Two distinct regions in Staphylococcus aureus GatCAB guarantee accurate tRNA recognition

Abstract

In many prokaryotes the biosynthesis of the amide aminoacyl-tRNAs, Gln-tRNA(Gln) and Asn-tRNA(Asn), proceeds by an indirect route in which mischarged Glu-tRNA(Gln) or Asp-tRNA(Asn) is amidated to the correct aminoacyl-tRNA catalyzed by a tRNA-dependent amidotransferase (AdT). Two types of AdTs exist: bacteria, archaea and organelles possess heterotrimeric GatCAB, while heterodimeric GatDE occurs exclusively in archaea. Bacterial GatCAB and GatDE recognize the first base pair of the acceptor stem and the D-loop of their tRNA substrates, while archaeal GatCAB recognizes the tertiary core of the tRNA, but not the first base pair. Here, we present the crystal structure of the full-length Staphylococcus aureus GatCAB. Its GatB tail domain possesses a conserved Lys rich motif that is situated close to the variable loop in a GatCAB:tRNA(Gln) docking model. This motif is also conserved in the tail domain of archaeal GatCAB, suggesting this basic region may recognize the tRNA variable loop to discriminate Asp-tRNA(Asn) from Asp-tRNA(Asp) in archaea. Furthermore, we identified a 3(10) turn in GatB that permits the bacterial GatCAB to distinguish a U1-A72 base pair from a G1-C72 pair; the absence of this element in archaeal GatCAB enables the latter enzyme to recognize aminoacyl-tRNAs with G1-C72 base pairs.

Figure 1.

(A) Overall structure of the full-length GatCAB. GatA and GatC are colored blue and magenta, respectively. GatB contains three domains: the cradle, the helical and the tail domain (light green, dark green and yellow green, respectively). Mg atom (black) in the cradle domain and C-terminal His-tag (grey) are also shown. (B) The structure of the GatB tail domain. The residues involved in the hydrophobic core are shown as a grey stick model, along with hydrogen bonds between the inter-domain loop and the tail domain (indicated as dashed lines). L472 is indicated as a grey sphere model. (C) Superposition of the S. aureus tail domain (yellow green) and YqeY-C (cyan), where the Lys rich motif is indicated as a stick model. (D) Structural based sequence alignments of the tail domain of YqeY-like proteins. Secondary structure of S. aureus tail domain is indicated at top, and that of YqeY-C is indicated at bottom (α, α-helix; β, β-strand; η, 3₁₀- turn). The number at top is corresponding to S. aureus GatB sequence. The residues constructing the hydrophobic core of S. aureus tail domain are marked with filled triangles. The Lys rich motif and the GXXAXGX motif are shown as cyan box and magenta box, respectively. The species aligned are as follows: STAAM S. aureus, METTH, M. thermautotrophicus; DEIRA, D. radiodurans; PYRAB, P. abyssi; BACSU, B. subtilis.

Figure 2.

Comparison with tRNA^Gln and tRNA^Glu. (A) Secondary structures of tRNA^Gln tRNA^Glu from S. aureus, E. coli and T. thermophilus. The G18:C56 base pair are colored green. The two guanines conserved in the tRNA D-loop are enclosed with black boxes. U8, A14–G19, A21, C48 and U54–G57, which employed on superposing of tRNA^Gln and tRNA^Glu, are shown as black large characters. U47 of tRNA^Gln and U20_B of tRNA^Glu are colored red. (B) Superposition of the E. coli tRNA^Gln and T. thermophilus tRNA^Glu. Orange and cyan ribbon diagram indicate E. coli tRNA^Gln and T. thermophilus tRNA^Glu, respectively. Supplemental nucleotides of the variable loop of E. coli tRNA^Gln and the D-loop of T. thermophilus tRNA^Glu are shown as thick stick models. The U8–A14–A21 base-triple, the conserved ‘Levitt’ pair and tRNA D-loop and TΨC -loop associating nucleotides are indicated as thin stick models.

Figure 3.

(A) Surface representation with a ribbon diagram of S. aureus tail domain is shown, and is colored by the sequence conservation among bacterial GatB using the program ConSurf 3.0: from low to high (cyan to white to magenta). The conserved hydrophobic pocket and the Lys rich motif are shown as yellow and purple dashed circles, respectively. (B) Solvent-accessible surface with a ribbon diagram of S. aureus tail domain is shown in the same orientation as (A) and is colored according to the electrostatic potential calculated by the program APBS running on Pymol (blue for positively charged and red for negatively charged). (C) The S. aureus tail domain docking model. S. aureus tail domain is shown as in (B). Orange and cyan ribbon diagram indicate E. coli tRNA^Gln and T. thermophilus tRNA^Glu, respectively. Supplemental nucleotides of the variable loop of E. coli tRNA^Gln and the D-loop of T. thermophilus tRNA^Glu are shown as a stick model. The helical domain from the S. aureus GatB:tRNA^Gln docking model (described below) shown together. The inter-domain loop between the helical and the tail domain is shown as dashed lines.

Figure 4.

(A) Experimental and computed SAXS scattering data. (Upper) the tRNA-bound GatCAB, (lower) tRNA-free GatCAB. The logarithm of the scattering intensity is plotted against the momentum transfer s=4πsinθ/λ, where 2θ is the scattering angle and λ=1.0 Å is the X-ray wavelength. The plots are displaced along the ordinate for better visualization. Red, cyan and pink curves indicated the computed scattering from tRNA-bound GatCAB model after the tail domain fitting, before the tail domain fitting, and tRNA-free GatCAB, respectively. (B) Comparison of the model of the S. aureus GatB:tRNA^Gln complex (left) and the crystal structure of the M. thermautotrophicus GatE:tRNA^Gln complex (right). S. aureus GatB, M. thermautotrophicus GatE and M. thermautotrophicus tRNA^Gln are shown as green, magenta and grey ribbon diagram, respectively. The A1–U72 base pair of M. thermautotrophicus tRNA^Gln is shown as a stick model. The 3₁₀ turn and the hydrophobic core of S. aureus GatB and the short loop of M. thermautotrophicus GatE are also shown. (C) Structural based sequence alignments of the cradle domain of bacterial GatB, archaeal GatB and GatE. The number at top is corresponding to S. aureus GatB sequence. Secondary structure of S. aureus GatB is indicated at top, and that of M. thermautotrophicus GatE is indicated at bottom. The 3₁₀ turn of bacterial GatB and the corresponding region of archaeal GatB and GatE are shown as cyan box. The species aligned are as follows: CHLTR, Chlamydia trachomatis; THETH, Thermus thermophilus HB8; METJA, Methanococcus jannaschii; AERPE, Aeropyrum pernix; SULSO, Sulfolobus solfataricus; ARCFU, Archaeoglobus fulgidus.