The structure of the plakin domain of plectin reveals a non-canonical SH3 domain interacting with its fourth spectrin repeat - PubMed (original) (raw)

The structure of the plakin domain of plectin reveals a non-canonical SH3 domain interacting with its fourth spectrin repeat

Esther Ortega et al. J Biol Chem. 2011.

Abstract

Plectin belongs to the plakin family of cytoskeletal crosslinkers, which is part of the spectrin superfamily. Plakins contain an N-terminal conserved region, the plakin domain, which is formed by an array of spectrin repeats (SR) and a Src-homology 3 (SH3), and harbors binding sites for junctional proteins. We have combined x-ray crystallography and small angle x-ray scattering (SAXS) to elucidate the structure of the central region of the plakin domain of plectin, which corresponds to the SR3, SR4, SR5, and SH3 domains. The crystal structures of the SR3-SR4 and SR4-SR5-SH3 fragments were determined to 2.2 and 2.95 Å resolution, respectively. The SH3 of plectin presents major alterations as compared with canonical Pro-rich binding SH3 domains, suggesting that plectin does not recognize Pro-rich motifs. In addition, the SH3 binding site is partially occluded by an intramolecular contact with the SR4. Residues of this pseudo-binding site and the SR4/SH3 interface are conserved within the plakin family, suggesting that the structure of this part of the plectin molecule is similar to that of other plakins. We have created a model for the SR3-SR4-SR5-SH3 region, which agrees well with SAXS data in solution. The three SRs form a semi-flexible rod that is not altered by the presence of the SH3 domain, and it is similar to those found in spectrins. The flexibility of the plakin domain, in analogy with spectrins, might contribute to the role of plakins in maintaining the stability of tissues subject to mechanical stress.

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Figures

FIGURE 1.

Crystal structure of the SR3-SR4 and SR4-SR5-SH3 regions of the plakin domain of plectin. A, schematic representation of the domain structure of plectin and a close up view on the structure of the plakin domain. The three proteins characterized in this study are aligned underneath. B and C, two orthogonal views of ribbon representations of the crystal structures of the SR3-SR4 (B) and the SR4-SR5-SH3 (C) regions. The SR4 is shown in equivalent orientations in both panels.

FIGURE 2.

The intramolecular interface between the SR4 and the SH3 domain of plectin. A, footprint of the SH3 domain and the upstream linker on the surface of the SR4. Residues in SR4 that participate in the intramolecular contact are labeled on the surface and are colored according to the percentage of the accessible surface buried as defined by the protein interfaces, surfaces, and assemblies service (PISA) (65): dark blue for residues mostly occluded (≥70% buried area) and light blue for residues partially occluded (≥20% and <70% buried area). The backbone of the upstream linker (purple) and the SH3 domain (orange) is partially shown as a wire, and the side chains of the main residues that contact the SR4 are shown as sticks and are labeled with arrows. The positions of the three α-helices of the SR4 are labeled on top of the surface and the amino-to-carboxyl direction indicated with arrowheads. B and C, stereo representations of the intramolecular contacts between the SR4 (blue), the SH3 (orange), and the upstream linker (purple). The two lateral views in panels B and C are related by a 180° rotation around a vertical axis.

FIGURE 3.

Structure of the putative binding site of the SH3 domain. A, ribbon representation of the SH3 domain of plectin. The side chains of residues in positions equivalent to those that form the binding pockets in canonical SH3 domains are shown as sticks. The side chain of Glu-846 was not modeled beyond its Cβ atom due to lack of well-defined electron density; an arbitrary conformation is shown here. B, multiple sequence alignment of the SH3 domain of human plectin (Uniprot Q15149–2) with those of other human plakins: BPAG1e (Q03001-3), MACF1 (Q9UPN3-2), desmoplakin (P15924), envoplakin (Q92817), and periplakin (O60437); plakins from invertebrates: VAB-10A from Caenorhabditis elegans (Q86NF8) and Shot from Drosophila melanogaster (A1Z9J1); and two human α-spectrins: αI-spectrin (erythrocyte, P02549), and αII-spectrin (brain, Q13813). The secondary elements of the plectin structure are shown above the alignment. Conserved residues that form the binding pockets in α-spectrin are shown with blue boxes, whereas residues of plakins unlikely to sustain binding in a canonical manner are shown with light red boxes.

FIGURE 4.

SAXS analysis of the SR3-SR4-SR5-SH3 region. A, scattering profile of SR3-SR4-SR5-SH3 (open circles). The theoretical scattering curve of the atomic model (dashed line) shows a good fit to the experimental data. B, pair-distance distribution curves estimated from the SAXS data using the program GNOM (solid line) and calculated for a hydrated model of the atomic structure (dashed line).

FIGURE 5.

Similar domain arrangement in the SR4-SR5-SH3 of plectin and the complex formed by βI-spectrin and ankyrin R. A, worm representation of the structure of the SR4-SR5-SH3 of plectin; the orientation is the same as in Fig. 1_C. B_, structure of the ZU5-ANK domain of ankyrin R bound to the SR14-SR15 of βI-spectrin (PDB code 3KBT); the SR13, which is present in the crystal structure of the complex but does not contact the ZU5-ANK domain, has been omitted. C, orthogonal views of the ankyrin/βI-spectrin and the plectin structures after superposition of the SR14 of βI-spectrin onto the SR4 of plectin. D, close-up view of the SH3/SR4 and ZU5-ANK/SR14 interfaces in plectin and the ankyrin/βI-spectrin complex, respectively. The side chain of Val-881 in the SH3 of plectin and Arg-948 in the ZU5-ANK domain occupy equivalent positions and are shown as sticks. For clarity, only a segment of the backbones around Val-881 and Arg-948 are shown.

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The structure of the plakin domain of plectin reveals a non-canonical SH3 domain interacting with its fourth spectrin repeat - PubMed (original) (raw)