Biochemical and biophysical characterization of the RVB-1/RVB-2 protein complex, the RuvBL/RVB homologues in Neurospora crassa
Introduction
The RVB proteins, also known as RUVBL1/RUVBL2, Pontin/Reptin, and TIP48/TIP49 belong to the AAA+ (ATPases Associated with various cellular Activities) protein superfamily that are present in archaea and eukaryotes. They are essential proteins in eukaryotes [[1], [2], [3]] and are described to participate in a wide range of cellular processes [4,5]. They are composed of two paralogs, Rvb1 and Rvb2 in the yeast Saccharomyces cerevisiae, that interact with each other forming the Rvb1/2 complex. According to structural studies, both proteins are organized in three domains, in which the I and III domains comprise the catalytic ATPase core, while the II domain is a DNA binding domain also involved in protein-protein interactions [6,7].
The proteins from different organisms have been extensively characterized either individually or as a protein complex. In general, both orthologs are described to exhibit ATPase and helicase activities; however, some controversial results are described by different groups depending on if they are characterized individually or not. For example, very low levels of ATPase activity and no helicase activity were described for the human RUVBL1/RuvBL1 [1,6]; however, the ATPase, but not the helicase activity, was detected when the RUVBL1/RUVBL2 complex was characterized [8,9]. The three-dimensional crystal structure was first solved for the human RuvBL1 showing a hexameric ring-shaped structure [6]. Later, the crystal structure of the human truncated protein complex (R1ΔDII/R2ΔDII) revealed a dodecamer consisting of two heterohexameric rings with alternating unities of each monomer [7]. The crystal structure of the full-length Rvb1/Rvb2 protein complex was solved with proteins from the thermophilic fungus Chaetomium thermophilum (ctRvb1/ctRvb2), which showed that both proteins assemble in dodecameric states consisting of alternating Rvb1 and Rvb2 molecules [10]. Two possible dodecameric assemblies were described depending on the region of interaction, suggesting that the capability of both proteins of forming different oligomeric states may affect the cellular pathways in which they are involved.
Although described as associated to a large variety of cellular processes, the exact molecular function of the proteins remains unclear. They are known as components of large protein complexes, such as the INO80, SWR-C/SRCAP, and TIP60/NuA4 chromatin remodeling complexes [[11], [12], [13], [14]]. In addition to DNA remodeling, the Rvbs proteins are described as associated to transcriptional processes, since they interact with the TATA-box binding protein, TBP [15], and various transcription factors resulting in the regulation of a variety of downstream processes. In humans, they are described to modulate the activity of transcriptional regulators such as c-Myc and β-catenin, which play roles in cancer development and progression [16]. Additionally, they are overexpressed in multiple types of cancer, and thus are considered as targets for the development of new therapeutic anticancer drugs [13,17].
In a previous study, we identified the RuvB-like helicase 1 protein (ORF NCU03482, the S. cerevisiae Rvb1 ortholog, named here as RVB-1) in Neurospora crassa using an approach to search for proteins able to bind to the gsn promoter, the gene encoding glycogen synthase, the regulatory enzyme in glycogen synthesis [18]. In the present work, we performed the characterization of the RVB-1 and RVB-2 proteins and of the RVB-1/RVB-2 complex from N. crassa. Both are essential proteins in N. crassa, and the mutant strains (Δrvb-1het and Δrvb-2het) show several phenotypic defects. Both proteins were individually and co-expressed in E. coli, and their biochemical and biophysical characteristics were analyzed. The RVB-1/RVB-2 complex exhibits ATPase activity and requires the N. crassa importin-α (NcIMPα) to be translocated to nucleus since their nuclear localization sites (NLS) showed high in vitro affinity to NcIMPα. In addition, the complex binds dsDNA and DNA largely increases its low ATPase activity. The analytical size-exclusion chromatography (aSEC) and the aSEC coupled to multi-angle light scattering (SEC-MALS) analyses revealed that the APO complex predominantly exists as a dimer in solution although hexamers were also observed. Nucleotides strongly influence the oligomerization state, while ATP favors the formation of hexamers, ADP favors the formation of multimeric states, likely dodecamers. Supporting this, MD simulations suggest that ADP or ATP may induce the oligomerization of RVB-1 and RVB-2 by an intermonomer cooperativity mechanism. The findings reported here extend the information regarding these proteins and reveal the fungus as a potential organism to investigate additional functions of this protein family.
Section snippets
Neurospora crassa strains and growth conditions
The FGSC #2489 (A), FGSC #9718 (mus52::bar+, a) and FGSC #9568 (mus52::hph+, a) strains used as wild-type, and the FGSC #13658 (hel-1, NCU03482, Δrvb-1het) and FGSC #13216 (hel-2, NCU06854, Δrvb-2het) heterokaryon mutant strains were purchased from the Fungal Genetics Stock Center (FGSC, University of Missouri, Kansas City, MO, USA, http://www.fgsc.net) [19]. The strains were maintained on solid Vogel's minimal (VM) medium [20], pH 5.8 containing 2% sucrose at 30 °C. Growth of mutant strains at
The ORFs NCU03482 and NCU06854 encode the Neurospora crassa RVB-1 and RVB-2 proteins, respectively
The NCU03482 protein was previously identified in a biochemical approach designed to isolate proteins able to bind to the gsn promoter, which encodes glycogen synthase [18]. The gene is annotated as “RuvB-like helicase 1” in the N. crassa genome database (http://fungidb.org/fungidb/app/record/gene/NCU03482). To identify the RVB-2 paralog, a BLASTP search in the N. crassa genome database using the Saccharomyces cerevisiae Rvb2 protein as query, retrieved the ORF NCU06854 as encoding the fungus
Discussion
Since their first identification, the RVB proteins are described as apparently involved in distinct biological events, with focus in the cancer context; however, their precise functional roles are not completely understood. As members of the AAA+ protein superfamily, they are active ATPases; moreover, they may also exhibit DNA helicase activity. A number of studies have reported their characterization using recombinant proteins produced either individually or together in a complex and the
Conclusions
In the present study we characterize the RVB-1 and/RVB-2 proteins from the N. crassa filamentous fungus. They are essential proteins, and the heterokaryotic strains exhibit phenotypic defects related to growth and development. The high instability of the individually produced recombinant proteins suggests that they may function in vivo as a protein complex. Biochemical and biophysical approaches, used to evaluate their structural properties, show that they are mainly present as a dimer in
Funding
Funding for this work was provided by São Paulo Research Foundation (FAPESP, Brazil) grants 2008/57566-8 and 2013/24705-3 (to MCB) and 2017/26131-5 (to JCB) and Coordenação de Aperfeiçoamento de Pessoal de Ensino Superior (CAPES, Brazil) for fellowship to JEMC. MCB is a Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil) fellow.
Author contributions
JEMC performed most of the experiments and analyzed data. JEMC, VTRK and SLRJ performed the CD, aSEC, ATPase and SEC-MALS experiments under supervision of JCB. JEMC and PAM worked on proteins cloning and expression. ACB worked on ITC experiments and analyses under supervision of MRMF. AASG worked on molecular modeling and molecular dynamic simulations under supervision of MRMF. FZF contributed to cloning and protein expression. MCB wrote the manuscript with contributions of JCB, MRMF and AASG.
Declaration of competing interest
The authors declare no competing interest related to this manuscript.
Acknowledgements
We would like to thank the Fungal Genetics Stock Center (Manhattan, Kansas, MO, USA) for the N. crassa strains. We also thank Dr. Ana Paula Ulian Araújo, Instituto de Física de São Carlos, USP, São Carlos, SP, Brazil, for providing the pRSFDuet-1 plasmid and for the discussions. We also thank Antonio Tarcisio Delfino for technical assistance.
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