Cancer-Associated Mutants of RNA Helicase DDX3X Are Defective in RNA-Stimulated ATP Hydrolysis

Abstract

The DEAD-box RNA helicase DDX3X is frequently mutated in pediatric medulloblastoma. We dissect how these mutants affect DDX3X function with structural, biochemical, and genetic experiments. We identify an N-terminal extension (“ATP-binding loop”, ABL) that is critical for the stimulation of ATP hydrolysis by RNA. We present crystal structures that suggest the ABL interacts dynamically with ATP and confirm the interaction occurs in solution by NMR chemical shift perturbation (CSP) and isothermal calorimetry (ITC). DEAD-box helicases require interaction between two conserved RecA-like helicase domains, D1 and D2 for function. We use NMR CSP to show that DDX3X interacts specifically with double-stranded RNA (dsRNA) through its D1 domain, with contact mediated by residues G302 and G325. Mutants of these residues, G302V and G325E, are associated with pediatric medulloblastoma. These mutants are defective in RNA-stimulated ATP hydrolysis. We show that DDX3X complements the growth defect in a ded1 temperature-sensitive strain of S. pombe, but the cancer-associated mutants G302V and G325E do not complement and exhibit protein expression defects. Taken together, our results suggest that impaired translation of important mRNA targets by mutant DDX3X represents a key step in the development of medulloblastoma.

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

“…In addition, there are regions adjacent to the helicase core conserved between DDX3, Ded1, and Vasa/DDX4 (Fig. 1A) (5,25). The N-terminal extension (NTE; residues 132-168; Fig.…”

“…1B) has no predicted structure but is highly positively charged (pI ϳ12). Recent structural and biochemical work demonstrated that DDX3 constructs containing the NTE are competent for ATP hydrolysis (25), but it is unknown how this activity compares with full-length human DDX3.…”

“…DDX3X is also one of the most frequently mutated genes in the highly malignant brain tumor medulloblastoma (21-24). In medulloblastoma, many mutations are predicted to inactivate DDX3, and some have been demonstrated to diminish activities in vitro (17,25).DEAD-box proteins are defined by 12 different motifs that function in ATP binding or hydrolysis and RNA binding, or couple ATP and RNA binding (1). Outside of these conserved motifs, each DEAD-box protein subfamily has unique tails that lie N-or C-terminal to the helicase core and contain elements that define the unique properties of that subfamily.…”

“…DDX3X is also one of the most frequently mutated genes in the highly malignant brain tumor medulloblastoma (21)(22)(23)(24). In medulloblastoma, many mutations are predicted to inactivate DDX3, and some have been demonstrated to diminish activities in vitro (17,25).…”

“…Similarly, prior structural work has truncated one (25) or both (33) of the Ded1/DDX3 subfamilyspecific regions down to the boundary of the helicase core, but it is unknown how active these truncations are when compared with full-length DDX3. Moreover, previous structures of DDX3 are of either inactive constructs (33) or point mutants (25), so the relevance of the crystallized conformations is unclear. Therefore, crucial details of the regions uniquely conserved within the Ded1/DDX3 subfamily and the conformational landscape of active, wild-type DDX3 remain incompletely understood.…”