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

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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‐tRNALeu or Phe‐tRNAPhe 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‐tRNALeu or Phe‐tRNAPhe, is accommodated in a highly hydrophobic pocket, with a shape and size suitable for hydrophobic amino‐acid residues lacking a branched β‐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.