. 1 and Fig. S2). In addition to these fluorescent probes, mutant GTPases and GTP analogues were used to block specific rearrangements and thus isolate each conformational intermediate (10, 11). We can block the earlyclosed rearrangement by leaving out GTP (Fig. 1; ref. 14); this allows us to isolate the early intermediate and characterize its kinetics and stability. Mutations in the catalytic loop, SRP A144W or SR A335W, allow a stable closed complex to form but block its rearrangement to the activated complex (11, 18). The nonhydrolyzable GTP analogue 5′-guanylylimido-diphosphate (GppNHp) allows most of the rearrangements to occur but inhibits GTP hydrolysis (10, 11). Using these tools, we determined how the SRP and SR GTPases use their conformational changes to respond to cargo loading.

Cargo Accelerates Assembly of a Stable SRP·SR Complex over 100-fold.

As cargo, we purified stalled ribosome-nascent chain complexes (RNCs) bearing the N-terminal 74 aa of the model SRP substrate FtsQ (1921). SRP·SR complex assembly was monitored using FRET in the presence of GppNHp. Comparison of the time courses for complex assembly shows 3 differences between free and cargo-loaded SRP (Fig. 2A): (i) the initial rates are much faster with cargo-loaded SRP; (ii) the kinetics of complex formation with cargo-loaded SRP is bi-phasic with a burst phase, suggesting the accumulation of an intermediate; (iii) at completion of the reaction, FRET plateaus at a lower value for cargo-loaded SRP, suggesting a change in the equilibrium stability of the final SRP·SR complex. These effects are further characterized below.

Fig. 2.
Cargo changes the kinetics of SRP–SR interaction. (A) Time courses for SRP·SR complex assembly with GppNHp in the absence (black) or presence of 10 nM (blue) and 50 nM (red) RNC, using 10 nM SRP and 100 nM SR to mimic physiological protein ...

An observed rate constant for complex formation (kobsd) at any protein concentration is the sum of the complex assembly and disassembly rate constants (Eq. 1; 22)

equation image

To isolate the effect of cargo on complex assembly, we measured the observed rate constants as a function of SR concentration; the slope of this concentration dependence gives the association rate constant, kon (Eq. 1; Fig. 2B). The value of kon is 4.4 × 104 M−1·s−1 in the absence of cargo, consistent with previous measurements (10). In the presence of cargo, the complex formation rate constant is 100–400-fold faster (Fig. 2B and SI Text Fig. S3A). Thus, the cargo-loaded SRP has a substantial kinetic advantage over the free SRP to form a complex with the SR, ensuring efficient delivery of cargo to the target membrane.

Cargo Stabilizes the Early Intermediate by Two Orders of Magnitude.

The biphasic kinetics with a burst phase during complex formation with cargo-loaded SRP suggests the accumulation of an intermediate (Figs. 2A and and33A, blue). A likely candidate to account for this burst is the early intermediate, which forms quickly and has a lower FRET value than the subsequent complexes (Fig. 1; ref. 14). To test this notion, we blocked the earlyclosed rearrangement and isolated the early complex by performing complex assembly in the absence of nucleotide (Figs. 1 step 2 and and33A, green). Both the rate and the magnitude of FRET changes for assembly of the early intermediate agree well with those of the burst phase during complex assembly with GppNHp (Fig. 3A). This provides strong evidence that in the presence of cargo, the early intermediate accumulates substantially during complex assembly.

Fig. 3.
Cargo stabilizes the early intermediate. (A) Comparison of the time courses for SRP·SR complex formation for cargo-loaded SRP in the absence (green) and presence of 100 μM GppNHp (blue). Data were obtained using 20 nM SRP, 100 nM SR, and ...

The early intermediate, which lacks stabilizing interactions from the γ-phosphate of GTP, is very unstable without cargo (5, 14), hence it cannot accumulate under the nanomolar concentrations of SRP and SR used here (Fig. 2A, black). Therefore, it was surprising to detect its accumulation with cargo-loaded SRP. This observation suggests that the cargo strongly stabilizes this intermediate. To test this hypothesis, we determined the equilibrium and kinetic stability of the early complex with and without cargo. Indeed, the cargo stabilizes the early complex over 50-fold, lowering its equilibrium dissociation constant (Kd) from 4–10 μM (14) to 80 ± 4 nM (Fig. 3B, squares) and decreasing its dissociation rate constant (koff, derived from the y-intercept in Fig. 3C) from 62 ± 2 s−1 to 1.6 ± 0.1 s−1.

Stabilization of the early intermediate explains the faster rate of SRP·SR complex assembly with GppNHp for cargo-loaded SRP (Fig. 2B). Without cargo, formation of the highly labile early intermediate is not sufficient to give a stable SRP·SR complex; to obtain a stable complex, the early intermediate needs to rearrange to the closed complex. However the early intermediate dissociates quickly and less than 2% of the population rearranges to form the closed complex (koff = 62 ± 2 s−1 vs. krearrange = 1.03 ± 0.02 s−1; ref. 14). This gives rise to the slow rate constant for formation of a stable closed complex between free SRP and SR. In contrast, for cargo-loaded SRP the early intermediate is stabilized over 50-fold. Thus forming the early complex (Fig. 1 step 1) is sufficient to give a relatively stable SRP·SR complex under physiological SRP and SR concentrations (200–400 nM; ref. 23). Furthermore, the cargo·SRP·SR early complex dissociates with much slower kinetics (Fig. 3C, koff = 1.6 ± 0.1 s−1), giving this intermediate a much longer lifetime to undergo subsequent rearrangements. Both of these effects contribute to the faster rate of assembling a stable GTPase complex with cargo-loaded SRP in the presence of GppNHp.

Cargo Stalls the SRP·SR Complex at Earlier Conformational Stages.

The different FRET end points in Fig. 2A suggest that the stability of the final SRP·SR complex is also altered by the cargo. To test this hypothesis, we compared the equilibrium stability of the SRP·SR complex assembled in GppNHp with and without cargo using SRP C235 labeled with acrylodan (Fig. 1 and Fig. S1). Equilibrium titrations using this probe showed that the cargo destabilizes the closed/activated complexes four-fold, increasing its Kd from 10 ± 2 nM to 40 ± 4 nM (Fig. 4A). A similar destabilizing effect was observed using the FRET probes, with the Kd of the closed/activated complexes increasing from 14 ± 3 nM without cargo to 60 ± 7 nM with cargo-loaded SRP (SI Text Fig. S4). An additional probe that specifically monitors the activated complex, acrylodan-labeled SR C356 (Fig. 1 and Fig. S2), also confirmed that the cargo destabilizes the activated complex (Fig. 4B). In summary, the results from all three fluorescence probes showed that, in contrast to the large stabilizing effect of the cargo on the early intermediate, the subsequent conformations during the SRP–SR interaction are destabilized by the cargo.

Fig. 4.
Cargo destabilizes the closed and activated states during SRP·SR interaction. (A) Equilibrium titration of the SRP·SR complex assembled in GppNHp with (■) and without (●) RNC using acrylodan-labeled SRP C235. Nonlinear ...

Thus the cargo significantly alters the conformational rearrangements in the SRP·SR complex (Fig. 4C). Without cargo, the closed and activated states are >400-fold more stable than the early intermediate, therefore the equilibrium for the earlyclosed rearrangement is extremely favorable (Fig. 4C, Krel = 400–1000). In contrast, in the cargo·SRP·SR complex, this rearrangement is 200-fold less favorable (Fig. 4C, Krel = 1.3–22). Thus, in the cargo·SRP·SR complex, a substantial fraction of the GTPase complex is still in the early conformation (30–40%) even in the presence of GppNHp. This conformational heterogeneity of the GTPase complex in the presence of cargo is consistent with previous EM analysis that showed that, whereas the SRP is well-resolved in the RNC·SRP complex, upon addition of the SR and GppNHp, the electron density for both the SRP and SR GTPase domains are no longer visible (24). Thus, both the biochemical and structural analyses highlight the dynamic nature of the GTPase complex when it is bound to the cargo.

The SRP·SR complex can use the earlyclosed rearrangement to drive cargo unloading during protein targeting (Fig. 4D). Initially, cargo loading stabilizes the early intermediate 50-fold (Fig. 4D, Kd and Kd′). Correspondingly, the interaction of cargo with the SRP should be stabilized to the same extent in the early intermediate (Fig. 4D, KdRNC′/KdRNC = Kd′/Kd = 50). Using the KdRNC value of ~ 1 nM (25, 26), the stability of cargo bound to the early intermediate (KdRNC′) to be 20 pM. Although this effect could enhance the initial recognition and delivery of cargo to the membrane, such strong binding will block the subsequent unloading of cargo from the SRP. This problem is circumvented by the 200-fold destabilizing effect of cargo on the earlyclosed rearrangement (Figs. 4 C and D, Krel and Krel′). Correspondingly, the interaction of cargo with the SRP would also be weakened 200-fold by this rearrangement (Fig. 4C, KdRNC″/KdRNC = Krel′/Krel), thus priming the cargo for subsequent unloading. This model is supported by mutational analyses that showed that mutant GTPases defective in the earlyclosed rearrangement severely block protein translocation (18). The observation that mutants defective in the closedactivated rearrangement inhibit protein translocation further suggests that this last rearrangement is also essential for cargo unloading (18). Therefore, both rearrangements within the GTPase complex provide essential driving forces to help unload the cargo from the SRP to the PCC, thus initiating protein translocation.

Because cargo disfavors the rearrangements to form the activated complex, one would predict that stimulated GTP hydrolysis, which occurs from the activated complex, would also be impaired. To test this notion, we compared the GTPase reaction rate from the SRP·SR complex in the presence and absence of cargo. In the absence of cargo, the GTPase rate of free SRP is significantly stimulated by the addition of the SR (Fig. 5, circles). The reaction rate reaches a plateau of 0.79 s−1 at saturating SR concentrations, representing the GTPase rate constant from the SRP·SR complex (Fig. 5, circles). In the presence of cargo, significantly less GTPase stimulation was observed (Fig. 5, squares). Intriguingly, two plateaus were observed for the GTPase reaction in the presence of cargo (Fig. 5, squares), suggesting the presence of two populations of cargo·SRP·SR complexes: one population, which forms at low SR concentrations (below 50 nM), hydrolyzes GTP at a rate constant of 0.064 s−1; the second population, which forms at higher SR concentrations (above 1 μM), hydrolyzes GTP at a rate constant of 0.11 s−1 (Fig. 5, squares). Although the nature of this heterogeneity is unclear at present, in both of these populations the GTPase activity is repressed by the RNC (12- and 8-fold for the first and second populations, respectively). The effect of cargo in reducing the GTP hydrolysis rate is specific to the SRP·SR complex as the cargo does not affect the basal GTP hydrolysis rate of the free SRP (SI Text Fig. S5). Thus the cargo also delays GTPase activation in the SRP·SR complex. This effect, which we term “stalling,” would provide an important time window that allows the SRP to unload the cargo before GTP hydrolysis drives irreversible complex disassembly, as discussed below.

Fig. 5.
Cargo delays activation of GTP hydrolysis in the SRP·SR complex. GTPase rate constants were measured using 40 nM SRP and 100 μM GTP in the absence (■) and presence (●) of 100 nM RNC. The data in the absence of cargo were ...


We showed here that cargo loading substantially alters the free energy landscape of the SRP–SR interaction cycle (Fig. 6A). Without cargo (black), assembly of a stable SRP·SR complex is slow because it requires rearrangement from an unstable early intermediate [Fig. 6A, ΔGcomplex = ΔGearly + ΔG; (14)]. Further, the stable SRP·SR complex has a short lifetime because as soon as it is formed, rapid activation of GTP hydrolysis drives its irreversible disassembly (8). The cargo uses a remarkably simple solution to these problems, by stabilizing the early intermediate (Fig. 6A, ΔΔG = −2.4 kcal/mol) and disfavoring the closed and activated states (Fig. 6A, ΔΔG ≥ +0.8 kcal/mol). This accelerates complex assembly (Fig. 6A, ΔΔG = −2.8 kcal/mol), and prolongs the lifetime of the SRP·SR complex because of delayed GTP hydrolysis (Fig. 6A, ΔΔG = +1.3–1.5 kcal/mol). The rate-limiting step of the SRP–SR interaction cycle changes from the earlyclosed rearrangement with free SRP to GTP hydrolysis with cargo-loaded SRP.

Fig. 6.
Conformational changes during the SRP–SR interaction respond to cargo loading and regulate protein targeting. (A) Rate constants and free energy profiles for the SRP–SR interaction in the absence (black) and presence (red) of cargo. A ...

These cargo-induced effects allow the SRP and SR to use each of their conformational rearrangements to regulate a distinct step during protein targeting (Fig. 6B). At the beginning of each targeting cycle, cargo loading (Fig. 6B step 1) allows the SRP to assemble a stable complex with SR >100-fold faster (Fig. 6B step 2). This ensures rapid delivery of cargo to the membrane (15, 27) and avoids futile interactions between the free SRP and SR. In the early intermediate, the cargo is locked in the SRP·SR complex with very high affinity (Fig. 4D, KdRNC′ ~ 20 pM), allowing the SRP to effectively compete with cellular chaperones for binding the cargo. Subsequent GTPase rearrangements to the closed and activated conformations weaken the interaction of cargo with the SRP (Fig. 6B steps 3 and 4; cf. Fig. 4D) and thus help the SRP to switch from a cargo-binding mode to a cargo-release mode, to unload the cargo to the PCC (Fig. 6B step 4). Once in the activated conformation, and especially after cargo release, rapid GTP hydrolysis drives the disassembly and recycling of both the SRP and SR (Fig. 6B step 5).

The mechanism proposed here (Fig. 6B) focuses on the GTP-bound SRP and SR because the high cellular concentration of GTP compared to GDP (~900 μM and 100 μM in bacteria, respectively) predicts that over 90% of both GTPases are bound with GTP. Minor pathways are also possible in which empty-site or GDP-bound forms of the SRP and SR first form the early intermediate to deliver cargo to the membrane surface, followed by rapid binding or exchange of GTP to drive the subsequent steps (15, 27); these pathways are not depicted in Fig. 6B for clarity.

The most intriguing effect of cargo is “stalling,” that is, the delay of GTPase activation by ~8–12-fold (Fig. 6B step 4). A similar effect was suggested from studies of the mammalian system where before the addition of the PCC, a stable cargo·SRP·SR complex persists in the presence of GTP, suggesting that the cargo may also delay GTP hydrolysis in the mammalian SRP·SR complex (28). We suggest that stalling creates an important time window during which the SRP ensures the efficiency and fidelity of protein targeting via either or both of the following mechanisms. First, stalling could provide a spatial checkpoint for the target membrane and/or the PCC. Before the SR associates with the PCC, stalling prevents premature GTP hydrolysis that would irreversibly disassemble the SRP·SR complex and thus help avoid abortive targeting reactions (Fig. 6B step 6). Interaction of SR with the PCC may trigger the rearrangement to the closed and activated states and initiate cargo unloading (28). The PCC also competes with the SRP for interacting with the RNC (20, 21, 24, 29), which could further drive the transfer of cargo from the SRP to the PCC (28, 30). Alternatively or in addition, stalling could provide a fidelity checkpoint. Many of the effects of the cargo described here are observed only with RNCs but not with empty ribosomes (SI Text Fig. S6), establishing the importance of the signal sequence. It could be envisioned that cargos with weaker signal sequences could not effectively stall the SRP·SR complex, and thus are more likely to be rejected via premature GTP hydrolysis (Fig. 6B step 6). In this way, GTP hydrolysis could be used to improve the fidelity of protein targeting akin to kinetic proofreading mechanisms used by elongation factor GTPases (31).

Materials and Methods


The Eschericia coli SRP and SR GTPases (Ffh and FtsY, respectively) and 4.5S RNA were expressed and purified using established procedures (8, 18). Most of the fluorescence experiments used the FtsY (47–497) construct. This truncated FtsY construct behaves similarly to full length FtsY in its ability to interact with the SRP and to respond to the cargo (SI Text Fig. S3). The GTPase reactions with and without cargo was determined with full length FtsY. Mutant proteins were constructed using the QuikChange procedure (Stratagene), and were expressed and purified by the same procedure as that for the wild-type protein. Fluorescent dyes DACM, BODIPY-FL, and acrylodan were from Invitrogen. 70S ribosomes and RNCs were purified as described previously (19, 32) and in the SI Text.

Fluorescence Labeling.

For FRET measurements, maleimide derivatives of coumarin and BODIPY-FL were used to label single-cysteine mutants of the SRP and SR, respectively, as described (14). Labeling of the SRP and SR with acrylodan followed the same procedure except that the labeling reaction was carried out using a 30-fold excess of dye over protein for over 12 h at 4 °C. Absorbance of acrylodan (ε391 = 20,000 M−1·cm−1) was used to determine the concentration of labeled protein. The efficiency of labeling reaction was typically greater than or equal to 90% for both proteins. The background, estimated from the labeling of the SRP and SR lacking cysteines using the same procedure, is less than 3%.

Fluorescence Measurement.

All measurements were carried out at 25 °C in assay buffer [50 mM KHEPES, pH 7.5, 150 mM KOAc, 10 mM Mg(OAc)2, 2 mM DTT, 0.01% Nikkol] on a Fluorolog-3 spectrofluorometer (Jobin Yvon) as described (8, 14). FRET measurements were carried out using an excitation wavelength of 380 nm and an emission wavelength of 470 nm. FRET efficiency was calculated as described (14). Fluorescence emission spectrum of the SRP (or SR) labeled with acrylodan was measured using an excitation wavelength of 370 nm. Fluorescence emission at 500 nm was monitored for equilibrium titrations using acrylodan-labeled protein.

Pulse–chase experiments were carried out using unlabeled protein to trap any dissociated SRP or SR (10). Fast reactions were measured on a Kintek stop-flow apparatus (10). The incubation time during equilibrium measurements was calculated based on the SRP·SR complex assembly rate (10, 14), and varies from 5 min for fast reactions (early complex assembly and complex assembly in the presence of cargo) to several hours (complex assembly with GppNHp in the absence of cargo).

GTPase Assay.

The GTPase assay to measure the stimulated GTP hydrolysis reaction between the SRP and SR were carried out and analyzed as described (8). Multiple turnover reactions were carried out at 25 °C with a small, fixed amount of free or cargo-loaded SRP and increasing concentrations of SR; 100 μM GTP (doped with trace γ-32P-GTP) was present in the reaction to saturate both GTPase sites. The data presented in Fig. 5 was representative of four experiments. Previous studies have established that the GTPase reaction rate is rate-limited by SRP·SR complex formation at sub-saturating SR concentrations, whereas at saturating SR concentrations, the reaction is rate-limited by GTP hydrolysis or a slow conformational change preceding GTP hydrolysis (8). The release of products, including dissociation of GDP, Pi, and disassembly of the GDP·SRP·SR·GDP complex, are not rate-limiting in this GTPase assay (8).

Supplementary Material

Supporting Information:


We thank Sandra Schmid, Douglas C. Rees, Raymond Deshaies, Nathan Pierce, and members of the Shan laboratory for comments on the manuscript. This work was supported by a National Institutes of Health grant GM078024 to S.S., and by the Swiss National Science Foundation (SNSF) and the NCCR Structural Biology program of the SNSF to N.B. S.S. was supported by a career award from the Burroughs Welcome Foundation, the Henry and Camille Dreyfus foundation, the Beckman Young Investigator award, and the Packard and Lucile award in science and engineering. X.Z. was supported by a fellowship from the Ulric B. and Evelyn L. Bray Endowment Fund.


The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0808573106/DCSupplemental.


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