Transcript Dystrophin and utrophin Protein

The Importance of
Utrophin and Dystrophin
• Dystrophin is the protein damaged in
many cases of muscular dystrophy.
• Duchenne Muscular Dystrophy, the
commonest and most severe form, is
caused by the absence of dystrophin and
normally leads to death by early
adulthood. It is X-linked.
• Becker Muscular Dystrophy arises from
point mutations or small deletions in
dystrophin and has a range of severity.
• Dystrophin is found only in muscle cells,
but utrophin, an autosomal homologue of
69% similarity over more than 3500
residues, is found in all tissues
• Increasing the amount of utrophin in
animal models of Duchenne Muscular
Dystrophy partially corrects for the defect
in dystrophin
Cellular function of
Utrophin and Dystrophin
•
•
•
Dystrophin and Utrophin bind to the actin cytoskeleton
just under the plasma membrane with the N terminal end
They bind to an assembly of proteins at the plasma
membrane known as the dystrophin associated protein
complex
This complex of proteins binds to the laminins of the
extracellular matrix forming a link between the actin
cytoskeleton and the extracellular matrix, which is
thought to provide a shock absorber role to the cell
maintaining the integrity of the membrane during muscle
contraction
T itle:
(DAP-diag.eps)
Creator:
Adobe Il lus trator(T M) 7.0
Preview:
T his EPS pic ture was not s aved
with a preview i nc luded i n i t.
Comment:
T his EPS pic ture wi ll pri nt to a
Pos tScri pt printer, but not to
other types of printers.
Domain Organisation
• Utrophin and dystrophin can be divided
into three region, an N terminal region that
binds actin, a long middle section of
spectrin like coiled-coil repeats and a C
terminal region which has various motifs
involved in protein-protein interactions.
This region interacts with the Dystrophin
Associated Protein Complex
Utrophin
Actin-binding
region
28 261
Plasma membrane protein
interaction region
2813
3434
CH1 CH2 Spectrin like coiled coil repeats WW Cys rich Coiled coil
1 246
3052
Dystrophin
3685
Actin Binding Region
• The actin binding region was localised to
the N terminal region in the ~250 amino
acids before the spectrin-like repeats begin
• The utrophin actin binding region binds
actin with a stoichiometry of 1:1 in
sedimentation assays and with a
dissociation constant of 58M. This is
weaker than that reported for whole
dystrophin and one of the spectrin repeats
of dystrophin, but not the equivalent
region of utrophin has been shown to bind
actin (1)
• Biochemical studies have identified three
actin binding sequences (ABS1,2,3). Two
of these were peptides that showed
changes in the NMR linewidth on addition
of F actin (ABS1 and ABS 3) (2,3). ABS2
was identified as the difference between a
proteolytic fragment that bound actin and
one that did not (4).
Calponin Homology
Domains
• The N Terminal region of about 250 amino
acids was identified as being homologous
to a region found in a range of proteins
that bind the cytoskeleton including actinin, -spectrin and fimbrin (see next
page)
• These actin binding regions show a weak
sequence motif repeat of 120 amino acids.
This motif is also found in a single copy in
a number of proteins including calponin
and is known as a calponin homology
domain.
• Although some of the single calponin
homology domain proteins bind actin, for
the family of two domain actin binding
regions both domains are required for full
actin binding activity. Isolated CH1
domains have some affinity for actin alone
but not isolated CH2 (5).
Actin Binding Region
Family
Phylogenetic Analysis
• The first calponin homology domains of
the pair (CH1) form one phylogenetic
group, the second (CH2) form a second
group and the single domains a third group
T itle:
phyl ip.ps
Creator:
Phylip
Preview:
T his EPS pic ture was not s aved
with a preview i nc luded i n i t.
Comment:
T his EPS pic ture wi ll pri nt to a
Pos tScri pt printer, but not to
other types of printers.
Structure of the utrophin
CH2 domain
•
•
•
•
•
•
•
•
•
•
Human utrophin 144-261 expressed as a non fusion
protein in E.coli.
Space group P21 a=63.92Å b=32.21Å c=65.36Å ß=
116.3o. 2 molecules in asymmetric unit.
Data Daresbury Station 9.5 Wavelength 1.00 Å
Molecular replacement (AMORE) with Spectrin CH2
domain (Matti Saraste)(6)
Resolution
20-2.0 Å
Completeness 99.5% Rmerge 0.032
Rref 0.185, Rfree 0.257
Model Chain A 147-254, B 151-258 173 Water
Published (7). PDB 1BHD
4 main helices 3 roughly parallel α3, α4 and α6 and one
roughly perpendicular α1 as first seen in spectrin CH2
(6). Smaller helices vary between domains
T itle:
Creator:
MolSc ript v2.0.1, Copyright (C) 1997 Per J . Kraul is
Preview:
T his EPS pic ture was not s aved
with a preview i nc luded i n i t.
Comment:
T his EPS pic ture wi ll pri nt to a
Pos tScri pt printer, but not to
other types of printers.
Structure of the utrophin
actin-binding region
•
•
•
•
Human Utrophin 28-261expressed without a fusion
partner in E.coli
Space Group C2: a=150.15Å, b=55.19Å, c=80.28 Å
ß=106.0o. 2 molecules in asymmetric unit
SeMet MAD BM14 ESRF. 10 Se in ASU found using
SHELXS. Refined against remote wavelength 0.900 Å
Resolution 24-3.0 Å
Completeness 96.7%
Rmerge 0.057
Rref 0.198 Rfree 0.258
•
Model A+B 31-256 12 Water PDB 1QAG
•
•
T itle:
Creator:
MolSc ript v2.0.1, Copyright (C) 1997 Per J . Krauli s
Preview:
T his EPS pic ture was not s aved
with a preview i nc luded i n i t.
Comment:
T his EPS pic ture wi ll pri nt to a
Pos tScri pt printer, but not to
other types of printers.
Structure of the dystrophin
actin-binding region
•
•
•
•
•
•
•
•
Human dystrophin 1-246 expressed with a C terminal
Tag in E.coli
Space Group P1: a=59.690Å b=79.330Å c=81.950 Å
α=61.08o β=78.22o γ=70.54o. 4 molecules in AU.
Data Trieste 5.2R.Wavelength 1.00 Å
MR (Amore) using utrophin CH1 from chain A/CH2
from chain B.
Resolution 40-2.6 Å
Completeness 95.4%
Rmerge 0.051
Rref 0.234 Rfree 0.262
Model A+B+C+D 9-246 29 Water
T itle:
Creator:
MolSc ript v2.0.1, Copyright (C) 1997 Per J . Kraul is
Preview:
T his EPS pic ture was not s aved
with a preview i nc luded i n i t.
Comment:
T his EPS pic ture wi ll pri nt to a
Pos tScri pt printer, but not to
other types of printers.
Other structures
from this actin-binding
region family
• Spectrin CH2 domain- First calponin
homology domain solved. Matti Saraste
and coworkers EMBL (6). PDB 1AA2
• First actin binding region from fimbrin.
Steven Almo and coworkers (8). PDB
1AOA. This was the first structure of a
CH1 and CH2 together. Monomer in the
asymmetric unit.
•
Fimbrin is different from the other members of the
family in having two actin binding regions (4 CH
domains) on the same chain and is most divergent in
sequence. It has an insertion of 13 amino acids between
CH1 and CH2 domains relative to utrophin/dystrophin.
9 of these are disordered in the crystal structure but
inspection of the distances to the symmetry related
copies confirms that this linker must fold back and the
two CH domains in the tight complex in the crystal come
from the same chain
• α-Actinin Poster P05.04.006 Uwe Sauer.
We have not seen any details of this
Individual CH domains
superimposed
•
Superposition of CH domains. Utrophin CH1 yellow
CH2 red, Dystrophin CH1 cyan, CH2 black, spectrin
CH2 blue, Fimbrin CH1 purple, CH2 green. As well as
the insertion between domains fimbrin has a large
inserted loop in each domain.
Alignment and
secondary structure
•
Residues conserved in 6 out of the 7 species are boxed.
The conserved tryptophan is involved in the inter CH
domain interface. The DG is a tight turn between helix α
2 and α3. The conserved Asp and Lys are in the
interdomain interface in CH2 but surface exposed in
ABS2 presumably for actin binding in CH1.
Dimers Superimposed
•
The top picture shows
fimbrin in green
superimposed on the
dimer of utrophin in red
and yellow and
dystrophin in blue and
cyan. The CH1/CH2
complex superimposes
closely
•
The lower picture
shows the second
CH1/CH2 complex of
utrophin in red and
yellow and dystrophin
in blue and cyan when
the first are
superimposed as above.
There is a rotation of
about 70 degrees in the
orientation of the
domains.
Domain swapping
•
•
•
Several examples of the same or similar proteins having
a conserved interface, which is in one case intrachain and
in another interchain are now known. This is known as
3D-domain swapping (9).
In many cases these are thought to be artefacts of
crystallisation conditions but in other cases, particularly
of homologous rather than identical proteins there is
thought to be a functional and evolutionary link.
Gel filtration, NMR and analytical ultracentrifugation
data all indicate that the utrophin actin binding region is
monomeric in solution, so it is probable that the linker
refolds to give a utrophin monomer that resembles the
fimbrin crystal structure. We cannot totally rule out the
dimer purely being an artefact of crystallisation.
• Eisenberg Model of Domain swapping
Closed
Monomers
Open
monomer
3D Domain
swapped dimer
Evolved Domain
swapped dimer
Actin Binding Models
•
•
A pseudo atomic model for fimbrin binding to actin has
been proposed based on building the atomic model of Factin (10) and the fimbrin actin binding region crystal
structure into a helical EM reconstruction. The simplest
model for utrophin binding to actin would be similar to
this (left)
An alternative model would be to have the extended
utrophin monomer seen in the crystal structure binding
actin. This does allow ABS1 and ABS3 which are
largely buried in the CH domain interface in the fimbrin
like structure to interact with actin directly.
T itle:
Creator:
BobSc ript (C) 1993 Per Krauli s, 1996 Robert Esnouf
Preview:
T his EPS pic ture was not s aved
with a preview i nc luded i n i t.
Comment:
T his EPS pic ture wi ll pri nt to a
Pos tScri pt printer, but not to
other types of printers.
EM Reconstruction
• The figure below shows two fittings of
utrophin CH domains to the difference
map of an EM helical reconstruction of
actin subtracted from a utrophin actin
binding domain-actin complex
reconstruction. There is some
rearrangement from the crystal structure to
obtain the fit on the right hand side but it is
clearly better than the fimbrin like
reconstruction (left).
Clinical Mutations
• Deletions of exon 3 (32-62) in green and exon 5
(89-119) in cyan which each remove large parts of
CH1 cause severe dystrophic symptoms.
• There are three point mutations that cause Becker
Dystrophy that map to the actin-binding region
• L54R introduces a charged residue into a
hydrophobic environment and is likely to disrupt
the actin binding region. Dystrophin is still found
at the plasma membrane but severe symptoms are
seen. Mutation in red surrounding atoms in grey.
• A168D again introduces a charged residue in a
hydrophobic pocket.and Y231N removes steric
bulk from a hydrophobic core. These lie in CH2
and cause less severe symptoms.
CH2
T itle:
T itle:
Creator:
MolSc ript v2.0.1, Copyright (C) 1997 Per J . Kraul is
Preview:
T his EPS pic ture was not s aved
with a preview i nc luded i n i t.
Comment:
T his EPS pic ture wi ll pri nt to a
Pos tScri pt printer, but not to
other types of printers.
CH1
Creator:
MolSc ript v2.0.1, Copyright (C) 1997 Per J . Kraul is
Preview:
T his EPS pic ture was not s aved
with a preview i nc luded i n i t.
Comment:
T his EPS pic ture wi ll pri nt to a
Pos tScri pt printer, but not to
other types of printers.
Summary
• We have determined the crystal structures
of the CH2 domain of utrophin and the
actin binding regions (CH1+CH2) of
utrophin and dystrophin.
• In contrast to the first fimbrin actin
binding region, which is a monomer, both
utrophin and dystrophin actin-binding
regions crystallise as a dimer.
• The dimer found in both the dystrophin
and utrophin crystals suggests an
alternative model for actin binding from
that seen in the EM reconstruction of
fimbrin
• Our EM helical reconstruction of the
utrophin actin-binding region bound to
actin confirms our alternative model for
actin binding
Acknowledgements and
References
•
•
•
•
•
•
•
•
•
•
•
As well as the authors of the poster for their various
contributions, we would like to thank Dr John Berriman,
Dr Linda Amos and Dr Tony Crowther for their
contribution to the EM reconstruction and the staff of
Daresbury, ESRF and Trieste synchrotrons particularly
Dr Andrew Thompson (ESRF) for assistance with data
collection
Pictures drawn with Molscript (Kraulis), Bobscript
(Esnouf), Alscript (Barton) and Raster3d (Merrit)
This work was supported by the Muscular Dystrophy
Group (now the Muscular Dystrophy Campaign). Data
collection was supported by the MRC, ESRF and EU.
References
1) Amann,K.J., Renley, B.A. & Ervasti,J.M. (1998). J. Biol. Chem. 273, 2841928423.
2) Levine, B.A., Moir, A.J.G., Patchell, V.B. & Perry, S.V. (1990). FEBS Lett. 263,
159-162.
3) Levine, B.A., Moir, A.J.G., Patchell, V.B. & Perry, S.V. (1992). FEBS Lett. 298,
44-48.
4) Bresnick, A.R., Warren, V. & Condeelis, J. (1990). J. Biol. Chem. 265, 9236-9240.
5) Gimona, M. & Winder, S.J. (1998). Curr. Biol. 8, R674-R675.
6) Djinovic-Carugo, K., Banuelos, S. & Sarraste, M. (1997). Nature Struct. Biol. 4,
175-179.
7) Keep, N.H., Norwood, F.L.M., Moores, C.A., Winder, S.J. & Kendrick-Jones, J.
(1999). J. Mol. Biol. 285, 1257-1264.
•
•
•
8) Goldsmith, S.C., Pokala, N., Shen, W., Federov, A.A., Matsudaira, P. & Almo, S.C.
(1997) Nature Struct. Biol. 4, 708-712
9) Schulunegger, M.P., Bennett, M.J. & Eisenberg, D. (1997). Adv. Protein Chem. 50,
61-122.
10) Hanein, D. et al., & Matsudaira, P. (1998). Nature Struct. Biol. 5, 787-792