Peptydil tRNA hydrolase 2

8/14/2019 Peptydil tRNA hydrolase 2

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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 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



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 toeukarya (mitochondria) and to eubacteria (lateral gene transfer )



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



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 sametime?

-Any interdependence (cross-

talk)?



Ptrh2 mediates apoptosis via a

cytoplasmic interaction with AES

• Yeast-two-hybrid screeningPtrh2

dimer

interface

•Protein-protein interaction: Pthr2 crystal dimer



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

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

Peptidyl-tRNA hydrolase

Ptrh2

(mitochondria)

backbone

electropos.

electroneg.

Ptrh1

(cytoplasm)



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

Km (µΜ)kcat (s-1)Enzyme

10.1S155A

n/an/aS87G, S92G,

R99G

--triple mut.

900.08D145A100.02K81A

0.221.3WT



% 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)



2’

3’

Ptrh substrate isomerizes between the

2’ and 3’ positions of the ribosespontaneously at room temperature



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



Chemical synthesis of a 2’-substituted

non-hydrolizable substrate analogue



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



Chemical synthesis of a 3’-substituted

non-hydrolizable substrate analogue



Summary: a bifunctional protein

CatalysisProtein-proteinStructure

TranslationApoptosisFunction

MitochondriaCytoplasmLocalization

Ptrh2Ptrh2Name



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.)



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



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

Peptydil tRNA hydrolase 2

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