The Crystal Structure of the Ternary Complex of Phenylalanyl-tRNA Synthetase with tRNA^Phe and a Phenylalanyl-Adenylate Analogue Reveals a Conformational Switch of the CCA End

Abstract

The crystal structure of the ternary complex of (alphabeta)(2) heterotetrameric phenylalanyl-tRNA synthetase (PheRS) from Thermus thermophilus with cognate tRNA(Phe) and a nonhydrolyzable phenylalanyl-adenylate analogue (PheOH-AMP) has been determined at 3.1 A resolution. It reveals conformational changes in tRNA(Phe) induced by the PheOH-AMP binding. The single-stranded 3' end exhibits a hairpin conformation in contrast to the partial unwinding observed previously in the binary PheRS.tRNA(Phe) complex. The CCA end orientation is stabilized by extensive base-specific interactions of A76 and C75 with the protein and by intra-RNA interactions of A73 with adjacent nucleotides. The 4-amino group of the "bulged out" C75 is trapped by two negatively charged residues of the beta subunit (Glubeta31 and Aspbeta33), highly conserved in eubacterial PheRSs. The position of the A76 base is stabilized by interactions with Hisalpha212 of motif 2 (universally conserved in PheRSs) and class II-invariant Argalpha321 of motif 3. Important conformational changes induced by the binding of tRNA(Phe) and PheOH-AMP are observed in the catalytic domain: the motif 2 loop and a "helical" loop (residues 139-152 of the alpha subunit) undergo coordinated displacement; Metalpha148 of the helical loop adopts a conformation preventing the 2'-OH group of A76 from approaching the alpha-carbonyl carbon of PheOH-AMP. The unfavorable position of the terminal ribose stems from the absence of the alpha-carbonyl oxygen in the analogue. Our data suggest that the idiosyncratic feature of PheRS, which aminoacylates the 2'-OH group of the terminal ribose, is dictated by the system-specific topology of the CCA end-binding site.

The Role of a Novel Auxiliary Pocket in Bacterial Phenylalanyl-tRNA Synthetase Druggability

“…Crystal Structure of P. aeruginosa PheRS-A variety of PheRS crystal structures from E. coli and Thermus thermophilus have been previously described, including the apo enzyme, complexes with either tRNA Phe or the substrates phenylalanine and AMP, and with the phenylalanyl-adenylate intermediate (12,(42)(43)(44). Moreover, two PheRS structures from the Gram-positive pathogen S. haemolyticus with a phenyl-thiazolylurea-sulfonamide inhibitor (compound 1a) have been determined (27).…”

Toward understanding phosphoseryl-tRNA Cys formation: The crystal structure of Methanococcus maripaludis phosphoseryl-tRNA synthetase

“…4]. In the structures of T. thermophilus PheRS bound to phenylalanyl-adenylate, or a nonhydrolyzable analog (29,30), the side chains of some of these conserved residues contact the adenylate base (F216 and E218), adenylate phosphate (R204), and carbonyl (R204). Because these residues are conserved, it is plausible that they mediate contacts in SepRS similar to those observed in PheRS.…”

Crystal Structure of Human Mitochondrial PheRS Complexed with tRNAPhe in the Active “Open” State

“…[7][8][9][10][11][12] Loss of the ABD in human cytosolic enzyme and a novel structural module at the N-terminus of the catalytic α-subunit are indicative of variations in tRNA Phe binding and recognition modes, as compared to bacterial enzymes. In eukaryotes, protein synthesis occurs in numerous cell compartments, including the cytoplasm, organelles, and probably the nucleus.…”