Dystrophin project
Dystrophin is one of a number of large cytoskeleton associated proteins that connect between various cytoskeletal elements and often are tethered to the membrane through other transmembrane protein complexes. These cytolinker proteins often provide structure and support to the cells where they are expressed, and mutation in genes encoding these proteins frequently gives rise to disease. Dystrophin is no exception in any of these respects, providing connections between a transmembrane complex known as the dystrophin glycoprotein complex and the underlying cytoskeleton. The most established connection and possibly the most important is that to F-actin, but more recently evidence has been forthcoming of connections to membrane phospholipids by our group (see below), intermediate filaments and microtubules.
Moreover it is becoming increasingly clear that the multiple spectrin-like repeats in the centre of the molecule, that had hitherto been thought to be largely redundant, harbor binding activities that have a significant impact on dystrophin functionality. This functionality is particularly apparent when assessed by the ability to rescue the dystrophic phenotype in mdx mice.
Our group has been interested in structural and functional aspects of dystrophin since about 10 years after the pioneering work of the Gratzer group which was the first to show that the 2nd dystrophin repeat actually binds to membrane phospholipids comprising phosphatidylserine and phosphatidylcholine, i.e. by electrostatic forces. This was an interesting finding implying that direct binding to the membrane lipids could occur in vivo in line with results from erythrocyte and brain spectrin. We have refined and extended their primary findings on repeat 2 by showing that tryptophan residues as well as strong electrostatic forces are involved in the binding, which overall depends upon lipid packing (Le Rumeur et al., 2003, 2007). This lipid binding property is also shared by other parts of the central coiled-coil region. The proximal coiled-coil region encompassing repeat 1 to 3 binds strongly to lipids by contrast to the distal coiled-coil region from repeat 20 to 24 which does not bind to lipid (Legardinier et al., 2008). Finally, we studied the large central coiled-coil region from repeat 4 to 19 by designing multi-repeat constructs and showed that all constructs from repeat 4 to 19 in 3 repeat constructs are able to bind to anionic phospholipids (Legardinier et al., 2009).
As it was not possible to produce single repeats from dystrophin, it was not possible to map these interacting properties as was done previously for spectrin repeats. In the case of spectrin, single repeats were designed and produced and it appears that a small number of them from both erythrocyte and brain spectrin are able to bind to lipids. Even though not mentioned by the authors, it is striking that the lipid binding properties were correlated with the lowest thermal stabilities of the repeats from spectrin. In the case of dystrophin repeats, such a correlation is not possible, as single repeats could not be produced. However, it is worth noting that the range of thermal stabilities of dystrophin single repeats 2 and 23 and multi-repeats was always smaller (50 to 68°C) than the range reported for spectrin repeats (37 to 75°C). But again, spectrin does not exist in vivo as a monomer but always as a dimer and the dimerization could be a stabilizing factor which is unlikely to be the case for dystrophin repeats. Thus dystrophin repeats may need to be intrinsically more stable than spectrin repeats, or stabilized by other interactions, because of the lack of dimerization. We extended the knowledge about the lipid-binding properties of the two sub-domains spanning R1-3 and R20-24 by monolayer experiments in collaboration with the “Institut de Physique de Rennes”. Atomic force microscopy and phase modulated-IR spectroscopy were used at the interface of lipid monolayer (Vié et al., 2010). We studied the adsorption behavior of both sub-domains at the air/lipid interface in a Langmuir trough in order to highlight differences in interfacial properties. Surface-pressure measurements, atomic force microscopy and PM-IRRAS are used in a Langmuir experiment with anionic monolayer at to different surface pressures. R1-3 is present in high amounts at the interface, being arranged in clusters representing 3.3% of the surface at low pressure. By contrast, R20-24 is present at the interface in small amounts bound only by a few electrostatic residues to the lipid film while the major part of the molecule remains floating in the sub-phase. Then for R1-3, the electrostatic interaction between the proteins and the film is enhanced by hydrophobic interactions. At higher surface pressure, the number of protein clusters increases and becomes closer in both cases implying the electrostatic character of the binding. These results indicate that even if the repeats exhibit large structural similarities, their interfacial properties are highly contrasted by their differential anchor mode in the membrane. Our work provides strong support for distinct physiological roles for the spectrin-like repeats and may partly explain the effects of therapeutic replacement of dystrophin deficiency by minidystrophins.
After these works, we made use of rare missense pathogenic mutations in the dystrophin gene and analyzed the biochemical properties of the isolated repeat 23 bearing single or double mutations E2910V and N2912D found in mu
scle dystrophy with severity grading. No dramatic effect on secondary and tertiary structure of the repeat was found in mutants compared with wild type as revealed by circular dichroism and NMR. Thermal and chemical unfolding data from circular dichroism and tryptophan fluorescence show significant decrease of stability for the mutants, and stopped-flow spectroscopy shows decreased refolding rates. The most deleterious single mutation is the N2912D replacement, although we observe additive effects of the two mutations on repeat stability. Based on three dimensional structures built by homology molecular modeling, we discuss the modifications of the mutation-induced repeat stability.We conclude that the main forces involved in repeat stability are electrostatic inter-helix interactions that are disrupted following mutations. This study represents the first analysis at the protein level of the consequences of missense mutations in the human dystrophin rod domain. Our results suggest that it may participate in mechanical weakening of dystrophin-deficient muscle (Legardinier et al., 2009b).
All this work is included in a review that we written with Steve Winder from the Universisty of Sheffield (Le Rumeur et al., 2010) about dystrophin and where we present our new hypothesis for the functional role of dystrophin in the muscle cell.
All this work is included in a review that we written with Steve Winder from the Universisty of Sheffield (Le Rumeur et al., 2010) about dystrophin and where we present our new hypothesis for the functional role of dystrophin in the muscle cell.