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Signal Recognition Particle-Receptor Complex Structure Solved Print

The signal recognition particle (SRP) is a ubiquitous ribonucleoprotein (RNP) complex that delivers membrane and secretory proteins to the cell membrane in prokaryotes and in eukaryotes to the endoplasmic reticulum (ER), an organelle that forms a network of protein and lipid synthesizing factories. This process, called co-translational protein targeting, is an essential and evolutionarily conserved pathway for delivering nascent proteins to the eukaryotic ER. To learn more about co-translational protein targeting, researchers from UC Berkeley and the Swiss Federal Institute of Technology collaborated to determine the crystal structure of the prokaryotic SRP:SR complex arrested in the cargo release state at 3.9 Å resolution.

Signal Recognition
Made Simple

The signal recognition particle (SRP) is a ribonucleoprotein complex in which the RNA and protein components are highly conserved across all kingdoms of life. Responsible for directing protein traffic within a cell, and then allowing proteins to be secreted from the cell, the SRP plays an integral role in the process of co-translational protein targeting.

A ribosome, an organelle that produces proteins, forms a complex with the protein it is synthesizing, or translating. This ribosome-nascent chain complex (RNC) binds the SRP, delaying protein translation until the ribosome-bound SRP reaches the SRP receptor in the membrane of a eukaryotic cell’s endoplasmic reticulum, or a prokaryote’s plasma membrane.

The cleavage of two guanosine triphosphate (GTP) molecules then occurs without interference or help from any external activation factors. This action is coupled to the final step of this process, wherein the SRP is released from its receptor and from the RNC, allowing translation to resume while the protein sequence moves through the translocation channel.

An SRP contains ribonucleic acids (RNA) and proteins. When an SRP recognizes the signal sequence of a nascent polypeptide, it forms a complex with its membrane-associated receptor (SR), and delivers the translating ribosome, the cargo, to the translocation channel (translocon). Despite extensive investigation over the last three decades, several fundamental questions remained unanswered: How does the SRP RNA stimulate hydrolysis, or cleavage, of guanine triphosphate (GTP) of the SRP:SR complex? Why is this GTPase activation, which leads to GTP hydrolysis, essential for protein targeting? How is the cargo transfer to the translocon coupled to GTP hydrolysis by Ffh, a component of the SRP, and the SRP receptor (SR) FtsY?

 

(A) -Top view of the SRP:SR complex. Ffh is colored in blue, RNA in gray, FtsY (SR) is shown in green. The atoms of the two GTP non-hydrolyzable analogue molecules (GMP-PCP) are displayed as yellow spheres. (B) Side view of the SRP:SR complex. N denotes the N domain, G denotes the G domain, M denotes the M domain, L is the flexible linker, and F denotes the finger loop. The linker is displayed together with the Fo-Fc difference electron density (blue mesh, contoured at 3σ). The 2Fo-Fc electron density for the entire complex is shown as gray mesh contoured at 1σ.

Data for the SRP:SR complex in question were obtained at ALS Beamline 8.2.2, as well as at the Swiss Light Source in Villigen, Switzerland. The newly solved structure reveals that the GTPase domains of the SRP protein Ffh and the SR surprisingly bind the distal end of the SRP hairpin RNA. For the first time, the structure of the linker region is shown as a long α-helix connecting the Ffh’s NG domain, which are responsible for GTP binding, with the Ffh M domain, responsible for binding the signal sequence polypeptide. The crystal structure of the SRP:SR together with previous structural and biochemical studies demonstrates that the SRP complex can exist in distinct conformational states depending on the orientation of the linker between the NG and M domains of Ffh.

Analysis also indicates that the GTPase region of the SRP RNA is in close proximity to the center of the NG-domain structure and is highly conserved. The conservation also includes a flipped out C83 base present in all bacterial SRP RNAs in the distal region of the molecule. Kinetic analyses involving truncation of the SRP RNA and mutation of the flipped base show that the RNA distal end specifically stimulates GTP hydrolysis in the SRP:SR complex. Remarkably, the GTP-controlled reorganization of the SRP structure simultaneously exposes the signal sequence in the M domain and frees up the ribosomal binding site for translocon binding.

(A) - Overall view of the interaction of Ffh and FtsY proteins (displayed as ribbons and colored as in Fig. 1) with the RNA (represented as a contoured surface colored according to the conservation with bright yellow being 100% conserved and white not conserved). GMP-PCP are shown as yellow spheres. (B) Close-up view of the interaction of the conserved flipped C83 with the interface of Ffh and FtsY. RNA is displayed as sticks colored in gray with C83 colored in green. Ffh and FtsY residues that interact directly with C83 are displayed as sticks and colored as in Fig. 1. GMP-PCP residues are represented as sticks colored with white carbons, yellow oxygens, pink nitrogens, and green phosphates.

These results provide critical information regarding the conformational changes that drive the SRP cycle. They define the SRP RNA as a bifunctional molecule. It first promotes SRP:SR complex formation using the tetraloop, which facilitates the formation of the NG heterodimer comprising the NG domain of Ffh and of FstY each bound to a GTP molecule. Second, the RNA recruits the GTP-activated heterodimer NG domain to its opposite end. According to this mechanism, the conformational changes necessary for signal sequence handoff to the translocon are coupled to GTP hydrolysis without the need for any external GTPase activating factor. According to this mechanism, the conformational changes necessary for signal sequence handoff to the translocon are coupled to GTP hydrolysis without the need for any external GTPase activating factor. The SRP targeting cycle therefore involves a large rearrangement of Ffh using the SRP RNA as a platform to control the conformational state of the protein in each step of the cycle.

Figure 3. Schematic depiction of the sequence of conformational changes involved in the SRP cycle. (A) SRP recognizes the RNC and the M domain interacts with the signal peptide. (E) In the final step, signal sequence is transferred from the M-domain to the translocon (Sec YEG) and the distal region of the RNA promotes GTP hydrolysis and subsequent Ffh and FtsY dissociation.

 


 

Research conducted by: S.F. Ataide, N. Schmitz, and N. Ban (Institute of Molecular Biology and Biophysics, Zurich, Switzerland), A. Ke (Cornell University), K. Shen and S. Shan (California Institute of Technology), and J.A. Doudna (Howard Hughes Medical Institute, UC Berkeley, and Berkeley Lab).

Research funding: U.S. Department of Energy (DOE), Office of Basic Energy Sciences (BES). Operation of the ALS is supported by DOE BES.

Publication about this reserach: S.F. Ataide, N. Schmitz, K. Shen, A. Ke, S. Shan, J.A. Doudna, and N. Ban, "The Crystal Structure of the Signal Recognition Particle in Complex with Its Receptor," Science 331, 881 (2011).

ALS Science Highlight #242

 

ALSNews Vol. 328