|Signal Recognition Particle-Receptor Complex Structure Solved|
|Monday, 27 February 2012 15:06|
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.
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?
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.
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.
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