Structural Basis for Dodecameric Assembly States and Conformational Plasticity of the Full-Length AAA+ ATPases Rvb1·Rvb2

Abstract

As building blocks of diverse macromolecular complexes, the AAA+ ATPases Rvb1 and Rvb2 are crucial for many cellular activities including cancer-related processes. Their oligomeric structure and function remain unclear. We report the crystal structures of full-length heteromeric Rvb1·Rvb2 complexes in distinct nucleotide binding states. Chaetomium thermophilum Rvb1·Rvb2 assemble into hexameric rings of alternating molecules and into stable dodecamers. Intriguingly, the characteristic oligonucleotide-binding (OB) fold domains (DIIs) of Rvb1 and Rvb2 occupy unequal places relative to the compact AAA+ core ring. While Rvb1's DII forms contacts between hexamers, Rvb2's DII is rotated 100° outward, occupying lateral positions. ATP was retained bound to Rvb1 but not Rvb2 throughout purification, suggesting nonconcerted ATPase activities and nucleotide binding. Significant conformational differences between nucleotide-free and ATP-/ADP-bound states in the crystal structures and in solution suggest that the functional role of Rvb1·Rvb2 is mediated by highly interconnected structural switches. Our structures provide an atomic framework for dodecameric states and Rvb1·Rvb2's conformational plasticity.

PRMT5-Dependent Methylation of the TIP60 Coactivator RUVBL1 Is a Key Regulator of Homologous Recombination

“…While this approach enriched for a well-established PRMT5 cofactor (MEP50), it also identified the AAA+ ATPase RUVBL1 as a potential binding protein/substrate (Figure 3A). RUVBL1 and its binding partner RUVBL2 can form homohexameric assemblies (Matias et al., 2006, Petukhov et al., 2012) as well as a large dodecamer complex consisting of two hetero-hexameric rings with alternating RUVBL1 and RUVBL2 monomers (Gorynia et al., 2011, Lakomek et al., 2015). This protein complex predominantly functions as part of a number of high molecular weight nuclear complexes involved in chromatin remodeling, histone tail modification, and histone exchange.…”

“…In five independent experiments, digestion with Asp-N consistently detected the presence of a methyl group on a single residue, R205 (Figure S3F). Residue R205 is highly conserved among higher and lower eukaryotes (Lakomek et al., 2015), implying functional significance. We therefore mutated R205 to lysine and validated our mass spectrometry finding of R205 as a site of methylation by in vivo [ 3 H]-methyl methionine labeling of cells (Figure 3E).…”

“…Recent insights into the solved hetero-hexameric structure of chaetomium thermophilum ( ct ) full-length RUVBL1/RUVBL2 complex revealed that the domain II (DII) region, containing R205 in human RUVBL1 and its equivalent R206 in human RUVBL2, can adopt widely different orientations with respect to the ATPase core (Lakomek et al., 2015), confirming the flexibility originally inferred from the homohexameric RUVBL1 structure (Matias et al., 2006). In human and ct RUVBL1, R205 is exposed to the solvent and is therefore accessible to PRMT5 for methylation (Figure S4A).…”

Functional characterization and architecture of recombinant yeast SWR1 histone exchange complex

“…The best quality model, representing the most compact conformation of SWR1, was chosen for docking of Rvb1/2 atomic coordinates (PDB accession code: 4WVY (22)). The Rvb1/2 ring density could be distinguished clearly from the rest of the SWR1 complex (Figure 6A).…”

“…The sixth monomer was less extended, with DII apparently in an intermediate conformation between the corresponding domain in the Rvb1 and Rvb2 subunits in the 4WVY model. The end of DII contains a structurally conserved OB domain, while the preceding DII linker is flexible (22). Thus, to obtain a better fit for the Rvb1 ring we performed a rigid body fit of the OB domain into the SWR1 density using the Phenix software (24) along with the other subunits.…”