All class II aminoacyl-tRNA synthetases (aaRSs) are known to be active as functional homodimers, homotetramers, or heterotetramers. However, multimeric organization is not a prerequisite for phenylalanylation activity, as monomeric mitochondrial phenylalanyl-tRNA synthetase (PheRS) is also active. We herein report the structure, at 2.2 A resolution, of a human monomeric mitPheRS complexed with Phe-AMP. The smallest known aaRS, which is, in fact, 1/5 of a cytoplasmic analog, is a chimera of the catalytic module of the alpha and anticodon binding domain (ABD) of the bacterial beta subunit of (alphabeta)2 PheRS. We demonstrate that the ABD located at the C terminus of mitPheRS overlaps with the acceptor stem of phenylalanine transfer RNA (tRNAPhe) if the substrate is positioned in a manner similar to that seen in the binary Thermus thermophilus complex. Thus, formation of the PheRS-tRNAPhe complex in human mitochondria must be accompanied by considerable rearrangement (hinge-type rotation through approximately 160 degrees) of the ABD upon tRNA binding.

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The existence of three types of phenylalanyl-tRNA synthetase (PheRS), bacterial (alphabeta)(2), eukaryotic/archaeal cytosolic (alphabeta)(2), and mitochondrial alpha, is a prominent example of structural diversity within the aaRS family. PheRSs have considerably diverged in primary sequences, domain compositions, and subunit organizations. Loss of the anticodon-binding domain B8 in human cytosolic PheRS (hcPheRS) is indicative of variations in the tRNA(Phe) binding and recognition as compared to bacterial PheRSs. We report herein the crystal structure of hcPheRS in complex with phenylalanine at 3.3 A resolution. A novel structural module has been revealed at the N terminus of the alpha subunit. It stretches out into the solvent of approximately 80 A and is made up of three structural domains (DBDs) possessing DNA-binding fold. The dramatic reduction of aminoacylation activity for truncated N terminus variants coupled with structural data and tRNA-docking model testify that DBDs play crucial role in hcPheRS activity.

The crystal structure of Phenylalanyl-tRNA synthetase from E. coli (EcPheRS), a class II aminoacyl-tRNA synthetase, complexed with phenylalanine and AMP was determined at 3.05 Å resolution. EcPheRS is a (ab) 2 heterotetramer: the ab heterodimer of EcPheRS consists of 11 structural domains. Three of them: the N-terminus, A1 and A2 belong to the a-subunit and B1-B8 domains to the b subunit. The structure of EcPheRS revealed that architecture of four helix-bundle interface, characteristic of class IIc heterotetrameric aaRSs, is changed: each of the two long helices belonging to CLM transformed into the coil-short helix structural fragments. The N-terminal domain of the a-subunit in EcPheRS forms compact triple helix domain. This observation is contradictory to the structure of the apo form of TtPheRS, where N-terminal domain was not detected in the electron density map. Comparison of EcPheRS structure with TtPheRS has uncovered significant rearrangements of the structural domains involved in tRNA Phe binding/translocation. As it follows from modeling experiments, to achieve a tighter fit with anticodon loop of tRNA, a shift of~5 Å is required for C-terminal domain B8, and of~6 to 7 Å for the whole N terminus. EcPheRSs have emerged as an important target for the incorporation of novel amino acids into genetic code. Further progress in design of novel compounds is anticipated based on the structural data of EcPheRS.

Aminoacyl-tRNA synthetases (aaRSs) are a canonical set of enzymes that specifically attach corresponding amino acids to their cognate transfer RNAs in the cytoplasm, mitochondria, and nucleus. The aaRSs display great differences in primary sequence, subunit size, and quaternary structure. Existence of three types of phenylalanyl-tRNA synthetase (PheRS)—bacterial (αβ)2, eukaryotic/archaeal cytosolic (αβ)2, and mitochondrial α—is a prominent example of structural diversity within the aaRSs family. Although archaeal/eukaryotic and bacterial PheRSs share common topology of the core domains and the B3/B4 interface, where editing activity of heterotetrameric PheRSs is localized, the detailed investigation of the three-dimensional structures from three kingdoms revealed significant variations in the local design of their synthetic and editing sites. Moreover, as might be expected from structural data eubacterial, Thermus thermophilus and human cytoplasmic PheRSs acquire different patterns of tRNAPhe anticodon recognition.

Human cytosolic phenylalanyl-tRNA synthetase (hcPheRS) is responsible for the covalent attachment of phenylalanine to its cognate tRNA Phe . Significant differences between the amino-acid sequences of eukaryotic and prokaryotic PheRSs indicate that the domain composition of hcPheRS differs from that of the Thermus thermophilus analogue. As a consequence of the absence of the anticodon-recognizing B8 domain, the binding mode of tRNA Phe to hcPheRS is expected to differ from that in prokaryotes. Recombinant hcPheRS protein was purified to homogeneity and crystallized. The crystals used for structure determination diffracted to 3.3 Å resolution and belonged to space group C2, with unit-cell parameters a = 362.9, b = 213.6, c = 212.7 Å , = 125.2. The structure of hcPheRS was determined by the molecular-replacement method in combination with phase information from multiwavelength anomalous dispersion.

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