Structural ensemble of the 10 lowest energy structures.
Solution structure of TA1092, a ribosomal protein S24e from Thermoplasma acidophilum
Article first published online: 19 JUN 2006
Copyright © 2006 Wiley-Liss, Inc.
Proteins: Structure, Function, and Bioinformatics
Volume 64, Issue 4, pages 1095–1097, 1 September 2006
How to Cite
Jeon, B.-Y., Jung, J., Kim, D.-W., Yee, A., Arrowsmith, C. H. and Lee, W. (2006), Solution structure of TA1092, a ribosomal protein S24e from Thermoplasma acidophilum. Proteins, 64: 1095–1097. doi: 10.1002/prot.21045
- Issue published online: 2 AUG 2006
- Article first published online: 19 JUN 2006
- Manuscript Accepted: 30 MAR 2006
- Manuscript Received: 16 MAR 2006
- NRL program of MOST NRDP. Grant Number: M1-0203-00-0020
- Korea Research Foundation Grant (funded by the Korean Government (MOEHRD). Grant Number: KRF-2004-005-C00129
The ribosome is a large ribonucleoprotein complex that catalyzes protein synthesis. In archaea, the ribosome is composed of a small 30S subunit and a large 50S subunit. The 30S subunit of the archaea ribosome is composed of a 16S rRNA and 28 ribosomal proteins. Genes that encode ribosomal proteins consist of multiple processed pseudogenes, which are dispersed through the genome. Recent advances in ribosome structure1 suggest close molecular interactions between rRNA and ribosomal protein, within ribosomal subunits. However, the structural role of ribosomal components during various stages of translation remains to be characterized.
Here we report the solution structure of TA1092, a member of the S24e protein family from Thermoplasma acidophilum.2 Members of the S24e protein family are components of the 30S subunit in both archaea and eukaryotes. Because the detailed structure of ribosomal protein S24e is not yet available for model organisms, the structure of TA1092 will expand our knowledge and understanding of 30S ribosomal protein structure–function for different species.
Methods and Materials.
The TA1092 gene was cloned into the expression vector, pET15b, and overexpressed in Escherichia coli strain BL21 (DE3) (Novagen Inc., Madison, WI). The cells were grown on a minimal M9 medium, with 15NH4Cl and 13C-glucose to obtain uniformly labeled 15N/13C- protein. The protein was purified by immobilized metal affinity chromatography, and the N-terminal His-Tag was cleaved by tobacco etch virus (TEV) protease, for use in NMR experiments. The final protein sample was concentrated to 1.5 mM, in 50 mM potassium phosphate, 0.1 mM NaN3, and 90% H2O/10% 2H2O buffer solution.
2D-[1H-15N] HSQC, HNCA, HNCACB, CBCA(CO)NH, H(CC)(CO)NH, HCCONH, 3D 15N-edited NOESY (tmix = 200 ms) and 3D 13C-edited NOESY3 (tmix = 200 ms) spectra were obtained using Bruker DRX 500, 800 and Varian INOVA 500 MHz spectrometers. The resulting data were processed using NMRPipe.4 Backbone assignments of TA1092 were obtained using Sparky,5 and side-chain proton resonances were identified using H(CC)(CO)NH and HCCH-TOCSY spectra. Dihedral angle restraints were derived from chemical shift analysis of Cα, Cβ, CO, and Hα, which were performed using the TALOS program.6
Results and Discussion.
NMR structures were determined from the following: 1141 NOEs [307 intraresidues, 377 sequentials, 134 medium ranges (2 = |i − j| = <4), 323 long ranges (|i − j| > 4)], 58 distance restraints (for backbone hydrogen bonds) and 129 dihedral angle restraints. The low energy structures were optimized using a water refinement protocol, to obtain final structures. The root-mean-square deviation (RMSD) values of the 10 structures with the lowest energy were determined to be 0.81 ± 0.26 Å, for the structural regions (Table I). The solution structure shows that the N-terminal region forms a compact folded structure, while the C-terminal end (residues 85–98) is very flexible [Fig. 1(A)]. The structure is classified as an α + β fold, with a ββαββα order [Fig. 1(B)]. The first helix (α1) adjoins its counterpart strands (β1:β2, β3:β4) and is anchored to one side of the β-sheet [Fig. 1(C)]. The C-terminal helix (α2) is flanked by β-sheets and moves independently.
|Sequential (|i − j| = 1)||377|
|Medium range (2 ≤ |i−j| ≤ 4)||134|
|Long range (|i − j| > 4)||323|
|Dihedral angle restraints|
|CNS energy (kcal/mol)|
|Van der Waals||−192.15|
|Backbone atoms||All heavy atoms|
|All residuesb||1.41 ± 0.33 Å||2.33 ± 0.43 Å|
|Ordered regionsc||0.81 ± 0.26 Å||1.67 ± 0.51 Å|
|Ramachandran plot (%)d|
|Residues in most favored regions||84.6|
|Residues in additional allowed regions||11.7|
|Residues in generously allowed regions||2.8|
|Residues in disallowed regions||0.9|
Previous reports have suggested that most ribosomal proteins have common structural features, such as N-terminal globular domains and extension of the C-terminal disordered region.12 The structural data base search by DALI13 provided eight structural homologues (Z-score > 2.0). The best score was for the S24E protein, which is derived from Methanosarcina mazei (1XN9, Z-score 6.6), and has a 31% sequence identity with TA1092. The S24e protein from M. mazei showed a common molecular topology with TA1092, specifically the relative orientation of the β-sheet and α1, which are very similar to those of M. mazei. The major structural differences are found in β3 and the α2 helix [Fig. 2(B)]. Even though β3 of TA1092 is much shorter than that of M. mazei, the overall molecular shape of TA1092 is very similar to that of M. mazei.
Highly conserved residues [Asn11 (β1), Arg16 (β2), and Tyr72 (β4)] among the S24e protein family are all found as solvent exposed forms that generate a hydrophilic paddle [Fig. 2(A)]. We hypothesize that these residues have an important effect on protein function. This implies that Asn11, Arg16, and Tyr72 residues might interact directly with rRNA, via a hydrogen bonding network. In addition, our structure suggests that hydrophilic residues, located in the β-sheet region, could be involved in the stabilizing the protein/rRNA complex, by intercalating with the rRNA molecule. The conserved basic residues (Arg89, Lys93, Lys95, Lys98), located near the flexible C-terminal tail, could allow an intermolecular interaction with the phosphate group of rRNA, thus providing extra selectivity for binding of TA1092 with the rRNA molecule.
Note: Both the backbone and side-chain chemical shifts for TA1092 have been deposited in the BioMagResBank (accession code BMRB-6989). Coordinates for the 10 structures, and the average energy minimized structure, have been deposited in the RCSB PDB with accession code 2G1D.
- 5SPARKY 3. San Fransicso, CA: University of California; 2004., .
- 11The PyMOL molecular graphics system. San Carlos, CA: DeLano Scientific; 2002..