Nmd3 binds stoichiometrically with the 60S subunit
To examine the interaction of Nmd3 with 60S subunits, we used a rapid coimmunoprecipitation technique. A fixed amount of epitope-tagged subunits (60S-Rpl25-13xmyc) was bound to protein A beads and incubated with increasing amounts of maltose-binding protein (MBP)–Nmd3. After binding, the beads were washed extensively, and bound proteins were eluted and separated by SDS-PAGE. As the ratio of Nmd3 to 60S was increased, the amount of Nmd3 bound to 60S increased accordingly, reaching a maximum of 1:1, even at 81-fold excess of Nmd3 relative to 60S (). This result suggests that Nmd3 binds to the 60S subunit as a monomer and to a single site on the subunit.
Figure 1. In vitro binding of MBP-Nmd3 to 60S but not 80S ribosomes. (A) Increasing amounts of MBP-Nmd3 were incubated with Rpl25-myc–containing 60S subunits and immunoprecipitated with anti-myc antibody and protein A beads. Bound proteins were eluted in (more ...)
The Nmd3 protein used in this work was expressed as a fusion to MBP. MBP-Nmd3 fully complemented an nmd3
deletion mutant (unpublished data), indicating that the fusion protein is functional in vivo. We found that cleavage of MBP from Nmd3 destabilized the protein and reduced 60S binding. Consequently, all work was performed with the intact fusion protein. Previously, we showed reconstitution of the Nmd3–60S complex using a GST-Nmd3 fusion (Ho et al., 2000a
). However, because this protein dimerizes 60S subunits (unpublished data), we used it in this work only as a control for specificity of RNase footprinting.
Nmd3 binds to free 60S subunits but not to 40S subunits or 80S complexes in vivo, suggesting that its binding site may be blocked by the presence of the 40S subunit (Ho and Johnson, 1999
). To test whether this result could be recapitulated in vitro, we compared the binding of Nmd3 to purified 60S subunits versus 80S ribosomes. MBP-Nmd3 was incubated alone, with 60S subunits, or with preformed 80S ribosomes, and reactions were separated by centrifugation through a sucrose cushion. Under these conditions, free Nmd3 remained entirely in the supernatant (, lanes 1 and 2), whereas in the presence of 60S subunits, Nmd3 quantitatively cosedimented with the subunits (, lanes 3 and 4). In contrast, Nmd3 did not cosediment with preformed 80S ribosomes, but rather remained in the supernatant fraction (, lanes 5 and 6).
Localization of Nmd3 protein on the 60S subunit
To obtain a more detailed picture of how Nmd3 interacts with the large subunit, we used cryo-EM and single particle image reconstruction. Cryo-EM maps depicting the 60S subunit alone () and in complex with the adaptor protein () were obtained at resolutions of 18 Å and 16 Å, respectively. A comparison of the two maps clearly shows an extra density attached to the intersubunit side of the large subunit covering the region extending from the Rpl1 stalk base to the P-protein stalk base of the 25S ribosomal RNA (rRNA; ).
Figure 2. Visualization of MBP-Nmd3 binding to the 60S subunit. (A) Intersubunit side view of the control 60S subunit. (B) Intersubunit view of the segmented 60S part of the MBP-Nmd3–60S reconstruction. Significant conformational changes are seen in the (more ...)
This extra density visualized in the cryo-EM map was computationally separated by a segmentation procedure using SPIDER (system for processing image data from EM and related fields; Frank et al., 1996
). Because the Nmd3 protein was purified with the MBP tag, it is expected that the extra density contains both MBP and Nmd3 as the intact fusion protein. Indeed, the molecular mass of this extra density calculated from the volume it occupies (~110 kD at threshold value 42; mean density and variability [σ] of the MBP-Nmd3–bound 60S subunit map are 6 and 31, respectively, using 0.82 D/Å3
as protein density) is substantially larger than the known molecular mass of Nmd3 (59 kD) and close to the expected size of MBP-Nmd3 (103 kD). In addition, biochemical results () rule out the possibility of the presence of two copies of Nmd3. Therefore, the additional mass can be attributed to the presence of the MBP tag (molecular mass ~44 kD). These results suggest that the entire MBP-Nmd3 fusion protein is visualized in the cryo-EM map. The current resolution does not allow us to model MBP into the mass attributed to MBP-Nmd3. The position of Nmd3 at the interface of the 60S subunit is incompatible with subunit joining, which is consistent with the observation that Nmd3 does not bind to the 80S ribosome in vivo (Ho and Johnson, 1999
) or in vitro ().
The conformational changes in the MBP-Nmd3–bound 60S subunit relative to the control 60S subunit are shown in . Significant displacement was observed in the following regions: (a) the base of the L1 stalk, (b) the GAC and the sarcin–ricin loop (SRL; domain VI of the 25S rRNA), and (c) the CP and the region around the peptidyl-transfer center. In all of these regions, ribosome density was shifted toward the MBP-Nmd3 density. Of particular note, the cleft between the CP and the GAC was narrower in the complex than in the control map. Overall, the changes on the intersubunit side of the 60S subunit can be likened, in their tendency, to the grip of a hand around an object (MBP-Nmd3) on its palm (primary binding sites are H69 and H95 of 25S rRNA; for brevity, rRNA helices of 25S rRNA will be denoted by “H”).
The L1 stalk, containing protein Rpl1, is seen in the open position (Valle et al., 2003
) in both complex and control maps in which the protein part (Rpl1) of the mushroom-shaped head is partially visible. In contrast, the extended part of the acidic P-protein stalk region and the protein Rpl12 (L11p) at the stalk base are neither visible in the control nor in the 60S subunit map of our complex.
Biochemical characterization of 60S subunit–ligand interactions
To seek supporting evidence for the position of MBP-Nmd3 on the 60S subunit, helices appearing to make contact with the mass assigned to MBP-Nmd3 in the cryo-EM map were probed for altered sensitivity to RNaseV1, a nuclease specific for double-stranded RNA. In these assays, 60S subunits were incubated alone, with MBP-Nmd3, or with GST-Nmd3 and treated with RNaseV1. The GST-Nmd3 reactions were used to control for MBP-specific effects. After RNaseV1 treatment, the rRNA was extracted, and reverse transcription was performed using radio-labeled primers. Primer extension reactions were compared with a DNA sequencing ladder to identify the positions of cleavages.
Protection by both MBP- and GST-Nmd3 against RNaseV1 was observed for H38 at four positions (). Two of these positions (bases 1045 and 1054) correspond to E. coli 23S bases 908 and 920. MBP- and GST-Nmd3 binding also protected three positions in H95 against cleavage. These positions (3003, 3009, and 3047) correspond to E. coli 23S bases 2637, 2643, and 2680 (). A strong enhancement of RNaseV1 cleavage was observed with both Nmd3 fusion proteins (i.e., GST- and MBP-Nmd3) at three positions in H69, nt 2253, 2256, and 2259 (corresponding to E. coli 23S nt 1913, 1916, and 1919). Furthermore, a GST-Nmd3–specific protection was seen in H69 at position 2265 (corresponding to E. coli 23S position 1925), and an MBP-Nmd3–specific RNaseV1 protection was observed at position 2142 of H65 (corresponding to E. coli 23S nt 1784). Primer extension analysis did not reveal significant changes in other regions of 25S, 5.8S, or 5S rRNA (unpublished data).
Figure 3. rRNA protection: MBP-Nmd3 interaction with helices H38, H65, H69, and H95 of 25S. (A) 60S subunits were incubated with no protein, MBP-Nmd3, or GST-Nmd3 and treated with RNaseV1. The rRNA was extracted, and primer extension reactions were performed to (more ...)
H38 is part of 25S rRNA domain II, which accounts for most of the solvent-side surface of the large subunit. However, the tip of this helix (A-site finger), adjacent to the CP, protrudes toward the subunit interface side and participates in the formation of the intersubunit bridge B1a. In contrast, H65 and H69 belong to domain IV, which accounts for most of the intersubunit surface of the large subunit. H69 is positioned at the center of the large subunit interface and participates in the formation of two essential intersubunit bridges, B2a and B2b (Yusupov et al., 2001
). H65 is also exposed to the subunit surface on the intersubunit side. H95 (SRL; rRNA domain VI) is situated below the P-protein stalk base region (Ban et al., 2000
), and part of it is exposed to the solvent ( and ). Based on the protection assay results, a tentative identification of the positions for MBP and Nmd3 in the density can be made. Our results enable us to suggest that the SRL/CP proximal part of the differential mass observed in the cryo-EM structure accounts for Nmd3, whereas the distal part close to H65 likely represents the MBP portion of the fusion protein (see next section).
Identification of Nmd3–60S subunit interactions
Overall, the MBP-Nmd3 density appears as a complex, extended mass stretching from the Rpl1 stalk base side of the 60S subunit to the P-protein stalk base (). Connections between the MBP-Nmd3 complex and the large subunit are clearly visible in three places (marked as C1, C2, and C3 in and ). To determine the molecular details of interaction between the large subunit and the MBP-Nmd3 density, the quasiatomic model of the 60S subunit previously derived by cryo-EM and homology modeling (Spahn et al., 2001
) was used (PDB ID 1S1I
). This model was aligned, as a rigid body, to the 60S subunit within the map of the complex. The analysis of the ligand–subunit interactions is consistent with the biochemical results: the anchoring region (C2) of the center of the MBP-Nmd3 mass on the ribosome was determined as being formed at the H69 region of 25S rRNA (). Additionally, contact C1 with the part of rRNA domain IV that contains H65 and contact C3 with the H95/SRL region were identified. Based on MBP and Nmd3 molecular masses and our biochemical data, we expect the MBP-Nmd3 density in the cryo-EM map to be roughly split evenly between the two, with the Nmd3 part being closest to H69/SRL. We suggest Nmd3 to be directly interacting with H95 and H38 of the 60S subunit at the C3 point of contact (; ; and ). The C2 point of contact involves the central part of the MBP-Nmd3 density ( and ). This region of the density is most likely at the borderline between Nmd3 and MBP. This interpretation is supported by the fact that in , we observe multiple H69 sites of altered RNaseV1 sensitivity for both MBP- and GST-Nmd3 (suggesting Nmd3 interaction) along with a protection specific for the GST-Nmd3 control (suggesting the GST protein tag to be interacting). The C1 point of contact is at the end of the combined MBP-Nmd3 density closest to Rpl1 and is therefore expected to represent solely the interaction between MBP and the 60S subunit. Indeed, we observe an MBP-specific protection from RNaseV1 cleavage in H65 strongly supporting this interpretation.
Figure 4. MBP-Nmd3 interaction with the 60S subunit. (A and B) Close-up view of the quasiatomic structure of the 60S subunit (PDB ID 1S1I; Spahn et al., 2001), with the MBP-Nmd3 density showing connections with the rRNA helices (A) and nearby proteins (B). (C–E) (more ...)
Cryo-EM maps at the resolution achieved in this study cannot confirm the identity of interacting nucleotides suggested by the protection assay. However, to participate in the interaction with the MBP-Nmd3 mass, these nucleotides are required to be exposed on the subunit interface. Examination of the 50S subunit (PDB ID 2AW4
) within the E. coli
70S ribosome crystal structure (Schuwirth et al., 2005
) elucidated that, indeed, all of the nucleotides identified in the protection assay were situated at the intersubunit side of the rRNA helices concerned (). However, except for the base 1919 (E. coli
number) on H69, none of the nucleotides were totally exposed on the surface (). The fact that they are accessible to the binding by MBP-Nmd3 must be attributed to a difference in the conformation of the relevant helices caused by subunit association. In fact, H38 and H69 are disordered in the crystal structure of the isolated 50S subunit (Ban et al., 2000
), suggesting that these helices are initially in a variety of different conformations and stabilized only upon subunit joining. In addition, the observed change in large subunit conformation upon MBP-Nmd3 binding indicates that an induced-fit mechanism might be at work.
A thread of density at the P-protein stalk base side of the MBP-Nmd3 density is visible (, asterisk). It is likely that this part of the density does not belong to MBP-Nmd3 but rather represents a conformational change in this region of the ribosome caused by ligand binding. Apparently, the nearest neighbors of MBP-Nmd3 among the large subunit proteins are Rpl23 (L14 family), Rpl9 (L6), Rpl12 (L11), and Rpl10 (L10e; ).
The cryo-EM map presented in this study is of Nmd3 (fused with MBP) in complex with a mature 60S subunit. However, during ribosome assembly in eukaryotes, Nmd3 initially binds to pre-60S particles in the nucleus to direct their export to the cytoplasm (Ho et al., 2000b
; Gadal et al., 2001
). After export, the pre-60S particle undergoes a series of maturation steps involving the release of trans-acting factors and the assembly of certain ribosomal proteins (Panse and Johnson, 2010
) and culminates in the release of Nmd3 (Lo et al., 2010
). The release of Nmd3 depends on the presence of ribosomal protein Rpl10 and the activity of the GTPase Lsg1 (Hedges et al., 2006
). Thus, at the time of Nmd3 release, the subunit is presumably mature. The complex that we have reconstituted, which contains Rpl10 (unpublished data), may represent a late intermediate of 60S maturation, after Rpl10 loading but before Nmd3 release. We have also previously suggested that both Nmd3 and Lsg1 can bind to mature subunits as well as nascent subunits (Ho et al., 2000a
). Considering that Nmd3 appears to be the last factor released from the nascent subunit, its binding to mature subunits could simply be a reversal of this step, possibly as a means of inhibiting 60S function under certain conditions, for example during stress response.
With the binding of Nmd3, the interface of the 60S subunit containing Rpl10 is apparently pulled toward the ligand. The resulting conformational change of the 60S subunit may reflect strain induced by Nmd3 binding. Although there is no direct interaction between the isolated mass and the 60S subunit at the Rpl10 region visible at this resolution of the cryo-EM map, the morphological features at the Rpl10-binding site and H38 (A-site finger) regions in the 60S subunit appear different in the complex as compared with the control 60S subunit map (). However, the resolution of the maps does not allow us to model this protein accurately inside the density. We speculate that the conformation of the Rpl10-binding site changes upon GTP hydrolysis on Lsg1, allowing the 60S subunit to relax into the more open position. This relaxation may facilitate proper accommodation of Rpl10 and the coordinated release of Nmd3 ().
Figure 5. Schematic representation of the proposed mechanism for Nmd3 release. The Nmd3-bound 60S subunit in the cytoplasm presents a different conformation compared with the unbound 60S subunit, characterized as the result of a closing motion, as if gripping the (more ...)