The crystal structure of the zinc phosphodiesterase from Escherichia coli provides insight into function and cooperativity of tRNase Z-family proteins

Abstract

The elaC gene product from Escherichia coli, ZiPD, is a 3' tRNA-processing endonuclease belonging to the tRNase Z family of enzymes that have been identified in a wide variety of organisms. In contrast to the elaC homologue from Bacillus subtilis, E. coli elaC is not essential for viability, and although both enzymes process only precursor tRNA (pre-tRNA) lacking a CCA triplet at the 3' end in vitro, the physiological role of ZiPD remains enigmatic because all pre-tRNA species in E. coli are transcribed with the CCA triplet. We present the first crystal structure of ZiPD determined by multiple anomalous diffraction at a resolution of 2.9 A. This structure shares many features with the tRNase Z enzymes from B. subtilis and Thermotoga maritima, but there are distinct differences in metal binding and overall domain organization. Unlike the previously described homologous structures, ZiPD dimers display crystallographic symmetry and fully loaded metal sites. The ZiPD exosite is similar to that of the B. subtilis enzyme structurally, but its position with respect to the protein core differs substantially, illustrating its ability to act as a clamp in binding tRNA. Furthermore, the ZiPD crystal structure presented here provides insight into the enzyme's cooperativity and assists the ongoing attempt to elucidate the physiological function of this protein.

FIG. 1.

(A) Ribbon diagram of the crystal structure of the ZiPD homodimer at a resolution of 2.9 Å. Subunit A is red, subunit B is blue, and the zinc ions are represented by green spheres. (B) Active site cleft 1 of ZiPD homodimer formed jointly by subunits A and B. His248 and His 270, which are essential for cooperativity, are yellow, and the remaining zinc ligands are represented by cyan sticks.

FIG. 2.

Stereo view of ZiPD zinc coordination site with the experimental electron density map shown at 1σ above the mean. Zinc ions are represented by green spheres. Bond lengths (in angstroms) between Zn2 and His270 and between Zn2 and His248 are indicated to update previous spectroscopic and mutational analyses in which determining which of the two residues coordinates zinc was impossible.

FIG. 3.

Sequence alignment of tRNase Z-family proteins. ECOLI, E. coli ZiPD; BACSU, B. subtilis tRNase Z; TMAR, T. maritima tRNase Z. Conserved metal coordinating residues are highlighted in blue, and exosite residues are indicated by a yellow background. Secondary structure features are indicated by red arrows for β-strands and by blue cylinders for α-helices. unknown.

FIG. 4.

Comparison of the structures of tRNase Z-family proteins E. coli ZiPD, B. subtilis tRNase Z (subunits A and B) (8), and T. maritima tRNase Z (11). The RMSD values for the protein cores were calculated to be 1.0 Å for E. coli-B. subtilis (subunit B) using 254 carbon alpha atoms and 2.1 Å for E. coli-T. maritima using 241 carbon alpha atoms. The location of the exosite with respect to the protein core is indicated in the E. coli ZiPD diagram by dotted lines.

FIG. 5.

Superposition of ZiPD and B. subtilis tRNase Z carbon alpha chains. ZiPD is blue, and tRNase Z is yellow. The RMSD value between the exosites was calculated to be 1.5 Å using the carbon alpha atoms of 51 residues.

FIG. 6.

Comparison of active sites with and without PO₄ bound from E. coli ZiPD (A) and B. subtilis tRNase Z subunit B (bound to PO₄) (B). Hydrogen bonds to the phosphate molecule are magenta and highlight the key role of His248 and His270 in determining substrate orientation.