Cooperative binding of ATP and RNA induces a closed conformation in a DEAD box RNA helicase

Abstract

RNA helicases couple the energy from ATP hydrolysis with structural changes of their RNA substrates. DEAD box helicases form the largest class of RNA helicases and share a helicase core comprising two RecA-like domains. An opening and closing of the interdomain cleft during RNA unwinding has been postulated but not shown experimentally. Single-molecule FRET experiments with the Bacillus subtilis DEAD box helicase YxiN carrying donor and acceptor fluorophores on different sides of the interdomain cleft reveal an open helicase conformation in the absence of nucleotides, or in the presence of ATP, or ADP, or RNA. In the presence of ADP and RNA, the open conformation is retained. By contrast, cooperative binding of ATP and RNA leads to a compact helicase structure, proving that the ATP- and ADP-bound states of RNA helicases display substantially different structures only when the RNA substrate is bound. These results establish a closure of the interdomain cleft in the helicase core at the beginning of the unwinding reaction, and suggest a conserved mechanism of energy conversion among DEAD box helicases across kingdoms.

Fig. 1.

Conformation of YxiN in the absence of ligands. (A) Homology model of YxiN (residues 3–367) according to the structure of M. jannaschii DeaD (mjDeaD, PDB ID code 1hv8). The two RecA-like domains of the helicase core are separated by a large interdomain cleft. The positions used for fluorophore attachment, 108 and 115 in the N-terminal, and 224, 229, and 262 in the C-terminal RecA-like domain, are highlighted. (B) RNA unwinding by wild-type YxiN and mutants. All constructs show similar unwinding yields as wild-type YxiN after 30 min of incubation with RNA substrate. (C) FRET histograms (Upper) for donor–acceptor-labeled YxiN constructs. All histograms display a unimodal distribution of FRET efficiencies, E_FRET, consistent with a single conformation of YxiN in the absence of ligand. Lines show Gaussian distributions fitted to the data (see SI Table 6). The numbers denote the mean FRET efficiency. The table (Lower) summarizes the values for E_FRET, the experimental distances calculated from E_FRET, R_calc, in nm, and the distances calculated from the homology model in A (Cβ–Cβ), R_model.

Fig. 2.

Nucleotide or RNA binding does not influence the conformation of YxiN. (A) FRET histograms for YxiN* A115C/S229C (A488/A546), in the absence of ligands, and in the presence of ADP, ATP, and the nonhydrolyzable ATP analog ADPNP. (B) FRET histogram for YxiN* A115C/S229C (A488/A546) in the presence of 200 nM RNA substrate. RNA binding does not induce a conformational change of YxiN. Nucleotides or RNA do not affect the FRET efficiencies and thus do not induce a global conformational change. Lines show Gaussian distributions fitted to the data (SI Table 6). The numbers denote the mean FRET efficiency.

Fig. 3.

Cooperative binding of RNA and ADPNP induces a closure of the interdomain cleft. Neither ADP or ADPNP nor RNA binding show any effect on the helicase conformation, whereas binding of RNA and ADPNP decreases the FRET efficiency from ≈0.5 in the apoprotein to ≈0.8, corresponding to a change in interdye distance from 5.16 to 4.13 nm. No such effect is detected upon binding of ADP and RNA. Consequently, the structures of the helicase in the ATP and the ADP form are significantly different when RNA is bound. (A) FRET histograms for YxiN* S108C/S229C in the absence of ligands, in the presence of ADP or ADPNP, of RNA, or of ADP and RNA or ADPNP and RNA.(B) FRET histograms for YxiN* A115C/S229C in the presence of ADP and RNA or ADPNP and RNA. (C) Cooperative binding of ADPNP and RNA. mantADP (1 μM) (Left) or mantADPNP (1 μM) (Right) were titrated with YxiN (wild type) in the presence (filled symbols) and absence (open symbols) of 154-mer RNA. The K_D values determined are 23 (±5.9) μM (mantADP/YxiN) and 308 (±16) μM (ADPNP) in the absence, and 0.64 (±0.05) μM (mantADP) and 3.8 (±0.6) μM (ADPNP) in the presence of RNA, corresponding to a 35-fold increase in mantADP affinity and a 80-fold increase in mantADPNP affinity when RNA is bound.

Fig. 4.

The closed conformation of the RNA helicase YxiN. Homology model of YxiN (1–354) based on the crystal structure of the DEAD box helicase Vasa in the presence of RNA and ADPNP (PDB ID code 2db3). The table below summarizes FRET efficiencies of the closed conformation from smFRET experiments for all constructs, the interdye distance calculated from E_FRET, R_calc, and the distance expected from the homology model (Cβ–Cβ), R_model, in nm.

Fig. 5.

“Switch-kink model” for DEAD box helicase-mediated RNA unwinding. The two RecA-like domains of the helicase core are depicted as large gray ovals (N, N-terminal domain), and the C-terminal domain mediating RNA binding is depicted as a small oval (C, C-terminal RNA-binding domain). The hairpin of the RNA substrate binds to the RNA recognition motif in the C-terminal domain, and ATP binds to the N-terminal RecA domain in the helicase core. Upon binding of RNA and ATP, YxiN adopts a closed conformation. The conformational change of the helicase core assembles the ATPase site and the RNA-binding site, establishing contacts with the adjacent double-helical region of the RNA. Introduction of a kink into the RNA backbone leads to local unwinding (“switch-kink” model). Upon ATP hydrolysis, YxiN returns to the open conformation. The affinity for RNA substrate is reduced, and the RNA is released. Kinking the RNA is not sufficient for unwinding, but an additional “power stroke” upon ATP hydrolysis is required to complete the catalytic cycle.