Jinwon Jung and In Young Yeo contributed equally to this work.
Solution structure of TA0895, a MoaD homologue from Thermoplasma acidophilum
Article first published online: 3 OCT 2006
Copyright © 2006 Wiley-Liss, Inc.
Proteins: Structure, Function, and Bioinformatics
Volume 65, Issue 4, pages 1055–1057, 1 December 2006
How to Cite
Jung, J., Yeo, I. Y., Hong, E., Yee, A., Arrowsmith, C. H. and Lee, W. (2006), Solution structure of TA0895, a MoaD homologue from Thermoplasma acidophilum. Proteins, 65: 1055–1057. doi: 10.1002/prot.21171
- Issue published online: 14 NOV 2006
- Article first published online: 3 OCT 2006
- Manuscript Accepted: 14 JUL 2006
- Manuscript Revised: 15 JUN 2006
- Manuscript Received: 4 APR 2006
- NRL program of MOST NRDP. Grant Number: M1-0203-00-0020
- Korean Government (MOEHRD). Grant Number: KRF-2004-005-C00129
Thermoplasma acidophilum is a thermoacidophilic archaeon that thrives at a temperature of 59°C and a pH of 2, conditions in which few organisms are viable. It is one of the smallest archaeal organisms with plasma membrane.1 The T. acidophilum genome encodes for the 90-residue, hypothetical protein TA0895 (TrEMBL ID: Q9HJR9). BLAST2 search indicates that TA0895 has 21% sequence identity with the small subunit of E. coli molybdopterin synthase (MoaD) and it has many homologues in archea, including T. volcanium, G. sulfurreducens, P. horikoshii, P. furiosus, and T. kodakarensis [Fig. 1(A)]. Here, we present the solution structure of TA0895 and it suggests evidence that TA0895 protein shares its function with E. coli MoaD.
Methods and Materials.
TA0895 was cloned into the pET-15b expression vector, and the recombinant protein was expressed in E. coli BL21 (DE3) cells (Novagen). This vector encodes for 18 additional amino acids which confer a six-histidine affinity tag (His-tag) at the N-terminal and Gly and Ser residues at the C-terminal. The recombinant TA0895-expressing bacteria were grown on a minimal M9 medium containing 15NH4Cl (to obtain 15N-labeled protein) or 13C-glucose, 15NH4Cl (to obtain 15N, 13C-labeled protein) at 37°C until the optical density reached 0.6 at 600 nm. Protein expression was induced with 1 mM IPTG for 5 h at 37°C. The recombinant protein was purified using immobilized-metal affinity chromatography followed by gel filtration chromatography. The concentration of the final NMR sample was about 1.0–1.5 mM. The condition of all NMR samples was as follows: 1.5 mM13C-, 15N-, or 15N-labeled protein, 50 mM potassium phosphate, and 0.01% NaN3 in 90% H2O and 10% D2O at pH 7.4.
All NMR data was acquired at 298 K using Bruker DRX 500 or Varian INOVA 600 MHz spectrometers. [1H-15N] HSQC, HNCA, HNCACB, CBCA(CO)NH, HNHA, (H)CC(CO)NH, H(CC)(CO)NH, 3D 15N-edited NOESY (τm = 150 ms), and 3D 13C-edited NOESY (τm = 150 ms) spectra were acquired for the structure analysis. Acquired FIDs were processed by NMRPipe,7 and the spectra were analyzed with Sparky.8 Backbone assignments were performed using AUTOASSIGN9 and were manually analyzed with Sparky. Backbone dihedral angle restraints were derived from chemical shifts of the backbone atoms using TALOS.10 Hydrogen bond restraints were determined based on slow exchanging amide protons identified by recording 1H-, 15N HSQC after dissolving the lyophilized 13C/15N-labeled protein sample in 100% D2O. Hydrogen bond acceptors were identified from the ensemble structure calculated without distance restraints of hydrogen bonds.11 Structure calculations were performed using both manually and automatically assigned NOE restraints with CYANA.12 The restraints used for the final structure refinement are 1047 distance constraints from NOEs, 84 from hydrogen bonds, and 109 dihedral angle restraints. No distance and angle violation was observed for structure calculations. The final 20 energy-minimized structures with the lowest target function energy were selected for further analysis. Structures were analyzed and validated using the program PROCHECK.13
Results and Discussion.
The average root-mean-square deviation (RMSD) values relative to the mean coordinates of 20 representative conformers [Fig. 1(B)] were determined to be 0.47 ± 0.15 Å for the backbone atoms and 0.93 ± 0.08 Å for all heavy atoms, respectively (Table I). The solution structure of TA0895 is composed of four β-strands and three α-helices organized in an βαβααββ order [Fig. 1(C)]. Most of residues involved in the hydrophobic surface were found between the β-sheets (β1, β3, and β4) and second helix (α2). Residues Leu30, Leu31, Leu34, and Tyr38 of α2 interact with residues on the β-sheets (Val2, Val4, and Tyr6 from β1; Val56, Ile58, and Val 60 from β3; and Ile81 and Leu83 from β4) via hydrophobic contacts.
|Sequential (|i − j| = 1)||316|
|Medium range (2 ≤ |i − j| ≤ 5)||178|
|Long range (|i − j| > 5)||282|
|Hydrogen bond restraintsa||84|
|Dihedral angle restraints|
|Mean CYANA target functionb||1.22 ± 0.05 Å2|
|RMS deviations from the average coordinate|
|Backbone atoms||0.47 ± 0.15 Å|
|All heavy atoms||0.93 ± 0.08 Å|
|Ramachandran analysis (%)|
|Residues in most favored regions||83.9|
|Residues in additional allowed regions||13.6|
|Residues in generously allowed regions||2.5|
|Residues in disallowed regions||0|
To find structural homologues, DALI14 search was performed. Most similar structure from data base search is 1V8C, a structure of MoaD related protein from Thermus thermophilus HB8 with Z score of 10.1. The second one is MoaD from E. coli (1FMA) with Z score of 7.3. Ubiquitin is also found with Z score of 4.5. The structural similarity between MoaD and ubiquitin was previously reported.15 BLAST search on PDB reports another structural homologue, which is a homologue protein (1VJK) from P. furiosus. Both structures were well superimposed with RMSD of 2.48 Å for Cα.
E. coli MoaD and TA0895 display 21% sequence identity and 52% sequence similarity. If these two proteins share structural homologues, TA0895 might represent a MoaD homologue protein with similar molecular and cellular functions of MoaD. NMR structure shows that the overall topology of TA0895 is almost the same as the structure of MoaD. When the structures of these two proteins are superimposed, backbone RMSD is calculated as 2.392 Å for the best-fitted 74 pairs of Cα atoms [Fig. 1(D)]. Additionally, TA0895 and MoaD share their amino acid sequences. Multiple sequence alignments of TA0895 with its homologue proteins suggest four highly conserved residues, showing that the consensus sequence is PPXXGG [Fig. 1(A)]. In MoaD, the carboxyl group of the last glycine residue is considered as a sulfur donor in a thiocarboxylate reaction during molybdopterin synthesis. Its long and flexible C-terminal tail has penetrated deeply into its interacting proteins, such as MoeB(PDB:1JW9_D)15 or MoaE(PDB:1FMA_D),16 where it is placed at their active sites. TA0895 also has a similar long and flexible tail which might be essential for interaction with its partner proteins. In addition, many of the hydrophobic residues involved in the interactions between MoaD and MoaE or MoeB are both sequentially and structurally well-conserved. For example, the F7, L52, L59, and F75 residues of E. coli MoaD are known to interact with MoaE or MoeB. The corresponding residues in TA0895 (Y7, I57, M68, and F84) have similar chemical properties and they are located in the same positions in the 3D structure. Beside TA0895 belongs to thiaminS (ThiS) family according to Pfam,17 the structure of ThiS and MoaD is quite different. They have similar topology with ubiquitin, however, MoaD has a helix between first two β-strands and the helix between β2 and β3 is kinked unlike ubiquitin or ThiS. Therefore, it seems to be reasonable to distinguish between MoaD and ThiS by Conserved-Domain Database.18 All these results lead us to suggest that TA0895 shares some of the same functions as E. coli MoaD, representing a MoaD homologue found in Thermoplasma acidophilum.
(Note: Chemical shifts for TA0895 have been deposited in BioMagResBank (accession code: BMRB-6982) and coordinates for the 20 structures have been deposited into RCSB PDB with accession code 2G1E).
- 6The PyMOL molecular graphics system. San Carlos, CA: DeLano Scientific; 2002..
- 8SPARKY 3. San Francisco, CA: University of California; 1999., .