Figure 1.
The archeal Signal Recognition Particle.
(A) Simplified schematic of the archaeal signal recognition particle from Pyrococcus furiosus. (B) The sequence and organization of the core of the SRP RNA are shown with helices 6 and 8, the respective binding sites for the proteins SRP19 and SRP54/Ffh. For SRP54/Ffh the M domain, responsible for both SRP RNA and signal sequence recognition, is connected to the NG domain with the GTPase activity, through a flexible linker (in magenta). Although the NG of SRP54 domain has also been shown to interact loosely with the core of the SRP RNA, for the sake of clarity this is not represented on this schematic.
Table 1.
X-ray data collection and structure refinement statistics.
Figure 2.
The SRP54 from Pyrococcus furiosus.
(A) Sequence alignment of Ffh/SRP54 full-length proteins of known structure including Pyrococcus furiosus, Thermus aquaticus, Methanococcus jannaschii and Sulfolobus solfataricus. α-helices and conserved motifs of the SRP/SR-GTPase subfamily are labeled. The N, G and M domains are indicated, as is the linker region between the G and M domains. (B) Overall structure of the monomer of Pfu-SRP54. The secondary structure elements are indicated. The bound-GDP nucleotide is represented in sticks and the disordered finger loop (FL) schematized as a dashed line. The distance between the end of the NG domain (Leu296 at the C-terminus of helix α7) and the N-terminus of the M domain (Gly326 at the C-terminus of helix α9) is 44 Å.
Figure 3.
An extended conformation of the linker in Pfu-SRP54.
(A) Arrangement of the non-crystallographic dimer of Pfu-SRP54•GDP in the tetragonal asymmetric unit. The NG catalytic cores, linkers and M domains are labeled and respectively colored in green, yellow and red. The non-crystallographic two-fold axis is represented on both views. The two views are perpendicularly related. (B) Stereo view showing the final 2.5 Å resolution 2mFo-DFc Fourier difference likelihood-weighted electron density map contoured at 1.2σ in the linker region between the NG and M domains. The strictly conserved residues involved in GDP binding are labeled and the hydrogen bonds drawn. The conserved residues in the R292XLGXGD298 motif of the GM linker are labeled. Residues from the NG domain, its C-terminal α7 helix and the linker are respectively colored in green, pink and yellow.
Figure 4.
Conformational variability of the different Ffh/SRP54 proteins.
(A) and (B) Conformational variability of the linker in the Ffh/SRP54 GTPases. (A) Superposition of the Pfu- and Ssol- free SRP54 structures. The NG domains (in grey) have been superposed to emphasize the different conformation adopted by the M domains (in red) and the G to M linkers (in yellow). The C terminal helices α7 of the G domains are highlighted (in pink). (B) Superposition of the Pfu-SRP54 and the Mja-SRP emphasizing the clash between the Mja-SRP RNA•SRP19 and the Pfu-M domain. In both figures Pfu-SRP54 is shown in the same orientation. The position of the glycine residues acting as “pivot points” is indicated with a red asterisk. (C) and (D) Conformational changes in the M domain. (C) The Pfu-M domain is shown superposed with the M domain as observed in the Ec, Taq and Mja structures. (D) The Pfu-M domain is superposed with the Ssol-M domain. As a reference the backbones of the SRP RNA from Ec, Mja and Ssol are shown in white. α helices are labeled according to the secondary structure assignment of Pfu-SRP54. The arrows emphasize the rearrangement and shift in position for the helix α10 when Taq, Ssol and Pfu structures are compared. In both figures the Pfu-M domain is shown in the same orientation.
Figure 5.
Stereo view showing the product GDP bound in the active site of Pfu-SRP54.
All residues shown are strictly conserved in all SRP-GTPases. Helices have been labeled. Hydrogen bonds between the residue Asp250, the nucleotide specificity determinant, and the guanine ring are indicated. In contrast with one of the Taq-Ffh•GDP complex structures the sidechain of Arg193 is solvent exposed and does not establish a salt bridge with the Asp137 sidechain. This catalytic aspartate is located about 6.3 Å away from the GDP β-phosphate.
Figure 6.
The GM linker couples the catalytic site of the GTPase and the signal peptide-binding domain.
Based on the comparison between the Pfu-SRP54 GDP and Taq-FtsY•FfhNG•GMPPCP complexes, upon repositioning of helix α7, the basic residues Arg288 and Arg292 become buried at the NG interface: An ion pair forms between Glu282 and Arg288 and the sidechain of Arg292 interacts with and stabilizes the DARGG motif backbone. Rearrangement of a cluster of hydrophobic residues also propagates structural changes between the linker and the GTP binding site. The arrows emphasize the motions of all the conserved residues listed. (A) and (B) show the same area in the Pfu-SRP54•GDP complex and in the Taq-FtsY•FfhNG complex bound to GMPPCP.
Figure 7.
The SRP19 from Pyrococcus furiosus.
(A) Sequence alignment of archeal SRP19 of known structures including Pyrococcus furiosus, Archaeglobus fulgidus and Methanococcus jannaschii. The secondary structure elements of Pfu-SRP19 are indicated. For the sake of clarity, the human SRP19 sequence [44] is not shown. (B) Two views of the monomer of free Pfu-SRP19 (in green and raspberry). The α helices, β strands and loops are labeled accordingly. The superposed structure of Mja-SRP19 as observed in the Mja-SRP complex is shown (in grey) to emphasize the overall rigidity of the protein backbones and the rearrangement of loop L2 upon binding to the SRP RNA. In free Pfu-SRP19 the L2 loop is disordered (dotted lines) whereas in Mja-SRP19 bound to SRP RNA it refolds and adopts an helical conformation.
Figure 8.
(A) Arrangement of the Pfu-SRP19 “tetramer” as observed in the asymmetric unit of the monoclinic crystal form. Two different orientations are shown. Each monomer is colored differently. The α helices, β strands and loops are labeled accordingly. (B) Stereo view of the 1.8 Å resolution 2mFo-DFc Fourier difference likelihood-weighted electron density map contoured at 1.5σ in the loop L1 region of one monomer, water molecules are represented as spheres. For the sake of clarity symmetry related molecules are colored in grey.
Figure 9.
Closing of loop L2 upon SRP19 binding to the SRP RNA and its implications for the sequential assembly of the archeal SRP.
(A) Superposition of the six crystallographically independent monomers of free Pfu-SRP19. The backbone trace is colored according to the atomic displacement factors. Dark blue corresponds to 15 Å2, green corresponds to 40 Å2 and red corresponds to 65 Å2. (B) Superposition of Pfu-SRP19 on the Mja-SRP19 as observed in the full Mja-SRP structure. Pfu-SRP19 and Mja-SRP19 are colored in green and red respectively. The Mja-SRP RNA is represented in white. The arrow emphasizes the rearrangement that the loop L2 is likely to undergo upon SRP19 binding to the RNA. (C) Model summarizing the role of SRP19 in the sequential assembly of the archeal SRP. The core of the archeal Mja-SRP RNA is shown, the nucleotides in the regions of the SRP RNA undergoing rearrangements during association are highlighted in green (primary SRP19 binding site) and orange (primary SRP54 binding site). As free SRP19 binds, through a reciprocal induced-fit mechanism, its disordered L2 loop folds (pink arrow). As was shown previously [9], the two SRP RNA regions where SRP19 (green arrow) and SRP54 (orange arrow) bind undergo concerted rearrangements, base pairs are splayed out and the RNA backbone is reconfigured resulting in a high affinity site for SRP54 M domain binding. Following docking of its M domain, the SRP54 NG domain may dock to the RNA backbone as observed in the Mja-SRP structure [20].