Crystal Structure of a Human Peptidyl-tRNA Hydrolase Reveals aNew Fold and Suggests Basis for a Bifunctional Activity*
Received for publication, October 19, 2003, and in revised form, November 21, 2003Published, JBC Papers in Press, December 5, 2003, DOI 10.1074/jbc.M311449200
Jose M. de Pereda‡§, William F. Waas
, Yiwen Jan‡, Erkki Ruoslahti‡, Paul Schimmel
,and Jaime Pascual‡
Program on Cell Adhesion and Signal Transduction, Cancer Center of the Burnham Institute, and the
Department of Chemistry and Molecular Biology, The Scripps Research Institute, La Jolla, California 92037
Peptidyl-tRNA hydrolase (Pth) activity releases tRNAfrom the premature translation termination productpeptidyl-tRNA. Two different enzymes have been re-ported to encode such activity, Pth present in bacteriaand eukaryotes and Pth2 present in archaea and eu-karyotes. Here we report the crystallographic structureof the
Pth2 at a 2.0-Å resolution as well asits catalytic properties. In contrast to the structure of
foldwitha four-stranded antiparallel
-sheet in its core sur-rounded by two
-helices on each side. This arrange-ment of secondary structure elements generates a foldnot previously reported. Its catalytic efficiency is com-parable with that reported for the archaeal
Pth2 and higher than that of the bacterial
Pth. Several lines of evidence target the activesite to two close loops with highly conserved residues.This active site architecture is unrelated to that of
Pth. In addition, intermolecular contacts in thecrystal asymmetric unit cell suggest a likely surfacefor protein-protein interactions related to the Pth2-mediated apoptosis.
During the protein translation process, a significant propor-tion of the ribosomes that initiate mRNA readout do not reachthe stop codon. Peptidyl-tRNA molecules may dissociate fromthe mRNA template causing a premature end of the process (1,2). Accumulation of peptidyl-tRNAs reduces the efficiency of translation by sequestering tRNAs and impairing the initiation(3). Prokaryotic and eukaryotic cells show an enzymatic activ-ity that releases the peptidyl moiety from the tRNA, calledpeptidyl-tRNA hydrolase (Pth),
allowing the free tRNA andpeptide to be reused in protein synthesis (4, 5). First identifiedin
, genes encoding Pth have been found inbacteria and eukaryotes but not in archaea.
Pth crystalstructure has been solved and exhibits an
fold formed bythree layers with a mixed five-strand
-sheet at the core. Itscatalytic parameters have been measured, and several residuesaffecting the 5
-phosphate and 3
-end recognition of the pepti-dyl-tRNA substrate have been mapped (6, 7).Recently, a Pth-like activity has been identified in
and characterized in
, both archaebacteria (8, 9). The new enzyme called Pth2is shorter in length and lacks any significant sequence homol-ogy to Pth. It coincides with the Pfam data base entry “Unchar-acterized Protein Family 0099” (UPF0099) found in archaeaand eukaryotes but not in bacterial genomes. Pth2 lacks anydetectable homology to solved three-dimensional structures.
Pth2 displays a greater catalytic efficiencytoward
Pth (9, 6). Inaddition, both enzymes differ in their mode of recognition of several tRNA features, like the 1–72-base pair mismatch in
or the presence of a phosphorylated 5
-end.Human Pth2, also called Bcl-2 inhibitor of transcription 1(Bit1), possesses an N-terminal signaling sequence involved inits mitochondrial localization,
indicating that it may serve tomaintain efficient protein translation there. To understand thestructural basis of Pth2 function, we report the crystal struc-ture of human Pth2 and the characterization of its hydrolaseactivity. Pth2 structural fold is unrelated to that of Pth. Itbelongs to the
class of proteins containing three layers witha twisted mixed
-sheet formed by four strands.
assaysdemonstrate that human Pth2 is active, showing a catalyticefficiency higher than that of Pth. Analysis of the electrostaticproperties of the structure, as well as the conservation of reac-tive residues and their position on the three-dimensional struc-ture, delineates a putative active site.
MATERIALS AND METHODS
Subcloning, Overexpression, and Protein Purification—
Based on asequence alignment of homologous domains, the first 62 residues of the179-amino acid-long human sequence do not belong to the enzyme andare likely involved in mitochondrial localization. Consequently we de-fined the human Pth2 as the 63–179 fragment. The DNA-coding frag-ment was obtained by standard PCR methods using full-length humanPth2 clone as template (GenBank
/EBI accession number AF151905)and subcloned into the NcoI/BamHI sites of the pET15b bacterial ex-pression vector (Novagen), thus adding no tag to the protein. DNAsequencing corroborated the identity of the inserted fragment. Theligated plasmid was transformed into
BL21(DE3) strain. Cellswere grown at 37 °C in LB medium, and overnight protein expression at15 °C was carried out upon addition of 0.5 m
-galactopyranoside. Overexpression and solubility were assessed by pro-tein extraction with B-PER reagent (Pierce) followed by SDS-PAGE.The fragment 63–179 was highly expressed and soluble. Cell pellets* This work was supported by an institutional grant from the NCI,National Institutes of Health, by Grants CA82713 and CA79984 (toE. R.) and Grant GM15539 from the National Institutes of Health, andby a grant from the National Foundation for Cancer Research (to P. S.).The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked“
” in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.
The atomic coordinates and structure factors (code 1Q7S) have beendeposited in the Protein Data Bank, Research Collaboratory for Struc-tural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
§ Present address: Centro de Investigacio´n del Cancer, University of Salamanca-CSIC, Campus Unamuno s/n, 37007 Salamanca, Spain.
To whom correspondence should be addressed. Tel.: 858-646-3100;Fax: 858-646-3196; E-mail: firstname.lastname@example.org.
The abbreviation used is: Pth, peptidyl-tRNA hydrolase.
Y. Jan, M. Matter, J. Pai, J. Pilch, M. Komatsu, Y. Chen, E. Ong, M.Fukuda, and E. Ruoslahti, submitted for publication.
Vol. 279, No. 9, Issue of February 27, pp. 8111–8115, 2004 © 2004 by The American Society for Biochemistry and Molecular Biology, Inc.
Printed in U.S.A.
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