Conformational transitions regulate the exposure of a DNA-binding domain in the RuvBL1–RuvBL2 complex

Abstract

RuvBL1 and RuvBL2, also known as Pontin and Reptin, are AAA+ proteins essential in small nucleolar ribonucloprotein biogenesis, chromatin remodelling, nonsense-mediated messenger RNA decay and telomerase assembly, among other functions. They are homologous to prokaryotic RuvB, forming single- and double-hexameric rings; however, a DNA binding domain II (DII) is inserted within the AAA+ core. Despite their biological significance, questions remain regarding their structure. Here, we report cryo-electron microscopy structures of human double-ring RuvBL1–RuvBL2 complexes at ∼15 Å resolution. Significantly, we resolve two coexisting conformations, compact and stretched, by image classification techniques. Movements in DII domains drive these conformational transitions, extending the complex and regulating the exposure of DNA binding regions. DII domains connect with the AAA+ core and bind nucleic acids, suggesting that these conformational changes could impact the regulation of RuvBL1–RuvBL2 containing complexes. These findings resolve some of the controversies in the structure of RuvBL1–RuvBL2 by revealing a mechanism that extends the complex by adjustments in DII.

Structure of Yin Yang 1 Oligomers That Cooperate with RuvBL1-RuvBL2 ATPases

“…Purified HisRuvBL1 was resolved as a mixture of oligomers and free subunits, whereas His-RuvBL2 did not oligomerize under our experimental conditions. On the other hand, His-RuvBL1-RuvBL2 and RuvBL1-RuvBL2 formed hetero-oligomeric complexes with an approximate 1:1 ratio that we interpreted as hexamers and dodecamers based on previous information (27). We found that Strep-II-YY1 interacted with His-RuvBL1 but not His-RuvBL2 under our experimental conditions after pulling down the Strep-II tag and eluting the retained His-RuvBL1 or His-RuvBL2 with D-desthiobiotin (Fig.…”

“…In the indicated cases, SEC was performed using a Superdex 200 PC 3.2/30 (GE Healthcare) or Superdex 200 10/300 GL (GE Healthcare) column equilibrated in 50 mM Tris-HCl, pH 7.4, 250 mM NaCl, 10% (v/v) glycerol. His-RuvBL1-RuvBL2 and RuvBL1-RuvBL2 were produced as described (27).…”

“…DII is a unique insertion connected to the ATPase core, resembling an OB-fold, and recombinant DII domains of RuvBL1 interact with ssDNA, dsDNA, and ssRNA in vitro (23)(24)(25). Using cryo-EM we recently solved the structure of human RuvBL1-RuvBL2 complexes assembled as hetero-dodecamers composed of two hexameric rings interacting back-to-back (27). These dodecamers co-existed in two different conformations whose functional significance is still unclear.…”

“…RuvBL1 and RuvBL2 assemble homohexameric rings on their own, whereas its co-expression results in a mixture of hetero-hexameric and dodecameric complexes that are believed to be functional forms of these ATPases in the cell (23)(24)(25). We purified His-RuvBL1, His-RuvBL2, and HisRuvBL1-RuvBL2 complexes and also RuvBL1-RuvBL2 where the tag was removed (27) (Fig. 6A), and the oligomeric state of each sample was characterized by SEC (Fig.…”

The ATPase Activity of Reptin Is Required for Its Effects on Tumor Cell Growth and Viability in Hepatocellular Carcinoma

“…However, we show here that cells reconstituted with the Reptin Walker B mutants expressed normal levels of Pontin such as those reconstituted with WT siRNA-resistant Reptin (20), thus ruling out that some of the effects seen with the mutants might be due to the loss of Pontin. Although the issue is not completely settled, the most recent evidence suggests that human Reptin and Pontin are organized in heterohexamers (24)(25)(26). It is thus likely that, when expressed in the presence of endogenous Reptin and Pontin, Walker B mutants will replace WT Reptin within hexamers and can thus exert a dominant negative effect.…”