Structural basis for RNA-duplex unwinding by the DEAD-box helicase DbpA

The DbpA/hp-HP92/ADPBeF₃ complex represents a trapped unwinding intermediate. (A) Fluorescence-based unwinding assays in the presence of different ATP analogs. A 5′ fluorescein (green star) labeled 9mer RNA is hybridized to an RNA containing HP92 (top). Unwinding can be followed by a decrease in fluorescence intensity (bottom). Mono-exponential fits to the fluorescence time traces are shown in black for ATP, ADP/BeF₃, and ATPγS. (B) Ile region of methyl TROSY spectra of ILMVA-labeled DbpA in the free state (black) and bound to hp-HP92 RNA (blue). (C) Sequence plots of chemical shift perturbations (CSPs) induced by binding of the hp-HP92 RNA (top) and differences between CSPs (ΔCSPs) induced by binding of the ss-HP92 RNA and the hp-HP92 (bottom). DbpA domains are indicated at the top. (D) Methyl TROSY spectra of DbpA/hp-HP92 complex prior to (blue) and after addition of ADP/BeF₃ (orange). (E) Sequence plots of CSPs induced by binding of ADP/BeF₃ to the DbpA/hp-HP92 RNA complex (top) and ΔCSPs between binding of ADP/BeF₃ to the ss-HP92/DbpA and to the hp-HP92/DbpA complexes (bottom). (F) ΔCSPs from (E) plotted onto a model of the closed state of DbpA bound to ssRNA (Wurm et al. 2021) (red, large ΔCSPs, blue, small ΔCSPs). The ssRNA is shown in orange. (G) Sequence of hp-HP92 RNA. Nucleotides with assigned imino proton signals are numbered and shown in red (HP92) or green (substrate hairpin). (H) ¹H-1D imino proton spectra (left) and ¹H¹⁵N-HMQC spectra (right) of uridine ¹⁵N-labeled hp-HP92 RNA prior to (black) and after addition of DbpA (blue). (I) Same spectra as in (H), but for the hp-HP92/DbpA complex prior to (blue) and after addition of ADP/BeF₃ (orange).