SRP RNA controls a conformational switch regulating the SRP-SRP receptor interaction

Abstract

The interaction of the signal-recognition particle (SRP) with its receptor (SR) mediates co-translational protein targeting to the membrane. SRP and SR interact via their homologous core GTPase domains and N-terminal four-helix bundles (N domains). SRP-SR complex formation is slow unless catalyzed by SRP's essential RNA component. We show that truncation of the first helix of the N domain (helix N1) of both proteins dramatically accelerates their interaction. SRP and SR with helix N1 truncations interact at nearly the RNA-catalyzed rate in the absence of RNA. NMR spectroscopy and analysis of GTPase activity show that helix N1 truncation in SR mimics the conformational switch caused by complex formation. These results demonstrate that the N-terminal helices of SRP and SR are autoinhibitory for complex formation in the absence of SRP RNA, suggesting a mechanism for RNA-mediated coordination of the SRP-SR interaction.

Figure 1

Structural and schematic representations of the FtsY and Ffh constructs used in this study. (a) Left, ribbon representation of the crystal structure of E. coli FtsY (a subset (residues 204–495) of PDB 2QY9 is shown). FtsYΔN1 begins at residue 221; residues up to 220 are red. Right, ribbon representation of the crystal structure of the T. aquaticus Ffh NG domain (residues 1–298, PDB 2FFH) with amino acids 1–8, truncated in FfhΔN1, in red. The G domains are dark gray and the N domains are light gray. The orientation of the individual proteins in relation to their position in the structure of the Ffh–FtsY complex (b) is indicated. (c) Domain map of FtsY and Ffh with positions of truncations indicated by arrows. The color scheme is the same as in a and b.

Figure 2

The N-terminal helices of Ffh and FtsY inhibit Ffh–FtsY association in the absence of 4.5S RNA. (a) Truncation of helix N1 increases the rate of Ffh–FtsY association in the absence of 4.5S RNA. Observed binding rates are plotted as a function of Ffh concentration for Ffh–FtsYΔN1 without RNA (−RNA) (●), FfhΔN1–FtsY −RNA (■) and Ffh-FtsY −RNA (◆). Lines are fits to the equation k_obs = k_on[Ffh] + k_off in a, b and c, and wild-type (WT) references are included in multiple figures for comparison. (b) The FfhΔN1–FtsYΔN1 complex forms nearly as rapidly in the absence of 4.5S RNA as the Ffh–FtsY complex forms in the presence of 4.5S RNA. Observed binding rates are plotted as a function of Ffh concentration for Ffh–FtsY +RNA (◇), FfhΔN1–FtsYΔN1 −RNA (▲) and Ffh-FtsY −RNA (◆). (c) Binding of FfhΔN1 and FtsYΔN1 in the presence of 4.5S RNA. Observed binding rates are plotted as a function of Ffh concentration for Ffh–FtsY +RNA (◇), Ffh–FtsYΔN1 +RNA (○), FfhΔN1–FtsYΔN1 +RNA (△) and FfhΔN1–FtsY +RNA (□). (d) Summary of binding rates. On rates for each Ffh–FtsY pair are plotted in the presence and absence of 4.5S RNA. Note the log scale. Error bars represent the standard error of the linear fit to the equation k_obs = k_on[Ffh] + k_off.

Figure 3

The N-terminal helices of Ffh and FtsY stimulate Ffh–FtsY complex dissociation in the presence of 4.5S RNA. (a) Bar graphs representing the dissociation rate constants (k_off) for disassembly of the Ffh–FtsY complex −RNA (dark gray) and +RNA (light gray). The k_offs were measured by forming complexes in the presence of GppNHp and trapping dissociated proteins with GDP. Data were fit to a single-exponential equation, and error bars represent the standard error of the fit. (b) Plot of equilibrium dissociation constants, ± RNA. K_d values were calculated by the equation K_d = k_off/k_on. Note the log scale axes. WT, wild type.

Figure 4

The N-terminal helix of FtsY represses its basal GTPase activity. (a) Plot of observed rates from single-turnover GTPase assays measuring GTP-hydrolysis rate as a function of FtsY (◆) or FtsYΔN1 (●) concentration. A fit of the data to the equation k_obs = k_cat[FtsY]/(K_M + [FtsY]) gave k_cat of 0.00979 ± 0.0028 min⁻¹ for FtsY and 0.662 ± 0.24 min⁻¹ for FtsYΔN1. (b) Single-turnover GTPase assays were performed for Ffh (◆) or FfhΔN1 (■) as a function of increasing concentrations of Ffh. A fit of the data to the equation k_obs = k_cat[Ffh]/(K_M + [Ffh]) gave k_cat of 0.0876 ± 0.012 min⁻¹ for Ffh and 0.305 ± 0.031 min⁻¹ for FfhΔN1. (c) Plot of stimulated GTP-hydrolysis rates for Ffh–FtsY complexes +RNA (light gray) or −RNA (dark gray). Rates were measured as pulse chase experiments as described in Methods. Error bars are standard errors of the fit to a single-exponential equation.

Figure 5

FtsYΔN1 assumes an ‘Ffh-bound’ conformation in the presence of GppNHp. NMR spectra of ¹³C methyl Ile-Leu-Val–labeled FtsY₂₀₄ and FtsYΔN1 are overlayed. FtsYΔN1 + GppNHp is shown in red as a reference. FtsY₂₀₄ + GppNHp (a) and FtsY₂₀₄ + GppNHp + Ffh (b) are shown in blue. Insets, magnifications of a region of the spectra shown above. Notice that several peaks that are unmatched in a have partners in b (a subset of these peaks from a particularly well-resolved region of the spectrum is marked with arrows).

Figure 6

Binding of Ffh to FtsY exposes the N-terminal helix of FtsY. (a) Western blot showing limited proteolysis of FtsY either alone or in complex with SRP (Ffh + 4.5S RNA). A low-molecular-weight band marked with an arrow appears specifically when SRP is bound. (b) Western blots showing fine mapping of the location of the cleavage site in FtsY. Truncation variants of FtsY were subjected to limited proteolysis in the presence of SRP with either GppNHp (allowing complex formation) or GDP (preventing complex formation). The low-molecular-weight band is indicated by an arrow. (c) Proteolysis of FtsY takes place between residues Ser116 and Leu117. The sequence of the N-terminal helix of the FtsY NG domain is shown with an arrow marking the cleavage site as determined by N-terminal sequencing.

Figure 7

Model for Ffh–FtsY structural rearrangement upon complex formation. (a) Ribbon representations of FtsY and Ffh in unbound form (PDB 2QY9 and PDB 2FFH, respectively). Helix N1 of both proteins is shown in red. Note that, in the unbound form, residue Lys453 of FtsY and residue Arg255 of Ffh (both shown in stick form in red) protrude into the dimerization interface, conceptually represented by a dashed line. (b) Ribbon representation of the Ffh–FtsY complex (PDB 1OKK). In the bound form, Lys453 of FtsY and Arg255 of Ffh move away from the interface, into the space formerly occupied by helix N1.

Figure 8

Thermodynamic model describing the mechanism of SRP RNA control of the interaction of the SRP and SR. Free-energy diagrams for interaction of Ffh and FtsY wild type (WT) and N-terminal truncation variants with and without 4.5S RNA. The free energy of activation is calculated from the observed association and dissociation rate constants (k) using the equation ΔG^‡ = −RT ln(hk/k_BT), where h is Planck’s constant, k_B is the Boltzmann constant, T is the absolute temperature and R is the universal gas constant. For forward reactions, a standard state of 1 μM was used to calculate free-energy changes. Cartoons depict Ffh and FtsY with circles representing the GTPase domain and lines representing the N-terminal four-helix bundle. The N1 helices are shown in red. Ffh additionally is shown with the M domain and the 4.5S RNA (hairpin). 4.5S RNA is shown interacting with helix N1 of Ffh and FtsY in the transition-state complex in a manner that is dependent on helix N1 of Ffh.