Enzymatic properties of an

apoptotic protein
Jaime Pascual
Burnham Institute
 Outline
• Multifunctional proteins

• Peptidyl-tRNA hydrolase 2
(formerly known as Bcl-2 inhibitor of
transcription 1)

• Protein-protein interaction vs. catalysis

• Summary / Model
 Multifunctional proteins
• Multifunctional proteins is a mechanism to
increase organism complexity while
keeping a similar number of genes

• Implies that a single polypeptide performs

several functions at different times and/or
places

• In terms of 3D structure, a single fold has

to accommodate two or more active sites
 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

 Ptrh2 multiple sequence alignment
• Ptrh2 highly conserved protein evolving from archaea to
eukarya (mitochondria) and to eubacteria (lateral gene transfer)
 Ptrh2 biophysical study
• circular dichroism & NMR & X-ray
1
H-15N HSQC spectrum

50
Far-UV CD spectrum

[θ ] MR (x1000 deg cm2/dmol)

30

10

-10

-30
190 200 210 220 230 240
wavelength (nm)
 Ptrh2 structure shows a new fold
• New α/β fold: mixed β-sheet with 2 α-helices per side
 Active site(s):

-Physically separated?

-Both accessible at the same

time?

-Any interdependence (cross-

talk)?
 Ptrh2 mediates apoptosis via a
cytoplasmic interaction with AES
• Yeast-two-hybrid screening
Ptrh2
dimer
interface

•
Protein-protein interaction: Pthr2 crystal dimer
 Peptidyl-tRNA hydrolase
Function: recycle peptidyl-tRNA from abortive translations

Enzyme: carboxylic ester hydrolase

Reaction: peptidyl-tRNA + H2O -> peptide + tRNA

 Peptidyl-tRNA hydrolase
• 13 genes are present in human mitochondrial DNA

electropos.

electroneg.

backbone

Ptrh1 Ptrh2
(cytoplasm) (mitochondria)

• 2 enzymes: different sequence, fold & catalytic param.

 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

Catalytic parameters of human Ptrh2
Enzyme kcat (s-1) Km (µΜ)
WT 1.3 0.22
K81A 0.02 10
D145A 0.08 90
S155A 0.1 1

triple mut. - -

S87G, S92G, n/a n/a

R99G
Apoptosis induced by Ptrh2 48 h after transfection
(HEK 293T cells)
50

40 P =0.006
% Apoptosis

30

20

10

0
Ptrh2 (WT) Ptrh2 triple mut. Vector (pCMVmyc)

2 experiments
(3 replicates each)
Ptrh substrate isomerizes between the
2’ and 3’ positions of the ribose
spontaneously at room temperature

2’

3’
 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. Active site of Thr-tRNA synth.

with 2’-substituted substrate with 3’-substituted substrate
analogue analogue
Chemical synthesis of a 2’-substituted
non-hydrolizable substrate analogue
 Co-crystal structure of Ptrh2 bound to a
2’-substrate analog
β3-β4 loop
free
bound
arch.
human

free
human

Crystal dimer conformation Conformational change of

similar to that the β3-β4 loop between
observed for the free protein the free & bound protein
Chemical synthesis of a 3’-substituted
non-hydrolizable substrate analogue
Summary: a bifunctional protein
Name Ptrh2 Ptrh2

Localization Cytoplasm Mitochondria

Function Apoptosis Translation

Structure Protein-protein Catalysis

 Model of Ptrh2 function:
enzyme or de-repressor depending on location
 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.)
 Working hypothesis
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
AES lacks WD repeats (can’t repress) but
through Q-rich region can oligomerize
with TLE (and de-repress)

Groucho protein domain composition

Amino terminal Enhancer of Split

shows a Gln-rich domain at its N-term.