Thermodynamic properties distinguish human mitochondrial aspartyl-tRNA synthetase from bacterial homolog with same 3D architecture
Supplementary
Figures
Anne
Neuenfeldt,
Bernard
Lorber,
Eric
Ennifar,
Agnès
Gaudry,
Claude
Sauter,
Marie
Sissler,
and
Catherine
Florentz*
Architecture
et
Réactivité
de
l'ARN,
Université
de
Strasbourg,
CNRS,
IBMC,
15
rue
René
Descartes
67084
Strasbourg
Cedex,
France
1
Supplementary
Figure
S1.
2
C.
Figure
S1.
Primary
and
secondary
structures
of
the
investigated
aminoacyltRNA
synthetases
and
tRNAs.
(A)
Secondary
structures
of
HsaDRS2
and
EcoDRS
(1,2).
(B)
Primary
sequence
alignments
of
the
two
recombinant
synthetases.
Secondary
structure
domains
are
boxed
using
the
same
color
code
as
in
(A)
and
as
in
the
crystal
structure
presented
in
Figure
1
of
the
main
text.
Additional
DRS
sequence
alignments
are
displayed
in
ref.
(1,3).
Residues
that
are
involved
in
specific
and
direct
(i.e.
not
mediated
by
water
molecules)
contacts
with
tRNA
bases
in
the
E.
coli
complex
(see
Table
II
in
(4))
are
boxed
in
green
if
conserved
or
in
orange
if
different
in
EcoDRS
and
HsaDRS2.
Additional
positively
charged
residues
in
HsaDRS2
in
the
neighborhood
of
the
tRNA
binding
cleft
are
boxed
in
blue.
(C)
Sequences
of
Eco
tRNAAsp
(5)
and
Hsa
mt‐tRNAAsp
(6,7).
Major
and
minor
aspartate
identity
determinants
for
aminoacylation
by
cognate
aspartyl‐tRNA
synthetases
(2,8,9)
are
emphasized
by
bold
and
light
squares
respectively.
3
Supplementary
Figure
S2.
15
dh (nm)
HsaDRS2
10 EcoDRS
5
0
0
5
10
concentration (mg/ml)
Figure
S2.
DLS
properties
of
HsaDRS2
and
EcoDRS.
Variation
of
the
apparent
hydrodynamic
diameter
of
DRSs
as
function
of
protein
concentration.
Measurements
were
conducted
in
ITC
buffer
at
20°C
(see
main
text).
4
Supplementary
Figure
S3.
Figure
S3.
From
the
initial
to
the
final
HsaDRS2
model.
(A)
First
electron
density
map
(EDM)
calculated
after
molecular
replacement
(MR)
in
the
region
of
the
bacterial
insertion.
The
MR
model
is
shown
in
pink,
the
final
structure
in
orange.
(B)
Final
EDM
with
initial
and
final
model
(same
color
code).
The
arrows
indicate
the
direction
of
the
rigid‐body
displacement
of
the
bacterial
insertion
during
DEN‐refinement
(see
methods).
(C)
Stereographic
view
of
the
class
II
anti‐parallel
beta‐sheet
of
one
monomer
of
HsaDRS2
colored
in
cyan
(second
monomer
in
dark
blue)
in
the
final
EDM
(sigmaa
weighted
2FoFc
maps
contoured
at
1.2
σ).
5
Supplementary
Figure
S4.
Figure
S4.
Comparison
of
HsaDRS2
monomers
(stereoviews).
(A)
Superposition
of
the
four
HsaDRS2
monomers:
HsaDRS2
dimers
are
slightly
asymmetric
due
to
differences
in
the
extra‐domain.
Monomer
A
(yellow)
superimposes
better
with
C
(orange),
B
(blue)
with
D
(green)
(see
Table
S5
for
corresponding
rmsd
values).
(B)
Superposition
of
respective
insertions
(same
color
code)
showing
an
excellent
match
and
revealing
a
rigid‐ body
movement
of
the
insertion.
6
Supplementary
Table
S5.
Comparison
of
DRS
structures
(rmsd
in
Å)
_____________________________________________________________________________________________________
HsaDRS2
monomers*
HsaDRS2_A
HsaDRS2_B
HsaDRS2_C
HsaDRS2_B
0.81
HsaDRS2_C
0.31
0.77
HsaDRS2_D
0.71
0.24
0.73
HsaDRS2
bacterial
insertions**
HsaDRS2_A
HsaDRS2_B
HsaDRS2_C
HsaDRS2_B
0.019
HsaDRS2_C
0.014
0.019
HsaDRS2_D
0.017
0.014
0.018
HsaDRS2
vs
EcoDRS
monomers***
HsaDRS2‐core
HsaDRS2‐cat
HsaDRS2‐insertion
EcoDRS_1C0A
1.54
1.34
1.52
EcoDRS_1EQR
1.39
1.27
1.54
_________________________________________________________________________________________________
*Two
independent
dimers
are
observed
in
the
asymmetric
unit,
monomers
A,
B
for
dimer
1,
and
monomers
C,
D
for
dimer
2.
Monomers
are
compared
pair‐wise
along
the
full
set
of
residues.
**The
comparison
of
monomers
is
restricted
to
the
bacterial
insertion
domain
(residues
328‐457)
***Comparison
of
the
full
set
of
monomers
from
HsaDRS2
with
that
of
EcoDRS.
Residues
along
three
domains
were
compared,
namely
the
DRS
“core”
corresponding
to
the
anticodon
binding
domain,
the
hinge
domain
and
the
catalytic
domains
(411
superimposed
residues);
the
catalytic
domain
“cat”
(297
superimposed
residues)
and
the
bacterial
insertion
(105
superimposed
residues).
Values
are
averaged
over
the
monomer
copies
present
in
each
structure:
3
copies
in
1EQR
for
free
EcoDRS,
1
in
1C0A
for
tRNA‐ bound
EcoDRS,
4
in
4AH6
for
free
HsaDRS2.
7
Supplementary
Figure
S6.
Figure
S6.
Dimer
interfaces
of
HsaDRS2
(top)
and
EcoDRS
(bottom).
On
the
left,
the
surface
of
one
monomer
is
shown
in
white,
residues
involved
in
dimer
contacts
are
represented
as
colored
patches
and
the
second
monomer
as
a
transparent
blue
surface.
On
the
right,
the
first
monomer
alone,
rotated
by
110°
to
visualize
the
dimer
interface.
The
buried
surface
is
comparable
in
the
monomers:
5500
and
5100
Å2
for
HsaDRS2
and
EcoDRS,
respectively.
Although
the
HsaDRS2
buried
area
is
10%
larger,
it
presents
a
lower
density
of
hydrogen
bonds
(orange
patches)
and
salt
bridges
(blue
and
red
patches
for
positively
and
negatively
charged
residues,
respectively)
than
the
bacterial
enzyme.
The
EcoDRS
interface
is
stabilized
by
a
total
of
70
hydrogen
bonds
and
28
salt
bridges,
whereas
their
number
in
HsaDRS2
is
60
and
20,
respectively.
The
analysis
was
performed
using
the
Protein
interfaces,
surfaces
and
assemblies
service
PISA
at
European
Bioinformatics
Institute
(http://www.ebi.ac.uk/pdbe/prot_int/pistart.html).
8
Supplementary
Figure
S7.
A1.
A2.
Samples
DRS
DRS
+
Asp
(1
mM)
DRS
+
ATP
(1
mM)
DRS
+
Asp
+
ATP
(1
mM
each)
DRS
+
AMP‐pCp
(1
mM)
DRS
+
AspOH
(1
mM)
DRS
+
GlnOH
(1
mM)
DRS
+
GluOH
(1
mM)
DRS
+
TyrOH
(1
mM)
DRS
+
AspSA
(1
mM)
DRS
+
GlnSA
(1
mM)
DRS
+
GluSA
(1
mM)
DRS
+
TyrSA
(1
mM)
HsaDRS2
Tm
(±1°C)
45
45
44
47
45
46
45
45
45
55
45
45
45
9
EcoDRS
Tm
(±1°C)
59
59
59
59
59
64
60
60
59
81
61
63
61
B.
D.
Samples
C.
HsaDRS2
Tm
(±1°C)
EcoDRS
Tm
(±1°C)
45
46
47
46
48
49
50
45
45
45
59
60
59
60
62
64
66
59
58
59
DRS
DRS
+
15%
glycerol
DRS
+
20%
glycerol
DRS
+
5%
sucrose
DRS
+
10%
sucrose
DRS
+
20%
sucrose
DRS
+
30%
sucrose
DRS
+
5
mM
DTT
DRS
+
CaCl2
(50
nM
to
5
mM)
DRS
+
MgCl2
(50
nM
to
5
mM)
Figure
S7.
Thermal
denaturation
of
HsaDRS2
as
evaluated
by
differential
scanning
fluorescence
(Thermofluor
approach).
(A)
Thermostability
of
HsaDRS2
and
EcoDRS.
(A1)
Typical
melting
profiles
in
the
presence
of
various
compounds.
(A2)
Summary
Table.
Components
were
as
follows:
aminoacyl‐adenylate
analogues:
asp
for
aspartic
acid,
AMP‐pCp
for
adenylyl‐methylene‐ diphosphonate,
AspSA
for
5’‐O‐[N‐(L‐aspartyl)sulfamoyl]
adenosine
(abbreviated
Asp‐ AMS
in
main
text);
GluSA,
GlnSA,
TyrSA
for
other
sulfamoyl‐adenylates;
AspOH,
GluOH,
TyrOH
for
amino
alcool‐adenylates.
Notice
the
unique
stabilizing
effect
of
the
adenylate
analogue
AspSA
on
both
enzymes.
(B)
Thermostability
of
variant
HsaDRS2(5XV48)
presenting
different
N‐
and
C‐termini
over
7
to
11
amino
acids
as
compared
to
HsaDRS
(8).
(C)
Melting
profiles
of
HsaYRS2
(prepared
according
to
ref.
(9))
and
of
a
structural
variant
of
HsaDRS2
(EcoHinge)
with
partial
replacement
of
the
hinge
domain
(see
Supplementary
Fig.
S1).
Residues
F157EIKNFVKK165
from
wild‐type
HsaDRS2
have
been
replaced
by
residues
L110DSNHVN116
from
EcoDRS.
(D)
Influence
of
various
biochemical
components
on
melting
temperatures
measured
under
different
biochemical
conditions.
10
Supplementary
Figure
S8.
Figure
S8.
Thermal
denaturation
of
DRSs
monitored
by
synchrotron
radiation
circular
dichroism
(SRCD).
(A)
SRCD
spectra
(average
of
3
measurements
–
see
Methods)
of
HsaDRS2
(left)
and
EcoDRS
(right)
at
temperatures
ranging
from
20
to
80°C.
Positive
and
negative
peaks
of
ellipticity
characteristic
of
α/β
structures
decrease
while
the
temperature
increases,
which
indicates
the
melting
of
the
secondary
structure.
(B)
Evolution
of
SRCD
signal
at
209
nm
(mininum
of
initial
spectra
at
20°C):
a
transition
from
folded
to
unfolded
state
is
observed
above
40°C
and
56°C
for
human
and
bacterial
enzymes,
respectively,
in
agreement
with
the
analyses
performed
in
dynamic
light
scattering
(DLS)
and
differential
scanning
fluorimetry
(DSF).
11
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
Bonnefond,
L.,
Fender,
A.,
Rudinger‐Thirion,
J.,
Giegé,
R.,
Florentz,
C.
and
Sissler,
M.
(2005)
Toward
the
full
set
of
human
mitochondrial
aminoacyl‐tRNA
synthetases:
characterization
of
AspRS
and
TyrRS.
Biochemistry,
44,
4805‐4816.
Fender,
A.,
Sauter,
C.,
Messmer,
M.,
Pütz,
J.,
Giegé,
R.,
Florentz,
C.
and
Sissler,
M.
(2006)
Loss
of
a
primordial
identity
element
for
a
mammalian
mitochondrial
aminoacylation
system.
J.
Biol.
Chem.,
281,
15980‐15986.
Giegé,
R.
and
Rees,
B.
(2005)
In
Ibba,
M.,
Francklyn,
C.
and
Cusack,
S.
(eds.),
AminoacyltRNA
Synthetases.
Landes
Bioscience,
Georgetown,
TX,
Vol.
chapter
19,
pp.
210‐226.
Eiler,
S.,
Dock‐Bregeon,
A.C.,
Moulinier,
L.,
Thierry,
J.‐C.
and
Moras,
D.
(1999)
Synthesis
of
aspartyl‐tRNAAsp
in
Escherichia
coli‐a
snapshot
of
the
second
step.
EMBO
J.,
18,
6532‐6541.
Sekiya,
T.,
Mori,
M.,
Takahashi,
N.
and
Nishimura,
S.
(1980)
Sequence
of
the
distal
tRNAAsp1
gene
and
the
transcription
termination
signal
in
the
Escherichia
coli
ribosomal
RNA
operon
rrnF
(or
G).
Nucleic
Acids
Res.,
8,
3809‐3827.
Anderson,
S.,
Bankier,
A.T.,
Barrel,
B.G.,
de
Bruijn,
M.H.L.,
Coulson,
A.R.,
Drouin,
J.,
Eperon,
J.C.,
Nierlich,
D.P.,
Roe,
B.A.,
Sanger,
F.
et
al.
(1981)
Sequence
and
organization
of
the
human
mitochondrial
genome.
Nature,
290,
457‐465.
Messmer,
M.,
Pütz,
J.,
Suzuki,
T.,
Suzuki,
T.,
Sauter,
C.,
Sissler,
M.
and
Florentz,
C.
(2009)
Tertiary
network
in
mammalian
mitochondrial
tRNAAsp
revealed
by
solution
probing
and
phylogeny.
Nucleic
Acids
Res.,
37,
6881‐6895.
Gaudry,
A.,
Lorber,
B.,
Messmer,
M.,
Neuenfeldt,
A.,
Sauter,
C.,
Florentz,
C.
and
Sissler,
M.
(2012)
Redesigned
N‐terminus
enhances
expression,
solubility,
and
crystallisability
of
mitochondrial
enzyme.
Protein
Engineering,
Desing
and
Selection,
25,
473‐481.
Bonnefond,
L.,
Frugier,
M.,
Touzé,
E.,
Lorber,
B.,
Forentz,
C.,
Giegé,
R.,
Rudinger‐ Thirion,
J.
and
Sauter,
C.
(2007)
Tyrosyl‐tRNA
synthetase:
the
first
crystallization
of
a
human
mitochondrial
aminoacyl‐tRNA
synthetase.
Acta
Crystallographica
Section
F,
F63,
338‐341.
12
