Solution structure of the dimerization domain of ribosomal protein P2 provides insights for the structural organization of eukaryotic stalk

Abstract

The lateral stalk of ribosome is responsible for kingdom-specific binding of translation factors and activation of GTP hydrolysis that drives protein synthesis. In eukaryotes, the stalk is composed of acidic ribosomal proteins P0, P1 and P2 that constitute a pentameric P-complex in 1: 2: 2 ratio. We have determined the solution structure of the N-terminal dimerization domain of human P2 (NTD-P2), which provides insights into the structural organization of the eukaryotic stalk. Our structure revealed that eukaryotic stalk protein P2 forms a symmetric homodimer in solution, and is structurally distinct from the bacterial counterpart L12 homodimer. The two subunits of NTD-P2 form extensive hydrophobic interactions in the dimeric interface that buries 2400 A(2) of solvent accessible surface area. We have showed that P1 can dissociate P2 homodimer spontaneously to form a more stable P1/P2 1 : 1 heterodimer. By homology modelling, we identified three exposed polar residues on helix-3 of P2 are substituted by conserved hydrophobic residues in P1. Confirmed by mutagenesis, we showed that these residues on helix-3 of P1 are not involved in the dimerization of P1/P2, but instead play a vital role in anchoring P1/P2 heterodimer to P0. Based on our results, models of the eukaryotic stalk complex were proposed.

Figure 1.

Molecular mass determination of P2 and NTD-P2 by static light scattering. Purified P2 and NTD-P2 were loaded to Superdex 75 (GE healthcare) gel filtration column and analysed by static light scattering. The molecular mass of P2 and NTD-P2 were determined to be 22.5 and 13.5 kDa, respectively (monomer molecular masses are 11.5 and 7.2, respectively), showing that both of them form dimer in solution.

Figure 2.

Dimerization mechanism of NTD-P2. (A) Topology of helices in symmetric homodimer NTD-P2. NTD-P2 homodimer consists of four helices from each chain. Noteworthy, helix-3 is located away from the dimeric interface formed by helices 1, 2 and 4. (B) Stereo-diagram showing the close-up view of the dimeric interface. (C) Residues in the dimeric interface are highly conserved (shaded black, A5, L8, L9, I26, L27, V30, I32, I55). Secondary structure elements are indicated above the alignment. (D) Intermolecular NOEs were obtained from the three dimensional ¹³C F₁-filtered, F₃-edited NOESY-HSQC experiment (25) acquired on an asymmetrically labelled NTD-P2 sample. Selected 2D F₁-F₃ plane at ¹³C frequency (24.2 ppm) of L9 CD2 was shown. L9 HD2 was found to have intermolecular NOE cross peaks to S6 HA, I26 HA, A5 HB, V30 HG2 and I32 HD.

Figure 3.

P1 and P2 form 1: 1 heterodimer spontaneously and their N-terminal domain is responsible for dimerization. (A) P1/P2 complex was formed by directly mixing P1 and P2. P2 (lane 1) was mixed with P1 (lane 2) in 1: 1 molar ratio. The presence of an up-shifted band in native gel electrophoresis (15% gel in Tris/Glycine buffer at pH 8.8) in lane 3 indicates the formation of P1/P2 complex. (B) The protein mixture of P1 and P2 was also loaded to Superdex 200 (GE Healthcare) gel filtration column and analysed by static light scattering. A single peak of P1/P2 complex was eluted, and the eluted protein was analysed by 15% SDS–PAGE (inset). The molecular mass of the P1/P2 complex was determined to be 24 ± 1 kDa (monomer molecular masses of P1 and P2 are 11.5 and 11.6 kDa, respectively), showing that they form 1: 1 heterodimer in solution. (C) P1/P2 heterodimer is more stable than P2 homodimer. Conformational stability of P2 homodimer and P1/P2 heterodimer was determined by urea-induced denaturation experiment. Mid-point of transition and m-values were 1.7 ± 0.2 M and 3.9 ± 0.4 kJ/mol/M for P2 homodimer, and 4.6 ± 0.3 M, 2.8 ± 0.4 kJ/mol/M for P1/P2 heterodimer. Free energy of unfolding were 7 ± 1 kJ/mol for P2 homodimer and 13 ± 2 kJ/mol for P1/P2 heterodimer. (D) N-terminal domain of P1 and P2 forms 1: 1 heterodimer in solution. Purified NTD-P1/NTD-P2 complex was loaded to Superdex 75 column. The molecular mass of the complex estimated by static light scattering was 14.8 ± 0.5 kDa, suggesting the formation of 1: 1 heterodimer. The inset shows SDS–PAGE analysis of purified NTD-P1/NTD-P2 complex. The theoretical molecular masses of monomeric NTD-P1 and NTD-P2 are 7.5 and 7.2 kDa, respectively.

Figure 4.

Conserved hydrophobic residues on helix-3 of P1 is responsible for P0 binding but not for P1/P2 dimerization. (A) Three conserved charged residues (D37, R38 and K41) on helix-3 of human P2 were found to be substituted by conserved hydrophobic residues (F42, W43 and L46) in P1. A triple substituted variant of P1 (P1TM, F42D/W43R/L46K) was constructed to test the role of these residues in P-complex formation. (B) P1TM can interact with P2 to form 1: 1 heterodimer. P1TM was mixed with P2 in 1: 1 molar ratio, and was then loaded to Superdex 200 (GE Healthcare) gel filtration column and analysed by static light scattering. A single peak of P1TM/P2 complex was eluted, and the eluted protein was analysed by 15% SDS–PAGE (inset). The molecular mass of P1TM/P2, estimated by static light scattering, suggests the formation of 1: 1 heterodimer. (C) Conserved residues on helix-3 of P1 are involved in binding P0. Poly-histidine-tagged P0 (HisP0) was refolded alone (lanes 1 and 2), with P1/P2 (lanes 3 and 4), with P1 (lanes 5 and 6), with P2/P2 (lanes 7 and 8) or with P1TM/P2 (lanes 9 and 10). After refolding, the protein samples were centrifuged. The pellet (lanes 1, 3, 5, 7 and 9) and the soluble fractions (lanes 2, 4, 6, 8 and 10) were analysed by 15% SDS–PAGE. (D) P0, P1 and P2 form pentameric P-complex in 1: 2: 2 ratio. The refolded complex of P0/P1/P2 (Figure 4C, lane 4) was first purified by metal-chelating chromatography and then loaded to Superdex 200 (GE Healthcare) gel filtration column and analysed by static light scattering. The molecular mass of the P-complex was determined to be 80 kDa, which is consistent with the stoichiometry of P0:P1:P2 = 1: 2: 2 for HisP0, P1 and P2 having molecular masses of 34, 11.5 and 11.6 kDa, respectively.

Figure 5.

Structural comparison among eukaryotic, bacterial and archaeal stalk proteins. The structure of human P2 homodimer (green) is compared with the structure of bacterial (Thermotoga maritima, orange) L12 homodimer (4,40,41) and of archaeal (Pyrococcus horikoshii, cyan) P1 homodimer (5). The C-terminal spine helices of bacteria L10 and archaeal P0 are in purple and magenta, respectively.

Figure 6.

Structural organization of eukaryotic stalk complex. (A) Proposed topological arrangement of eukaryotic stalk complex. The two P1/P2 heterodimers are arranged in such a way that their helix-3 of P1 are facing each other. This arrangement is consistent with our results that the conserved residues on helix-3 of P1 are vital to the formation of the eukaryotic stalk complex. (B) The structural model of the dimerization domain of eukaryotic stalk complex was fitted into the cryo-EM map of the canine 80S ribosome (35) by CHIMERA (36). P0, helix-3 of P1 and P2 are colour coded in magenta, red and green, respectively. The C-terminal domain (CTD) of P-proteins, which is extending out from the dimerization domain of the P-complex via a flexible linker (dotted line), should be in vicinity to make interactions with translation factors.