Structural and functional insights into a dodecameric molecular machine – The RuvBL1/RuvBL2 complex

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Abstract

RuvBL1 (RuvB-like 1) and its homolog RuvBL2 are evolutionarily highly conserved AAA+ ATPases essential for many cellular activities. They play an important role in chromatin remodeling, transcriptional regulation and DNA damage repair. RuvBL1 and RuvBL2 are overexpressed in different types of cancer and interact with major oncogenic factors, such as β-catenin and c-Myc regulating their function. We solved the first three-dimensional crystal structure of the human RuvBL complex with a truncated domain II and show that this complex is competent for helicase activity. The structure reveals a dodecamer consisting of two heterohexameric rings with alternating RuvBL1 and RuvBL2 monomers bound to ADP/ATP, that interact with each other via the retained part of domain II. The dodecameric quaternary structure of the R1ΔDII/R2ΔDII complex observed in the crystal structure was confirmed by small-angle X-ray scattering analysis.

Interestingly, truncation of domain II led to a substantial increase in ATP consumption of RuvBL1, RuvBL2 and their complex. In addition, we present evidence that DNA unwinding of the human RuvBL proteins can be auto-inhibited by domain II, which is not present in the homologous bacterial helicase RuvB. Our data give new insights into the molecular arrangement of RuvBL1 and RuvBL2 and strongly suggest that in vivo activities of these highly interesting therapeutic drug targets are regulated by cofactors inducing conformational changes via domain II in order to modulate the enzyme complex into its active state.

Introduction

RuvBL1 and RuvBL2 are implicated in many cellular pathways (Jha and Dutta, 2009) and function predominantly as part of chromatin remodeling complexes. Most chromatin remodeling complexes are typically considered to be transcriptional regulators, but different roles have recently been assigned beyond transcription, such as cell cycle checkpoint activation, DNA repair and replication, telomere regulation, centromere stability and chromosome segregation (Morrison and Shen, 2009). The assumption that these complexes play a key role in many essential cellular processes beyond transcription, suggests the existence of a sophisticated regulatory system modulating the chromatin remodeling activity of each complex for each different role. How these regulatory mechanisms modulate such functional diversity has been a puzzling feature of chromatin remodeling.

RuvBL1 and RuvBL2 are part of various ATP-dependent chromatin remodeling complexes, such as INO80, SWR1, p400 and TIP60 (Ikura et al., 2000, Jonsson et al., 2001, Jonsson et al., 2004, Kusch et al., 2004, Mizuguchi et al., 2004, Samuelson et al., 2005, Shen et al., 2000). These ubiquitously expressed proteins (Bauer et al., 1998) belong to the AAA+ family of ATPases (ATPases associated with diverse cellular activities) (Neuwald et al., 1999). This class of ATPases includes nucleic acid processing enzymes, chaperones and proteases that contain conserved motifs for ATP-binding and hydrolysis such as the Walker A and Walker B boxes (Walker et al., 1982), sensor residues and the Arg-finger. AAA+ proteins use the hydrolysis of ATP to exert mechanical forces, and this has been shown to be essential for the biological activity of RuvBL1 and RuvBL2 (Feng et al., 2003, Jonsson et al., 2004, Wood et al., 2000). RuvBL1 and RuvBL2 share 43% sequence identity and 65% sequence similarity and are homologs of the bacterial DNA-dependent ATPase and helicase RuvB (Putnam et al., 2001, Yamada et al., 2001). However, the original data reporting DNA helicase activities of RuvBL1 (Makino et al., 1999) and RuvBL2 (Kanemaki et al., 1999) were not reproducible with the purified wild-type proteins (Ikura et al., 2000, Qiu et al., 1998). Although RuvBL1 and RuvBL2 share structural features with bacterial RuvB (Matias et al., 2006), domain II is not present in the bacterial homolog and had to be truncated in the RuvBL proteins used for structural analysis in this work.

A link between RuvBL1, RuvBL2 and cancer has been established in the last decade. Both proteins interact with transcription regulators known to be involved in oncogenic pathways, such as β-catenin and c-Myc. Among the transcription factors with oncogenic potential, c-Myc is one of the most frequent sites of mutation in human cancer (Cole, 1986), while β-catenin has a key role in Wnt signaling via effects on T-cell factor (TCF)-mediated transcription (Bauer et al., 1998, Bauer et al., 2000, Feng et al., 2003).

It was shown that RuvBL1 is required for the transforming effect of c-Myc (Wood et al., 2000), the viral oncoprotein E1A (Dugan et al., 2002) and β-catenin (Feng et al., 2003). Studies from different groups report an overexpression of RuvBL1 and RuvBL2 in several types of cancer, such as bladder cancer, melanoma, non-small cell lung cancer, gastric cancer and colon cancer (Dehan et al., 2007, Lauscher et al., 2007, Rousseau et al., 2007). A differential proteomic analysis of human hepatocellular carcinoma revealed an overexpression of RuvBL1 and RuvBL2, and both proteins were considered markers of poor prognosis (Blanc et al., 2005, Huber et al., 2008). These findings imply that both proteins are not only of general interest for oncologists, but might also represent highly effective therapeutic drug targets.

To date there is no crystal structure available for the RuvBL1/RuvBL2 complex, but electron microscopy (EM) studies of the yeast and human complex show that RuvBL1 and RuvBL2 form a dodecameric complex consisting of two structurally distinct hexameric rings (Puri et al., 2007, Torreira et al., 2008). The yeast model suggests that domain II forms the interaction site between two hexameric rings (Torreira et al., 2008), but neither study settled the issues of whether the rings are homo- or hetero-oligomeric and if they interchange depending on different chromatin remodeling functions. Furthermore, Gribun and co-workers proposed from EM studies a single heterohexameric ring structure for the yeast RuvBL1/RuvBL2 complex (Gribun et al., 2008). The differences between the EM structures suggest that RuvBL1 and RuvBL2 may be capable of forming various complexes. Previous work showed that the weak ATPase activity of RuvBL1 and RuvBL2 in vitro increased synergistically when the proteins formed a double-hexameric complex demonstrating that this is the enzymatically active form (Ikura et al., 2000, Puri et al., 2007). A recent study analyzing the oligomeric assembly of the human RuvBL1/2 proteins suggests that the RuvBL1/RuvBL2 complex forms single and double hexamers together with smaller forms and that truncation of domain II destabilizes the dodecamer formation (Niewiarowski et al., 2010).

The need to clarify both the oligomerization-function relationships and the ATP hydrolysis mechanism, prompted us to perform further structural studies. To address these questions, the crystal structure of the human RuvBL1/RuvBL2 complex with bound ATP/ADP has been determined. Since the full-length complex did not crystallize, mutants of RuvBL1 (R1) and RuvBL2 (R2) with a two-thirds truncation of the flexible domain II (Matias et al., 2006) were generated (R1ΔDII and R2ΔDII). Crystals of the selenomethionine derivative of the R1ΔDII/R2ΔDII complex diffracted to 3 Å resolution and led to the determination of the three-dimensional structure of the complex. These structural data combined with functional studies provide a possible mode of in vivo activation of these highly conserved proteins.

Section snippets

Protein purification

For biochemical studies, deletion mutants of RuvBL1 and RuvBL2 with truncations in their flexible domains II (Matias et al., 2006) were generated: R1ΔDII missing residues T127-E233 and R2ΔDII missing residues between E134-E237. A linker consisting of amino acids GPPG was inserted to replace the deleted region (see Supplementary Fig. S1). The RuvBL complexes were co-expressed in Escherichia coli BL21(DE3) using the pETDuet vector (Novagen) with RuvBL1 carrying a N-terminal 6xHis-tag and RuvBL2 a

Overall dodecameric structure of the RuvBL1ΔDII/RuvBL2ΔDII complex

The crystal structure of the R1ΔDII/R2ΔDII complex consists of dodecamers formed by two heterohexamers stacked on top of each other. Each hexamer is composed of alternating R1ΔDII and R2ΔDII monomers, as illustrated in Fig. 1a and b. The complex crystallized in space group C2221 with only one heterohexamer in the asymmetric unit. The two heterohexamers in the dodecameric structure are related by a crystallographic 2-fold rotation axis, so that each R1ΔDII and R2ΔDII monomer in one hexamer

Discussion

In this work, we have solved the first crystal structure of the biologically active RuvBL1/RuvBL2 complex at 3 Å resolution, which is a double hexameric ring composed of alternating RuvBL1 and RuvBL2 monomers. The dodecameric assembly observed in the crystal is supported by the crystal packing and SAXS results in solution. Unfortunately, the protein regions responsible for the interactions between two hexameric rings could not be modeled due to poor electron density. This can be rationalized by

Acknowledgments

We thank Peter Lindley, Bernard Haendler (Bayer Schering Pharma) and Carlos Frazão (ITQB-UNL) for helpful suggestions, discussions and comments on the manuscript. This work was supported by European Commission funding through the SPINE2-COMPLEXES project LSHG-CT-2006-031220. ESRF support for the ID-29 data collection and EU support for the SAXS measurements at X33 (FP7/2007-2013 under Grant agreement No. 226716) are also acknowledged.

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    Current address: UCLA, Department of Biological Chemistry, David Geffen School of Medicine, 615 Charles E. Young Drive South, Box 951737, BSRB#390C, Los Angeles, CA 90095-1737, United States.

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    Current address: European Molecular Biology Laboratory, Grenoble Outstation, 6 Rue Jules Horowitz, 38042 Grenoble Cedex 9, France.

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