Reconstitution of the human SRP system and quantitative and systematic analysis of its ribosome interactions

Abstract

Co-translational protein targeting to membranes depends on the regulated interaction of two ribonucleoprotein particles (RNPs): the ribosome and the signal recognition particle (SRP). Human SRP is composed of an SRP RNA and six proteins with the SRP GTPase SRP54 forming the targeting complex with the heterodimeric SRP receptor (SRαβ) at the endoplasmic reticulum membrane. While detailed structural and functional data are available especially for the bacterial homologs, the analysis of human SRP was impeded by the unavailability of recombinant SRP. Here, we describe the large-scale production of all human SRP components and the reconstitution of homogeneous SRP and SR complexes. Binding to human ribosomes is determined by microscale thermophoresis for individual components, assembly intermediates and entire SRP, and binding affinities are correlated with structural information available for all ribosomal contacts. We show that SRP RNA does not bind to the ribosome, while SRP binds with nanomolar affinity involving a two-step mechanism of the key-player SRP54. Ultrasensitive binding of SRP68/72 indicates avidity by multiple binding sites that are dominated by the C-terminus of SRP72. Our data extend the experimental basis to understand the mechanistic principles of co-translational targeting in mammals and may guide analyses of complex RNP-RNP interactions in general.

Figure 1.

Co-translational targeting by the SRP system. (A) Scheme for the mammalian SRP system and the targeting of RNCs to the translocon in the ER membrane. SRP consists of six proteins (numbered by molecular weight in kDa) bound to SRP RNA acting as scaffold. The Alu domain reaches into the factor binding site within the ribosomal 40S/60S subunit interface and the S domain binds to the signal sequence (ss) emerging from the polypeptide exit tunnel (exit) in the signal pre-handover state (left). The multi-domain SRP GTPase SRP54 recognizes the signal with its M domain and establishes the targeting complex consisting of its NG domain bound to the homologous NG domain of the SRP receptor SRα at a proximal ribosome binding site. SRP68/72 are large solenoids including RBD and protein-binding modules. Upon signal handover to the translocon (right), the GTP-dependent (T) stable targeting complex is activated at a distal SRP RNA site (5e and 5f loops; *: activation). The SRX domain of SRα regulates the membrane-anchored Ras-like SRβ GTPase (D: GDP). (B) SDS-PAGE gels of fully recombinantly reconstituted and purified SRP/SR complexes (Coomassie and methylene blue stained). SRPS, SRPS/SR and entire SRP can be purified to homogeneity in large scale up to milligram amounts. SRP54 is added either as full-length protein or as C-terminal deletion (ΔC) lacking the flexible signal-enclosing and exit-binding region.

Figure 2.

MST data for SRPS assembly complexes and SRP68/72 with the 80S ribosome. (A) SRPS assembly: SRP RNA, SRP19 and the binary complex thereof do not bind to the ribosome. The Alu domain including the SRP9/14 heterodimer is not a primary ribosomal-binding site. Low affinity SRPS binding to the ribosome is induced by the SRP68/72 heterodimer. The interaction involves more than the SRP68/72 RBD-domains previously characterized as ribosome binders at the C4-contact. (B) SRP68/72: Ribosome binding of heterodimer constructs. SRP72–RBD alone binds weakly and the large solenoidal parts of SRP68/72 contribute significantly. The flexible very C-terminus of SRP72 is relevant for ribosome binding. ^$: Binding of SRP72 is ultrasensitive (Hill-model with EC50 values), indicative for multiple binding sites and an avidity mechanism.

Figure 3.

MST data for SRP54 proteins and SRPS complexes. (A) SRP54: The conserved SRP key-player binds with high affinity (K_D1) to the ribosome. MST data at higher concentration show aggregation (gray dots). The binding curve for the stable SRP54ΔC construct, missing a flexible C-terminus adjusting between ribosome and signal sequences, is double-sigmoidal and reveals a high (K_D1) and low (K_D2) affinity binding event (^§: overall affinity). Homologous SRP54 proteins from bacteria (Escherichia coli) and chloroplasts (Arabidopsis thaliana) show a significantly reduced affinity to human ribosomes. (B) SRPS ternary complex: SRP54 assembly into the binary SRP RNA/SRP19 complex results in high affinity binding of the ternary complex. Binding of SRP54 is almost saturated (saturation curves are indicated with an asterisk) at used ribosome concentrations (given as vertical line), and K_D-values can only be estimated to be at lower concentrations. A Hill-model is used to optimally fit the saturation curve. (C) SRPS complexes: Ribosome binding of entire SRPS occurs way below the ribosome concentrations for both SRP54 and its truncated construct SRP54ΔC and saturation (kinking of the curve) is more pronounced.

Figure 4.

High and low affinity binding of SRP54 domains with the ribosome. (A) Two-site binding analysis of the SRP54ΔC–ribosome interaction using program PALMIST (30) (upper panel: fluorescence intensity data, lower panel: two-site fitted relative fluorescence with residuals). High affinity binding (K_D1) occurs below 100 nM, while low affinity is in the micromolar range (K_D2) and does not reach complete saturation. (B) Split sub-curve single binding event analyses using the single-site analysis software (MO.Affinity). Top: High affinity binding yields a value of roughly 75 nM and might correspond to M-domain binding. Bottom: Fitting of the high concentration data confirms the low micromolar affinity. Note: Data evaluation is performed at the same (early, 5 s) time windows for both types of analyses. (C) MST measurements of SRP54 domains. Upper panels: Raw MST data and binding curve for SRP54MΔC. The domain binds with high affinity with a K_D of 100 nM (K_D1). Lower panels: SRP54NG binds in the micromolar range (K_D2) as estimated from the SRP54ΔC measurement. Notes: (i) MST data for highest concentrations of SRP54NG and at late MST times show aggregation effects. (ii) A putative two-site binding of SRP54NG is not evaluated.

Figure 5.

MST data for entire SRP and SRP receptor complexes with and without signal. (A) Entire SRP: Fully assembled recombinant SRP binds with high affinity in the low nanomolar range to empty human ribosomes (Hill-fitted saturation curve). The original MST trace is shown as typical example, highlighting the data quality even at lowest fluorophore concentrations. (B) SRP receptor (SR) and SRPS/SR complexes: The SRαβ heterodimer binds to the ribosome by itself with high affinity. Interaction occurs via a positively charged RBR in the X-NG linker of SRα. All SRPS/SR complexes bind to the ribosome in the sub-nanomolar range. (C) SRPS/SR with signal sequences: SRPS/SR with SRP54 fused to a signal sequence binds at least as good as without signal (sharp kink in saturation curve). Ribosome binding using RNCs with a stalled translation and exposing an SRP substrate occurs with sub-nanomolar affinity and validates the MST setup. Due to the pre-handover state (bound signal before translocon docking), the TC is drawn at the proximal site of the SRP RNA. ^#: The RNCs (rabbit reticulocyte lysate ribosomes) behave differently in the MST measurements (highlighted in red).

Figure 6.

Scheme of SRP/SR/ribosome interactions in the SRP cycle. Left panel: SRP(S) strongly binds to the ribosome in the scanning state (individual contributions in nM determined by MST are discerned). SRP54 reveals a dual binding mode by its M (high affinity (1)) and NG domains (low affinity (2)). Contributions of the Alu domain and SRβ are not defined. SRP68/72 interactions (4) are ultrasensitive, include more than the C4-contact (3) established by SRP72–RBD, and involve the C-terminus (C). Middle panel: in the pre-handover state (engaged) interactions are reinforced mainly by signal sequence recognition of SRP54M (1′) and SRP54NG is locked in place (12,14). Upon SR binding, ribosome affinity is as strong although the TC complex of the NG domains is known to dissociate from the ribosome (20). Right panel: Upon translocon docking, the TC relocates and the signal is handed-over. No structure of any post-handover complex is available and individual contributions are elusive. TC re-localization induces a rotation of SRPS in respect to the ribosome and a modification of the C4-contact (3′) at the distal site (20,44). Only upon GTP-hydrolysis, SRP dissociates from the ribosome (indicated by asterisks) and the SRP cycle is completed. ^#: MST data for SRPS/SR indicate a very tight RNC interaction before GTP-hydrolysis occurs (although the Alu domain and translocon were missing in the assay).