Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST)* Department of Medical Genome Sciences, Graduate School of Frontier Sciences, University of Tokyo, Japan**
○Kyoko Suto* Yoshihiro Shimizu** Kozo Tomita*
The stability and degradation of proteins in living organisms plays a vital role in post-translational gene expression and is governed by the N1-end rule, where the N-terminal amino residue of a protein influences its metabolic stability. Proteins with N-terminal destabilizing amino acid residue are degraded by specific proteases. The aminoacyl-tRNA protein transferases are involved in this pathway in both eubacteria and eukarya.
Leucyl/Phenylalanyl-tRNA protein transferase (L/F-transferase) from eubacteria catalyzes the conjugation of Leu (and Phe) of Leu-tRNALeu (and Phe-tRNAPhe) to the N-terminus of a protein starting with basic amino acid residues (Arg or Lys). The proteins with the destabilizing Leu (or Phe) at their N-termini are degraded by an action of the ClpAp proteasome complex. Therefore, the L/F-transferase is an essential factor for initiating protein degradation and for the quality control of cellular proteins in eubacteria.
Here, we present the crystal structures of E. coli L/F-transferase and its complex with an aminoacyl-tRNA analog, puromycin. The structure of C-terminal domain of L/F-transferase is GNAT superfamily fold and the puromycin is accommodated in a highly hydrophobic pocket of the enzyme. The shape of the puromycin binding pocket is suitable only for hydrophobic amino acid residues with an unbranched b-carbon. The base moiety of puromycin stacks with several hydrophobic amino acid residues, but is not specific to adenine. The presented structure and structure-based mutagenesis analysis of L/F-transferase explain its substrate specificity. We also present a model for tRNA binding to L/F-transferase.
Ref. A. Varshavsky, Trends Biochem Sci. 30, 283-286, 2005.