NMR structure of the conserved novel‐fold protein TA0743 from <i>Thermoplasma acidophilum</i>

Thermoplasma acidophilum1 has one of the smallest known archaeal genomes. This organism is a thermo-acidophilic archaeon that is surrounded by only a plasma membrane. TA0743 is a 122-residue conserved hypothetical protein found in Thermoplasma acidophilum. Because neither functional nor structural information is available for this protein, the solution structure of TA0743 has been determined by NMR spectroscopy. Based on the solution structure and the amino acid sequence analysis, we propose that TA0743 is an uncharacterized protein with a novel structural fold. Therefore, our structure may serve as a starting point to study the structure–function of proteins with a similar fold in various species. The ORF of TA0743 was cloned into the expression vector pET-15b and was expressed in the Eschericha coli BL21 (DE3) strain (Novagen Inc.). The vector contains 18 extra amino acids with a histidine affinity tag (His-tag) at the N-terminus. Cells were grown in M9 media with 15NH4Cl and 13C-gluscose in order to produce an 15N/13C-labeled protein. Cells were grown at 37°C until an OD600 of 0.5 and then induced with 1 mM IPTG for 7h at 25°C. The protein was purified on a metal affinity column. The protein sample was further purified using gel filtration chromatography (Superdex 75). Finally, the protein was concentrated to 1.6 mM in 50 mM potassium phosphate, 0.1% NaN3, and 90% H2O/10% D2O solution (pH 6.5). All NMR spectra were recoded at 25°C on a Bruker AVANCE DRX500 spectrometer equipped with a pulse-field gradient triple-resonance probe or on a VARIAN UNITY600 spectrometer. NMR data were processed by the NMRPipe2 with linear prediction to double the number of points in the indirect dimensions for digital resolution improvement. The spectra were analyzed with Sparky.3 The backbone resonance assignments of TA0743 were performed with the HNCACB and CBCA(CO)NH spectra and confirmed with the HNCA4 spectrum. Backbone resonance assignments were analyzed with program MARS.5 Carbonyl carbons were assigned with HNCO spectrum and side-chain proton resonances were assigned with HCCH-TOCSY and (H)CCH-TOCSY spectra. 3D 15N-edited NOESY and 13C-edited NOESY spectra were acquired for NOE constraints. For structure calculation, the NOE peak assignment was obtained by both manual and automatic peak picking procedure. Backbone dihedral angle restraints were also derived from chemical shifts of the 1Hα, 13Cα, 13Cβ, CO, and NH using TALOS.6 The dihedral angle restraints were used for structure calculation using CYANA 2.0.7 A total of 1422 NOEs, 84 distance for hydrogen bonds, and 168 dihedral angle restraints were used for structure calculation. From 100 calculated structures, the 20 structures with the lowest target function energy were selected and finally analyzed by the program MOLMOL8 and pyMOL.9 The 20 final structures are well-converged with a root-mean-square deviation of 0.51 Å for ordered structural regions and 0.78 Å for all of the backbone heavy atoms [Fig. 1(A)]. The structural statistics associated with the 20 final structures are listed in Table I. TA0743 is determined as a two-layered α/β-sandwich structure [Fig. 1(B)] and the molecular topology is a βαββααβα fold. One layer comprises three α-helices [Gly14-Asp30 (α1), Pro60-Arg80 (α2), and Tyr83-Asp92 (α3)], while the other surface is composed of a four-stranded β-sheet with 1(↓), 3(↑), 2(↓), and 4(↓) order. Three α-helices (α1, α2, and α3) are located on the same side of the β-sheet [Fig. 1(B)]. The α3-helix, which is not connected to β-strands, is interconnected to α2. Two other helices (α2 and α3) form an HTH motif at an angle of about 90° to each other. This motif might provide a binding surface for interacting molecule. Interestingly, compared to residues in other secondary structural region, a number of charged polar residues (α2: Arg68, Lys69, Lys70, Arg72, Lys74, Arg80, and α3: Lys89, His91) are found near HTH region, suggesting that this region is also of functional importance. NMR solution structure of TA0743. (A) A stereo-view of the superposition of the final 20 structures over the energy-minimized average structure. (B) Ribbon diagram showing a molecular topology of TA0743. The β-strands and α-helices are shown in green and yellow, respectively. A homology search using the program DALI10 within the Protein Data Bank11 indicates that TA0743 shares weak similarity with 54 different proteins, however, no meaningful match of structural fold to TA0743 was detected from CATH.12 These results imply that TA0743 would be an uncharacterized protein with a novel structural fold. From BLAST13 database search, three proteins were identified with over 40% sequence identity with TA0743: NP111379 (Thermoplasma volcanium), EAM93965 (Ferroplasma acidarmanus), and YP023413 (Picrophilus torridus) (Fig. 2). The functions of all of these proteins are also unknown. Therefore, the structure of TA0743 could potentially serve as a model to characterize the structure–function of this novel fold family of proteins in other species. [Note: Both backbone and side-chain chemical shifts for TA0743 have been deposited in the BioMagResBank (accession code: BMRB-6755). Coordinates for the 20 structures and average energy-minimized structure have been deposited in the RCSB PDB with accession code 2ABY.] Sequence alignment of TA0743 from Thermoplasma acidophilum with three homologous proteins: NP 111379 (Thermoplasma volcanium), EAM93965 (Ferroplasma acidarmanus), and YP 023413 (Picrophilus torridus). The secondary structure of TA0743 determined by NMR is shown on the top of the sequences. All conserved residues are also highlighted.

Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.