Autoinhibitory Interdomain Interactions and Subfamily-specific Extensions Redefine the Catalytic Core of the Human DEAD-box Protein DDX3

Abstract

DEAD-box proteins utilize ATP to bind and remodel RNA and RNA-protein complexes. All DEAD-box proteins share a conserved core that consists of two RecA-like domains. The core is flanked by subfamily-specific extensions of idiosyncratic function. The Ded1/DDX3 subfamily of DEAD-box proteins is of particular interest as members function during protein translation, are essential for viability, and are frequently altered in human malignancies. Here, we define the function of the subfamily-specific extensions of the human DEAD-box protein DDX3. We describe the crystal structure of the subfamily-specific core of wild-type DDX3 at 2.2 Å resolution, alone and in the presence of AMP or nonhydrolyzable ATP. These structures illustrate a unique interdomain interaction between the two ATPase domains in which the C-terminal domain clashes with the RNA-binding surface. Destabilizing this interaction accelerates RNA duplex unwinding, suggesting that it is present in solution and inhibitory for catalysis. We use this core fragment of DDX3 to test the function of two recurrent medulloblastoma variants of DDX3 and find that both inactivate the protein in vitro and in vivo. Taken together, these results redefine the structural and functional core of the DDX3 subfamily of DEAD-box proteins.

Keywords: ATPase; RNA helicase; RNA-binding protein; X-ray crystallography; conformational change; crystal structure; medulloblastoma; molecular dynamics.

FIGURE 1.

Conserved regions specific to the Ded1/DDX3 subfamily of DEAD-box proteins. A, a linear diagram of the features of the Ded1/DDX3 family, including the core helicase DEAD and HELICc RecA-like domains, the Crm1-dependent nuclear export sequence (NES), and the eIF4E-binding site. Conserved regions specific to the Ded1/DDX3 subfamily are indicated in gray; GINF, RDYR, and WW refer to amino acid motifs. The NTE and CTE are indicated. B, sequence alignments showing the N- and C-terminal extensions found in the Ded1/DDX3 family and indicating construct boundaries used in this study and two previous crystal structures. Numbers correspond to human DDX3X.

FIGURE 2.

Subfamily-specific extensions to the DEAD-box core are essential for DDX3 function. A, RNA duplex unwinding rates at the indicated concentrations for four truncations of DDX3. Error bars indicate S.D. B, yeast growth assays for strains containing truncated copies of the essential gene DED1 demonstrate a requirement for the region conserved in the Ded1/DDX3 family. DED1 residues 16, 30, 85, 543, 561, and 604 correspond to DDX3 residues 23, 47, 122, 592, 607, and 661, respectively. Strains containing truncations of both tails were nonviable (data not shown).

FIGURE 3.

The 2.2 Å crystal structure of the conserved core of wild-type DDX3. A, the structure of DDX3 132–607 bound to AMP (blue) is shown along with the structure of DDX3 135–582 D354V (green; PDB 4PXA) and 168–582 (yellow; PDB 2I4I). Structures are aligned by the DEAD domain, highlighting the rotation of the C-terminal HELICc domain between the three structures. B, the partially closed state of DDX3 (blue) clashes with the RNA-binding site based on a comparison with the DEAD-box protein Vasa bound to RNA (Vasa: pink; RNA: gray).

FIGURE 4.

Mutations that destabilize the partially closed state accelerate duplex unwinding. A, contacts between the DEAD and HELICc domains oriented on the view as in Fig. 3 (upper left). Asp-354 and Glu-388 are separated from His-527 by 4.0 and 2.7 Å, respectively, and Asp-506 is separated from the backbone of Arg-276 by 2.8 Å. B, RNA duplex unwinding conducted at 1 μm protein with DDX3 132–607 and mutations indicated. GINF is a mutation of the GINF motif to AAAA (residues 157–160). Error bars indicate standard error of the fit parameter.

FIGURE 5.

Molecular dynamics simulations suggest a stable interdomain interface and transient ABL-ATP interactions. A, the partially closed structure is stable over a 100-ns trajectory. Separation between the side chains Glu-388 (C_δ) and His-527 (C_γ) is shown for apo DDX3 132–607 (black) and a closed-state model based off the Vasa structure (cyan; PDB 2DB3; see “Experimental Procedures”). RMSD, root mean square deviation. B, the 150′s α-helix forms stable interactions with the DEAD domain (colors as in A). Distance is measured between Phe-151 (C_γ) and Leu-197 (C_γ). C, the ABL makes transient, stable interactions with the adenine group of ATP. Black: distance between the Cγ of Phe-160 and the H2 of ATP; cyan: distance between the N_ζ of Lys-162 and the N3 of ATP. In A–C, distances are smoothed over a sliding window of 20 frames. D, structural model of the ABL interacting with ATP at 50 ns of simulation.

FIGURE 6.

Medulloblastoma-associated variants inactivate DDX3. A, motifs Ia and VI of human DEAD-box proteins with residues altered in the medulloblastoma indicated. B, RNA duplex unwinding conducted with DDX3 132–607 and recurrent medulloblastoma variants indicated. Error bars indicate standard error of the fit parameter. C, yeast with mutations at DED1 Arg-235 to alanine or lysine are tolerated, whereas R235Y, R492A, and R492H were nonviable (not shown). DED1 Arg-235 and Arg-492 correspond to DDX3 Arg-276 and Arg-534, respectively.