Solution structure of Archaeglobus fulgidis peptidyl-tRNA hydrolase (Pth2) provides evidence for an extensive conserved family of Pth2 enzymes in archea, bacteria, and eukaryotes

Abstract

The solution structure of protein AF2095 from the thermophilic archaea Archaeglobus fulgidis, a 123-residue (13.6-kDa) protein, has been determined by NMR methods. The structure of AF2095 is comprised of four alpha-helices and a mixed beta-sheet consisting of four parallel and anti-parallel beta-strands, where the alpha-helices sandwich the beta-sheet. Sequence and structural comparison of AF2095 with proteins from Homo sapiens, Methanocaldococcus jannaschii, and Sulfolobus solfataricus reveals that AF2095 is a peptidyl-tRNA hydrolase (Pth2). This structural comparison also identifies putative catalytic residues and a tRNA interaction region for AF2095. The structure of AF2095 is also similar to the structure of protein TA0108 from archaea Thermoplasma acidophilum, which is deposited in the Protein Data Bank but not functionally annotated. The NMR structure of AF2095 has been further leveraged to obtain good-quality structural models for 55 other proteins. Although earlier studies have proposed that the Pth2 protein family is restricted to archeal and eukaryotic organisms, the similarity of the AF2095 structure to human Pth2, the conservation of key active-site residues, and the good quality of the resulting homology models demonstrate a large family of homologous Pth2 proteins that are conserved in eukaryotic, archaeal, and bacterial organisms, providing novel insights in the evolution of the Pth and Pth2 enzyme families.

Figure 1.

(A) Ribbon diagram of the NMR structure of AF2095 for residues 1–112 colored by secondary structure. Disordered residues 113–123 were removed for clarity. (B) Superposition of the backbone (N,C,C′) atoms for the 30 best structures determined for AF2095 for residues 1–112. The disorder for residues Q79–I91 in the loop that connects β-strands β₃ and β₄ is evident by the large RMSD spread. The figures were generated with MOLSCRIPT (Kraulis 1991) and rendered with Raster3D (Merritt and Bacon 1997).

Figure 2.

Structures of A. fulgidis AF2095. (A) Carbon-α backbone of AF2095. Residues proposed to form the catalytic triad (Lys 19, Asp 80, Thr 90) are shown in sticks in green. (B) Electrostatic surface of AF2095, in the same orientation as in A. The electrostatic surface was calculated by using a salt concentration of 0.1 M, and the color scale is −5 kt (negative, red) to 5 kt (positive, blue). (C) Phylogenetic analysis of AF2095 performed with the program ConSurf (Glaser et al. 2003). Maroon indicates conserved; cyan, variable. Sequences used in the ConSurf analysis were obtained from a PSIBLAST search against the nonredundant database using an e-value <0.001 after four iterations. Conserved residues cluster to the β3–β4 loop and the N terminus of α1, as well as to the region containing a surface patch of positive electrostatic potential. The right column is rotated −45° about the Y-axis, relative to the left column.

Figure 3.

Ribbon diagrams of the aligned views of (A) human Pth2 (PDB ID 1q7s), (B) T. acidophilum TA0108 (PDB ID 1rlk), and (C) A. fulgidis AF2095 (GR4; PDB ID 1rzw).

Figure 4.

Phylogenic tree of Pth2 enzymes with <≥30% sequence similarity to AF2095, including homologs. For clarity, each major branch is colored separately, each organism is labeled archaea (A), eukaryote (E), and eubacteria (Eu), and the homologs are numbered sequentially.