The CryoEM Structure of the Ribosome Maturation Factor Rea1

The biogenesis of the 60S ribosomal subunit is initiated in the nucleus where rRNAs and proteins form pre-60S particles. These pre-60S particles mature by transiently interacting with various assembly factors. The ~5000 amino-acid AAA+ ATPase Rea1 (or Midasin) generates force to mechanically remove assembly factors from pre-60S particles, which promotes their export to the cytosol. Here we present three Rea1 cryoEM structures. We visualize the Rea1 engine, a hexameric ring of AAA+ domains, and identify an α-helical bundle of AAA2 as a major ATPase activity regulator. The α-helical bundle interferes with nucleotide induced conformational changes that create a docking site for the substrate binding MIDAS domain of Rea1 on the AAA+ ring. Furthermore, we reveal the architecture of the Rea1 linker, which is involved in force generation and extends from the AAA+ ring. The data presented here provide insights into the mechanism of one of the most complex ribosome maturation factors.


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Eukaryotic ribosome assembly is tightly controlled by more than 200 assembly factors to ensure 14 faithful protein synthesis [1]. During the initial stages of ribosome biogenesis, rRNAs, ribosomal 15 proteins, and assembly factors associate into nucleolar pre60S particles, which ultimately mature 16 into functional large ribosomal subunits in the cytosol [2]. The AAA+ (ATPases Associated with 17 various cellular Activities) family member Rea1 (or Midasin) consists of nearly 5000 amino acids 18 and generates force to mechanically remove assembly factors. Rea1 pulls out the Ytm1 complex 19 [3,4] to promote the transfer of pre-60S particles from the nucleolus to the nucleoplasm [5]. Rea1 20 also removes the assembly factor Rsa4 [6], which triggers a signalling pathway that ultimately 21 recruits RanGTP to pre-60S particles to export them to the cytosol [6,7].  removal might also indirectly remodel the important H89 rRNA helix of the peptidyltransferase 23 3 centre into its correct position [8][9][10]. Despite its crucial importance for pre-60S particle 1 maturation, the Rea1 structure and mechanism have remained largely enigmatic. 2 Rea1 consists of an N-terminal α-helical domain (NTD), a ring of six AAA+ domains, and 3 the C-terminal tail [6,11,12]. The Rea1 tail is subdivided into an α-helical linker region, a D/E 4 rich region, and a MIDAS (Metal Ion Dependent Adhesion Site) domain which is responsible for 5 the interaction with the Ytm1 and Rsa4 substrates [5,6,13]. Rea1-mediated Ytm1 and Rsa4 6 removal is ATP dependent [5,6]. In analogy to other AAA+ family members, it has been proposed 7 that ATP binding and hydrolysis in the AAA+ ring is coupled with conformational changes within 8 the Rea1 molecule that generate the force for assembly factor removal [6,13]. Pioneering negative 9 stain electron microscopy studies have revealed that the Rea1 tail can extend from the AAA+ ring 10 but is also able to adopt AAA+ ring proximal conformations [6]. The latter tail conformations 11 could bring the MIDAS domain close to its assembly factor substrates when Rea1 is bound to 12 pre60S particles [6]. It has been proposed that switching between these different tail conformations 13 might produce the force for the removal of Ytm1 and Rsa4 assembly factors [13]. However, in the 14 absence of high-resolution information, it is unclear what the structure of the Rea1 AAA+ engine 15 looks like, what the molecular architecture of the tail is and how Rea1 binds to its substrates. 16 17

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Overall structure 19 To provide insights into these open questions, we have determined the S. cerevisiae Rea1 structure 20 in complex with ADP by CryoEM, which revealed the NTD, the AAA+ ring, as well as the linker  In order to determine the degree of closure at the Rea1 AAA1 and AAA6 sites, we compared 8 them with closed nucleotide binding sites in a crystal structure of the Rea1 related AAA+ family 9 member dynein [17]. In the dynein structure, the AAA1 site is trapped in the ATP-hydrolysis 10 transition state due to the presence of ADP.vanadate. All catalytic residues of this site contact the 11 nucleotide and have the right conformation to support hydrolysis, indicating that the site is 12 completely closed [17]. We also carried out a comparison with the dynein AAA3 site, which is 13 bound to ADP and, although overall still closed, appears slightly more open than the AAA1 site. 14 The comparison was done by analysing the distance between the H4 α-helix, which carries the 15 arginine finger, and the closest phosphate group of the nucleotide bound to the Walker-A motif of 16 the H1 α-helix (Figure 2-Supplement 4A-D). This distance is around 8 Å in the case of the dynein 17 AAA1 site and around 12 Å in the case of the dynein AAA3 site. The equivalent distances in the 18 Rea1 AAA1 and AAA6 sites are around 10 Å and 8 Å, respectively. For the Rea1 AAA1 site, the 19 degree of closure is intermediate between the completely closed dynein AAA1 site and the "less 20 closed" dynein AAA3 site. The Rea1 AAA6 appears to be already fully closed in the ADP state. 21 In contrast to the AAA1 and AAA6 sites, the AAA2 and AAA5 nucleotide binding sites of Our structural analysis suggests that the AAA2L-H2α insert impairs the hydrolytic activity 12 of all functional Rea1 nucleotide binding sites. To provide additional evidence for its inhibitory 13 role, we deleted AAA2L-H2α and determined the ATPase activity of the mutant. In support of our 14 interpretation, the deletion of AAA2L-H2α increased the ATPase rate 10-15 fold compared to 15 wildtype Rea1 ( Figure 2F). The increase in ATPase activity was specific for the AAA2L-H2α 16 deletion mutant as deleting the AAA6L-H2α insert did not lead to a drastic change in ATPase 17 activity ( Figure 2F). The overall appearance of the Rea1 AAA+ ring can be described as consisting of two halves 4 adopting an "open" conformation [20]. The first half is formed by modules AAA6, AAA1, and 5 AAA2, which tightly associate because of the more closed AAA6 and AAA1 nucleotide binding

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A similar open AAA+ ring arrangement can be found for the dynein crystal structure [20]. 12 Here, the AAA2, AAA3, and AAA4 modules form the tightly associated first half due to the closed 13 AAA2 and AAA3 nucleotide binding sites. This first half is separated from the other AAA+ 14 modules by gaps between AAA2/AAA1 and AAA4/AAA5 [19,21]. The second half consists of 15 modules AAA5, AAA6, and AAA1 and its internal arrangement is highly similar to the situation 16 in Rea1. The most N-terminal AAA+ module of this half, AAA5, is separated from AAA6 by a 17 gap, and AAA6 is rotated towards AAA1 (Figure 2 The AAA+ ring -linker interface and the structure of the Rea1 linker 5 The AAA+ ring -linker interface is formed by the NTD, AAA6L, as well as the stem domain of 6 the linker ( Figure 3A). Within the AAA+ ring-linker interface the NTD acts as a scaffold that helps 7 to stabilize the interactions between AAA6L and the linker stem domain by contacting  H2α and the N-terminal stem domain region, which sits like a saddle on top of the NTD. In addition 9 to the linker stem, AAA6L also contacts the linker middle domain ( Figure 3A), which is located 10 right above the linker stem domain. One of the middle domain α-helices runs along the linker stem 11 towards the AAA+ ring, and a peptide region at its tip contacts the loop leading to a β-sheet that 12 packs against AAA6L ( Figure 3A,B). Additional contacts exist with the loop connecting this β-13 sheet to AAA6L H0 and the peptide linker that connects AAA6L S5 to the linker stem domain 14 ( Figure 3A). AAA6L is the only AAA+ ring part directly contacting the Rea1 linker, which puts 15 it in a key position for communicating ATP induced conformational changes from the AAA+ ring 16 into the linker. 17 In addition to the stem and middle domain, the Rea1 linker also comprises three α-helical 18 bundles at its top (top1-top3) ( Figure 1B,C). It emerges at an angle of roughly 120° from the AAA+ 19 ring ( Figure 1B). The 210 Å length of the complete linker fits well with the ~200 Å AAA+ ring 20 extension that bends towards the pre60S particle when Rea1 is bound to it [6], suggesting that the 21 linker is a key element for Rea1 tail remodelling. Previous negative stain EM studies have 22 identified a hinge region within the Rea1 tail that is involved in its remodelling [6]. Comparing interpretation of the AAA2L-H2α insert as an auto-inhibitory element that prevents movement 23 between the AAA+ modules of the ring. Since there was no evidence for linker remodelling in the 1 AMPPNP state, we decided to investigate the alternative APO and ATP nucleotide states by 2 negative stain electron microscopy. However, in these states we also did not observe linker 3 remodelling ( Figure 4-Supplement 2A,B). 4 We hypothesized that linker remodelling might be obscured by the auto-inhibitory AAA2L- difference is apparent for the AAA4 module. Since the AAA2L-H2α insert is no longer pushing 16 against AAA4L, the AAA4 module is free to move towards the central pore of the ring ( Figure   17 4D). Two rod shaped cryoEM densities emerge from AAA4L that were not present in the Rea1 18 AMPPNP map. We interpret these densities as two α-helices of the previously disordered AAA4L-19 H2α insert ( Figure 4B,E). The AAA4L-H2α insert occupies the area taken by AAA2L-H2α in the 20 Rea1 AMPPNP map ( Figure 4D). It is located in close proximity to the AAA5L H2 and AAA6L The first Rea1 high-resolution structure has allowed us to gain important insights into the 10 architecture and regulation of this essential molecular machine. The most prominent feature of the 11 Rea1 AAA+ ring is the AAA2L-H2α insert that sits like a plug in the central pore of the AAA+ 12 ring. In other AAA+ family members, such as the mitochondrial protease YME1, the disaggregase 13 Hsp106, or the unfoldase VAT, the AAA+ ring pore interacts with their respective substrates [22-14 24]. Instead of binding to the Ytm1 or Rsa4 substrates, the Rea1 AAA+ ring centre hosts a 15 structural element that regulates the ATPase activity. Its central position allows the AAA2L-H2α 16 insert to influence all conserved Rea1 ATP hydrolysis sites in parallel. The structural and ATPase 17 data presented here suggest that Rea1 exists in an auto-inhibited state with impaired hydrolytic 18 activity at the AAA2-AAA5 sites. However, previous work has established the importance of ATP 19 hydrolysis at these sites for yeast growth [16]. Furthermore, the AAA3 site has also been directly 20 implicated in Rsa4 assembly factor removal and pre60S particle export to the cytosol [11]. This 21 raises the question how Rea1 might be activated to carry out its essential function. Recent studies 22 have established that the AAA2L-H2α insert interacts with the Rix1 component of pre60S particles 23 13 to recruit Rea1 to its substrates [11]. We suggest here that this binding event relocates the AAA2L-1 H2α insert from the AAA+ ring pore to stimulate the Rea1 ATPase activity ( Figure 5). In support 2 of this idea, a recent cryoEM structure of a Rea1-pre60S particle complex has revealed a density 3 extending from the Rea1 AAA+ ring centre towards Rix1 that was interpreted as the AAA2L-H2α 4 insert [11]. 5 A key question of the Rea1 mechanism is how ATP induced conformational changes in the 6 AAA+ ring drive the remodelling of the linker to generate force for assembly factor removal. Our 7 analysis of the AAA+ ring -linker interface identified interactions between AAA6L, the linker 8 stem and helix extension of the middle domain ( Figure 5). It is conceivable that a movement of 9 AAA6L during ATP hydrolysis in the ring is transferred to the linker middle domain via the 10 interaction between AAA6L and the middle domain helix extension. A subsequent shift in the 11 position of the middle domain, with the linker stem as pivot point, would be suitable to disrupt the 12 architecture of the linker top to trigger a large scale remodelling event. 13 We determined Rea1 and Rea1_ΔAAA2L-H2α electron microscopy structures in the APO, 14 AMPPNP, ATP, and ADP nucleotide states to get insight into Rea1 linker remodelling. However, 15 we were not able to observe the expected large scale rearrangement of the linker with respect to 16 the AAA+ ring. The relative orientation between these Rea1 parts remained essentially the same 17 in all structures. We can only speculate about the reasons for this absence of linker remodelling. 18 One possibility would be that linker remodelling relies on critical interactions between Rea1 and 19 the pre60S particle so that it can only be observed when Rea1 is bound to pre60S particles. 20 Evidence for this hypothesis is provided by the Rea1-pre60S particle cryoEM structure [11]. The 21 AAA+ ring cryoEM density is much stronger than the linker cryoEM density. This suggests that 22 the linker becomes flexible with respect to the ring when Rea1 is bound to pre60S particles. CryoEM sample preparation and data acquisition 20 Gel filtration fractions of Rea1 and Rea1_ΔAAA2-H2α were diluted to 1 mg/ml using buffer B 21 (without glycerol) and ADP or AMPPNP were added to a final concentration of 3 mM. 3 μl of 22 sample were applied to glow-discharged holey carbon grids (Quantifoil Cu R2/2, 300-square-23 mesh) that were subsequently blotted for 7-8 s and flash frozen in liquid ethane using a manual 1 plunger. All cryoEM grid preparation was carried out at 4 ̊ C. All data was collected on an FEI 2 Titan Krios equipped with a Cs corrector and a Gatan K2-Summit detector (300 kV, 35-38 frames, 3 7-8 sec exposure, 1.09 Å /pixel, ~45-50 ē/Å 2) using a slit width of 20 eV on a GIF-quantum energy 4 filter (Gatan). All images were recorded in super-resolution counting mode using the automated 5 data collection software Serial EM [25] with a defocus range of 1.8 to 3.4 μm. In the case of the 6 Rea1_ΔAAA2-H2α AMPPNP state a Volta phase plate was used in combination with a target 7 defocus of 0.5 μm. The irradiated area on the VPP was changed every hour. particles were subjected to 3D refinement. The initial Rea1 model was obtained using the ab-initio 23 modelling function as implemented in Relion 2.1. The output X and Y origin information of the 1 3D refinement was used to obtain more accurate coordinates of the particles. The re-centred 2 particles were subjected to another round of 3D refinement. The obtained map was divided into 3 two parts covering the Rea1 linker region and the NTD-AAA+ ring region. We conducted focused 4 3D classification and 3D refinement of these individual parts as described [30]. The final 5 reconstructions of the NTD-AAA+ ring region and the linker were based on 35671 and 432556 6 particles, respectively. To aid the interpretation of the obtained maps, sharpening was carried out 7 by applying a negative B-factor that was either estimated using automated procedures within 8 Relion or manually set parameters. The Rea1 AMPPNP state was also reconstructed with Relion 9 2.1. The Rea1 ADP state map was low pass filtered and used as an initial reference for the first 3D 10 refinement step. Particles were aligned using the output of the initial refinement as a reference 11 followed by a focused refinement of the Rea1 linker part. The subsequent 3D classification was 12 performed post alignment using a large elliptic featureless mask encompassing the Rea1 AAA+ 13 ring to evaluate potential movement between the linker and the AAA+ ring. In all analysed classes,      Rea1 AAA ring, the H3 α-helices of AAA1L, AAA2L, AAA3L, AAA5L and AAA6L (red) point towards the pore of the AAA+ ring. The orientation of AAA4L (yellow) deviates from the orientation of the other AAA+ domains in the ring. It is rotated towards AAA5L as indicated by its H3 α-helix that points towards AAA5L rather than the AAA+ ring centre. For clarity only the AAAL domains of the AAA+ ring are shown and all inserts have been removed. B.
Comparison of Rea1 AAA+ ring with the ring from a dynein crystal structure in the ADP state.
For clarity, only the AAAL domains are shown. Left panel: In the Rea1, the AAA1L, AAA2L and AAA6L domains are more tightly associated and form one half of the AAA+ ring. AAA3L, AAA4L, and AAA5L form the more loosely associated second half. The AAA1L-AAA2L+AAA6L domain block is separated from the AAA3L-AAA5L block by large gaps between AAA2L and AAA3L as well as AAA6L and AAA5L (black arrow heads). The internal organization of the AAA3L-AAA5L half is characterized by the gap between AAA3L and AAA4L (grey arrow head), as well as the rotation of AAA4L towards AAA5L (both domains are shown in grey, with red H3 helices to indicate their relative orientation with respect to each other). Right panel: The dynein AAA+ ring (PDB-ID: 3VKG) is highly similar. Here, AAA2L-AAA4L form the more tightly packed first half of the ring. The weaker packed second half consists of AAA5L-AAA6L+AAA1L. Large gaps (black arrow heads) separate the two halves.
The internal organization of the second half is characterized by a gap between AAA5L and AAA6L (grey arrow head) and the rotation of AAA6L towards AAA1L (both highlighted in grey with H3 helices in red to indicate the orientation of AAA6L towards AAA1L).    structure of a complex between Rea1 and an Rsa4 containing pre60S particle revealed the location of the Rea1 AAA+ ring on the pre60S particle. The Rea1 linker was flexible with respect to the AAA+ ring as indicated by its weaker density. It also revealed the location of Rsa4 (green). An unidentified density was found to sit right above the Rsa4 MIDO domain. B.
Docking the AAA+ ring of Rea1_ΔAAA2-H2α APO would place the additional AAA+ ring