Heteronuclear NMR investigations of dynamic regions of intact Escherichia coli ribosomes

Abstract

15N-(1)H NMR spectroscopy has been used to probe the dynamic properties of uniformly (15)N labeled Escherichia coli ribosomes. Despite the high molecular weight of the complex ( approximately 2.3 MDa), [(1)H-(15)N] heteronuclear single-quantum correlation spectra contain approximately 100 well resolved resonances, the majority of which arise from two of the four C-terminal domains of the stalk proteins, L7/L12. Heteronuclear pulse-field gradient NMR experiments show that the resonances arise from species with a translational diffusion constant consistent with that of the intact ribosome. Longitudinal relaxation time (T(1)) and T(1 rho) (15)N-spin relaxation measurements show that the observable domains tumble anisotropically, with an apparent rotational correlation time significantly longer than that expected for a free L7/L12 domain but much shorter than expected for a protein rigidly incorporated within the ribosomal particle. The relaxation data allow the ribosomally bound C-terminal domains to be oriented relative to the rotational diffusion tensor. Binding of elongation factor G to the ribosome results in the disappearance of the resonances of the L7/L12 domains, indicating a dramatic reduction in their mobility. This result is in agreement with cryoelectron microscopy studies showing that the ribosomal stalk assumes a single rigid orientation upon elongation factor G binding. As well as providing information about the dynamical properties of L7/L12, these results demonstrate the utility of heteronuclear NMR in the study of mobile regions of large biological complexes and form the basis for further NMR studies of functional ribosomal complexes in the context of protein synthesis.

Fig. 1.

[¹H-¹⁵N] HSQC spectra of uniformly ¹⁵N labeled E. coli ribosomes. Spectra of (A) intact 70S; (B) 50S; (C) 30S ribosomes; and (D) overlay of spectra of the 50S and 30S subunits, acquired at 750 MHz in 10 mM KCl/10 mM MgCl₂/10 mM KHPO₄, pH 7.2, 303 K. Peaks in B are labeled according to the assignments of isolated L7/L12 protein, because the chemical shift values are essentially identical [the differences are less than ± 0.02 (¹H) and ±0.1 (¹⁵N) ppm (11)]. Crosspeaks likely to originate from A39, V40, and A41 in the interdomain hinge are labeled with an asterisk. The deviations of the chemical shifts of these signals from those of isolated L7/L12 are greater than for other labeled peaks, and multiple resonances are assigned for these residues in the isolated protein.

Fig. 2.

Pulsed-field gradient–NMR characterization of the intact ribosome. Relative diffusion coefficients of the 70S and 50S ribosomes and of the small monomeric proteins calcium-dependent protein kinase and CA. The data for CA-2 show the reduced diffusion resulting from CA being added to a solution containing the 50S subunit (CA-1 corresponds to measurements with the isolated protein). Assuming constant densities and spherical particles, then M₁/M₂ = (D₂/D₁)³ (where M₁ and M₂ are the molecular masses, and D₁ and D₂ are the diffusion coefficients of proteins 1 and 2, respectively). These values provide an estimate of the apparent molecular mass for the ribosomal particles in excess of 1 MDa.

Fig. 3.

¹⁵N-spin relaxation properties of ribosomal L7/L12. (A) T₁ and (B) T₂ relaxation times of L7/L12 incorporated in the 50S subunit. T₂ values are derived from the R_1ρ experiments, as detailed in Supporting Text. Average errors, 8% and 12%, for the T₁ and T₂ relaxation times, respectively, are relatively large through low S/N ratios resulting from the need to minimize accumulation times to avoid sample degradation (see text). Residues removed by the R₁/R₂ filter (see Results) are marked x at the bottom. (C) Values of the apparent rotational correlation time measured from the relaxation times. Errors in values are ≈±1.4 ns, based on the average errors in the T₁ and T₂ values given above; error bars for have, however, been omitted for clarity. Average values for the α-helices are indicated with thick lines and their standard deviations as thin lines above and below each helical region. (D) Order parameters, S² (based on an average of 13.6 ns). Values for residues 90, 100, 106, and 107 are omitted, because they are affected by conformational exchange.

Fig. 4.

Representation of the orientation of L7/L12 on the ribosome. (A) Orientation of the CTD of an L7/L12 protein derived from rotational anisotropy analysis of relaxation data. L7/L12 is positioned within the cone in an extended conformation [modified version of Protein Data Bank (PDB) 1ctf, with the hinge residues (43–51) modeled as a random coil]. Helical angle orientations are 66°,63°, and 32° for helices I, II, and III, respectively, relative to the y axis and the long axis of the diffusion tensor. (B) The hinge and CTD regions of L7/L12 (residues 43–120), modeled into the structure of the 50S subunit from T. thermophilus [(6) PDB 1giy] with L11 (in orange indicating the base of the stalk), rRNA (cyan), and the remaining ribosomal proteins (dark blue). L7/L12 is drawn in various colors and positioned in six orientations representing a full rotation around the principal axis (gray line) as seen in A. In this model, the cone representing the possible range of orientations of L7/L12 is placed in an arbitrary position similar to that of the ribosomal stalk, although the precise orientation of the CTD of L7/L12 relative to the ribosomal body is unknown.