36 37 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 0 / 2 1 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 0 / 2 1 Biological Cryo-Imaging Group eBIC Cryo-EMvisualises dynamicmolecularmachines Related publication: López-Perrote A., Hug N., González-Corpas A., Rodríguez C. F., SernaM., García-Martín C., Boskovic J., Fernandez-Leiro R., Caceres J. F. & Llorca O. Regulation of RUVBL1-RUVBL2 AAA-ATPases by the nonsense-mediatedmRNA decay factor DHX34, as evidenced by Cryo-EM. eLife 9 , 1–23 (2020). DOI: 10.7554/eLife.63042 Publication keywords: AAA-ATPases; DHX34; RUVBL1-RUVBL2; Human; Molecular biophysics; NonsensemediatedmRNA decay; Structural biology M essenger RNA (mRNA) is a single-strandedmolecule of RNA that corresponds to the genetic sequence of a gene. Inside a cell, mRNA is used as a template to build a protein. However, mRNAmolecules are prone to errors, and cells use quality-control processes called mRNA surveillance mechanisms to ensure the mRNA molecules (and hence proteins) are correct. Nonsense-mediated mRNA decay (NMD) is one of these quality-control mechanisms. Its role is to degrade mRNAs with premature termination codons (PTCs), which might otherwise produce truncated proteins. AAA-ATPases are proteins found in all organisms. They are essential for many cellular functions, including DNA replication, protein degradation and the regulation of gene expression. RUVBL1 and RUVBL2 are two closely related AAA-ATPases required for the initiation of the NMD pathway. However, how RUVBL1 and RUVBL2 regulate NMD and how interacting partners regulate the ATPase activity of RUVBL1 and RUVBL2 remain poorly understood. Researchers from the Spanish National Cancer Research Centre (CNIO) and the University of Edinburgh investigated which of the core NMD factors could form a direct complex with RUVBL1-RUVBL2 to determine the structure of the complex and study if the interaction affected the ATPase activity of the chaperone. To image these dynamic molecular machines to this level of detail, they used cryo-electronmicroscopy (cryo-EM). Access to the high-end instrumentation at the Electron Bio-Imaging Centre (eBIC) at Diamond Light Source was essential to solve these structures. Their results revealed the regulation of RUVBL1-RUVBL2 by a factor participating in the NMD pathway, and the core elements of their model may apply to other processes where these ATPases participate. RUVBL1 and RUVBL2 proteins are two closely related AAA-ATPases that are essential in a wide variety of cellular processes 1 . Their known functions can be broadly classified in at least three categories. On the one hand, they are constituent subunits of several chromatin remodelling complexes such as INO80, SWR1, SRCAP and TIP60 where they act as scaffolds to organise other subunits in the complexes 2 . On the other hand, RUVBL1 and RUVBL2 interact with RPAP3 and PIH1D1 proteins to constitute R2TP, a co-chaperone complex that works in concert with the HSP90 chaperone in the assembly and activation of a growing list of macromolecular complexes, including members of the Phosphatidylinositol 3-kinase-related kinase (PIKK) family members such as mTOR and ATR, and RNA polymerase II 3 . Finally, RUVBL1 and RUVBL2 have been found to work as chaperones in some processes without the involvement of HSP90 1 . RUVBL1 and RUVBL2 organise hetero-hexameric complexes of alternating subunits. Domains I and III of each protein assemble to form an hexameric ring with ATPase activity. Domain II (DII) from each subunit protrudes from the hexameric ring and they function as modules that can interact with different proteins and clients 2,3 . The ATPase activity of RUVBL1-RUVBL2 complexes is essential for most of their known cellular activities, but a clear understanding of how this is regulated and what its role in each of the processes where RUVBL1-RUVBL2 participates is missing. Nonsense-mediated mRNA decay (NMD) promotes the degradation of mRNAs containing premature termination codons (PTCs) to prevent the production of truncated proteins with potential deleterious effects 4 . NMD is also involved in fine-tuning gene expression by the regulation of some physiological transcripts. Initiation of the NMD response occurs during the first round of translation when the ribosome is stalled at a PTC, allowing the recruitment of several NMD factors including SMG1, UPF1 and the DHX34 helicase. Remodelling of this complex by interaction with additional NMD factors promotes SMG1-mediated phosphorylation of UPF1, triggering the recruitment of the RNA degradation machinery. RUVBL1 and RUVBL2 are essential to initiate the NMD response 5 . Their depletion reduced UPF1 phosphorylation and affected the degradation of a PTC-containing reporter transcript, but these defects were recovered by the wild-type proteins but not ATPase-deficient mutants. How RUVBL1-RUVBL2 regulates NMD remains to be elucidated. The present study starts characterising the mechanisms involved in the functions of RUVBL1-RUVBL2 in NMD, but in addition the NMD pathway was used as a model to study the regulation of RUVBL1-RUVBL2 ATPase activity by the interaction with other proteins. A novel interaction of RUVBL1- RUVBL2 with the DHX34 helicase, a factor essential to initiate NMD, was described using cryo-EM in combination with biochemical and cellular experiments. The RUVBL1-RUVBL2-DHX34 complex was identified in cellular immunoprecipitation experiments and reconstituted in vitro . Imaging of the complex by cryo-EM revealed that DHX34 attaches to the face of the RUVBL1- RUVBL2 ring containing the DII domains (Fig. 1a). Whereas the density for the RUVBL1-RUVBL2 hexameric ring was well defined in the 2D averages of the complex, smeared signal for DHX34 and the DII domains denoted the flexible attachment of DHX34 to the chaperone. Accordingly, the 3D reconstruction of the complex revealed DHX34 interacting with all RUVBL subunits at the DII face of the hexameric ring but the resolution of DHX34 in the complex was insufficient for atomic modelling (Fig. 1b). In contrast, the structure of the ATPase ring of RUVBL1-RUVBL2 in the complex was determined by omitting flexible regions using focus refinement (Fig. 2). Large conformational rearrangements were identified in RUVBL1- RUVBL2 upon binding of DHX34 by comparing to the structure of the unliganded RUVBL1-RUVBL2. The DII domains in both RUVBL1 and RUVBL2 subunits were slightly displaced, but major changes were found at the N-terminus of all three RUVBL2 subunits. An N-terminal segment of both RUVBL1 and RUVBL2 contains two histidine residues that are involved in the binding to nucleotides. When bound to nucleotide this region acts as a“gatekeeper”that blocks accessibility to the nucleotide binding site, traps the nucleotide and hampers nucleotide exchange. In complex with DHX34, this N-terminal region of all three RUVBL2 subunits is disordered and missing from the cryo-EM map (Fig. 2). Interestingly, DHX34 interacts with all RUVBL1 and RUVBL2 subunits but only the N-terminus of RUVBL2 subunits were affected by these large conformational changes. As a consequence, all RUVBL2 subunits lost the nucleotide whereas RUVBL1 subunits showed ADP trapped by the N-terminal region in the cryo-EM structure. By measuring how DHX34 affects the ATPase hydrolysis by RUVBL1-RUVBL2 using a combination of wild type and mutant proteins, a clear inhibitory effect of DHX34 in the ATPase activity of RUVBL2 subunits but not RUVBL1 was observed, suggesting that DHX34 traps RUVBL2 in a conformation that is incapable of binding to ATP. Thanks to this work together with previous research, a model starts to emerge of how the ATPase activity of the RUVBL1-RUVBL2 chaperone is regulated. Protruding DII domains serve as a hub capable of interacting with different partners, and these domains can communicate this “signal” as conformational changes at the N-terminus of RUVBL2 subunits and regulate the access to the ATP-binding site. In NMD, conformational changes induced by DHX34 trap the RUVBL2 subunits in a nucleotide-free conformation unable to interact back with nucleotides, thus inhibiting their ATPase activity. Hypothetically other proteins could work to stimulate ATP hydrolysis if an open access to the nucleotide pocket is compatible with binding to a new ATP molecule and the subsequent closure of the“gatekeeper”N-terminal segment, but examples of this have not been described yet. Further work is needed to understand how different clients may regulate RUVBL1-RUVBL2 differently, if RUVBL1 and RUVBL2 ATPase activity is coordinated and to define what is the actual role of this ATPase activity in the cellular functions of these chaperones. References: 1. Jha S. et al. RVB1/RVB2: Running Rings around Molecular Biology. Mol. Cell 34 , 521–533 (2009). DOI: 10.1016/j.molcel.2009.05.016 2. Dauden M. I. et al. RUVBL1–RUVBL2 AAA-ATPase: a versatile scaffold for multiple complexes and functions. Curr. Opin. Struct. Biol. 67 , 78–85 (2021). DOI: 10.1016/j.sbi.2020.08.010 3. Martino F. et al. RPAP3 provides a flexible scaffold for coupling HSP90 to the human R2TP co-chaperone complex. Nat. Commun. 9 , 1501 (2018). DOI: 10.1038/s41467-018-03942-1 4. Hug N. et al. Mechanism and regulation of the nonsense-mediated decay pathway. Nucleic Acids Res. 44 , 1483–1495 (2015). DOI: 10.1093/nar/ gkw010 5. Izumi N. et al. AAA+ proteins RUVBL1 and RUVBL2 coordinate PIKK activity and function in nonsense-mediated mRNA decay. Sci. Signal. 3 , ra27--ra27 (2010). DOI: 10.1126/scisignal.2000468 Funding acknowledgement: This work was funded by grant SAF2017-82632-P to OL by the Spanish Ministry of Science, Innovation and Universities (MCIU/AEI), co-funded by the European Regional Development Fund (ERDF); the support of the National Institute of Health Carlos III to CNIO; grants Y2018/BIO4747 and P2018/ NMT4443 from the Autonomous Region of Madrid and co-funded by the European Social Fund and the European Regional Development Fund to the activities of the group directed by OL. This work was also supported by the Human Frontiers Science Project (HFSP) grant RGP0031/2017 to OL. Corresponding author: Dr Oscar Llorca, Structural Biology Programme, Spanish National Cancer Research Centre (CNIO),
[email protected] Figure 1: (a) Representative 2D class averages of the RUVBL1-RUVBL2-DHX34 complex. Top views (top panels) and side views (bottom panels); (b) Side view of the cryo-EM reconstruction of RUVBL1-RUVBL2-DHX34. RUVBL1 is coloured in green, RUVBL2 in orange, and DHX34 in grey. Figure 2: Conformational changes of RUVBL1-RUVBL2 hexameric ring upon DHX34 binding. Top panel shows the atomic structure of RUVBL1-RUVBL within the cryo-EMmap as a transparent density. ADP is bound to RUVBL1 subunits. Bottom panel shows a close-up view of the nucleotide binding pocket of RUVBL2 after DHX34 binding (orange) superimposed to the structure of free RUVBL2 (PDB 2XSZ) in transparent grey colour. The disordered N-terminal segment of RUVBL2, missing in the cryo-EMmap, is indicated with an orange dotted line. A displaced ADP molecule indicates the release of the nucleotide from its binding site after DHX34 binding.