Structural And Functional Analysis Insights Into The DEAD-box RNA Helicase Dbp2

RNA helicase are motor proteins that are widely existed in all kingdoms of life,using ATP hydrolysis to regulate the RNA structure and RNA-protein complexes（RNP）.DEAD-box proteins are the largest family of RNA helicases,and they are involved in almost all metabolic processes associated with RNA.Human DDX5 and yeast Dbp2 represent a subfamily of DEAD-box RNA helicase,the DDX5/Dbp2 subfamily proteins are involved in transcriptional regulation,energy metabolism,and cell signaling pathways.Recent studies have suggested that the DDX5 protein is closely associated to the occurrence and development of many cancers.In addition,small molecule inhibitors of DDX5 have been developed for treatment of breast cancer patients,so the DDX5 is very important for biomedical research.At present,only the crystal structure of Rec A1 of DDX5 has been obtained,but the structure of other domains of DDX5 protein are still unknown.The helicase core of the DEAD-box protein consists of 13 conserved motifs that play a role in ATP binding,hydrolysis,and RNA binding.In addition to these conserved motifs,each DEAD-box subfamily protein has unique terminal tails that locate at the N-and C-terminus of the helicase core.Since the terminal tails of the DEAD-box subfamily proteins are specific,and the structure of the helicase core are very similar,it is necessary to study the function of the specific terminal tails of the DDX5/Dbp2 subfamily proteins in detail.Dbp2 from Saccharomyces cerevisiae was selected for the research material and the Dbp2protein was successfully expressed and purified by the E.coli expression system.The biochemical activities of Dbp2 protein suggested that it is a bona fide RNA helicase.Then,bioinformatics analysis of protein sequence,truncated proteins and limited proteolysis to obtain the compact fragment for crystallographic studies,in order to obtain the crystal structure of Dbp2.In addition,we systematically investigated the function of terminal domains of Dbp2 and the molecular mechanisms by which Dbp2 remolding RNA structure using fluorescence anisotropy,stopped-flow,sm FRET,fluorescent labeling of proteins,and fluorescence spectroscopy experiments.The main results are as follows:（1）The article obtained the first crystal structure of the helicase core of Dbp2^53-496 and in complex with ADP,representing apo-state and ADP-bound post-hydrolysis state.A unique CTE conformation and a conserved NTE conformation have been observed in the crystal structure of Dbp2^53-496,and in vitro mutagenesis experiments have further confirmed that NTE and CTE regulate the ATPase activity of Dbp2.（2）Based on observation of the conformational changes of the Dbp2 helicase core in crystal structures,we first demonstrate the global conformation changes in the helicase core of full-length Dbp2 in solution using sm FRET and fluorescent labeling of helicase core of full length Dbp2.The helicase core of Dbp2 undergoes dynamic conformation changes in solution.The conformation of the helicase core is a closed state upon binding of the ATP analogue and ss RNA.Moreover,it was further demonstrated that the Dbp2 helicase core is locked into a closed conformation by intramolecular crosslinking,suggesting that Dbp2 depends on the conformational changes of the helicase core to exert helicase activity.（3）The research finally obtained the structure of full-length Dbp2 protein in solution by small-angle X-ray scattering experiment,the full-length Dbp2 underwent dynamic changes in solution,due to the flexibility in the N-and C-tails,and in the linker between the helicase core.（4）The helicase core of Dbp2 has almost no activity,while the disordered N-and C-tails confer full helicase activity to the Dbp2 protein.Subsequently,the molecular mechanism of RNA remodeling through the N-and C-terminal tails of Dbp2 was revealed using fluorophore-labeled terminal tails and adjacent helicase core in combination with sm FRET,providing an important biochemical basis for Dbp2 participating in RNP remodeling in vivo.（5）Dbp2 possesses a dual function for different conformations of G4 DNA,destabilizing the folded G4 and inducing further folding of the unfolded G4 sequence.Similarly,DDX5 can unfold G4 DNA,but it exhibits a weaker G4 folding-promoting activity relative to Dbp2.This study suggests that Dbp2 may be involved in the remodeling of the short loop G4 in the Saccharomyces cerevisiae genome.Taken together,this article investigated the structure and function of the yeast Dbp2protein,providing an important molecular mechanism for Dbp2 to participate in RNA and RNP remodeling.The crystal structure of the helicase core of Dbp2 is critical in the search for seeking small molecule inhibitors of DDX5-associated cancers.