Solution structure of human P1*P2 heterodimer provides insights into the role of eukaryotic stalk in recruiting the ribosome-inactivating protein trichosanthin to the ribosome

Abstract

Lateral ribosomal stalk is responsible for binding and recruiting translation factors during protein synthesis. The eukaryotic stalk consists of one P0 protein with two copies of P1•P2 heterodimers to form a P0(P1•P2)2 pentameric P-complex. Here, we have solved the structure of full-length P1•P2 by nuclear magnetic resonance spectroscopy. P1 and P2 dimerize via their helical N-terminal domains, whereas the C-terminal tails of P1•P2 are unstructured and can extend up to ∼125 Å away from the dimerization domains. 15N relaxation study reveals that the C-terminal tails are flexible, having a much faster internal mobility than the N-terminal domains. Replacement of prokaryotic L10(L7/L12)4/L11 by eukaryotic P0(P1•P2)2/eL12 rendered Escherichia coli ribosome, which is insensitive to trichosanthin (TCS), susceptible to depurination by TCS and the C-terminal tail was found to be responsible for this depurination. Truncation and insertion studies showed that depurination of hybrid ribosome is dependent on the length of the proline-alanine rich hinge region within the C-terminal tail. All together, we propose a model that recruitment of TCS to the sarcin-ricin loop required the flexible C-terminal tail, and the proline-alanine rich hinge region lengthens this C-terminal tail, allowing the tail to sweep around the ribosome to recruit TCS.

Arginine Residues on the Opposite Side of the Active Site Stimulate the Catalysis of Ribosome Depurination by Ricin A Chain by Interacting with the P-protein Stalk

“…Because other RIPs, such as Stx1 (30,31) and TCS (29,33), also bind to the stalk to depurinate the SRL and contain basic residues at similar positions, the orientation of the active site toward the SRL by stalk binding may be a general mechanism applicable to other RIPs that interact with the stalk. Consistent with this model, recent results indicate that the level of depurination by TCS depends on the length of the hinge region within the C-terminal tail of the P-proteins (32).…”

“…Previous studies showed that trichosanthin (TCS), a single chain RIP (29), and the A 1 chain of Shiga toxin 1 (Stx1) (30,31) interact with the conserved CTD fragment of P0, P1, and P2. A recent solution structure of the fulllength P1/P2 heterodimer showed a helical N-terminal domain and an unstructured C-terminal tail, which is required for the depurination activity of TCS (32). The structure of a peptide corresponding to the last 11 amino acids of the stalk proteins in a complex with TCS has been determined (33).…”

Crystal Structure of Ribosome-Inactivating Protein Ricin A Chain in Complex with the C-Terminal Peptide of the Ribosomal Stalk Protein P2

“…Furthermore, based on our NMR structure of the stalk protein complex of P1/P2 heterodimer and biochemical analyses, we demonstrated that the flexible C-terminal tail of eukaryotic ribosome stalk can form an arm-like structure and extend with a radius up to ~125 Å [18,19,20], hinting that the complex can help recruiting RIPs towards the core of the ribosome upon RIP-P protein interaction. Apart from interacting with RIPs, several studies have reported that the conserved C-terminal motif of ribosomal P proteins is crucial for binding translation factors or interacting with elongation factors in bacterial [21,22], archaeal [11] and eukaryotic ribosomes [23].…”

Functional divergence between the two P1–P2 stalk dimers on the ribosome in their interaction with ricin A chain

“…In archaea, the ribosomal stalk is a heptamer, L10–(L12) 6 [10], whereas in eukaryotes, it is a pentamer, P0–(P1–P2) 2 [11]. Despite a wealth of crystallographic [5,12–19], NMR [20,21], cryo-EM [17,22,23] and SAXS [24,25] studies, the L12/P stalk represents the last structure on the ribosome for which the architecture and precise molecular function still remain poorly established.…”