Structural dynamics of AAA + ATPase Drg1 and mechanism of benzo-diazaborine inhibition

Abstract

The type II AAA + ATPase Drg1 is a ribosome assembly factor, functioning to release Rlp24 from the pre-60S particle just exported from nucleus, and its activity in can be inhibited by a drug molecule diazaborine. However, molecular mechanisms of Drg1-mediated Rlp24 removal and diazaborine-mediated inhibition are not fully understood. Here, we report Drg1 structures in different nucleotide-binding and benzo-diazaborine treated states. Drg1 hexamers transits between two extreme conformations (planar or helical arrangement of protomers). By forming covalent adducts with ATP molecules in both ATPase domain, benzo-diazaborine locks Drg1 hexamers in a symmetric and non-productive conformation to inhibits both inter-protomer and inter-ring communication of Drg1 hexamers. We also obtained a substrate-engaged mutant Drg1 structure, in which conserved pore-loops form a spiral staircase to interact with the polypeptide through a sequence-independent manner. Structure-based mutagenesis data highlight the functional importance of the pore-loop, the D1-D2 linker and the inter-subunit signaling motif of Drg1, which share similar regulatory mechanisms with p97. Our results suggest that Drg1 may function as an unfoldase that threads a substrate protein within the pre-60S particle.

Comprehensive structural characterization of the human AAA+ disaggregase CLPB in the apo- and substrate-bound states reveals a unique mode of action driven by oligomerization

“…From the improved map of the NBD ring, the substrate density in the central channel could be unambiguously identified ( S4D Fig ). In general, the helical arrangement of subunits within a hexamer is highly similar to other substrate-bound AAA+ ATPases [ 7 , 25 , 28 – 30 ].…”

“…For most of the AAA + proteins, this mutation would greatly slow down ATP hydrolysis but not ATP binding. Structural studies of a few AAA+ unfoldases/disaggregases showed that this mutation often resulted in a co-purification of endogenous peptide in the central channel [25][26][27][28]. Therefore, we prepared CLPB E425Q mutant from E. coli cells (S2C and S2D S5B).…”

Analysis of the Conformational Landscape of the N-Domains of the AAA ATPase p97: Disentangling the Continuous Conformational Variability in Partially Symmetrical Complexes

“…The structures of p97 and its yeast homolog bound to substrates show a large change in conformation in the D1 and D2 domains, with splitting of the rings into a washer-like conformation and a staggered arrangement of the domains along the unfolded substrate [ 17 , 18 , 51 , 52 , 53 , 54 ]. Co-factors recruit the substrate to p97, yet how they promote substrate engagement remains unknown at the mechanistic level, as they are mostly not resolved in the substrate-engaged conformations due to their intrinsic flexibility.…”