The Structure of the Plakin Domain of Plectin Reveals a Non-canonical SH3 Domain Interacting with Its Fourth Spectrin Repeat

Esther Ortega,Rubén M Buey,Arnoud Sonnenberg,José M De Pereda

doi:10.1074/jbc.m110.197467

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.

Highlights

Plectin is a highly versatile plakin that associates with intermediate filaments [3], microtubules [4], and actin fibers [5], and crosslinks these cytoskeletal networks [5,6]
Plectin connects intermediate filaments to membrane-associated complexes. In stratified epithelia such as the skin, plectin is localized at the hemidesmosomes, which are junctional complexes that link the intermediate filaments to the basement membrane, and links the integrin ␣6␤4 and the bullous pemphigoid antigen 2 (BPAG2, known as type XVII collagen or BP180) to the cytokeratins [7,8]
Initial analysis of the sequence of the plakin domain suggested that this region contains multiple ␣-helical bundles [27], some of which were later identified as spectrin repeats (SR) [28]

Summary

Introduction

Plectin is a highly versatile plakin that associates with intermediate filaments [3], microtubules [4], and actin fibers [5], and crosslinks these cytoskeletal networks [5,6]. The plakin domain of plectin and other plakins contain protein-protein interaction sites and they are important for the localization of plakins at junctional complexes In plectin this region harbors binding sites for the integrin ␤4 subunit [24], the cytoplasmic domain of BPAG2 [25], ␤-dystroglycan [13], ␤-synemin [15], and the tyrosine kinase Fer [26]. The C-terminal region contains six plakin repeat domains and harbors binding sites for intermediate filaments. The SR2 and SR3 of plectin are connected by a ϳ20residues long linker predicted to be non-helical, while repeats SR3 to SR9 occur contiguous in the plectin sequence as observed in the structure of the SR3-SR4 pair of BPAG1 [29]. Our results have implications for the contribution of the plakin domain of plectin and other plakins to the stability of junctional complexes subject to mechanical forces

Results

Discussion

Conclusion