Structural basis of domain destabilization by dilated-cardiomyopathy-causing missense mutations in titin

Truncating variants in the titin gene are the most common cause of familial dilated cardiomyopathy (DCM), accounting for 25% of the genotype-positive cases. Similar to other genetic diseases triggered by gene loss of function, it has been proposed that destabilizing titin missense mutations can also result in DCM. Indeed, the highly destabilizing C3575S mutation targeting immunoglobulin-like (Ig) 21 domain of cardiac titin has been recently identified as a cause of DCM. However, the effects of the mutation on the structure of the parent domain and whether altered disulfide bond formation contribute to the destabilizing properties of the mutation remain unexplored. Here, using high-resolution X-ray crystallography, we report that the structure of the C3575S mutant domain is largely unperturbed (RMSD: 0.538 Å). In addition, using gel-shift assays, we have ruled out that the lower thermal stability of the mutant domain results from its inability to establish intramolecular disulfide bonds. Instead, we propose that the lower hydrophobicity of serine is not well accommodated in the core of mutant domains, causing their destabilization. Finally, we have observed that similar missense mutations, which cause a decrease in hydrophobicity in buried positions of titin Ig domains, are enriched in DCM patients. Altogether, our results illustrate that subtle physicochemical changes induced by titin missense mutations can cause domain destabilization, even in the absence of remarkable structural alterations, resulting in development of DCM.