PMC

Hill cooperativity analysis of the ATPase activity of human DDX3X. a ATPase activity of DDX3X was stimulated by 100 nM 22-bp double-stranded RNA (dsRNA) with a 25-nt 3ʹ overhang (Strand 1: 5ʹ-UCGUGGCAUUUCUGCGUCGUUCUUUUCUUUUCUUUUCUUUUCUUUUC-3ʹ; Strand 2: 5ʹ-GAACGACGCAGAAAUGCCACGA-3ʹ) at indicated concentrations of the helicase. Solid line is the best fits to the Hill equation (V_max = 7.4 ± 0.3 μM min⁻¹, K_1/2 = 320 ± 20 nM, H = 1.9 ± 0.2). b ATPase activity of DDX3X was stimulated by 100 nM 16-bp dsRNA with a 25-nt 3ʹ overhang (Strand 1: 5ʹ-GCGUCUUUACGGUGCUUAAAACAAAACAAAACAAAACAAAA-3ʹ; Strand 2: 5ʹ-AGCACCGUAAAGACGC-3ʹ) at indicated concentrations of the helicase. Solid line is the best fits to the Hill equation (V_max = 3.1 ± 0.4 μM min⁻¹, K_1/2 = 895 ± 120 nM, H = 1.7 ± 0.3). Error bars represent 1 SD. Source data are provided as a Source Data file

Unwinding of double-stranded RNA (dsRNA) by DEAD-box helicases. a Conformational difference between the apo-DDX3X (in green, PDB: 5E7I), DDX3X:dsRNA:DDX3X (in cyan and orange, this work), and Vasa:MgAMPPNP:ssRNA (in pink, PDB: 2DB3) structures. The structures are aligned based on Cα positions in D1. The D1 of the DDX3X:dsRNA:DDX3X structure is illustrated as a molecular surface, and those of other structures are not shown for clarity. The D2 of all three structures are shown as cartoon models (helices as cylinders, strands as arrows, and loops as tubes). The orientation of D2 relative to the fixed D1 at each state are indicated with a red arrow on the same α-helix in D2. b Represented by the apo (PDB: 5E7I), pre-unwound (this work), post-unwound (PDB: 2DB3), and post-release (PDB: 4PXA) states, the functional cycle explains a four-step mechanism of RNA duplex unwinding by DEAD-box helicases

Substrate recognition by the D1D2 core of DEAD-box helicases. a In stereo, the superimposition of the D2:dsRNA complex of Mss116p (in green, PDB entry 4DB2) with the D1D2:dsRNA:D1D2 complex of DDX3X (D1 in cyan, D2 in palecyan, RNA strands in red and blue, this work). The two structures are aligned based on D2 and are illustrated as cartoon models. The D1D2 core of DDX3X is also outlined with a transparent molecular surface in white. The double-headed arrow indicates a distance of ∼35 Å. b In stereo, the superimposition of the D1D2:ssRNA:MgAMPPNP complex of Vasa (protein in palecyan, RNA in blue, Mg in black, and AMPPNP in atomic color; PDB: 2DB3) with the D1D2:dsRNA:D1D2 complex of DDX3X (protein in cyan, RNA Strand 1 in blue and Strand 2 in red; this work). The two structures are aligned based on D1 and are illustrated as cartoon models. The D1D2 core of DDX3X is also outlined with a transparent molecular surface in white. RNA Strand 1 (in blue) is recognized by both D1 and D2 in the two structures. The single-stranded RNA (ssRNA) in the D1D2:ssRNA:MgAMPPNP structure (PDB: 2DB3) corresponds to part of the Strand 1 in the D1D2:dsRNA:D1D2 structure (this work)

Recognition of double-stranded RNA (dsRNA) by the D1D2 core of human DDX3X. a Illustration of major protein–RNA interactions with one D1D2 core (MolA, ribbon diagram in cyan/palecyan, overlapped with a transparent molecular surface) and dsRNA (Strands 1 and 2 in blue/red, overlapped with composite omit 2mFo-DFc electron density map contoured at 1σ). MolB is not shown for clarity. b D1 recognizes both the 2ʹ-OH groups and phosphate backbone of nucleotide residues 11 and 12 in the central region of RNA Strand 1. c D2 mainly recognizes the phosphate backbone of nucleotide residues 20–22 near the 3ʹ end of the same RNA strand. d Schematic representation of protein–RNA interactions observed in the D1D2:dsRNA:D1D2 structure

Overall structure of the D1D2:dsRNA:D1D2 assembly of human DDX3X. a Domain structure of human DDX3X and conserved sequence motifs of DDXs. The minimal active construct of DDX3X helicase core (the D1D2 core, residues 132–607) is used for structural studies. The 12 highly conserved DDX sequence motifs are color coded: red, ATP binding; blue, RNA binding; and orange, coordination between ATP and RNA binding. b Crystal structure of the D1D2:dsRNA:D1D2 assembly in two views. The complex contains two D1D2 cores (cyan/orange) and a 23-bp double-stranded RNA (dsRNA) (blue/red). c The D1D2:dsRNA:D1D2 structure fits well in the SAXS ab initio envelope. d Overlay of experimental scattering profiles (black) with back-calculated scattering profiles (red) for the D1D2:dsRNA:D1D2 complex. The inset shows the overlay of the crystal structure (red) with experimental (black) pair-distance distribution functions (PDDFs). Source data are provided as a Source Data file