Abstract
The eukaryotic P-stalk contains two P1-P2 protein dimers with a conserved C- terminal domain (CTD) critical for the interaction with external factors. To understand the role of the individual CTD of human P1/P2 proteins, we examined the interaction of reconstituted human P-protein complexes and C-terminally truncated forms with ricin A chain (RTA), which binds to the stalk to depurinate the sarcin/ricin loop (SRL). The interaction between P-protein complexes and RTA was examined by surface plasmon resonance, isothermal titration calorimetry, microscale thermophoresis and bio-layer interferometry. The P1-P2 heterodimer missing a CTD on P2 was able to bind RTA. In contrast, the P1-P2 heterodimer missing the CTD of P1 protein displayed almost no binding toward RTA. Very low interaction was detected between RTA and the non-truncated P2-P2 homodimer, suggesting that the structural architecture of the P1-P2 heterodimer is critical for binding RTA. The reconstituted pentameric human stalk complex had higher affinity for RTA than the P1-P2 dimer. Deletion of P1 CTD, but not P2 CTD reduced the affinity of the pentamer for RTA. These results highlight the importance of the heterodimeric organization of P1-P2 in the human stalk pentamer and functional non-equivalence of the individual P-protein CTDs in the interaction with RTA.
Highlights
The ribosomal stalk is a pentameric protein complex made up of universally conserved ribosomal protein uL10 and P1, P2 proteins, which form a pentameric architecture called the P-stalk, uL10-(P1-P2)[2], located on the large subunit of eukaryotic ribosome[1,2,3,4,5]
The obtained molecular masses of all complexes agreed with the calculated ones (Table 1) and showed that the recombinant proteins are in intact form, with one modification most likely indicating posttranslational removal of the first methionine[44, 49]
Since the optimal structural configuration of the P-stalk proteins is exhibited by heterodimeric and especially pentameric organization[4, 38, 44, 49], we examined here the interaction of RTA with the human P1-P2 heterodimer, which is regarded as the smallest functional component of the eukaryotic stalk
Summary
The ribosomal stalk is a pentameric protein complex made up of universally conserved ribosomal protein uL10 and P1, P2 proteins, which form a pentameric architecture called the P-stalk, uL10-(P1-P2)[2], located on the large subunit of eukaryotic ribosome[1,2,3,4,5]. The most prominent feature of the eukaryotic stalk proteins is a conserved amino acid element present at the C-terminal domain (CTD) of uL10 and P1/P2 proteins[5] This stretch of highly acidic and hydrophobic amino acids (EEEAKEESDDDMGFGLFD) is regarded as the main functional element of the stalk proteins and is involved in the interaction with translational GTPases and ribosome inactivating proteins (RIPs)[20]. The dissociation constant (KD) of RTA interaction with the yeast ribosomal stalk pentamer is in the low nanomolar range, indicating that P-proteins represent a primary landing platform for ricin to accelerate depurination of the SRL38. Using TCS as experimental model, the interaction site of P-proteins with RIPs was mapped to a conserved 11-mer peptide, SDDDMGFGLFD (P11) present at the CTD of all P proteins[32]. We show that the heterodimeric conformation of P1-P2 in the stalk pentamer represents an optimal binding site for RTA and present the first evidence that universally conserved CTD of P1 and P2 have an unequal role in their interaction with RTA
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