Table 1.
Crystallographic x-ray data collection and refinement statistics.
Figure 1.
Utrophin and dystrophin spectrin repeat one crystal structures.
Cartoon representations of A) Utr-SR1, B) Utr-SR1-L and C) Dys-SR1 structures colour-coded helix A blue, helix B green, helix C red and A–B and B–C loops in orange. The C-terminal extension of Utr-SR1-L is coloured yellow and the position of the sequence-defined repeat C-terminus is shown with a dashed white line.
Figure 2.
Sequence alignment of Utr-SR1 and Dys-SR1 colour-coded by amino acid conservation.
The upper secondary structure cartoon shows the Utr-SR1 and Dys-SR1 crystal structure heptad repeat phasing and helix boundaries (A helix blue, B helix green and C helix red). The heptad repeat and helix boundaries for the Koenig [13] and Winder [14] sequence alignments are shown in cyan and yellow cartoon representation respectively.
Figure 3.
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.
Figure 4.
A structure for Utr-SR1-SR2 modeled from the Utr-SR1 crystal structure and a homology model for Utr-SR2.
Ribbon representation of the hybrid Utr-SR1-SR2 model containing the experimentally determined Utr-SR1-L structure combined with an I-TASSER predicted model for Utr-SR2 superposed on the overlapping region (yellow). SR1 is colour coded A helix (dark blue), B helix (green), C helix (red), extended Utr-SR1-L region (yellow). SR2 is colour-coded helix A (mid-blue), helix B (green), helix C (brown). The orientation of the SR1 domain is approximately equivalent to figure 1. The close up view of the linker regions shows the heptad repeat phasing of L427 and L430 from Utr-SR1-L at the ‘a’ and ‘d’ heptad positions of helix A’ of SR2 forming coiled-coil interactions with the Utr-SR2 domain.
Table 2.
Comparison of Utr-SR1 and Dys-SR1 to selected spectrin repeat domain structures.
Figure 5.
Cα backbone superpositions of Utr-SR1 and Dys-SR1 with selected homologous spectrin repeat structures.
A) Utr-SR1 (blue) and B) Dys-SR1 (red) superimposed on α-actinin-2 repeat 2 (cyan, 1HCI) and α-spectrin repeat 16 (magenta, 1CUN) structures.
Figure 6.
Comparison of the Utr-SR1 and Dys-SR1 structures.
A) Cα backbone superposition of the two Utr-SR1 and the two Dys-SR1 molecules of their respective crystallographic asymmetric units: Utr-SR1; molecule A (blue), molecule B (green). Dys-SR1; molecule A (red), molecule B (orange). B) Superposition of Dys-SR1 molecules A (red) and B (orange) highlighting the difference in W354 and H392 sidechain rotamers involved in the usually conserved spectrin repeat core stacking interaction.
Figure 7.
Dys-SR1 and Utr-SR1 exhibit no F-actin binding activity.
A) Utr-SR1 and B) Dys-SR1 F-actin binding co-sedimentation assay SDS-PAGE gels. Lane 1; MW markers. Lanes 2–6; S and P are supernatant and pellet fractions; actin concentration is 8 µM. A) 1S, 1P; 20 µM Utr-SR1 & F-actin. 2S, 2P; 40 µM Utr-SR1 & F-actin. 3S, 3P; F-actin & 20 µM Filamin B (FLNB) actin binding domain (ABD) as a positive control found in the pellet fraction bound to F-actin. B) 1S, 1P; 20 µM Dys-SR1 & F-actin. 2S, 2P; 40 µM Dys-SR1 & F-actin. 3S, 3P; 20 µM FLNB-ABD & 8 µM F-actin. Utr-SR1 and Dys-SR1 are found in the supernatant not associated with F-actin (which is in the pellet). Empty gel lanes are left between each sample to prevent any adjacent band overlap. C) Utr-SR1 and D) Dys-SR1 structural electrostatic surface representations. The left hand views of each pair have an orientation approximately equivalent to figure 1, the right hand views are rotated ∼180° around the vertical axis. The surface has been colour coded by electrostatic potential, red −0.5 V to blue 0.5 V.