Crystal structures of leucyl/phenylalanyl-tRNA-protein transferase and its complex with an aminoacyl-tRNA analog

Abstract

Eubacterial leucyl/phenylalanyl-tRNA protein transferase (L/F-transferase), encoded by the aat gene, conjugates leucine or phenylalanine to the N-terminal Arg or Lys residue of proteins, using Leu-tRNA(Leu) or Phe-tRNA(Phe) as a substrate. The resulting N-terminal Leu or Phe acts as a degradation signal for the ClpS-ClpAP-mediated N-end rule protein degradation pathway. Here, we present the crystal structures of Escherichia coli L/F-transferase and its complex with an aminoacyl-tRNA analog, puromycin. The C-terminal domain of L/F-transferase consists of the GCN5-related N-acetyltransferase fold, commonly observed in the acetyltransferase superfamily. The p-methoxybenzyl group of puromycin, corresponding to the side chain of Leu or Phe of Leu-tRNA(Leu) or Phe-tRNA(Phe), is accommodated in a highly hydrophobic pocket, with a shape and size suitable for hydrophobic amino-acid residues lacking a branched beta-carbon, such as leucine and phenylalanine. Structure-based mutagenesis of L/F-transferase revealed its substrate specificity. Furthermore, we present a model of the L/F-transferase complex with tRNA and substrate proteins bearing an N-terminal Arg or Lys.

Figure 1

Overall architecture of E. coli L/F-transferase. (A) Stereo view of the E. coli L/F-transferase structure. The NH₂-terminal domain (residues 2–62) and the COOH-terminal domain (residues 63–232) are colored blue and green, respectively. The puromycin bound to the hydrophobic pocket is colored yellow. (B) Topology diagram of L/F-transferase. The rimmed elements in the COOH-terminal domain (α3–α5) and (β5–β12) are common to the GNAT superfamily fold. The α-helices and β-strands in the COOH-terminal domains are colored red and yellow, respectively. (C) Comparison of the structures of E. coli L/F-transferase (left), W. viridescens FemX (wvFemX; middle, PDB accession number 1P4N; Biarrotte-Sorin et al, 2004) and S. aureus FemA (saFemA; PDB accession number 1LRZ; Benson et al, 2002). The COOH-terminal domain of L/F-transferase is topologically similar to the domain 2′s of wvFemX and saFemA. The conserved α-helices and β-strands in L/F-transferase, wvFemX and saFemA, are colored red and yellow, respectively.

Figure 2

Sequence alignment of the L/F-transferases from E. coli (Aat_E. coli), Xylella fastidosa (Aat_X-fast; accession number ZP_00683190.1), Pseudomonas aeruginosa (Aat_P. aeru; accession number ZP_00204849.1), Synechocystis (Aat_Synech; accession number NP_440931.1), and Mesorhizobium loti (Aat_M. loti; accession number NP_102051.1). The sequence of R-transferase from Plasmodium falciparum (ATEL_P. fa; accession number NP_473045.1). The secondary structure elements of E. coli L/F-transferase are indicated above the alignment. The α-helices and β-strands in the NH₂-terminal domain are colored blue, and those of the COOH-terminal domain are colored red and yellow, respectively, as shown in Figure 1B.

Figure 3

Recognition of the puromycin by E. coli L/F-transferase. (A) Chemical structure of puromycin (left) and that of the 3′-ends of Leu-tRNA^Leu and Phe-tRNA^Phe (middle and right, respectively). The amino-acid moiety and the base moiety are colored pink and blue, respectively. (B) ∣Fo−Fc∣ omit map of puromycin (contour level 3.0σ). (C) Recognition of the p-methoxybenzyl group and the puromycin base by the hydrophobic pocket, as shown by a surface model. (D) Ribbon model of (C). The hydrophobic amino acid involved in the recognition of the p-methoxybenzyl group and the base moiety of puromycin are colored green and blue, respectively. (E) The C-shaped edge of the hydrophobic pocket is composed of continuous amino-acid residues (Gly155-Glu156-Ser157-Met158; colored yellow and highlighted). The α-, β- and γ-carbons of puromycin are also shown.

Figure 4

In vitro activity assays for various mutants of L/F-transferase. (A) The activity of [¹⁴C]Leu incorporation into α-casein from [¹⁴C]Leu-tRNA^Leu by mutant L/F-transferases (see Materials and methods). The upper gel shows the [¹⁴C]Leu-labeled α-casein. The lower graph shows the quantification of the relative intensity of the labeled product of the upper gel, where the incorporation of [¹⁴C]Leu into α-casein by wild-type L/F-transferase was taken as 1.0. The bars on the graph indicate the s.d. of more than three independent experiments. (B) The activity of [¹⁴C]Phe incorporation into α-casein from [¹⁴C]Phe-tRNA^Phe by mutant L/F-transferases, as in (A).

Figure 5

Recognition of the aminoacyl-moiety by L/F-transferase. Recognition of the side chains of Leu (A) and Phe (B) by L/F-transferase. The α- and β-carbons of Leu and Phe were superimposed onto those of puromycin (colored orange). Discrimination of Ile (C) and Val (D) by L/F-transferase. The α- and β-carbons of Ile and Val were superimposed as in (A) and (B). The steric hindrance between the branched methyl groups at the β-carbons of Ile and Val. The C-shaped edge of the hydrophobic pocket is colored yellow. The chemical structures of each amino acid are depicted in parentheses.

Figure 6

Model of aminoacyl-tRNA binding to L/F-transferase. (A) Two views of a ribbon diagram of the docking model of L/F-transferase and tRNA. The tRNA backbone is shown as a green line (upper two panels). Two views of the L/F-transferase-tRNA complex model, showing the surface colored according to its calculated electrostatic potential (lower panel; blue, positively charged +8KT; red, negatively charged –8KT). The electrostatic surface model was calculated by the program APBS (Baker et al, 2001). The upper view displays the top side of the complex of front views. (B) Electrostatic potentials of wvFemX (upper) and saFemA (lower). The marked regions on the diagrams show the positively charged regions corresponding to α2 of L/F-transferase and predicted as RNA binding regions. (C) The simultaneous tRNA binding to L/F-transferase (colored green) and EF-Tu (colored pink). The tRNA phosphate backbones are shown as a green line and a pink line for the L/F-transferase-tRNA complex and the EE-Tu-tRNA complex, respectively. The EF-Tu and Cys-tRNA^Cys complex structure was from PDB 1B23 (Nissen et al, 1999). The 3′-terminus of Cys-tRNA^Cys was manually modeled to accommodate the puromycin binding pocket of L/F-transferase.