Crystal structure of the actin-binding region of utrophin reveals a head-to-tail dimer

Keep, Nicholas H.; Winder, Steven J.; Moores, Carolyn A.; Walke, Stefan; Norwood, Fiona; Kendrick‐Jones, John

doi:10.1016/s0969-2126(00)88344-6

Cited by 92 publications

(125 citation statements)

References 51 publications

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“…As estimated by the frictional coefficient, utrophin fragments assumed an elongated shape in the solution and became increasingly elongated as more spectrin-like repeats were added (Table I). This observation is consistent with the available molecular dimensions (16,28) and electron microscopy images of utrophin (12).…”

Section: Resultssupporting

confidence: 91%

“…Utr261 cosedimented with F-actin in a saturable manner but with low affinity and localized to stress fibers when microinjected into fibroblasts (13,14). The crystallographic study of Utr261 revealed that its two CH domains are separated by an extended ␣-helix, suggesting that this actin binding region of utrophin may be more flexible than was concluded previously from the crystal structure of the analogous domain of fimbrin (15,16). Electron microscopy and image reconstruction analysis of Utr261 complexed with actin filaments established that the flexibility of CH domains within the utrophin amino terminus results in two different modes or stoichiometries of utrophin-actin interaction (17).…”

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confidence: 83%

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Identification of Spectrin-like Repeats Required for High Affinity Utrophin-Actin Interaction

Rybakova

Ervasti

2005

Journal of Biological Chemistry

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Most studies aimed at characterizing the utrophinactin interaction have focused on the amino-terminal tandem calponin homology domain. However, we recently reported evidence suggesting that spectrin-like repeats of utrophin also participate in binding to actin. Here we expressed several recombinant fragments encoding the utrophin amino-terminal domain alone or in combination with various numbers of spectrin-like repeats. We further quantitatively characterized the actin binding properties of each recombinant utrophin fragment using a high-speed sedimentation assay. To evaluate the capacity of each protein to stabilize actin filaments, we compared the effect of utrophin recombinant fragments and full-length utrophin on 6-propionyl-2-(N,N-dimethylamino)naphthalene actin depolymerization. Our results suggest that, whereas the amino-terminal domain is essential for primary interaction between utrophin and actin, spectrin-like repeats have additive effects on the affinity and stoichiometry of binding. Our data indicate that the amino-terminal domain and first 10 consecutive spectrin-like repeats recapitulate the actin binding activity of full-length utrophin more faithfully than the amino-terminal domain alone. These findings support the model for lateral association of utrophin along the actin filament and provide the molecular basis for designing the most effective utrophin "mini-genes" for treatment of dystrophinopathies.

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Section: Resultssupporting

confidence: 91%

mentioning

confidence: 83%

Identification of Spectrin-like Repeats Required for High Affinity Utrophin-Actin Interaction

Rybakova

Ervasti

2005

Journal of Biological Chemistry

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“…Based on crystal structures showing a variable linkage between CH domains, it has been proposed that tandem CH domains are highly dynamic in both structure and function (13,17,18). Our study provides high-resolution structural information on one of these tandem CH domains in solution, showing that it is in equilibrium between two distinct conformations.…”

Section: Discussionmentioning

confidence: 70%

Large-scale opening of utrophin’s tandem calponin homology (CH) domains upon actin binding by an induced-fit mechanism

Lin

Próchniewicz

James

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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We have used site-directed spin labeling and pulsed electron paramagnetic resonance to resolve a controversy concerning the structure of the utrophin-actin complex, with implications for the pathophysiology of muscular dystrophy. Utrophin is a homolog of dystrophin, the defective protein in Duchenne and Becker muscular dystrophies, and therapeutic utrophin derivatives are currently being developed. Both proteins have a pair of N-terminal calponin homology (CH) domains that are important for actin binding. Although there is a crystal structure of the utrophin actin-binding domain, electron microscopy of the actin-bound complexes has produced two very different structural models, in which the CH domains are in open or closed conformations. We engineered a pair of labeling sites in the CH domains of utrophin and used dipolar electron-electron resonance to determine the distribution of interdomain distances with high resolution. We found that the two domains are flexibly connected in solution, indicating a dynamic equilibrium between two distinct open structures. Upon actin binding, the two domains become dramatically separated and ordered, indicating a transition to a single open and extended conformation. There is no trace of this open conformation of utrophin in the absence of actin, providing strong support for an induced-fit model of actin binding.pulsed EPR | spectroscopy | cryo-EM U trophin is a homolog protein of dystrophin that has shown high therapeutic promise for the treatment of muscular dystrophy (1). It is endogenously found in fetal or regenerating muscle but is replaced by dystrophin, the defective protein in Duchenne and Becker muscular dystrophies, as the muscle matures (2). Up-regulation of utrophin in mdx mice, which lack dystrophin, has been shown to rescue its dystrophic phenotype, improving muscle morphology and function (1, 3). The full-length protein is not required to improve dystrophic pathology in mdx mice; i.e., substantial internal truncations in utrophin can be tolerated (4). These internally truncated constructs for muscular dystrophy therapeutics support the importance of actin binding by the N-terminal portions of either dystrophin or utrophin (5). Utrophin (395 kD) and dystrophin (427 kD) both contain highly homologous N-terminal actin-binding domains (ABD1), consisting of a pair of calponin homology (CH) domains. Despite additional actin-binding regions identified in the central spectrin-type repeats (6), microutrophin constructs with high potential for clinical applications rely almost exclusively on the N-terminal CH domains for actin interaction (7,8). Therefore, understanding the structural interaction between utrophin CH domains and actin has become crucial for the rational development of therapeutic constructs.More generally, there is an urgent need for a structural blueprint of CH domain-actin complexes for the entire spectrin superfamily of actin-binding proteins (e.g., fimbrin and α-actinin), of which dystrophin and utrophin are members. The diversity of crystal structur...

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“…This type of ABD is not restricted to this family; more distantly related versions are found in other types of actin-binding proteins, such as filamin (Korenbaum and Rivero, 2002), but the lack of spectrin repeats excludes these other proteins from the spectrin superfamily. The structure of the ABD in dystrophin and utrophin has been solved, revealing a bundle of α-helices arranged in an extended head-to-tail dimer (Keep et al, 1999;Norwood et al, 2000).…”

Section: Spectrin Superfamily Motifsmentioning

confidence: 99%

The `Spectraplakins': cytoskeletal giants with characteristics of both spectrin and plakin families

Röper

Gregory

Brown

2002

Journal of Cell Science

151

169

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IntroductionA recently discovered family of cytoskeletal proteins belongs within both the spectrin and plakin superfamilies. These superfamilies consist of proteins that contribute to the linkage between the plasma membrane and the cytoskeleton. Spectrin superfamily members bind and crosslink actin filaments and attach these to membrane receptors. Members of the plakin superfamily were first identified as components of desmosomes and hemidesmosomes, connecting the adhesion receptors to intermediate filaments, but they also can crosslink different cytoskeletal elements. At present we know of two mammalian genes that encode related members of the new family, BPAG1 and MACF1, and a single Drosophila gene, short stop (shot) (Figs 1 and 2). Each of these is also known by other names, which reflects their independent discovery by different groups: MACF1 is known as ACF7, MACF, MACF7, macrophin, trabeculin α and ABP620; BPAG1 is known as dystonin and MACF2; shot is known as kakapo, kopupu and groovin (Brown et al., 1995a;Bernier et al., 1996;Becker et al., 1997;Prout et al., 1997;Gregory and Brown, 1998;Strumpf and Volk, 1998;Walsh and Brown, 1998;Leung et al., 1999b; Okuda et al., 1999;Sun et al., 1999;Yang et al., 1999;Lee et al., 2000;Leung et al., 2001b;Sun et al., 2001). A single Caenorhabditis elegans gene can also be identified in the genomic sequence; this is currently annotated as three genes: ZK1151.1, ZK1151.2 and ZK1151.3 (C. elegans Sequencing Consortium, 1998). Because the proteins encoded by these genes share features with both spectrin and plakin superfamily members and may represent the evolutionary precursor of the plakins (see below), we refer to them as spectraplakins to highlight their dual nature. Here we discuss the possible functions of spectraplakins indicated by their sequences and by biochemical and genetic studies, extrapolating from the functions of homologous cytoskeletal proteins.Spectraplakins in various species were identified independently in quite different contexts. In the fly, the spectraplakin shot emerged from genetic screens aimed at identifying genes required for integrin-mediated adhesion (Prout et al., 1997;Gregory and Brown, 1998;Strumpf and Volk, 1998) or axon extension (Van Vactor et al., 1993;Lee et al., 2000). The vertebrate spectraplakin BPAG1/dystonin was identified as a protein targeted by the autoimmune disease bullous pemphigoid in humans (Minoshima et al., 1991) and as the gene affected in the disease dystonia musculorum in mice (Brown et al., 1995a;Guo et al., 1995). Diverse motifs within spectraplakin isoformsThe full complexity of the genes producing spectraplakins has gradually emerged. The first spectraplakin product characterised, BPAG1e, is a substantial protein of 2649 residues encoded by an 8.7 kb mRNA; yet this appears to be the smallest and simplest of the spectraplakin products (Fig. 3). The previous lack of awareness of the larger spectraplakins is likely to have led to erroneous conclusions being drawn in the older literature about the functions of th...

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Crystal structure of the actin-binding region of utrophin reveals a head-to-tail dimer

Cited by 92 publications

References 51 publications

Identification of Spectrin-like Repeats Required for High Affinity Utrophin-Actin Interaction

Identification of Spectrin-like Repeats Required for High Affinity Utrophin-Actin Interaction

Large-scale opening of utrophin’s tandem calponin homology (CH) domains upon actin binding by an induced-fit mechanism

The `Spectraplakins': cytoskeletal giants with characteristics of both spectrin and plakin families

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