Homodimerization of the G protein SRβ in the nucleotide-free state involves proline cis/trans isomerization in the switch II region

Abstract

Protein translocation across and insertion into membranes is essential to all life forms. Signal peptide-bearing nascent polypeptide chains emerging from the ribosome are first sampled by the signal-recognition particle (SRP), then targeted to the membrane via the SRP receptor (SR), and, finally, transferred to the proteinconducting channel. In eukaryotes, this process is tightly controlled by the concerted action of three G proteins, the 54-kD subunit of SRP and the ␣-and ␤-subunits of SR. We have determined the 2.2-Å crystal structure of the nucleotide-free SR␤ domain. Unexpectedly, the structure is a homodimer with a highly intertwined interface made up of residues from the switch regions of the G domain. The remodeling of the switch regions does not resemble any of the known G protein switch mechanisms. Biochemical analysis confirms homodimerization in vitro, which is incompatible with SR␣ binding. The switch mechanism involves cis͞trans isomerization of a strictly conserved proline, potentially implying a new layer of regulation of cotranslational transport.cotranslational transport ͉ proline isomerization ͉ signal-recognition particle receptor N ascent proteins, destined for secretion or membrane insertion, are first targeted to the endoplasmic reticulum (ER) in eukaryotes or the plasma membrane in bacteria. Targeting in eukaryotes occurs primarily via the conserved signal-recognition particle (SRP) pathway (1). This cyclic pathway starts with SRP, a ribonucleoprotein complex consisting of the 300-nucleotide SRP-RNA and six proteins, recognizing a ribosome-nascent chain (RNC) complex and arresting translation. In a second step, the stalled SRP-RNC complex docks with the ER membranebound SRP receptor (SR) (2, 3), which is dynamically associated with the protein-conducting channel (PCC or Sec61 complex) (4). Third, the RNC is detached from the SRP, binds to the PCC, and releases the nascent polypeptide chain into an aqueous pore in the channel (5, 6). Protein synthesis can then continue. Concomitantly, SRP and SR dissociate, ready for a new cycle of protein targeting. GTP binding and hydrolysis by three synchronized G proteins regulate the process and ensure unidirectionality (7,8).Intense investigation of the protein targeting process by x-ray crystallography and cryoelectron microscopy has lead to the structural characterization of domains and interactions (9, 10). As a result of these advances, several key steps are now relatively well understood. The recent cryoelectron microscopic structure of the SRP-RNC complex shows how the elongated SRP wraps around the large ribosomal subunit, binding the N-terminal signal sequence of the nascent chain at the exit tunnel via SRP54 at one end of the molecule and arresting translation by blocking the elongation-factor-binding site with its other end (11). SRP54, the only SRP protein with a bacterial homologue, has two functional domains; the C-terminal M domain is the signal sequence-binding interface, and the N-terminal composite NG domain has regulatory GTPase acti...

Structure of the GTPase and GDI domains of FeoB, the ferrous iron transporter of Legionella pneumophila

“…Interestingly, the position of this loop indicates a “closed conformation” of the GTPase site, which is usually only observed for the nucleotide‐bound state of GTPases and also for NFeoB Ec in its GDP and GTP states (PDB codes 3I8X and 3I92; NP et al, to be published elsewhere). However, a closed conformation of the G5 loop in nucleotide‐free GTPase structures is not unprecedented and has been observed in Rac1 [28] and SRβ [29] (Fig. 2D).…”

“…In GTPases, the binding of GTP induces conformational changes mediated by the flexible switch I and switch II regions that lead to an activation of the protein [25]. In contrast to structures of several other GTPases, the switch I region is well defined in NFeoB Lp (residues [24][25][26][27][28][29][30][31][32][33][34][35][36][37][38]. It includes the b2 E -strand , which is hydrogenbonded to b2 of the central b-sheet in an antiparallel fashion.…”

Initiation of Phage Infection by Partial Unfolding and Prolyl Isomerization

“…It has long been known that prolyl isomerizations play important roles as rate-determining steps in protein folding, but more recently, evidence has accumulated that prolyl isomerizations in folded proteins are involved in a multitude of regulatory processes (22)(23)(24)(25)(26)(27). Also, it was proposed that the time course of proline-controlled signaling events is modulated by prolyl isomerases such as cyclophilin or Pin1 (28,29).…”

Elucidation of the FKBP25-60S Ribosomal Protein L7a Stress Response Signaling During Ischemic Injury

“…FKBPs, as FK506-binding proteins, have in common peptidyl-prolyl cis/trans isomerase activity [4,5], but the precise role in ischemia damage remains largely unknown. Here, we found that FKBP25 translocated from the cytosol to nucleus under OGD and ONOO -treatment in endothelial cells, which is independent of the first 108 residues of the coding sequence of FKBP25.…”