Abstract 63: Missense Mutation Lys18Asn in Dystrophin that Triggers X-Linked Cardiomyopathy Affects Protein Structure, Decreases Protein Stability and Increases Protein Unfolding

Abstract

A genetic mutation in dystrophin that replaces lysine at amino acid position 18 by an asparagine (K18N) results in X-linked cardiomyopathy (XCM), however, the underlying molecular mechanisms are not known. Such knowledge might help in developing effective therapies to treat XCM. The first step in understanding disease-triggering mechanisms is to probe how mutation affects protein structure and function. The K18N mutation occurs in the N-terminal actin binding domain (N-ABD) of dystrophin. Here, we examined the effect of K18N on the structure, stability, and function of N-ABD. Fig. 1A shows the 15 N- 1 H HSQC NMR spectrum before and after the mutation. Each crosspeak in the spectrum represents the structure around an amino acid. Mutation affects the position of nearly all the crosspeaks. These changes are not just located at the mutation site, but were observed in regions far away from the mutation site and also in the three actin binding regions with which N-ABD interacts with actin. In addition, the mutation makes the protein more dynamic. These structural changes result in decreased actin binding function of N-ABD. Also, the K18N mutation decreases the stability by ΔΔG = 5.3 kcal/mol (Fig. 1B) and increases the rate of unfolding by 3.2 times (Fig. 1C), which exponentially increase the relative population of unfolded state that is more prone to proteolysis and hence will result in decreased levels of the functional protein. These results indicate that the physical mechanism by which the K18N mutation triggers the disease might be by affecting protein structure leading to decreased actin binding and by decreasing the net protein levels leading to decreased net function.