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. 2000 Jan 17;19(2):174-86.
doi: 10.1093/emboj/19.2.174.

Flexibility, conformational diversity and two dimerization modes in complexes of ribosomal protein L12

M C Wahl  1 , G P Bourenkov, H D Bartunik, R Huber
Affiliations

Flexibility, conformational diversity and two dimerization modes in complexes of ribosomal protein L12

M C Wahl et al. EMBO J. .

Abstract

Protein L12, the only multicopy component of the ribosome, is presumed to be involved in the binding of translation factors, stimulating factor-dependent GTP hydrolysis. Crystal structures of L12 from Thermotogamaritima have been solved in two space groups by the multiple anomalous dispersion method and refined at 2.4 and 2.0 A resolution. In both crystal forms, an asymmetric unit comprises two full-length L12 molecules and two N-terminal L12 fragments that are associated in a specific, hetero-tetrameric complex with one non-crystallographic 2-fold axis. The two full-length proteins form a tight, symmetric, parallel dimer, mainly through their N-terminal domains. Each monomer of this central dimer additionally associates in a different way with an N-terminal L12 fragment. Both dimerization modes are unlike models proposed previously and suggest that similar complexes may occur in vivo and in situ. The structures also display different L12 monomer conformations, in accord with the suggested dynamic role of the protein in the ribosomal translocation process. The structures have been submitted to the Protein Databank (http://www.rcsb.org/pdb) under accession numbers 1DD3 and 1DD4.

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Figures

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Fig. 1. Alignment of 21 representative bacterial L12 molecules. Sequence numbering is according to the T.maritima protein. The secondary structure elements as found in a full-length molecule of the present orthorhombic crystal structure are indicated by standard symbols underneath the alignment. Positions with a conservation value of ≥9 and ≥5 (ALSCRIPT; Barton, 1993) are shown in front of red and yellow backgrounds, respectively. The alignment was done with the PILEUP option of the Wisconsin package [version 9.1, Genetics Computer Group (GCG), Madison, WI] using default parameters. The figure was produced with ALSCRIPT (Barton, 1993).
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Fig. 2. (A) Experimental MAD electron density at 2.6 Å resolution for the I213 structure in the area of the hinge regions. (B) Annealed composite 2Fo–Fc ‘omit’ map at 2.0 Å resolution for the C2221 structure covering the same region. Both maps are contoured at the 1σ level. It is obvious that the flexible hinge is well defined in the electron density. Unless otherwise indicated, all figures were produced with the programs MOLSCRIPT/BOBSCRIPT (Kraulis, 1991) and RASTER3D (Merrit and Murphy, 1994).
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Fig. 3. (A) Monomer fold of L12. Residues pertaining to the NTD are colored violet, the hinge region is shown in cyan and the CTD in magenta. Secondary structure elements are labeled. (B) Association of two full-length L12 molecules (red and blue) and two N–terminal fragments (green and yellow). Molecules are labeled I–IV as referred to in the text. The main contact areas between the component molecules are labeled a–d. (C) Surface representation of the tetrameric L12 complex indicating the intimacy of the contacts and the complementary shapes of the components. (C) was produced with DINO (http://www.bioz.unibas.ch/~xray/dino).
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Fig. 4. Electrostatic surface potential for the monomer (A), dimer (B) and tetramer (C). A highly acidic surface is visible in the oligomers. The monomer displays several hydrophobic areas, especially in the N–terminal furrow, along the hinge helix and on the underside of the CTD. These patches are efficiently shielded from solvent in the oligomers. Regions used for contacts a–d (Figure 3) are indicated. The figure was produced with GRASP (Nicholls et al., 1991).
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Fig. 5. (A) Dimerization mode I showing the four-helix bundle between the α2 and α3 helical hairpins. (B) Dimerization mode II with helices α1, α2 and α3 in a five–helix bundle. The molecule coloring is as in Figure 3. Secondary structure elements are labeled and the directions of the helices are indicated by arrows.
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Fig. 6. (A) Side view of a model dimer that contains the monomers in the observed conformation of the full-length molecules and makes use of dimerization mode II. A severe clash of the hinge helices is obvious in the center. (B) Superposition of residues 1–40 of a full-length L12 molecule (blue) and an N–terminal fragment (yellow) showing the different strand directions and conformations past residues 30.
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Fig. 7. Highly conserved residues (green) of L12 mapped onto the surface of the core dimer. Views are from the top, side and bottom as indicated. The most extensive area of conserved side chains is visible around helix α5 (circled, compare with Figure 3). The conserved residues may be instrumental in the interaction with L10 (bottom view) and translation factors (side and top views). The figure was produced with GRASP (Nicholls et al., 1991).
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Fig. 8. Wire diagram of the core L12 dimer (molecule coloring as in Figure 3) with residues Glu11, Phe29 and Thr32 in space filling mode and the molecular surface indicated as a glassy envelope. The six residues emphasized, critical for the interaction with r–protein L10, line the N–terminal furrows, marking the path of the additional N–terminal fragments (green and yellow). The figure was produced with DINO (http://www.bioz.unibas.ch/~xray/dino).
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