Multiple conformational switches in a GTPase complex control co-translational protein targeting - PubMed (original) (raw)

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

Xin Zhang et al. Proc Natl Acad Sci U S A. 2009.

Abstract

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 x 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.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

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.

Fig. 2.

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 concentrations (23). (B) Cargo accelerates SRP·SR complex assembly with GppNHp by 100-fold. The data are fit to Eq. 1 and gave association rate constants (_k_on) of 3.7 ± 0.4 × 106 M−1·s−1 and 4.0 ± 0.3 × 104 M−1·s−1 with (■) and without (●) 60 nM RNC, respectively.

Fig. 3.

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 20 nM RNC. (B) Cargo stabilizes the early intermediate 50-fold. Equilibrium titration of the early complex assembled in the absence of GppNHp with (■) and without (●) 50 nM RNC. Nonlinear fits of data gave _K_d values of 80 ± 4 nM in the presence of RNC. (C) Cargo increases the kinetic stability of the early intermediate 40-fold. The data are analyzed as in part B and give _k_on = 1.0 ± 0.1 × 107 M−1·s−1 with cargo-loaded SRP, which is within two-fold of the value in the absence of RNC (_k_on = 5.6 ± 0.3 × 106 M−1·s−1) (14), and _k_off = 1.62 ± 0.1 s−1, which is 40-fold slower than that in the absence of RNC (_k_off = 60 ± 2 s−1) (14). The inset shows the data in the absence of RNC (adapted from ref. 14). Note the difference in scales between the two plots.

Fig. 4.

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 fits of data gave _K_d values of 10 ± 2 nM (without RNC) and 40 ± 4 nM (with RNC). (B) Relative fluorescence changes of acrylodan-labeled SR C356 in the presence and absence of cargo, obtained using 50 nM SRP and 15 nM labeled SR with 100 μM GppNHp. An accurate _K_d value could not be determined with this probe because of the large amount of cargo-loaded SRP that would be required to saturate labeled SR C356. (C) Equilibrium constants of the GTP-independent (_K_d−G) and GTP-dependent (_K_d+G) SRP·SR complexes with or without RNC. The equilibrium for rearrangement (_K_rel) were calculated from _K_rel = Kd-G/K d+G. (D) Thermodynamic analysis of the interaction of cargo with SRP at different conformational stages during the SRP–SR interaction.

Fig. 5.

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 fit to a single binding curve and gave a rate constant of 0.79 s−1 for GTP hydrolysis from the SRP·SR complex. The data in the presence of cargo is not consistent with a single binding curve and was fit to a model based on two populations of SRP·SR complexes that reacts at rate constants of 0.064 and 0.11 s−1.

Fig. 6.

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 standard state of 200 nM SRP is used to approximate cellular protein concentrations. Activation energies were calculated from the observed association and dissociation rate constants using Δ_G_‡ = -RT_ln(kh/k_B_T), where R = 1.987 cal·K−1·mol−1, h = 1.58 × 10−37 kcal·s−1, k_B = 3.3 × 10−27 kcal·K−1, and T = 298K. The relative energies of the different complexes were calculated from the observed equilibrium stabilities using Δ_G = -RT ln_K, where K is the equilibrium constant. ΔGearly is the free energy cost to form the early complex, Δ_G_‡ is the activation energy for the early_→_closed rearrangement. The sum of these two gives the overall energy barrier to form the closed complex (Δ_G_complex‡), which is lowered 2.8 kcal·mol−1 by the cargo. In contrast, the RNC increases the activation energy for GTP hydrolysis by 1.3–1.5 kcal·mol−1. (B) Proposed model for how the conformational changes during the SRP–SR interaction regulate protein targeting and translocation as described in text.

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