RUVBL1 and RUVBL2 are enzymes belonging to the AAA+ superfamily (ATPases associated with diverse cellular activities) that participate in essential cellular processes, including transcriptional regulation, DNA repair, mitotic spindle assembly, and telomerase complex assembly. They are important components of key multiprotein complexes such as the histone acetyltransferase Tip60 complex, chromatin remodeling complexes Ino80 and SWR-C [[1], [2], [3]](reviewed in [4]). Additionally, RUVBLs acquire distinct functions when associated with RPAP3 and PIH1D1, forming the R2TP complex (Rvb1/RUVBL1 and Rvb2/RUVBL2, along with Tah1/RPAP3, and Pih1/PIH1D1 (yeast/human nomenclature). This complex was first identified in yeast through a systematic genomic and proteomic screen for Hsp90 (90 kDa Heat shock protein) interactors [5]. Subsequently, it was also found in other eukaryotes, including humans, Drosophila melanogaster, and Plasmodium falciparum, exhibiting a highly conserved sequence despite some species-specific differences [[6], [7], [8], [9]].

Rvb1/RUVBL1 and Rvb2/RUVBL2 function as the catalytic core of the R2TP complex, while Tah1/RPAP3 and Pih1/Pih1D1 mediate the recruitment of client proteins and additional chaperones. The interaction between R2TP and Hsp90 underscores the essential cellular role of the complex, as Hsp90 is a central molecular chaperone known to interact with approximately 10 % of the proteome [10]. Together, R2TP and Hsp90 cooperate in the assembly and stabilization of several large macromolecular complexes, including L7Ae ribonucleoproteins, U5 snRNP, PIKK complexes, RNA polymerases, the MRN complex, and the TSC complex, all of which participate in critical cellular pathways [6,[11], [12], [13], [14], [15], [16], [17]] (for review see [18,19]).

Within the R2TP complex, RUVBL1 and RUVBL2 assemble into either hetero-hexameric or hetero-dodecameric structures, using their ATPase activity to drive the assembly and disassembly of macromolecular complexes. Structurally, RUVBL1 and RUVBL2 consist of three main domains, DI and DIII form part of the AAA+ domain, which harbors the ATP-binding motifs, while DII serves as an insertion domain (Fig. 1). The AAA+ domain, responsible for ATPase activity, forms a hexameric ring and contains the Walker A and Walker B motifs, which mediate ATP binding and hydrolysis, respectively. Additionally, three other motifs—Sensor I, Sensor II, and the Arginine finger—are essential for ATP hydrolysis (see Fig. 1B for details). In contrast, the insertion domain (DII) extends outward from the AAA+ ring and serves as a key interaction site for proteins. This domain contains an oligonucleotide/oligosaccharide-binding (OB) like-fold subdomain and an internal region comprising a helical bundle and a β-stalk [19] (Fig. 1B). DII has also been suggested to regulate of RUVBL's ATPase and helicase activities [20]. Further studies revealed that the interaction of DII with Tah1 and Pih1 in the yeast R2TP complex induces conformational changes in DII, ultimately modulating RUVBL1/2 activity [21]. More recently, it has been proposed that RUVBL1/2 regulate complex assembly via the binding of CS-domain (CHORD-containing proteins and SGT1 domain) proteins to their DII domains [22]. RUVBL1 and RUVBL2 are essential for cell survival and proliferation [[23], [24], [25], [26], [27]]. Additionally, they are overexpressed in several cancer cells (reviewed in [28]). Their essential role stems from their involvement in maintaining both genomic and proteomic homeostasis. Among the R2TP complex members, RUVBL1 and RUVBL2 are the most evolutionarily conserved across species, both structurally and functionally, further emphasizing their biological significance.

In this study, we characterized a novel RUVBL1/2 heterocomplex from Aedes aegypti, a major vector of endemic diseases such as Dengue, Zika, and Chikungunya. These arbovirus-driven diseases predominantly affect tropical regions; however, climate change has facilitated the expansion of A. aegypti into new territories, including parts of Europe and North America [29]. A deeper understanding of the mosquito's cellular biochemistry and proteome is therefore crucial for developing strategies to curb its spread and control these diseases. Gaining mechanistic insights into RUVBL function in A. aegypti, a species with limited molecular characterization, could contribute to the identification of novel molecular targets for vector control strategies. Moreover, this study marks the fifth organism in which the RUVBL1/2 complex structure has been characterized and only the second within the Animalia phylum, highlighting the broader significance of our findings, particularly for the field of structural biology and biophysics.