Unraveling the mechanism of RNA duplex unwinding by DEAD-box helicase DDX3X: Insights into Cooperativity and roles of protomers.

DDX3X, a human DEAD-box helicase involved in ATP-dependent unwinding of short RNA duplexes, plays a pivotal role in RNA metabolism, cancer progression, and HIV-1 infection. It is composed of an N-terminal region (N: residues 1-131), a helicase core containing two RecA-like domains (D1D2: residues 132-607), and a C-terminal tail (C: residues 608-662). Previous research has shown that D1D2 forms a pre-unwound complex with dsRNA, exhibiting two-molecule cooperativity for both RNA-unwinding and ATPase activities. However, the cooperative mechanism by which the full-length DDX3X (N-D1D2-C) unwinds RNA remains to be fully understood. Knowing that the C-terminal tail is crucial for oligomerization, we have created an N-truncated form of DDX3X (D1D2-C: residues 132-662) for further investigation. Our findings indicate that D1D2-C oligomerizes in the presence of RNA substrate, exhibits three-molecule cooperativity for RNA-unwinding activity, and displays two-molecule cooperativity for ATPase activity. Furthermore, D1 residue E186 and C-terminal tail are essential for the oligomerization of D1D2-C, enabling two types of subunit interface to form, one between two D1 domains (C-D2D1:D1D2-C) and the other between two C-terminal tails (D1D2-C:C-D2D1). These results offer new insights into the molecular mechanism of cooperative RNA unwinding by DDX3X.