NMR structure of the Vibrio vulnificus ribosomal protein S1 domains D3 and D4 provides insights into molecular recognition of single-stranded RNAs

Abstract

The ribosomal S1 protein (rS1) is indispensable for translation initiation in Gram-negative bacteria. rS1 is a multidomain protein that acts as an RNA chaperone and ensures that mRNAs can bind the ribosome in a single-stranded conformation, which could be related to fast recognition. Although many ribosome structures were solved in recent years, a high-resolution structure of a two-domain mRNA-binding competent rS1 construct is not yet available. Here, we present the NMR solution structure of the minimal mRNA-binding fragment of Vibrio Vulnificus rS1 containing the domains D3 and D4. Both domains are homologues and adapt an oligonucleotide-binding fold (OB fold) motif. NMR titration experiments reveal that recognition of miscellaneous mRNAs occurs via a continuous interaction surface to one side of these structurally linked domains. Using a novel paramagnetic relaxation enhancement (PRE) approach and exploring different spin-labeling positions within RNA, we were able to track the location and determine the orientation of the RNA in the rS1-D34 bound form. Our investigations show that paramagnetically labeled RNAs, spiked into unmodified RNA, can be used as a molecular ruler to provide structural information on protein-RNA complexes. The dynamic interaction occurs on a defined binding groove spanning both domains with identical β2-β3-β5 interfaces. Evidently, the 3'-ends of the cis-acting RNAs are positioned in the direction of the N-terminus of the rS1 protein, thus towards the 30S binding site and adopt a conformation required for translation initiation.

Figure 1.

Resonance assignment and backbone dynamics of rS1–D34. (A) The ¹H,¹⁵N-HSQC spectrum of ¹⁵N–rS1–D34 (1.3 mM) was acquired at 950 MHz and 303 K in 25 mM potassium phosphate (pH 7.2), 150 mM KCl, 5 mM DTT, 5% D₂O. The resonance assignment is annotated. Side chain NH resonances are assigned in blue. The backbone amide resonance of T221 is not visible at the given contour level. A schematic of the rS1 is shown on top. The rS1–D34 region is indicated with a box. (B) R₁, R₂ and {¹H}, ¹⁵N heteronuclear NOE experiments were acquired at 308 K and 600 MHz on a 250 μM ¹⁵N-labeled rS1–D34 sample. Relaxation rates and HetNOE values are given as function of protein sequence. Ambiguous resonances were excluded. Secondary structure elements are shown on the top and were predicted with TALOS-N (43). Flexible regions (HetNOE < 0.6, below black dashed line) are highlighted with blue boxes.

Figure 2.

NMR solution structure of rS1–D34. (A) Ensemble of 20 lowest-energy structures is shown with view on the RNA-binding interface (β2–β3–β5). The protein is represented as cartoon and color coded from N- (green) to C-terminus (blue). In the right panel the protein is rotated by 180°. (B) The topology of each domain is depicted and color coded. (C) Enhanced view of each domain is shown with focus on the RNA binding interface. The interacting residues are displayed.

Figure 3.

CSP mapping on rS1–D34 structure of all 5′-UTRs. The CSPs are color coded from blue (no CSP) to red (maximal CSP). Resonances under intermediate exchange conditions are shown in yellow. Enlarged structure in presence of 5.5 eq preQ1-(III)-14 is shown on the left-hand side in cartoon representation and transparent surface. Interacting amino acids are annotated. Maximal CSPs cluster around the same region for different RNAs. In case of preQ1(I)-14, response to increased concentration of RNA could not be observed after addition of the first RNA equivalent. With 5.5 eq RNA at a protein concentration [100 uM], no saturation could be achieved. This RNA oligomerizes to form G-quadruplex structures. At this stoichiometry, the key interacting residues (e.g. Y205/Y290) were not detected, as they were under intermediate exchange conditions.

Figure 4.

Percentage intensity reductions of paramagnetic samples relative to the diamagnetic reference. All samples contained rS1–D34 and four equivalents of RNA, while the paramagnetic samples were spiked with 10% spin-labeled RNA. Strong changes are highlighted from blue to red in the 3D structure. Overlapping peaks were excluded from the intensity plots and are shown in gray in the 3D structure. Most affected residues are annotated.