X-ray structure of full-length human RuvB-Like 2 – mechanistic insights into coupling between ATP binding and mechanical action

Abstract

RuvB-Like transcription factors function in cell cycle regulation, development and human disease, such as cancer and heart hyperplasia. The mechanisms that regulate adenosine triphosphate (ATP)-dependent activity, oligomerization and post-translational modifications in this family of enzymes are yet unknown. We present the first crystallographic structure of full-length human RuvBL2 which provides novel insights into its mechanistic action and biology. The ring-shaped hexameric RuvBL2 structure presented here resolves for the first time the mobile domain II of the human protein, which is responsible for protein-protein interactions and ATPase activity regulation. Structural analysis suggests how ATP binding may lead to domain II motion through interactions with conserved N-terminal loop histidine residues. Furthermore, a comparison between hsRuvBL1 and 2 shows differences in surface charge distribution that may account for previously described differences in regulation. Analytical ultracentrifugation and cryo electron microscopy analyses performed on hsRuvBL2 highlight an oligomer plasticity that possibly reflects different physiological conformations of the protein in the cell, as well as that single-stranded DNA (ssDNA) can promote the oligomerization of monomeric hsRuvBL2. Based on these findings, we propose a mechanism for ATP binding and domain II conformational change coupling.

Sorafenib as an Inhibitor of RUVBL2

“…Calculation of the radii of gyration resulted in values of 47.7 Å for apo RUVBL2, 47.9 Å for RUVBL2 + DMSO and 47.2 Å for RUVBL2 + Sorafenib. These values agree with previously observed dimensions for human RUVBL2 by X-ray crystallography (52 Å) [14]. To further investigate differences in the overall fold and flexibility of RUVBL2, dimensionless Kratky analysis was performed.…”

“…Further experiments performed on RUVBL2∆DII confirmed that the ATPase activity of RUVBL2 with truncation of its insertion domain was not inhibited by sorafenib; therefore, leading us to propose that the mechanism of action of sorafenib is mediated through its interaction with DII or that DII flexibility causes RUVBL2 to populate certain conformations to which sorafenib can bind. Recently, it was proposed that the RUVBL2 N-terminal segment may function as a lid for the nucleotide-binding site and the binding of ATP could induce the recruitment of DII by the N-terminal segment [14,16]. In this sense, binding of sorafenib to DII would influence the dynamics of DII, consequently affecting RUVBL2 nucleotide exchange rate.…”

“…RUVBL1 and RUVBL2 typically act as a heterohexameric complex, but also function independently [6]. Their sequences can be divided into three domains [7][8][9][10][11][12][13][14][15][16]: (i) an N-terminal αβα subdomain of the AAA+ domain (DI), (ii) an insertion domain unique to the RUVBL proteins (DII) and that is similar in structure to an oligonucleotide/oligosaccharide-binding (OB) fold, which is a ssDNA binding domain present in replication factor protein RPA, and (iii) an all α subdomain of the AAA+ domain (DIII).…”

Regulation of RUVBL1-RUVBL2 AAA-ATPases by the nonsense-mediated mRNA decay factor DHX34, as evidenced by Cryo-EM

“…The regulation of RUVBL1-RUVBL2 ATPase activity is poorly understood. The Ntermini of RUVBL1 and RUVBL2 contain two histidine residues, His 25 and His 27 in RUVBL2, that bind the nucleotide and contribute to maintain it within its binding pocket (Munoz-Hernandez et al, 2019;Silva et al, 2018). Here, cryo-EM reveals that the NMD factor DHX34 directly affects the conformation of RUVBL2 N-termini and decreases ATP hydrolysis.…”

“…We first analyzed the structure of the RUVBL1-RUVBL2 ring using a mask that removed the influence of DHX34 and the protruding OB-fold domains during image processing ( The most striking feature in the cryo-EM map is that the N-terminal regions that contribute to nucleotide binding (Munoz-Hernandez et al, 2019;Silva et al, 2018) are visualized in RUVBL1 but not in any of the three RUVBL2 subunits ( Figure 3A, B). We performed several experiments to fully verify that every RUVBL2 in the complex lacked density for the N-terminal region.…”