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8/14/2019 Peptydil tRNA hydrolase 2
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Enzymatic properties of an
apoptotic protein
Jaime Pascual
Burnham Institute
8/14/2019 Peptydil tRNA hydrolase 2
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Outline
• Multifunctional proteins
• Peptidyl-tRNA hydrolase 2
(formerly known as Bcl-2 inhibitor of
transcription 1)
• Protein-protein interaction vs. catalysis
• Summary / Model
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Multifunctional proteins
• Multifunctional proteins is a mechanism toincrease organism complexity whilekeeping a similar number of genes
• Implies that a single polypeptide performsseveral functions at different times and/or places
• In terms of 3D structure, a single fold hasto accommodate two or more active sites
8/14/2019 Peptydil tRNA hydrolase 2
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Peptidyl-tRNA hydrolase 2
(Ptrh2)
• Protein domain composition:
N-term mitochondrial localization signal (only in eukaryotes)
followed by the Ptrh2 domain(~110 amino acids)
• 3D structures available:
H. sapiens (X-ray), S. solfataricus (X-ray),
T. acidophilum (X-ray), A. fulgidis (NMR)
• New 3D fold: mixed α/β (ordered 2-1-4-3) protein
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Ptrh2 multiple sequence alignment
• Ptrh2 highly conserved protein evolving from archaea toeukarya (mitochondria) and to eubacteria (lateral gene transfer )
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Ptrh2 biophysical study• circular dichroism & NMR & X-ray
1H-15N HSQC spectrum
-30
-10
10
30
50
190 200 210 220 230 240
wavelength (nm)
[ θ
] M R
( x 1 0 0 0 d e g c m 2
/ d m o l )
Far-UV CD spectrum
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Ptrh2 structure shows a new fold
• New α/β fold: mixed β-sheet with 2 α-helices per side
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Active site(s):
-Physically separated?
-Both accessible at the sametime?
-Any interdependence (cross-
talk)?
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Ptrh2 mediates apoptosis via a
cytoplasmic interaction with AES
• Yeast-two-hybrid screeningPtrh2
dimer
interface
•Protein-protein interaction: Pthr2 crystal dimer
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Function: recycle peptidyl-tRNA from abortive translations
Enzyme: carboxylic ester hydrolase
Reaction: peptidyl-tRNA + H2O -> peptide + tRNA
Peptidyl-tRNA hydrolase
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• 13 genes are present in human mitochondrial DNA
• 2 enzymes: different sequence, fold & catalytic param.
Peptidyl-tRNA hydrolase
Ptrh2
(mitochondria)
backbone
electropos.
electroneg.
Ptrh1
(cytoplasm)
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Ptrh2 putative catalytic triad
• Comparison with classical carboxylic ester hydrolases:
nucleophile (Ser), acidic res. (Asp) & basic res. (His)
• Predicted active center for Ptrh2: Lys81-Asp145-Ser155
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Catalytic parameters of human Ptrh2
Km (µΜ)kcat (s-1)Enzyme
10.1S155A
n/an/aS87G, S92G,
R99G
--triple mut.
900.08D145A100.02K81A
0.221.3WT
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% A
p o p t o s
i s
Ptrh2 (WT) Ptrh2 triple mut. Vector (pCMVmyc )
50
P =0.00640
30
20
10
0
Apoptosis induced by Ptrh2 48 h after transfection
(HEK 293T cells)
2 experiments
(3 replicates each)
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2’
3’
Ptrh substrate isomerizes between the
2’ and 3’ positions of the ribosespontaneously at room temperature
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Aminoacyl-tRNA synthetases: post-transfer editing activity on mischarged
amino acids• Comparison with other carboxylic ester hydrolases:
– Editing activity of 2 families of aminoacyl-tRNA synthethases
Active site of Leu-tRNA synth.
with 2’-substituted substrate
analogue
Active site of Thr-tRNA synth.
with 3’-substituted substrateanalogue
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Chemical synthesis of a 2’-substituted
non-hydrolizable substrate analogue
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Co-crystal structure of Ptrh2 bound to a
2’-substrate analog
Crystal dimer conformation
similar to that
observed for the free protein
Conformational change of
the β3-β4 loop between
the free & bound protein
bound
human
free
human
free
arch.
β3-β4 loop
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Chemical synthesis of a 3’-substituted
non-hydrolizable substrate analogue
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Summary: a bifunctional protein
CatalysisProtein-proteinStructure
TranslationApoptosisFunction
MitochondriaCytoplasmLocalization
Ptrh2Ptrh2Name
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Model of Ptrh2 function:enzyme or de-repressor depending on location
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Acknowledgements
• Jose Pereda, Eugenio Santelli
• Bob Liddington
• Yiwen Jan, Ing Wei, Rania• Erkki Ruoslahti
• Anjali Mascarenhas (Univ. Illinois)
• Susan Martinis (Univ. Illinois)• Morten Grotli (Goteborg Univ.)
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Cell attached to ECM (normal state):Mitochondria is intact and Ptrh2 behaves as an enzyme;
in the cytoplasm, AES (de-repressor) shuttles to the nucleus,
hetero-oligomerizes with TLE1 (co-repressor)
and bHLH family of repressors do not bind to
the silencer region of the Bcl-2 gene, so Bcl-2 is transcribed
Cell detaches from ECM (apoptotic insult):
Mitochondria outer membrane leaks,
Ptrh2 is released to the cytoplasm,
hetero-dimerizes with AES;
in the nucleus, homo-oligomers of TLE1 accumulate
and bind bHLH protein repressors that silence Bcl-2 expression
Working hypothesis
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AES lacks WD repeats (can’t repress) but
through Q-rich region can oligomerize
with TLE (and de-repress)
Amino terminal Enhancer of Split
shows a Gln-rich domain at its N-term.
Groucho protein domain composition