Abstract
Plakin repeat domains (PRDs) are globular modules that mediate the interaction of plakin proteins with the intermediate filament (IF) cytoskeleton. These associations are vital for maintaining tissue integrity in cardiac muscle and epithelial tissues. PRDs are subject to mutations that give rise to cardiomyopathies such as arrhythmogenic right ventricular cardiomyopathy, characterised by ventricular arrhythmias and associated with an increased risk of sudden heart failure, and skin blistering diseases. Herein, we have examined the functional and structural effects of 12 disease-linked missense mutations, identified from the human gene mutation database, on the PRDs of the desmosomal protein desmoplakin. Five mutations (G2056R and E2193K in PRD-A, G2338R and G2375R in PRD-B and G2647D in PRD-C) rendered their respective PRD proteins either fully or partially insoluble following expression in bacterial cells. Each of the residues affected are conserved across plakin family members, inferring a crucial role in maintaining the structural integrity of the PRD. In transfected HeLa cells, the mutation G2375R adversely affected the targeting of a desmoplakin C-terminal construct containing all three PRDs to vimentin IFs. The deletion of PRD-B and PRD-C from the construct compromised its targeting to vimentin. Bioinformatic and structural modelling approaches provided multiple mechanisms by which the disease-causing mutations could potentially destabilise PRD structure and compromise cytoskeletal linkages. Overall, our data highlight potential molecular mechanisms underlying pathogenic missense mutations and could pave the way for informing novel curative interventions targeting cardiomyopathies and skin blistering disorders.
Highlights
Desmoplakin is a cytoplasmic desmosomal protein that plays a central role in intercellular adhesion [1]
All three wild-type plakin repeat domains (PRDs) were detected in the soluble fraction of the E. coli cells (Figure 2), indicative of a correctly folded protein
An examination of the desmoplakin PRD-B models encompassing G2338R and G2375R variants reveal that R2338 may clash with PR4 (Figure 6B, right panel), whereas R2375 is likely to protrude against the N-terminal PR-like motif (Figure 6C, right panel), leading to potential structural vulnerabilities within the PRD fold
Summary
(red with sphere) to a(right sharp turn in panel). (B) Assessing the effect of disease-associated variant G2338R on desmoplakin PRD-B. (red with sphere) to a(right sharp turn in panel). Introduction of R2056 leads to substantial clashes with the Nt-PR-like motif (right bon diagram of the desmoplakin PRD-B crystal structure shows that G2338 (red sphere) contributes panel). G2338R onclashes desmoplakin to a sharp in PR2 (left panel).ofIntroduction of R2338 variant leads to substantial with PR4PRD-B. G2375Rthat on desmoplakin of the PRD-BIntroduction crystal structure shows leads that G2375. Of R2338 to substantial clashes with to PR4 (right a sharp turn in PR3 (left panel). Ribbon diagram of thePRD-B desmoplakin crystal structure highlighting the electrostatic sharp turn in PR3
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