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Bit1/Ptrh2 structure

Bit1/Ptrh2 structure

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Published by jamie pascal
Crystal structure of the human peptidyl-tRNA hydrolase 2
Crystal structure of the human peptidyl-tRNA hydrolase 2

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Published by: jamie pascal on Feb 20, 2008
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05/08/2014

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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‡
 From the
 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
Homo sapiens
Pth2 at a 2.0-Å resolution as well asits catalytic properties. In contrast to the structure of 
 Escherichiacoli
Pth,
 H.sapiens
Pth2hasan
 / 
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
Sulfolobussolfataricus
Pth2 and higher than that of the bacterial
 E. coli
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 
 E. coli
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),
1
allowing the free tRNA andpeptide to be reused in protein synthesis (4, 5). First identifiedin
Escherichia coli
, genes encoding Pth have been found inbacteria and eukaryotes but not in archaea.
E. coli
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
Methano-caldococcus jannaschii
and characterized in
Sulfolobus solfa-taricus
, 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.
 S. solfataricus
Pth2 displays a greater catalytic efficiencytoward
E. coli
diacetyl-lysyl-tRNA
Lys
than
E. coli
Pth (9, 6). Inaddition, both enzymes differ in their mode of recognition of several tRNA features, like the 1–72-base pair mismatch in
 E. coli
tRNA
fMet
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,
2
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.
In vitro
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
TM
 /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
E. coli
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
M
isopropyl-1-thio-
-
D
-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
advertisement
” 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: pascual@burnham.org.
1
The abbreviation used is: Pth, peptidyl-tRNA hydrolase.
2
 Y. Jan, M. Matter, J. Pai, J. Pilch, M. Komatsu, Y. Chen, E. Ong, M.Fukuda, and E. Ruoslahti, submitted for publication.
T
HE
J
OURNAL OF
B
IOLOGICAL
C
HEMISTRY
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.
This paper is available on line at http://www.jbc.org
8111
 
coming from 1 liter were resuspended in 25 ml of lysis buffer (100 m
M
Tris pH 7.5) and sonicated three times. Taking advantage of the highisoelectric point of the protein (pI 9.3) and after centrifugation, filteredsupernatant was loaded into a cationic exchange column and purified innative conditions using a gradient of NaCl by standard fast proteinliquid chromatography methods. Protein eluted at a salt concentrationof 250 m
M
. Further purification was achieved by gel filtration methodsusing 20 m
M
Tris pH 7.0 and 150 m
M
NaCl as running buffer. Theprotein eluted at its monomeric position. Purity and identity of thefragment were checked by matrix-assisted laser desorption ionizationmass spectrometry techniques. Circular dichroism and differentialscanning calorimetry were used to confirm the presence of secondaryand tertiary structure.
Crystallization and Structure Determination
 —
Crystals grew in
24h at room temperature using sitting drop vapor diffusion techniques bymixing protein at 10 mg/ml with a solution containing 100 m
M
HepespH 7.5 with 20% (w/v) polyethylene glycol-10,000 and equilibratingagainst a reservoir of the same solution. For data collection, crystalswere transferred into cryoprotectant solutions consisting of motherliquor containing increasing concentrations of glycerol up to 20% (v/v)and flash-frozen in liquid nitrogen. Data from native and derivativecrystals were collected at 100 K at the Stanford Synchrotron RadiationLaboratory. Data were indexed, integrated, and scaled with DENZOand SCALEPACK programs (11).Phase information was obtained by multiple wavelength anomalousdiffraction using NaBr-soaked crystals (12). Positions of five bromineatoms were determined, and phases were calculated to a 2.8-
 Å
resolu-tion applying the software SOLVE (13). After phase improvement withthe program DM (14), a readily interpretable map was obtained. Basedon the experimental electron density map an initial model was builtwith the program TURBO-FRODO and refined by simulated annealingwith the CNS software package (15) against native data to a 2.0-
 Å
resolution. Further calculations using cycles of conjugate gradient min-imization and individual B-factor refinement alternated with manualmodelbuildingleadtothefinalmodel.Thestereochemicalqualityoftherefined model was verified with the PROCHECK software (16).
 Enzymatic Assay
 —
Hexahistidine-tagged
E. coli
lysyl-tRNA synthe-tase was overexpressed in MG1655 cells and purified by nickel-nitrilo-triacetic acid (Qiagen) affinity chromatography as described previously(17).F
IG
. 1.
Multiple sequence alignment of Pth2s from different archaeal and eukaryotic organisms.
Sequences shown are from
Homosapiens
,
Tetraodonnigroviridis
,
 Arabidopsisthaliana
,
Caenorhabditiselegans
,
 Dictyosteliumdiscoideum
,
 Schizosaccharomycespombe
,
 Drosophilamelanogaster
,
Methanocaldococcus jannaschii
,
Giardia lamblia
, and
Pyrococcus horikoshii
.
Coloring
of each column is according to chemicalproperty and conservation of the residue. The
topline
indicates the position and order of the regular elements of secondary structure for the humansequence (
b
, strand;
a
, helix). The
bottomline
labels reactive residues as either strictly conserved (*) or highly conserved (
).
 Numbering
correspondsto the human sequence (AF151905). The alignment was generated with the program ClustalX (24).F
IG
. 2.
Electron density maps of a fragment of the
1
-helix.
The refined structure of the helical residues is shown in stick form andusing standard atom colors.
A
, experimental solvent flattened map at a2.8-
 Å
resolution, contoured at 1
 
.
B
, 2
 F 
o
c
map with phasescalculated from the final model at 2.0
Å
, contoured at 1
 
. The figurewas generated with MOLSCRIPT (25) and rendered with Raster3D(10).
 Structure of Pth2
8112
 
The tRNA-charging reaction (1 ml) contained 50 m
M
HEPES (pH7.5), 2 m
M
ATP, 10 m
M
MgCl
2
, 2 m
M
dithiothreitol, 0.5 m
M
EDTA, 60
M
lysine, 6
M
[4,5-
3
H]lysine (80 Ci/mol supplied by Amersham Bio-sciences), and
E. coli
tRNA
Lys
(2.4 nmol, supplied by Sigma). Themixture was equilibrated at 27
°
C before the addition of lysyl-tRNAsynthetase (50 n
M
). After 45 min the lysyl-tRNA
Lys
reaction productwas purified by phenol:chloroform and ethanol precipitation. Amino-acylated tRNA
Lys
was then dissolved in a solution of 5 m
M
sodiumacetate (500
l, pH 5.5), dimethyl sulfoxide (100
l), and glacial aceticacid (200
l).
-acetylation of lysine was achieved with the addition of acetic anhydride (200
l) followed by incubation at 0
°
C for 1 h. Thereaction product was precipitated with ethanol. The precipitate wasredissolved in 200 m
M
sodium acetate (pH 5.5) containing 10 m
M
CuCl
2
and incubated for 1 h at 37
°
C to remove unacetylated lysine fromtRNA
Lys
. Free amino acid was cleared by ethanol precipitation. Resid-ual copper ions were extracted by chromatography over a column con-taining 300
l of Chelex 100 resin (Sigma) in 150 m
M
sodium acetate(pH 5.5) (18). Prior to use in kinetic assays the substrate was ethanol-precipitated, dried at room temperature, and dissolved in sterile H
2
O.Deacylation assays (200
l) were conducted at 27
°
C in 20 m
M
Hepes(pH 7.5), 50 m
M
KCl, 20 m
M
MgCl
2
, 2 m
M
dithiothreitol, 0.5 m
M
EDTA,0.5
g/ml bovine serum albumin, 30
5000 n
M
diacetyl-lysyl-tRNA
Lys
,andcatalyticamountsofhumanPth2.Reactionswereinitiatedwiththeaddition of enzyme. At set time points, aliquots (36
l) were removedand mixed with 500
l of trichloroacetic acid (5% w/v) on ice. Precipi-tated tRNA was pelleted by centrifugation. A portion of the supernatant(400
l) was then removed and mixed with scintillation fluid. Free[4,5-
3
H]diacetyl-lysine was quantified by liquid scintillation counting.Kinetic parameters were determined using initial rate methodology.Rates were recorded over a range of substrate concentrations.
m
and
k
cat
values were derived from iterative non-linear fitting of the theoret-ical Michaelis-Menten equation to the experimental values using theLevenberg-Marquardt algorithm (19).
RESULTS AND DISCUSSION
 A sequence similarity search using the human AF151905full-length sequence as query on the non-redundant sequencedata base with the Psi-Blast algorithm produced several reli-able hits below a cutoff E value of 1
e
20
. The sequencesbelonged to eukaryotes and archaea organisms but not to bac-teria. For the archaeal sequences the similarity extended onlyto the two-third C-terminal end of the query sequence suggest-ing that the first third of the protein in eukaryotes is a local-ization signal. That is the case for the human protein encodedby chromosome 17 and located in the mitochondria.
2
The con-served region (fragment 63
179 of the human protein) wasused to generate a multiple sequence alignment as shown inFig. 1. This fragment coincides with the Pfam data base entryUPF0099 (20). Because of its catalytic properties we will referto it as Pth2.The catalytic fragment (residues 63
179) was produced in
 E. coli
as a soluble protein and purified to homogeneity behav-ing as a monomer in solution. Its crystal structure was solvedby multiple wavelength anomalous diffraction techniques andrefined to a 2.0-
 Å
resolution (Fig. 2). The crystal contained twomolecules in the asymmetric unit. The final model has an
 R
work
of 21.1% and an
R
free
of 23.7% (Table I). It includes all residuesof the first molecule and all but Gly-147 and Arg-148 of thesecond. Regarding the stereochemical quality of the model, 94%of the main chain torsion angles of non-glycine residues lie in
T
 ABLE
I
 Summary of crystallographic analyses and refinement statistics
NativeNaBr multiple wavelength anomalous diffractionPeak Inflection Remote
Data collection
a
Wavelength (
 Å
) 1.0079 0.9195 0.8670 0.9199Resolution (
 Å
) 2.0 (2.07
2.00)
b
2.8 (2.9
2.8)
b
2.8 (2.9
2.8)
b
2.8 (2.9
2.8)
b
Measured reflections 168,771 42,087 42,451 38,958Unique reflections 24,275 16,149
c
16,166
c
15,969
c
Completeness (%) 99.7 (99.9)
b
99.1 (99.7)
b
99.2 (99.4)
b
98.5 (98.9)
b
 R
sym
(%) 6.6 (45.7)
b
10.2 (38.3)
b
9.8 (37.3)
b
9.7 (36.9)
b
 I 
 / 
 
 I 
27.7 (4.7)
b
7.9 (2.2)
b
8.8 (2.7)
b
7.5 (1.9)
b
Figure of merit after density modification 0.76 (0.56)
b
Refinement statisticsResolution range (
 Å
) 43.0
2.0Unique reflections (free) 22,580 (1674)
 R
work
(%) 21.1
 R
free
(%) 23.7Number of residues 232Number of solvent molecules 123 Average B value (
 Å
2
)Protein 29.3Solvent 36.7Root mean square deviation bond lengths (
 Å
) 0.008Root mean square deviation angles (
°
) 1.40
a
Space group,
P
4
3
2
1
2; cell dimensions,
a
b
61.6
Å
,
c
177.9
Å
; molecules in asymetric unit
2.
b
Numbers in parentheses correspond to the outer shell.
c
Keeping Bijvoet pairs separate.
R
sym
 I 
i
 I 
|
 I 
i
F
IG
. 3.
RibbondiagramofthecrystalstructureofhumanPth2.
Elements of regular secondary structure are labeled and numberedaccording to their position in the primary sequence.
-Helices are col-ored
purple
,
-strands
green
, and loops
gray
. The figure was generatedwith MOLSCRIPT (25) and rendered with Raster3D (10).
 Structure of Pth2
8113

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