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Transcript
Asymmetric flexibility of a homodimeric enzyme as shown by
molecular dynamics computations.
A case study of the cold-active Vibrio alkaline phosphatase.
Ásgeirsson, B.1, Renzetti, G.2, Invernizzi, G.2, Papaleo, E.2
2
1Science Institute, University of Iceland, Dunhaga 3, 107 Reykjavik, Iceland.
Department of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza della Scienza 2, 20126, Milan, Italy.
Introduc4on
Multiple all-atom explicit solvent molecular dynamics simulations were
employed in conjunction with different metrics to analyze the dynamics
patterns and the paths of intra- and intermolecular communication in a
cold-active alkaline phosphatase (VAP)1. Asymmetric dynamics have
been suggested to play a part in the catalytic cycle in homodimeric
alkaline phosphatases. A conformational change might be the rate-limiting
step since the chemical transformations are much faster than kcat2.
Asymmetric protein dynamics would influence protein function and
stability by modulating conformational changes consistent with half-ofthe-sites mechanism3,4. Here, we wanted to see if the symmetric crystal
structure of VAP would become asymmetrical in solution, a good
predictor of half-of the-sites mechanism.
Results
Fig.1 - Dynamic patterns of the two subunits had a different
distribution of intramolecular interactions and correlated motions
(rmsf).
Fig. 2 - VAP displayed a low number of intersubunit interactions.
Coupled motions between the two halves were also few.
Fig. 3 - Numerous salt-bridge clusters were observed with
asymmetric distribution in the two subunits .
Fig. 4 - Hub residues (those that link 3 or more other residues)
were nonsymmetrically distributed. Several were located in area
specific for VAP, i.e. the large loop (insert II).
Asymmetric
flexibility
paLerns
Cα
correla4ons
Fig.
2.
Significant
posi4ve
correla4ons
between
Cα
atoms.
The
posi=ve
correla=ons
are
shown
as
green
s=cks
connec=ng
the
Cα
atoms
of
the
average
structures
from
the
simula=ons.
The
two
subunits
A
and
B
are
shown
as
white
and
pale‐cyan
and
pink
cartoon,
respec=vely,
with
regions
of
differen=al
flexibility
highlighted
in
red
and
blue
according
to
Fig.
2.
The
cataly=c
residue
S65
and
the
metal
ions
Mg2+
and
Zn2+
are
shown
as
s=cks,
a
black
sphere
and
grey
spheres,
respec=vely
in
both
the
subunits.
The
panel
A
and
B
present
two
different
orienta=on
of
the
3D
structure
for
sake
of
clarity.
Salt‐bridge
clusters
Fig.
3.
Salt‐bridge
clusters
in
VAP.
(A)
The
salt‐bridges
are
mapped
on
the
3D
average
structure
from
simula=ons
as
s=cks
connec=ng
Cα
atoms
of
VAP.
The
shade
of
colors
indicate
the
persistence
degree
of
the
salt‐bridge
interac=ons
from
blue
(high
persistence)
to
light‐green
(low
persistence).
The
black
dots
indicate
the
loca=on
of
the
metal
ions.
The
different
clusters
of
spa=al
proximity
of
the
salt‐bridges
and
their
networks
are
indicated
by
different
colors
and
for
sake
of
clarity
only
the
most
populated
clusters
are
indicated
by
labels.
(B‐C)
The
intramolecular
salt‐bridges
and
heir
networks
have
been
mapped
on
the
individual
subunit
aXer
structural
alignment
of
the
subunits
in
order
to
compare
their
distribu=on
on
the
3D
structure
of
subunit
A
(panel
B)
and
B
(panel
C).
The
different
shade
of
colors
of
the
residues
involved
in
the
salt‐
bridges,
which
are
represented
as
spheres
corresponding
to
the
Cα
posi=on,
are
only
for
sake
of
clarity
and
are
not
related
to
clustering
of
the
interac=ons.
Hub
residues
Fig.
4.
Hub
residues
derived
from
Protein
Structure
Network
analyzes
of
VAP
MD
trajectories
are
shown
as
magenta
and
blue
spheres
for
subunit
A
and
B,
respec=vely.
The
four
inserts
(I
to
IV)
not
conserved
in
the
warm‐adapted
counterparts
are
in
green.
R129
and
S65
are
shown
as
s=cks.
Conclusions
•
Dynamic patterns of the two VAP subunits were asymmetric.
•
VAP subunits had a different distribution of intramolecular interactions
and correlated motions.
•
VAP displayed a low number of intersubunit interactions. Coupled
motions between the two halves were also few.
Figure
1.
(A)
The
average
rmsf
per‐residue
profiles
calculated
over
different
=me
windows
(whole
trajectory,
10
ns,
or
5
ns)
of
subunit
A
(purple
shade
of
colors)
and
B
(blue
shade
of
colors)
are
shown.
(B)
The
=me‐dependent
rmsf
profiles
calculated
on
=me‐windows
of
3
ns
of
subunit
A
(purple
shade
of
colors)
and
B
(blue
shade
of
colors)
show
the
progressive
changes
in
residue
mobili=es.
(C)
Regions
characterized
by
differen=al
flexibility
in
subunit
A
and
B
are
mapped
on
the
3D
structure.
In
par=cular,
regions
characterized
by
the
highest
flexibility
in
monomer
A
or
in
monomer
B
are
shown
in
red
and
blue,
respec=vely.
The
two
subunits
A
and
B
are
shown
as
white
and
pale‐cyan
and
pink
cartoon,
respec=vely.
The
four
inser=ons
characteris=cs
of
VAP
are
highlighted
by
different
colors
in
both
the
subunits.
The
cataly=c
residue
S65
and
the
metal
ions
Mg2+
and
Zn2+
are
shown
as
s=cks,
a
black
sphere
and
grey
spheres,
respec=vely
in
both
the
subunits.
(D‐E).
The
rmsf
profiles
have
been
mapped
on
the
3D
structure
of
monomer
A(D)
and
B
(E)
aXer
a
structural
alignment
of
the
two
subunits.
In
par=cular,
cartoon
shade
of
color
and
thickness
are
propor=onal
to
the
Cα
rmsf
values.
[email protected] [email protected]
Our results provide a structural rationale to support the half-of-thesites mechanism for VAP. This will help us to understand the
reaction mechanism of this metallo-phosphatase better and open the
door for studying how cold-adaptation has generated a cold–active
variant by mutagenesis. References
(1)
Papaleo
E,
Renze]
G,
Invernizzi
G,
Ásgeirsson
B.
Dynamics
fingerprint
and
inherent
asymmetric
flexibility
of
a
cold‐
adapted
homodimeric
enzyme.
A
case
study
of
the
Vibrio
alkaline
phosphatase.
Biochim
Biophys
Acta
(BBA)
‐
General
Subjects.
2013;1830(4):2970‐2980.
(2)
Halford, S. E., Bennett, N. G., Trentham, D. R., and Gutfeund, H. (1969) A substate-induced conformation
change in the reaction of alkaline phosphatase from Escherichia coli, Biochem J 114, 243-251.
(3) Chappelet-Tordo, D., Fosset, M., Iwatsubo, M., Gache, C., and Lazdunski, M. (1974) Intestinal alkaline
phosphatase. Catalytic properties and half of the sites reactivity, Biochemistry 13, 1788-1794.
(4)
Orhanovic, S., and Pavela-Vrancic, M. (2003) Dimer asymmetry and the catalytic cycle of alkaline phosphatase
from Escherichia coli, Eur J Biochem 270, 4356-4364.
Acknowledgement:
The
Icelandic
Centre
for
Research
and
University
of
Iceland
provided
financial
support