The acidic proteins P0, P1 and P2 assemble into a pentameric complex, which forms the characteristic protuberance that is termed the ribosomal stalk. In both eukaryotic and prokaryotic ribosomes, this stalk is essential for translation, as it binds a number of initiation and elongation factors (17–19
). Through mutagenesis analysis and protein–protein interaction studies, we have shown that TCS interacts with the conserved DDD motif of the C-terminal tail of P2 through K173, R174 and K177 (4
). Here, we report the structural evidence for this interaction by elucidating the crystal structure of the TCS–c11-P complex. In this complex, we find that a hydrophobic pocket at the C-terminal domain of TCS also takes part in the interaction, associating with the non-polar residues at the C-terminal tail of the P protein.
In the human ribosomal P proteins, the DDD motif is present in P1 and P2 but is replaced by DED in P0. This indicates that, the second Asp in this motif may not be important for the interaction. Consistent with this, the TCS–c11-P structure shows that the side chain of the second Asp (D3) in c11-P points toward the solvent and does not take part in the interaction with TCS. Since the other amino acids in c11-P are identical in the three P proteins, our observation of the interaction between TCS and c11-P may be applied to all of them.
On the surface of the ribosome, the negative electrostatic potential that is contributed by the phosphodiester backbone and the solvent-exposed acidic patches of ribosomal proteins covers much of the ribosomal surface. Recently, it has been found that the electrostatic interaction between ribotoxin and the ribosome leads to an unusually high catalytic efficiency; it is hypothesised that ribotoxins may bind to several sites on the ribosome and diffuse to the SRL (20
). Our structure of the complex also confirmed that charge-charge interactions are crucial for TCS to interact with the P protein. The present finding does not exclude the possibility that TCS may interact with other ribosomal proteins, although we have not found such interactions by yeast two-hybrid screening (22
). By passing rat liver extract through a TCS-sepharose column, a group has recently observed that TCS binds to ribosomal protein L10a with high affinity; whether this interaction contributes to the activity of TCS has not been determined (23
). By superimposing the TCS-SRL model on the eFE2-SRL model, we have previously found that the three basic residues (K173, R174 and K177) of TCS are in close proximity to the P-protein binding site of eEF2 (4
). Also, a model of the mammalian 60S ribosome, with the structure of P0 including up to the first Asp in the c11-P sequence, has just been released (24
). The distance between the targeted adenine (A4565) on SRL and P0 was found to be about 50 Å, and the distance between the concerned adenine and L10a was about 140 Å. This observation further supports the argument that the binding of TCS to P proteins would make the TCS readily accessible to the SRL. Since we have shown that the interaction between TCS and the P protein is needed for the full activity of TCS, this interaction should be crucial for TCS to act on the targeted adenine.
Pokeweed anti-viral protein, a type I RIP distinctly related to TCS and active on prokaryotic and eukaryotic ribosomes, has been found to interact with ribosomal protein L3 (25
) and does not need the C-terminus of the P protein in Trypaosoma cruzi
ribosomes for full activity (26
). On the other hand, a number of other type I and type II RIPs have been shown to access the ribosome through interactions with P proteins. With cross-linking experiments, the interaction between RTA and P0 of human lung carcinoma cells has been observed (27
). Also, NSBr-saporin has been cross-linked to a 30 kDa ribosomal protein, presumably P0, from the 60S yeast ribosome (28
), and several positively charged residues in the C-terminal region of SO6 have been protected from chemical modification in a SO6-ribosome complex (12
). Pepocin has been shown to require P proteins for the depurination of rRNA (29
). Recently, it has also been found that the C-terminal 11-aa of the P protein interacts directly with SLT-1A and RTA (5
), thus providing independent support to our structural observations of TCS and the c-11 peptide.
Using the TCS–c11-P complex as a reference and docking the c11-P peptide to RTA, SO6 and SLT-1A, we have found that the C-terminal domains of these RIPs share a common binding surface with the c11-peptide. The N-terminal charged residues of c11-P, in particular the side chains of Asp2 and Asp4 and the backbone oxygen of Asp3, form hydrogen bonds with the side chains of an α-helix of the RIP ( and ). Such hydrophilic interaction involving the N-terminal Asp appears to be crucial, as a C-7 peptide (MGFGLFD) did not interact with TCS, RTA or SLT-1A (4
). Similar to the case of TCS–c11-P, there is a well-defined hydrophobic pocket in RTA, SO6 and SLT-1A for the insertion of Phe 10 of the c-11P. In RTA, SO6 and SLT-1A, we have also found potential hydrogen bonds between them and residues in the C-terminal region of c11-P (). The additional interactions are attributed by the extended two stranded β-sheet in both TCS (residues 213–225) and RTA (residues 230–242) or loop in SO6 (residues 227–236). Such interactions are absent in SLT-A of which the C-terminal 80 residues fold in a completely different conformation. The potential interaction of C-11 peptide with PAP, which has not been shown to interact with P proteins, was also examined (Supplementary Figure 1
). In the N-terminal region of c11-P, only Asp4 is hydrogen bonded to the PAP (Supplementary Table 1
). Hence, the importance of the N-terminal Asp residues that was demonstrated in other RIP–c11-P complexes is not well supported in the PAP–c11-P model. Also, a clear molecular interaction surface on PAP, in particular a hydrophobic cleft for Phe 10 of c11-P, is absent, suggesting that the interaction between PAP and c11-P, if it occurs, may not be as strong or specific as that shown for TCS, RTA, SO6 and SLT-1A.
Figure 6. Stereo images of the molecular docking of c11-P peptide to RTA, SO6 and SLT-1A. (A) RTA, (B) SO6 and (C) SLT-1A. c11-P is shown in ball and stick representation and in green colour. Potential hydrogen bond interactions between the RIPs and the c-11 peptide (more ...)
Possible hydrogen bonds formed between the c11-P peptide and RTA, SO6 or SLT-1A
In conclusion, we have provided structural evidence of the interaction between TCS and the consensus C-terminal motif of ribosomal stalk proteins. This interaction is required for TCS to exert its full activity and may be generalised to other RIPs.
Coordinates of TCS–C11-P complex, the V232K/N236D and the K173A/R174A/K177A variants have been deposited with PDB accession codes 2JDL, 2JJR and 2VS6, respectively.