Journal of Molecular Biology
Volume 411, Issue 5, 2 September 2011, Pages 1049-1061
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The Nonlinear Structure of the Desmoplakin Plakin Domain and the Effects of Cardiomyopathy-Linked Mutations

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Abstract

Desmoplakin is a cytoplasmic desmosomal protein that plays a vital role in normal intercellular adhesion. Mutations in desmoplakin can result in devastating skin blistering diseases and arrhythmogenic right ventricular cardiomyopathy, a heart muscle disorder associated with ventricular arrhythmias, heart failure, and sudden death. The desmoplakin N-terminal region is a 1056-amino-acid sequence of unknown structure. It mediates interactions with other desmosomal proteins, is found in a variety of plakin proteins, and spans what has been termed the “plakin domain,” which includes residues 180–1022 and consists of six spectrin repeats (SRs) and an Src homology 3 domain. Herein we elucidate the architecture of desmoplakin's plakin domain, as well as its constituent tandem SRs. Small-angle X-ray scattering analysis shows that the entire plakin domain has an “L” shape, with a long arm and a short arm held at a perpendicular angle. The long arm is 24.0 nm long and accommodates four stably folded SRs arranged in tandem. In contrast, the short arm is 17.9 nm in length and accommodates two independently folded repeats and an extended C-terminus. We show that mutations linked to arrhythmogenic right ventricular cardiomyopathy (K470E and R808C) cause local conformational alterations, while the overall folded structure is maintained. This provides the first structural and mechanistic insights into an entire plakin domain and provides a basis for understanding the critical role of desmoplakin in desmosome function.

Introduction

Desmosomes are intercellular junctions of epithelial tissues and cardiac muscles. They resist mechanical stress and play a crucial role in the maintenance of tissue architecture.1 Desmoplakin, a member of the plakin family of cytolinker proteins,2 is of critical importance for desmosome structure and function. It is tripartite in nature, with head and tail domains flanking a central rod region (Fig. 1). The desmoplakin N-terminal region associates with other desmosomal proteins such as plakoglobin and plakophilin (PKP). The central coiled-coil region mediates dimerization, and the tail interacts with intermediate filaments through three homologous plakin repeat domains, designated A, B, and C (Fig. 1).4 Hence, desmoplakin provides a vital link between other proteins of the desmosome and the intermediate filament cytoskeleton. Severance of this link results in loss of cell–cell adhesion and severely compromises tissue integrity.5

Mutations in the desmoplakin gene result in an array of human diseases that affect the integrity of the skin or heart or, in some cases, both (reviewed by Lai-Cheong et al.6). Dominant heritable mutations in desmoplakin cause striate palmoplantar keratoderma7, 8 and arrhythmogenic right ventricular cardiomyopathy (ARVC), one of the most prevalent cardiomyopathies and a common cause of ventricular arrhythmias and sudden death. It afflicts 1 in 5000 people and is characterized by the replacement of cardiomyocytes in the right ventricle with fibrofatty tissues. Clinical manifestations include right ventricular enlargement and dysfunction, life-threatening arrhythmias, and sudden cardiac death.9 Pathogenic ARVC mutations occur throughout the desmoplakin gene, including the desmoplakin N-terminal region†.10 The latter includes a sequence that is predicted to be unstructured (residues 1–179), and the plakin domain, which is shared by other members of the plakin family of proteins, including envoplakin and periplakin.2 Two ARVC mutations (V30M and Q90R) are localized within the unstructured sequence and are known to affect binding of plakoglobin.11 However, the mechanisms through which plakin domain mutations compromise tissue integrity are unclear and would benefit from a structural understanding of this hot spot for pathogenic mutations.

The solution structure of the plakin domain, including its interdomain juxtapositions, flexible linkers, and stabilities, remains uncharacterized. The conserved architecture of the plakin domain contains a series of spectrin repeats (SRs) and a single Src homology 3 (SH3) domain of unknown function. Each SR is composed of a bundle of three helices, as revealed by crystal structures of the tandem SR sets of spectrin, bullous pemphigoid antigen 1, and plectin.3, 12, 13 Atomic force microscopy studies of tandem arrays of SRs reveal that they can fold and unfold individually and independently.14 How multiple SRs orient in solution remains poorly understood, as is the role of the noncanonical SH3 domain, which is found in all plakin domains. The homologous SH3 domain of plectin is atypical in that intramolecular contact with SR4 occludes its proposed binding site.15 Whether the repeats within the plakin domain form a rigid rod, flexible architecture, or modular scaffold for desmosomal protein docking remains to be defined.

In order to provide mechanistic insights into the desmoplakin architecture and the effects of ARVC mutations, we present the first structure of an entire plakin domain. This reveals an unprecedented “L”-shaped arrangement, with long and short arms consisting of quadruple and double SRs, respectively. Structural integrity is compromised by two independent ARVC mutations: one within the proposed occluded SH3 domain binding site and the other within desmoplakin's SR8. Although well-defined linear juxtapositions connect most SRs, a labile linker and a dramatic kink connect the SR6 and SR7 modules of desmoplakin, introducing a novel bend within the architecture of its plakin domain.

Section snippets

Biophysical properties of desmoplakin's SRs

In order to identify the structural and functional units of desmoplakin, we designed a series of constructs encompassing its plakin domain based on sequence conservation and limited proteolysis data (Fig. 1). Our structure-based sequence alignment (Supplementary Fig. 1) indicates that residues 654–770 constitute an SR, designated SR7, and that residues 883–1022 constitute a flexible region, designated CT (Fig. 1). By contrast, earlier sequence analysis3 suggested that SR7 was absent and that CT

Discussion

In this study, we have solved the structure of the entire plakin domain from the desmosomal protein desmoplakin. We show that the plakin domain structure has an extended but unexpectedly bent conformation, with one long arm and one short arm containing predominantly α-helical secondary structures. Furthermore, we show that two ARVC-causing mutations, including a novel mutation in SR8, cause changes in the conformation and stability of SR structures while maintaining their overall tertiary folds

Construct design

DNA encoding the wild-type plakin domain sequence from human desmoplakin (residues 180–1022) was cloned in-frame with DNA coding for GST in the expression vector pGEX-6P-1 (GE Healthcare). The plakin sequence was tagged at the 3′ end with DNA encoding residues SGHHHHHH. Residue Lys470 was altered to Glu (K470E), and residue Arg808 was altered to Cys (R808C) in the wild-type plakin domain construct to produce constructs PD-K470E and PD-R808C, respectively. Constructs encoding fragments of the

Acknowledgements

We thank Rosemary Parslow and Raul Pacheco-Gomez for advice and access to the Birmingham Biophysical Characterization Facility and the Wellcome-Trust-supported Henry Wellcome Building for Biomolecular NMR Spectroscopy, respectively. We are grateful to Dmitri Svergun and Manfred Roessle for discussions on SAXS data, William Weis for communicating results before publication, and the ARVC patients. This research was funded by the Medical Research Council (C.A.), the Biotechnology and Biological

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    Present address: K. Kami, Riken Systems and Structural Biology Center, Yokohama, Japan.

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