The Combination of X-Ray Crystallography and Cryo-Electron Microscopy Provides Insight into the Overall Architecture of the Dodecameric Rvb1/Rvb2 Complex

Abstract

The Rvb1/Rvb2 complex is an essential component of many cellular pathways. The Rvb1/Rvb2 complex forms a dodecameric assembly where six copies of each subunit form two heterohexameric rings. However, due to conformational variability, the way the two rings pack together is still not fully understood. Here, we present the crystal structure and two cryo-electron microscopy reconstructions of the dodecameric, full-length Rvb1/Rvb2 complex, all showing that the interaction between the two heterohexameric rings is mediated through the Rvb1/Rvb2-specific domain II. Two conformations of the Rvb1/Rvb2 dodecamer are present in solution: a stretched conformation also present in the crystal, and a compact conformation. Novel asymmetric features observed in the reconstruction of the compact conformation provide additional insight into the plasticity of the Rvb1/Rvb2 complex.

Regulation of Rvb1/Rvb2 by a Domain within the INO80 Chromatin Remodeling Complex Implicates the Yeast Rvbs as Protein Assembly Chaperones

“…We also observed both bent and straight conformations of the Rvb1/Rvb2/Ino80INS dodecamer in our 2D classes (Figure 5A). These features are similar to those reported in a recently published cryo-EM structure of Rvb1/Rvb2 dodecamers without an activator bound (Ewens et al, 2016; Silva-Martin et al, 2016), suggesting that Ino80INS binding does not substantially diminish the natural dynamics of the Rvbs.…”

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

“…1 ). The structure shows a hexameric arrangement of monomers similar to other structures in the RuvB-Like family 18 , 20 – 22 . The hexameric arrangement of hs RuvBL2 in solution was confirmed by small angle X-ray scattering (SAXS - see below) coupled to size exclusion chromatography, which unequivocally showed the formation of a complex with a mean radius of gyration of ca .…”

“…The ATPase core is 51 Å high, similar to 50 Å in RuvBL1 18 and 51 Å in the truncated dodecameric complex 22 . The hs RuvBL2 hexamer has a central channel 23 Å wide (similar to the Ct RuvBL1:RuvBL2 dimensions 20 , 21 , and slightly larger than the channel of hs RuvBL1, which is 20 Å wide) on its narrowest part and although a double-stranded B-DNA molecule could be tightly fitted (not shown), it has been clearly demonstrated biochemically that RuvBL2 can only bind single-stranded DNA 28 . To the best of our knowledge, similar studies have not been published concerning RuvBL1.…”

“…These observations led us to investigate the structural basis for their antagonistic activities. Available atomic resolution structures of RuvB-Like proteins include the human RuvBL1 hexamer 18 , the human RuvBL2 hexameric ATPase core 19 , the RuvBL1:RuvBL2 complex from the thermophilic fungus Chaetomium thermophilum 20 , 21 , and the human RuvBL1ΔDII:RuvBL2ΔDII dodecameric complex 22 . In the latter, the mobile domain II was removed for crystallization purposes.…”

The plant Pontin and Reptin homologues, RuvBL1 and RuvBL2a, colocalize with TERT and TRB proteins in vivo, and participate in telomerase biogenesis

“…In the AAA+ domain, the Walker A/B motifs are responsible for ATP binding and hydrolysis, while sensor I/II motifs sense whether the protein is bound to di‐ or triphosphates. Domain II (DII) corresponds to an insertion that is unique to RuvBL in comparison with other AAA+ family members (Silva‐Martin et al ., ).…”