Abstract
The AAA+ ATPase Drg1 is a ribosome assembly factor in yeast, and functions to release Rlp24, another assembly factor, from the pre-60S particle just exported from nucleus to initiate its further cytoplasmic maturation. Being a type II AAA+ protein with two ATPase domains (D1 and D2), its activity in ribosome assembly can be inhibited by a drug molecule diazaborine. In human, mutations of Drg1 homologue has been linked to a disease condition called epilepsy, hearing loss, and mental retardation syndrome. Although the general structure of Drg1 hexamer was recently reported, its complete structure and dynamic conformational rearrangements driven by ATP-hydrolysis are poorly understood. Here, we report a comprehensive structural characterization of Drg1 hexamers in different nucleotide-binding and benzo-diazaborine treated states. Our data show that Drg1 hexamers transits between two extreme conformations, characterized by a planar or helical arrangement of its six protomers. By forming covalent adducts with the ATP molecules in the active centers of both D1 and D2, benzo-diazaborine locks Drg1 hexamers in a more symmetric and non-productive conformation. In addition, we obtained the structure of a mutant Drg1 hexamer (Walker B mutations) with a polypeptide trapped in the central channel, representing a 3D snapshot of its functional, substrate-processing form. Conserved pore loops on the ATPase domains of Drg1 form a spiral staircase to interact with the substrate through a sequence-independent manner. These results suggest that Drg1, similar as Cdc48/p97, acts as a molecular unfoldase to remodel pre-60S particles, and benzo-diazaborine inhibits both the inter-protomer and inter-ring communication to disable the conformational cycling of Drg1 protomers required for the unfolding activity.
The Crystal Structure of the Ubiquitin-like Domain of Ribosome Assembly Factor Ytm1 and Characterization of Its Interaction with the AAA-ATPase Midasin
