Presentation on theme: "The Importance of Utrophin and Dystrophin Dystrophin is the protein damaged in many cases of muscular dystrophy. Duchenne Muscular Dystrophy, the commonest."— Presentation transcript:
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
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 Domain Organisation Spectrin like coiled coil repeatsWWCys rich Coiled coilCH1 CH2 Dystrophin Utrophin 124630523685 28261 28133434 Actin-binding region Plasma membrane protein interaction region
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).
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 Phylogenetic Analysis
Structure of the utrophin CH2 domain Human utrophin 144-261 expressed as a non fusion protein in E.coli. Space group P2 1 a=63.92Å b=32.21Å c=65.36Å ß= 116.3 o. 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
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=106.0 o. 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
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=61.08 o β=78.22 o γ=70.54 o. 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
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 F- actin (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.
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 CH1 CH2 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.
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, 28419- 28423. 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
Your consent to our cookies if you continue to use this website.