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 DEADbox proteins.DEAD-box proteins are ATP-dependent RNA-binding proteins that remodel RNA structures and RNA-protein complexes, stably clamp RNA, and promote fluidity within RNA granules (1-3). The human DEAD-box protein DDX3 (encoded by DDX3X) and its yeast ortholog Ded1p have been implicated in numerous functions including translation initiation (4 -12). Messenger RNA molecules containing especially long or structured 5Ј leader sequences are particularly sensitive to DDX3 activity (6,7,12,13). DDX3X is frequently mutated in numerous cancer types (5), such as chronic lymphocytic leukemia (14 -16), natural killer/T-cell lymphoma (17), head and neck squamous cell carcinoma (18,19), and lung cancer (20). 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. For example, DDX21 has a GUCT domain in its C-terminal extension (26, 27), DDX5 has tandem P68HR domains in its C-terminal extension, and DDX43 has a KH1 domain in its N-terminal extension. However, as the tails of each DEAD-box protein subfamily are idiosyncratic, whereas the cores are very similar (28,29), it is essential to study individual subfamilies of DEAD-box proteins in detail to understand the role of subfamily-specific tails.DDX3 is a member of the Ded1/DDX...

Targeting RNA helicases in cancer: The translation trap

“…However, more recent reports have found that the mutations have inhibitory effects on mRNA translation. Specific mutations occurring in medulloblastoma were found to result in reduced RNA unwinding activity[44], defects in RNA-stimulated ATP hydrolysis[45] and hyper-assembly of RNA stress granules, which have a general inhibitory effect on translation[46]. It was proposed that inhibition of translation potentially provides a survival advantage to medulloblastoma cells during progression.…”

Biochemical Differences and Similarities between the DEAD-Box Helicase Orthologs DDX3X and Ded1p

“…Like all DEAD-box RNA helicases, DDX3X contains a helicase core with two similar RecA-like domains that harbor 13 characteristic helicase sequence motifs, including the eponymous motif II, which reads D–E-A–D in single letter code (3, 4). The helicase core of DDX3X is flanked by an N-terminus with a nuclear export signal and regions of low sequence complexity and a C-terminus that also contains regions of low sequence complexity (Suppl.…”

“…The helicase core of DDX3X is flanked by an N-terminus with a nuclear export signal and regions of low sequence complexity and a C-terminus that also contains regions of low sequence complexity (Suppl. Fig.S1) (2, 4). …”

“…In these regions, the sequence varies more than in the helicase core (2, 5). Crystal structures of the helicase core of DDX3X have been reported (3, 4, 6). A recent structure revealed defined interactions between the two RecA-like domains of DDX3X that are specific to the DDX3X/Ded1p subfamily (4).…”

“…Crystal structures of the helicase core of DDX3X have been reported (3, 4, 6). A recent structure revealed defined interactions between the two RecA-like domains of DDX3X that are specific to the DDX3X/Ded1p subfamily (4). DDX3X and several of its orthologs have been shown to function in translation initiation, mRNA export and in other aspects of RNA metabolism (7–13).…”