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The crystal structures of dystrophin and utrophin spectrin repeats: implications for domain boundaries.

Muthu M, Richardson KA, Sutherland-Smith AJ - PLoS ONE (2012)

Bottom Line: Dystrophin and utrophin link the F-actin cytoskeleton to the cell membrane via an associated glycoprotein complex.This functionality results from their domain organization having an N-terminal actin-binding domain followed by multiple spectrin-repeat domains and then C-terminal protein-binding motifs.Furthermore we show that the first dystrophin and utrophin spectrin repeats have no affinity for F-actin in the absence of other domains.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular BioSciences, Massey University, Palmerston North, New Zealand.

ABSTRACT
Dystrophin and utrophin link the F-actin cytoskeleton to the cell membrane via an associated glycoprotein complex. This functionality results from their domain organization having an N-terminal actin-binding domain followed by multiple spectrin-repeat domains and then C-terminal protein-binding motifs. Therapeutic strategies to replace defective dystrophin with utrophin in patients with Duchenne muscular dystrophy require full-characterization of both these proteins to assess their degree of structural and functional equivalence. Here the high resolution structures of the first spectrin repeats (N-terminal repeat 1) from both dystrophin and utrophin have been determined by x-ray crystallography. The repeat structures both display a three-helix bundle fold very similar to one another and to homologous domains from spectrin, α-actinin and plectin. The utrophin and dystrophin repeat structures reveal the relationship between the structural domain and the canonical spectrin repeat domain sequence motif, showing the compact structural domain of spectrin repeat one to be extended at the C-terminus relative to its previously defined sequence repeat. These structures explain previous in vitro biochemical studies in which extending dystrophin spectrin repeat domain length leads to increased protein stability. Furthermore we show that the first dystrophin and utrophin spectrin repeats have no affinity for F-actin in the absence of other domains.

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Related in: MedlinePlus

Interactions formed by the C-terminus of utrophin and dystrophin spectrin repeat one domains.Structural representations of A) Utr-SR1, B) Utr-SR1-L and C) Dys-SR1 showing the burial of hydrophobic sidechains (stick representation) on the B helix (green) and A–B loop (orange) by the C-terminus of helix C (red). The A helix is coloured blue. The extended Utr-SR1-L C-terminus and sidechains of the SR2 heptad repeat (L427, L430) are coloured yellow. The protein main-chain is depicted in ribbon representation with key side-chains shown as sticks.
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pone-0040066-g003: Interactions formed by the C-terminus of utrophin and dystrophin spectrin repeat one domains.Structural representations of A) Utr-SR1, B) Utr-SR1-L and C) Dys-SR1 showing the burial of hydrophobic sidechains (stick representation) on the B helix (green) and A–B loop (orange) by the C-terminus of helix C (red). The A helix is coloured blue. The extended Utr-SR1-L C-terminus and sidechains of the SR2 heptad repeat (L427, L430) are coloured yellow. The protein main-chain is depicted in ribbon representation with key side-chains shown as sticks.

Mentions: Based on sequence analysis Dys repeat 1 has been previously defined as amino acids 338–446/7 [13], [14]. The compact structural domain of the Dys-SR1 (338–453) and Utr-SR1 (308–423) crystal structures is extended by six residues at the C-terminus of helix C. The Dys/Utr-SR1 structures finish with a break in the heptad repeat that coincides with a two residue gap in the Koenig sequence alignment [13], with the heptad repeat discontinuous from the C-terminus of helix C SR1 through the linker to helix A’ of SR2 although the helix itself is continuous into SR2 as evident from the extended Utr-SR1-L structure. The discontinuity of the heptad repeat at the C-terminal extension linker region is now explained as this region does not form helix-helix interactions; instead the extra helical turn and half at the C-terminus of helix C packs against the interior hydrophobic face of the SR1 A-B loop (fig. 3). The heptad repeat of Utr-SR1 helix C finishes with L422 (Dys L452) in the heptad ‘a’ position while the heptad repeat of SR2 helix A’ starts with L427 (Dys L457) in position ‘a’. From a tertiary domain structural perspective, including the linker region at the C-terminus of SR1 helix C rather than the N-terminus of helix A’ of SR2 results in a compact spectrin repeat structure for Utr/Dys-SR1. The extended C helix C-terminus is positioned to stabilise the hydrophobic face of the interior of the A–B loop; L415 (Dys L445) and L422 (Dys L452) in heptad ‘a’ repeat position and A418 (Dys V448) in ‘d’ position of the C helix pack against I338 (Dys I368), V342 (Dys V372) and V345 (Dys V375), from the A–B loop (fig. 3). The hydrophobic interface presented by the extension to helix C, provides a structural explanation of why longer spectrin repeat constructs than those suggested by sequence alignment are required for stable Utr/Dys spectrin repeats; shorter constructs would presumably leave the hydrophobic surface of the A–B loop solvent exposed reducing polypeptide stability. By homology the extended C-terminus of SR1 suggests that the compact domain structure for Dys-SR2 extends to 454–561/562 providing a rationale for the previously observed increased stability of the Dys-SR2 439–564 polypeptide relative to 439–553 and 448–556 [19], [21], [29]. A similar argument can be made with respect to the increased stability observed for an extended Dys-SR7-9 construct [30]. For β-spectrin the linker region between R8 and R9 interacts with both the A–B loop of R8 and the B’–C’ loop of R9 [35], so likewise for Utr and Dys the repeats can be considered to be overlapped by one and a half helical turns of the linker region from a structural domain perspective.


The crystal structures of dystrophin and utrophin spectrin repeats: implications for domain boundaries.

Muthu M, Richardson KA, Sutherland-Smith AJ - PLoS ONE (2012)

Interactions formed by the C-terminus of utrophin and dystrophin spectrin repeat one domains.Structural representations of A) Utr-SR1, B) Utr-SR1-L and C) Dys-SR1 showing the burial of hydrophobic sidechains (stick representation) on the B helix (green) and A–B loop (orange) by the C-terminus of helix C (red). The A helix is coloured blue. The extended Utr-SR1-L C-terminus and sidechains of the SR2 heptad repeat (L427, L430) are coloured yellow. The protein main-chain is depicted in ribbon representation with key side-chains shown as sticks.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3401230&req=5

pone-0040066-g003: Interactions formed by the C-terminus of utrophin and dystrophin spectrin repeat one domains.Structural representations of A) Utr-SR1, B) Utr-SR1-L and C) Dys-SR1 showing the burial of hydrophobic sidechains (stick representation) on the B helix (green) and A–B loop (orange) by the C-terminus of helix C (red). The A helix is coloured blue. The extended Utr-SR1-L C-terminus and sidechains of the SR2 heptad repeat (L427, L430) are coloured yellow. The protein main-chain is depicted in ribbon representation with key side-chains shown as sticks.
Mentions: Based on sequence analysis Dys repeat 1 has been previously defined as amino acids 338–446/7 [13], [14]. The compact structural domain of the Dys-SR1 (338–453) and Utr-SR1 (308–423) crystal structures is extended by six residues at the C-terminus of helix C. The Dys/Utr-SR1 structures finish with a break in the heptad repeat that coincides with a two residue gap in the Koenig sequence alignment [13], with the heptad repeat discontinuous from the C-terminus of helix C SR1 through the linker to helix A’ of SR2 although the helix itself is continuous into SR2 as evident from the extended Utr-SR1-L structure. The discontinuity of the heptad repeat at the C-terminal extension linker region is now explained as this region does not form helix-helix interactions; instead the extra helical turn and half at the C-terminus of helix C packs against the interior hydrophobic face of the SR1 A-B loop (fig. 3). The heptad repeat of Utr-SR1 helix C finishes with L422 (Dys L452) in the heptad ‘a’ position while the heptad repeat of SR2 helix A’ starts with L427 (Dys L457) in position ‘a’. From a tertiary domain structural perspective, including the linker region at the C-terminus of SR1 helix C rather than the N-terminus of helix A’ of SR2 results in a compact spectrin repeat structure for Utr/Dys-SR1. The extended C helix C-terminus is positioned to stabilise the hydrophobic face of the interior of the A–B loop; L415 (Dys L445) and L422 (Dys L452) in heptad ‘a’ repeat position and A418 (Dys V448) in ‘d’ position of the C helix pack against I338 (Dys I368), V342 (Dys V372) and V345 (Dys V375), from the A–B loop (fig. 3). The hydrophobic interface presented by the extension to helix C, provides a structural explanation of why longer spectrin repeat constructs than those suggested by sequence alignment are required for stable Utr/Dys spectrin repeats; shorter constructs would presumably leave the hydrophobic surface of the A–B loop solvent exposed reducing polypeptide stability. By homology the extended C-terminus of SR1 suggests that the compact domain structure for Dys-SR2 extends to 454–561/562 providing a rationale for the previously observed increased stability of the Dys-SR2 439–564 polypeptide relative to 439–553 and 448–556 [19], [21], [29]. A similar argument can be made with respect to the increased stability observed for an extended Dys-SR7-9 construct [30]. For β-spectrin the linker region between R8 and R9 interacts with both the A–B loop of R8 and the B’–C’ loop of R9 [35], so likewise for Utr and Dys the repeats can be considered to be overlapped by one and a half helical turns of the linker region from a structural domain perspective.

Bottom Line: Dystrophin and utrophin link the F-actin cytoskeleton to the cell membrane via an associated glycoprotein complex.This functionality results from their domain organization having an N-terminal actin-binding domain followed by multiple spectrin-repeat domains and then C-terminal protein-binding motifs.Furthermore we show that the first dystrophin and utrophin spectrin repeats have no affinity for F-actin in the absence of other domains.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular BioSciences, Massey University, Palmerston North, New Zealand.

ABSTRACT
Dystrophin and utrophin link the F-actin cytoskeleton to the cell membrane via an associated glycoprotein complex. This functionality results from their domain organization having an N-terminal actin-binding domain followed by multiple spectrin-repeat domains and then C-terminal protein-binding motifs. Therapeutic strategies to replace defective dystrophin with utrophin in patients with Duchenne muscular dystrophy require full-characterization of both these proteins to assess their degree of structural and functional equivalence. Here the high resolution structures of the first spectrin repeats (N-terminal repeat 1) from both dystrophin and utrophin have been determined by x-ray crystallography. The repeat structures both display a three-helix bundle fold very similar to one another and to homologous domains from spectrin, α-actinin and plectin. The utrophin and dystrophin repeat structures reveal the relationship between the structural domain and the canonical spectrin repeat domain sequence motif, showing the compact structural domain of spectrin repeat one to be extended at the C-terminus relative to its previously defined sequence repeat. These structures explain previous in vitro biochemical studies in which extending dystrophin spectrin repeat domain length leads to increased protein stability. Furthermore we show that the first dystrophin and utrophin spectrin repeats have no affinity for F-actin in the absence of other domains.

Show MeSH
Related in: MedlinePlus