Journal of Molecular Biology
The Nonlinear Structure of the Desmoplakin Plakin Domain and the Effects of Cardiomyopathy-Linked Mutations
Graphical Abstract
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
References (47)
- et al.
Desmosome structure, composition and function
Biochim. Biophys. Acta
(2008) - et al.
Plakins in development and disease
Exp. Cell Res.
(2007) - et al.
The structure of a tandem pair of spectrin repeats of plectin reveals a modular organization of the plakin domain
J. Mol. Biol.
(2007) - et al.
Genetic diseases of junctions
J. Invest. Dermatol.
(2007) - et al.
Striate palmoplantar keratoderma resulting from desmoplakin haploinsufficiency
J. Invest. Dermatol.
(1999) - et al.
Structural analysis of the plakin domain of bullous pemphigoid antigen 1 (BPAG1) suggests that plakins are members of the spectrin superfamily
J. Mol. Biol.
(2007) - et al.
States and transitions during forced unfolding of a single spectrin repeat
FEBS Lett.
(2000) - et al.
The structure of the plakin domain of plectin reveals a non-canonical SH3 domain interacting with its fourth spectrin repeat
J. Biol. Chem.
(2011) - et al.
Structure of the human desmoplakins. Implications for function in the desmosomal plaque
J. Biol. Chem.
(1990) - et al.
Crystal structure of a rigid four-spectrin-repeat fragment of the human desmoplakin plakin domain
J. Mol. Biol.
(2011)
Thermofluor-based high-throughput stability optimization of proteins for structural studies
Anal. Biochem.
Global rigid body modeling of macromolecular complexes against small-angle scattering data
Biophys. J.
A biophysical map of the dystrophin rod
Biochim. Biophys. Acta
Crystal structure of the alpha-actinin rod reveals an extensive torsional twist
Structure
Structures of two repeats of spectrin suggest models of flexibility
Cell
Conformational stabilities of the structural repeats of erythroid spectrin and their functional implications
J. Biol. Chem.
Pathway shifts and thermal softening in temperature-coupled forced unfolding of spectrin domains
Biophys. J.
A two-amino acid mutation encountered in Duchenne muscular dystrophy decreases stability of the rod domain 23 (R23) spectrin-like repeat of dystrophin
J. Biol. Chem.
Nuclear magnetic resonance studies of mutations at the tetramerization region of human alpha spectrin
Blood
Macromolecular size-and-shape distributions by sedimentation velocity analytical ultracentrifugation
Biophys. J.
Multi-resolution contour-based fitting of macromolecular structures
J. Mol. Biol.
The desmoglein-specific cytoplasmic region is intrinsically disordered in solution and interacts with multiple desmosomal protein partners
J. Mol. Biol.
Structures of two intermediate filament-binding fragments of desmoplakin reveal a unique repeat motif structure
Nat. Struct. Biol.
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Desmoplakin is required for epidermal integrity and morphogenesis in the Xenopus laevis embryo
2019, Developmental BiologyCitation Excerpt :Full-length (Desmoplakin) Dsp protein and mRNA sequences for Homo sapiens Desmoplakin I (NP_004406), Mus musculus Desmoplakin (NP_076331) and Danio rerio Desmoplakin isoform X1 (XP_001919901) X. laevis Dsp.L (XB-GENE-866134, Genome Build 9.1, http://www.xenbase.org), were aligned using the LALIGN tool (EMBL-EBI) and the EMBOSS Water tool (Smith-Waterman algorithm) (EMBL-EBI) was used to determine similarity. Protein domains were based on those identified in the human Dsp protein (Al-Jassar et al., 2011; Green et al., 1990; Virata et al., 1992). Embryos were processed for EM at the VCU microscopy core using standard protocols.
The structure of the plakin domain of plectin reveals an extended rod-like shape
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2016, Methods in EnzymologyCitation Excerpt :The SR7–8 model and the crystal structure of SR3–6 fit the short and long arms of the DP(180–1022) SAXS envelope (Fig. 2). Limited proteolysis of DPNT produces stable fragments corresponding to SR3–6 and SR7–8 (Al-Jassar et al., 2011; Choi & Weis, 2011), implying a flexible linker between SR3–6 and SR7–8 corresponding to the unmodeled “elbow” in the SAXS envelope. Recently, the ensemble optimization method of Bernardo, Mylonas, Petoukhov, Blackledge, & Svergun (2007) was used to investigate the flexibility of the SR6–SR7 hinge (Al-Jassar, Bernado, et al., 2013).
Structural insights into the activation of the RhoA GTPase by the lymphoid blast crisis (Lbc) oncoprotein
2014, Journal of Biological ChemistryMechanistic basis of desmosome-targeted diseases
2013, Journal of Molecular BiologyCitation Excerpt :Pathogenic mutations at conserved SH3 positions including S299R, N375I, I445V, and S507F are clustered near the SR4 binding site and are predicted to disrupt core stability and interdomain contact or both. Another mutation, K470E, occupies an exposed loop position and does not cause significant destabilization of the domain [121,122]. Other pathogenic mutations scattered along the length of the SR3–6 rod include R222L, D230N, N287K, N375I, E422K, S442F, I445V, N458Y, Y494F, S507F, S597L, and H618P (Fig. 5).
Towards a Better Understanding of Genotype–Phenotype Correlations and Therapeutic Targets for Cardiocutaneous Genes: The Importance of Functional Studies above Prediction
2022, International Journal of Molecular Sciences
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Present address: K. Kami, Riken Systems and Structural Biology Center, Yokohama, Japan.