Crystal Structure of the Human AAA+ Protein RuvBL1

Abstract

RuvBL1 is an evolutionarily highly conserved eukaryotic protein belonging to the AAA ؉ -family of ATPases (ATPase associated with diverse cellular activities). It plays important roles in essential signaling pathways such as the c-Myc and Wnt pathways in chromatin remodeling, transcriptional and developmental regulation, and DNA repair and apoptosis. Herein we present the three-dimensional structure of the selenomethionine variant of human RuvBL1 refined using diffraction data to 2.2 Å of resolution. The crystal structure of the hexamer is formed of ADP-bound RuvBL1 monomers. The monomers contain three domains, of which the first and the third are involved in ATP binding and hydrolysis. Although it has been shown that ATPase activity of RuvBL1 is needed for several in vivo functions, we could only detect a marginal activity with the purified protein. Structural homology and DNA binding studies demonstrate that the second domain, which is unique among AAA ؉ proteins and not present in the bacterial homolog RuvB, is a novel DNA/RNA-binding domain. We were able to demonstrate that RuvBL1 interacted with single-stranded DNA/RNA and double-stranded DNA. The structure of the RuvBL1⅐ADP complex, combined with our biochemical results, suggest that although RuvBL1 has all the structural characteristics of a molecular motor, even of an ATP-driven helicase, one or more as yet undetermined cofactors are needed for its enzymatic activity.RuvBL1 is a ubiquitously expressed protein (1) that plays important roles in chromatin remodeling, transcription, DNA repair, and apoptosis (2, 3). The significant evolutionary conservation of RuvBL1 from yeast to man strongly suggests that it mediates important cellular functions. RuvBL1 was originally identified by several unrelated approaches and is also known as TIP49a 3 (TATA-binding protein-interacting protein) (1, 4), Rvb1p (5), TAP54␣ (TIP60-associated protein) (3), and Pontin52 (1). The RuvBL1 gene is essential for viability in the yeast Saccharomyces cerevisiae (6), Drosophila melanogaster (7), and in Caenorhabditis elegans.4 A number of chromatin-remodeling complexes contain RuvBL1, like INO80 in yeast and human (8), which is involved in transcription and DNA repair (2), and p400 in animal cells (9). It was demonstrated that yeast Rvb1p is required for the catalytic activity of the INO80 chromatin-remodeling complex (8). RuvBL1 is also an essential component of the human histone acetylase/chromatin-remodeling complex TIP60 (3), which consists of at least 14 distinct subunits and displays histone acetylase activity on chromatin, ATPase, DNA helicase, and structural DNA binding activities.Generally, chromatin-remodeling complexes regulate chromatin structure and are critical for DNA-based transactions in the cell (10, 11). Acetylation of nucleosomal histones leads to relaxation of chromatin structure, thus enabling various transcription factors to gain access to chromatin and to interact with DNA (12, 13). As part of a chromatin-remodeling complex, yeast Rvb1p regulates the transcri...

On helicases and other motor proteins

“…also possesses an OB-fold at the N-terminal side that is internally fused to the ATPase domain [53]. This OB-fold may play a similar role to that of MCM.…”

Control of transcription by Pontin and Reptin

“…This Pontinspecific "domain 2" shows similarity to the ssDNA binding domain of the replication factor RPA. Indeed, both full length Pontin and the isolated domain 2 were shown to bind in vitro to ssDNA, as well as to dsDNA and to RNA (13). Like bacterial RuvB and other AAA+ helicases (2), Pontin monomers assemble to form a hexameric ring (see Fig.…”

“…The structure of human Pontin has recently been solved to 2.2 A resolution (13). Pontin consists of three distinct domains (Fig.…”

Genetic Suppressors ofCaenorhabditis elegans pha-4/FoxAIdentify the Predicted AAA Helicaseruvb-1/RuvB

“…We isolated a single allele of ruvb-1, px34, which carried a lysine substitution for glutamate at position 371; we obtained no alleles of ruvb-2. A comparison of C. elegans ruvb-1 with the crystal structure for human RuvbL1 (Matias et al 2006) suggests that the px34 mutation lies at the interface between RUVB molecules within the hexameric complex (see Figure 6C). Intriguingly, multiple dominant-negative alleles of RuvB have been generated in bacteria, and these map to the interface between two RuvB molecules in the hexameric ring (Iwasaki et al 2000;Putnam et al 2001).…”

“…Second, we detected a C-to-T transition within ruvb-1 that was predicted to change the conserved glutamate at position 371 to a lysine ( Figure 6B). This mutation was located after the conserved Walker A and Walker B motifs required for ATP binding of the helicase and four residues before a conserved Arginine finger motif thought to regulate ATP hydrolysis and DNA binding (Putnam et al 2001;Ohnishi et al 2005;Matias et al 2006). These data demonstrate that px34 is an allele of ruvb-1.…”