br The SRP SR Heterodimer GTPase
The SRP/SR Heterodimer: GTPase Tangos Drive Co-Translational Protein Targeting SRP and SR mediate a universally conserved protein targeting pathway responsible for the delivery of 25–30% of newly synthesized proteome to the eukaryotic endoplasmic reticulum (ER) or the bacterial plasma membrane 11, 12, 13. This process begins when a nascent membrane or secretory protein emerges from a translating ribosome. These proteins carry hydrophobic signal sequences or transmembrane domains (TMDs) near the N terminus that are recognized, together with the ribosome, by SRP. Via the interaction of SRP with SR, the ribosome–nascent chain complex (RNC) is delivered to the membrane. There, the RNC is transferred to the SecYEG (or Sec61p in eukaryotes) protein translocation channel, or translocon, which allows the nascent protein to integrate into or translocate across the membrane. Efficient and accurate co-translational protein targeting requires spatial and temporal control, which is provided by the two highly homologous GTPases, SRP54 and SR (Figure 1, Figure 2). Both proteins contain a P-loop GTPase domain, termed the G domain, which contains the GI–GIV sequence motifs conserved in most GTPases 14, 15. In both proteins, an N-terminal four-helix bundle termed the N domain packs against the G domain to form a structural and functional unit called the NG domain (Figure 2A, top structures) 16, 17. In addition to the NG domain, SRP54 contains a methionine-rich M domain (Figure 2A top, dark blue) that provides umbelliferone for the SRP RNA and for signal sequences or TMDs on the nascent polypeptide 18, 19, 20. Bacterial SR has an acidic A domain that mediates interactions with phospholipids 21, 22 and possibly with the SecYEG translocon . Eukaryotic SR is anchored on the ER membrane via association of the SRα subunit (containing the NG domain) with SRβ, an integral membrane protein 22, 23, 24, 25. Unlike the Ras-type GTPases, free SRP and SR do not exhibit significant conformational changes between the apo-, GDP-, and GTP-bound states 16, 17, 26, 27, 28. Further, their NG domains contain wide-open nucleotide binding sites (Figure 2, top panel) 16, 17, consistent with biochemical data showing that these GTPases exhibit weak nucleotide affinities and GDP–GTP exchange rates that are 104–106-fold faster than those of Ras-type GTPases 29, 30, 31, 32. Thus, there is no need to recruit an external GEF to turn these GTPases to the ‘on’ state. Moreover, the GII motif, which contains multiple catalytic residues, is disordered and suboptimally aligned with the bound GTP in free SRP and SR, consistent with their low basal GTPase rates . Notably, GTP hydrolysis is enhanced over 104-fold when SRP and SR assemble a complex 32, 33. Thus, there is also no need to recruit an external GAP to turn these GTPases to the ‘off’ state. These features strongly suggest that SRP and SR use a mode of regulation distinct from the classical GTPase switch paradigm. A combination of molecular genetics, fluorescence spectroscopy, and structural analyses, primarily focused on the bacterial SRP and SR, demonstrated that their GTPase cycle is instead driven by multiple conformational changes during SRP–SR dimerization, which culminates in reciprocal GTPase activation (Figure 2A). Free SRP and SR are in an inactive ‘open’ conformation suboptimal for binding one another (Figure 2A, top) 32, 34, 35. Their interaction begins with a transient ‘early’ intermediate, which is primarily stabilized by electrostatic attractions between their N domains and by contacts of the GGAA tetraloop of the SRP RNA with a conserved lysine on the SR G domain (Figure 2A, right structure) 36, 37, 38. Subsequent rearrangements in both proteins, involving adjustments at the NG domain interface, generate a stable ‘closed’ complex in which the G and N domains of SRP and SR together form an extensive interaction surface (Figure 2A, bottom structure) 37, 39, 40. The two GTP molecules also hydrogen bond across the dimer interface, further stabilizing the ‘closed’ complex and conferring its specificity for GTP. Finally, cooperative rearrangements in the GII motifs of both proteins bring key catalytic residues into close contact with the two bound GTP molecules, forming a composite active site at the dimer interface (Figure 2A, Step 4) 39, 40, 41. These movements of the GII motifs are coupled to a 100Å movement of the SRP–SR NG-domain dimer from the tetraloop to the 5′, 3′ distal site of the SRP RNA, where a conserved cyanine base inserts into and further optimizes the GTPase active site to generate the ‘activated’ complex (Figure 2A, left structure) 42, 43, 44. Stimulated GTP hydrolysis then drives the disassembly and recycling of the complex (Figure 2A, Step 5).