Solution structures of DEAD-box RNA chaperones reveal conformational changes and nucleic acid tethering by a basic tail

Abstract

The mitochondrial DEAD-box proteins Mss116p of Saccharomyces cerevisiae and CYT-19 of Neurospora crassa are ATP-dependent helicases that function as general RNA chaperones. The helicase core of each protein precedes a C-terminal extension and a basic tail, whose structural role is unclear. Here we used small-angle X-ray scattering to obtain solution structures of the full-length proteins and a series of deletion mutants. We find that the two core domains have a preferred relative orientation in the open state without substrates, and we visualize the transition to a compact closed state upon binding RNA and adenosine nucleotide. An analysis of complexes with large chimeric oligonucleotides shows that the basic tails of both proteins are attached flexibly, enabling them to bind rigid duplex DNA segments extending from the core in different directions. Our results indicate that the basic tails of DEAD-box proteins contribute to RNA-chaperone activity by binding nonspecifically to large RNA substrates and flexibly tethering the core for the unwinding of neighboring duplexes.

Fig. 1.

DEAD-box proteins Mss116p and CYT-19 and nucleic acid substrates. (A) Schematic representations of the domain architectures of Mss116p, CYT-19, and deletion mutants. Mss116p consists of a mt targeting sequence, which is cleaved in vivo and absent in the constructs used here (white); an N-terminal extension, which corresponds to the N-terminus of the mature proteins (NTE; dark blue); a helicase core of two RecA-like domains (domain 1 and domain 2; light blue and green, respectively), which are joined by a flexible linker (gray); a structured C-terminal extension (CTE; orange); and a basic hydrophilic tail (C-tail; red). CYT-19 consists of the same elements but with a shorter NTE. (B) The crystal structure of the closed-state helicase core and the CTE of Mss116p [Protein Data Bank (PDB) ID code 3I6I (22)] with domains colored as in A. The bound single-stranded U₁₀-RNA and the ATP-analogue are shown in yellow and black, respectively. (C) Schematic representations of nucleic acid substrates. RNA and DNA nucleotides are shown in yellow and gray, respectively, and nucleic-acid secondary structure was predicted using RNAfold (http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi).

Fig. 2.

SAXS analysis of Mss116p in the open state without substrates. SAXS data are shown for full-length Mss116p (dark blue), Mss116p/ΔC-tail (light blue), Mss116p/ΔNTE (green), and Mss116p/ΔNTE + ΔC-tail (red). (A) Scattering profiles, which are displaced along the logarithmic axis for visualization, are shown as the logarithm of the scattering intensity, I (black dots), as a function of the momentum transfer, q = 4π sin(θ)/λ, where 2θ is the scattering angle and λ is the X-ray wavelength. The solid curves overlaying the SAXS data are the expected scattering profiles of the corresponding BUNCH models (see below). (B) Normalized distance distribution functions calculated from the scattering profiles using the program AUTOGNOM (27). (C–F) Ab initio and rigid-body SAXS reconstructions of the open state of full-length Mss116p (C), Mss116p/ΔC-tail (D), Mss116p/ΔNTE (E), and Mss116p/ΔNTE + ΔC-tail (F). Low-resolution envelopes calculated by DAMMIN are shown separately (Upper) and superposed onto atomic models determined by BUNCH (Lower). In this and other figures, protein domains are colored as in Fig. 1 and views are rotated by 90 ° about the vertical axis for each model.

Fig. 3.

SAXS analysis of Mss116p ternary complexes with ssRNA and a nonhydrolyzable ATP analog. (A) Scattering profiles and (B) normalized distance distribution functions for full-length Mss116p (dark blue), Mss116p/ΔC-tail (light blue), Mss116p/ΔNTE (green), and Mss116p/ΔNTE + ΔC-tail (red) bound to U₁₀-RNA and ADP-BeF_x. In (A), the scattering profiles are shown as black dots, and the solid curves overlaying the data are the expected scattering profiles of the corresponding BUNCH models, except for the Mss116p/ΔNTE + ΔC-tail complex where the overlay is the expected scattering profile calculated from the corresponding X-ray crystal structure using CRYSOL (29). (C–F) SAXS reconstructions of full-length Mss116p (C), Mss116p/ΔC-tail (D), Mss116p/ΔNTE (E), and Mss116p/ΔNTE + ΔC-tail (F) in the closed state. Low-resolution envelopes calculated by DAMMIN (Upper) are colored as in (A) and atomic models (Lower) are shown aligned inside the DAMMIN envelope (gray).

Fig. 4.

Binding modes of Mss116p to large nucleic acid substrates. (A) Scattering profiles and (B) normalized distance distribution functions for Mss116p/ΔNTE (green) and Mss116p/ΔNTE + ΔC-tail (red) bound to ADP-BeF_x and either RNA–DNA duplex 1 (solid lines) or RNA-DNA duplex 2 (dashed lines). In (A), the scattering profiles are shown by black dots, and the colored lines represent the fit of the ab initio model of the complex obtained by MONSA. (C–F) Ab initio multiphase reconstructions from the SAXS data in (A) of complexes of Mss116p/ΔNTE + ΔC-tail with either RNA–DNA duplex 1 (substrate 1; C) or RNA–DNA duplex 2 (substrate 2; D) and complexes of Mss116p/ΔNTE with either RNA–DNA duplex 1 (E) or RNA–DNA duplex 2 (F). Two-phase models of protein (colored as above) and nucleic acid (yellow) were reconstructed by MONSA (Upper). The ssRNA regions of these substrates were assumed to bind within the helicase core in the orientation observed in the crystal structure of the closed state of Mss116p (Fig. 1B). This information was used to place manually atomic models for protein and nucleic acid inside the corresponding SAXS envelopes (Lower).

Fig. 5.

SAXS analysis of CYT-19. (A–C) SAXS data for full-length CYT-19 (green) and CYT-19/ΔC-tail (red) in the absence of ligands. (A) Normalized distance distribution functions. (B) and (C) low-resolution envelopes calculated by DAMMIN (Upper) and BUNCH atomic models (Lower), which are colored as in Fig. 1 and aligned inside the DAMMIN envelope (gray). BUNCH models were generated using a homology model of CYT-19 that is based upon its sequence similarity to Mss116p (see SI Methods). (D–F) SAXS data for CYT-19 bound to U₁₀–RNA and ADP-BeF_x, shown in the same arrangement as in (A–C). For the minimal CYT-19/ΔC-tail complex, the DAMMIN envelope in (F) is aligned to the homology model for CYT-19. (G–I) SAXS data for full-length CYT-19 bound to large nucleic acid substrates. (G) Normalized distribution functions for CYT-19-ADP-BeF_x bound to RNA-DNA-duplex 1 (solid green line) and RNA-DNA-duplex 2 (dashed green line). (H and I) Ab initio multiphase reconstructions of CYT-19 in complex with RNA–DNA duplex 1 (substrate 1) and RNA–DNA duplex 2 (substrate 2), respectively. Two-phase models of protein (green) and nucleic acid (yellow) were constructed by MONSA (Upper) and atomic models for protein and nucleic acid were manually placed inside the corresponding SAXS envelopes (Lower).

Fig. 6.

The tethering range observed for the basic tail of Mss116p when bound to nucleic acid substrates. (A) Range of motion of Mss116p’s flexibly attached C-tail. The cone shows the lower limit for the region of space over which the C-terminal tail of Mss116p can bind nucleic acid, as indicated by MONSA reconstructions of complexes with chimeric substrates containing duplex DNA extensions. (B) Model of Mss116p interacting via the C-tail with the Tetrahymena group I intron ribozyme (http://www-ibmc.u-strasbg.fr/upr9002/westhof/index.html). The model shows that, when anchored by the flexible C-tail, the helicase core of Mss116p can act at numerous sites over a wide region of the RNA.