Mechanisms of activity of DEAD-box helicases studied by NMR: a dynamic view.

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The mechanisms of communication between the ATP-binding and the RNA-binding sites in DEAD-box helicases are unknown. These mechanisms are at the basis of the enzyme activities and likely determine whether the DEAD-box protein acts as a helicase, an RNA clamp or an RNA chaperone.In this project we aim at elucidating the dynamic and structural codes at the basis of the activity of the Bombyx mori DEAD-box protein Vasa involved in the piRNA pathway. Using NMR, we will study the dynamics over a range of timescales, spanning from picoseconds to seconds, of the Vasa protein and an impaired mutant thereof in different functional states. We will identify which residues are involved in the communication between the ATP-binding and hydrolysis and the RNA-binding site and characterize them in terms of both structure and dynamics. For the structural part, we will focus only on those elements whose chemical shifts change across the different protein states. The dynamic information that we will obtain will be translated into thermodynamic parameters, in order to understand the energetics of the Vasa activity on a molecular basis. To this end calorimetric experiments for the binding of both the nucleotide and the RNA will be conducted in parallel to the NMR experiments.We anticipate that our results will reveal the path of cross-talk between the D1 and D2 domains of the Vasa protein. The resulting model will be confirmed by experiments in vivo in collaboration with the Pillai laboratory in Grenoble. Here, the behavior of newly designed Vasa mutants, with altered dynamics and thermodynamics, will be studied in BmN4 cells, in terms of their ability to support the ping-pong amplification cycle of piRNAs. The in vivo results with the designed Vasa mutants will reveal whether in the piRNA pathway the protein acts as a helicase, as an RNA clamp or as both.This project addresses an important class of enzymes involved in nearly all aspects of RNA metabolism, from transcription, to translation and mRNA decay. RNA helicases are ubiquitous in all three kingdoms of life and viruses also encode for some of these proteins. Mutations and deregulation of DEAD-box helicases have been linked to disease states, as for example cancer. Understanding the mechanisms underlining the function and substrate recognition of these enzymes prepares the field for the development of drugs acting on this still unexplored target.