The crystal structure of the signal recognition particle in complex with its receptor

Abstract

Cotranslational targeting of membrane and secretory proteins is mediated by the universally conserved signal recognition particle (SRP). Together with its receptor (SR), SRP mediates the guanine triphosphate (GTP)-dependent delivery of translating ribosomes bearing signal sequences to translocons on the target membrane. Here, we present the crystal structure of the SRP:SR complex at 3.9 angstrom resolution and biochemical data revealing that the activated SRP:SR guanine triphosphatase (GTPase) complex binds the distal end of the SRP hairpin RNA where GTP hydrolysis is stimulated. Combined with previous findings, these results suggest that the SRP:SR GTPase complex initially assembles at the tetraloop end of the SRP RNA and then relocalizes to the opposite end of the RNA. This rearrangement provides a mechanism for coupling GTP hydrolysis to the handover of cargo to the translocon.

Fig. 1. Structure of Signal Recognition Particle in complex with its Receptor

(A) Top view of the SRP:SR complex. Ffh is colored in blue, 4.5S RNA in gray, FtsY (SR) is shown in green. The atoms of the two GMP-PCP molecules are displayed as red spheres. (B) Side view of the SRP:SR complex rotated 70° relative to the view in (A). 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. (C) Visualization of the 2Fo−Fc electron density contoured at 1σ and the stick representation of the SRP:SR complex. (D) Side view of the SRP:SR complex with the contour of a 2Fo−Fc unbiased omit map calculated for linker region residues 300–330 of Ffh. The linker is displayed as a tube together with the difference density Fo−Fc electron density contoured at 3σ, shown as green mesh. The 2Fo−Fc electron density for the entire complex is contoured at 1σ and displayed as gray mesh. The cover radius used for the figure had a cutoff of 2.6 Å for the 2Fo−Fc and Fo−Fc.

Fig. 2. Interaction of the Ffh-SR NG domain with a conserved flipped base at 5′-3′ distal end of the 4.5S RNA

(A) Secondary structure of the D. radiodurans 4.5S RNA with conserved residues indicated in red (sequence alignment displayed in fig. S4). Base pairings are indicated as (−) and non-canonical interactions are indicated as (#), bulged residues are unpaired. (B) Overall view of the interaction of Ffh and FtsY (displayed as ribbons and colored as in Fig. 1A) with the 4.5S RNA (represented as a contoured surface colored according to the conservation indicated in fig. S4). GMP-PCP molecules are shown as red spheres. (C) Close-up view of the interaction of the conserved flipped C83 with the interface of Ffh and FtsY. 4.5S RNA is displayed as sticks colored in gray with C83 colored in orange. Ffh and FtsY residues that interact directly with C83 are displayed as sticks and colored as in (B). GMP-PCP residues are represented as sticks colored with white carbons, red oxygens, blue nitrogens and orange phosphates.

Fig. 3. Major contact area of the Ffh:FtsY NG domain with 5′,3′-region of the 4.5S RNA

(A) Surface representation of the SRP:SR complex in the 5′,3′-region of the 4.5S RNA. (B) Surface representation of the separated Ffh and FtsY (rotate each to one direction) from the 4.5S RNA (maintained in the same orientation as in A). The interface between Ffh and FtsY is displayed in white in both proteins with GMP-PCP displayed as red spheres. The contact area of each protein to the RNA is indicated in gray. The FtsY contact area in the RNA is indicated in green and the Ffh contact area in blue, C83 is indicated in blue but contacted by both proteins.

Fig. 4. The SRP RNA distal end specifically stimulates GTP hydrolysis in the SRP:SR complex

(A) E. coli SRP RNA systematically truncated from the distal end that were used in this study (see fig. S7A for the sequence of the truncated RNA constructs). The FtsY contact area in the RNA is indicated in green and the Ffh contact area in blue, C86 is indicated in blue but contacted by both proteins. (B) Stimulated GTPase activity between SRP and FtsY with the SRP RNA mutants in part A. The data were fit to the Michaelis-Menten equation, and the kinetic constants k_cat and k_cat/K_m are summarized in figure S7B. (C) A significant defect in the GTPase rate of the SRP:FtsY complex was observed upon truncation of the SRP RNA from the 92mer to 82mer. Rate constants were from part B and normalized to that of the wild-type SRP RNA. (D) Truncation of the RNA distal end did not significantly affect SRP-FtsY complex formation rates (k_on) until the C loop is truncated (43mer). Values of k_on were derived from figure S7B. (E) Mutation of the conserved base C86 (C83 in D. radiodurans 4.5S RNA) impairs the ability of SRP RNA to stimulate GTPase hydrolysis of the SRP:FtsY complex. Rate constants were relative to that of the wild type SRP RNA and were derived from the data in figure S7D.

Fig. 5. Conformation of the SRP and SRP:SR structure on the ribosome showing the large rearrangement between cargo binding and cargo release modes

(A) The structure of the SRP:SR is superimposed on the SRP:RNC bound structure from cryo-EM reconstruction (33), with Ffh in the cryo-EM structure omitted. The signal peptide from the SRP:RNC structure was maintained (red surface) as a reference for the exit tunnel. The RNC is displayed as a white surface with the protein L23 highlighted in yellow. SRP:SR are presented as spheres with 4.5S RNA colored with dark gray for phosphate and ribose and light gray for bases, Ffh is blue and FtsY is green. (B) The cargo binding conformation of SRP in the SRP:RNC model structure from cryo-EM is indicated as spheres with Ffh colored in purple.

Fig. 6. Model of the SRP targeting cycle

Schematic depiction of the sequence of conformational changes involved in the SRP cycle (viewed in the membrane plane). (A) SRP recognizes the RNC and M-domain interacts with the signal peptide (finger loop is indicated in pink). (B) The N-domain interacts with L23 and the linker region folds on top of the signal peptide covering/shielding the M-domain. (C) SRP bound cargo (RNC) is transferred to the membrane vicinity via SRP interaction with SR. (D) GTP-dependent rearrangements in the SRP:SR complex enables detachment of the Ffh N-domain from L23 and the RNA tetraloop, and the NG domain complex relocates to the 5′,3′-end of the 4.5S RNA sandwiching the C83 (orange arrowhead) at the interface of the two G domains. This repositioning simultaneously exposes the signal sequence bound to the M-domain, and the L23 region of the ribosome for interactions with the translocon (Sec YEG), which is associated with the A-domain of FtsY (black tail). In the final step, signal sequence is transferred from the M-domain to the translocon and the distal region of the 4.5S RNA promotes GTP hydrolysis and subsequent Ffh and FtsY dissociation.